Delegates’ Abstracts (Students)

 

Date of Web Publication

10-Feb-2015

 

Correspondence Address:

Source of Support: None, Conflict of Interest: None

How to cite this URL:
. Delegates’ Abstracts (Students). J Carcinog [serial online] 2015 [cited 2021 Oct 13];14, Suppl S1:21-38. Available from: https://carcinogenesis.com/text.asp?2015/14/2/21/151182
S1 Alkylation damage induces transformation in three-dimensional breast acini cultures of non-malignant breast epithelial cells

Libi Anandi V ¹ , and Mayurika Lahiri <sup>1

1</sup> Indian Institute of Science Education and Research, Pune, India. Corresponding author: Mayurika Lahiri, Department of Biology, Indian Institute of Science Education and Research (IISER), Pune, India

Loss of cellular architecture and polarity of breast tissue is one of the early markers for onset of breast cancer. This loss in cellular morphology has been phenocopied using three-dimensional (3D) cultures of human mammary epithelial cells, MCF10A. MCF10A are immortalized, non-transformed and non-malignant human mammary epithelial cells that when grown in 3D matrices, exhibit a number of features of normal breast epithelium. The morphology of these acini gets disrupted in malignancy, such as an increase in size and elongation of acini. As transformation progresses, the acini loose their polarization and some may even form multi-acinar structures. Using this model system, it was observed that damage induced by N-methyl-N-nitrosourea (MNU),a SN1 type DNA methylating agent which forms the modified base O6-methylguanine,caused disruption of the well-formed polarized spheroid architectureas observed using various polarity markers such as .6-integrin, GM-130, laminin V and cell-cell junction markers such as .-catenin andE-cadherin. Interestingly, an up-regulation of Vimentin was also observed in the breast acini. Loss in the ability of the damaged cells to form tubular structures with cytoplasmic localization of Laminin V was observed in the Collagen-Matrigel invasion assays. A gain of an elongated morphology was also observed in the treated cells, an indication of attainment of a mesenchymal phenotype. The disrupted spheroids were observed to have fewer cells with larger nuclear volume but the overall spheroid volume was similar to that of the untreated. Thus, MNU has the potential to cause early transformation in MCF10A cells grown as 3D cultures. The model proposed here, thus can be further used to study early events of chemical-induced transformation.

S2 Identification of 7q21.12-22.3 candidate genes and their association with Src/Ras/Akt signaling cascades in gastric cancer

Sembulingam Tamilzhalagan, Muthulakshmi Muthuswami, Jayaprakash Periasamy, Kumaresan Ganesan*

Cancer Genetics Laboratory, Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, India. *Corresponding author: Kumaresan Ganesan, Email: kumar@oncocellomics.org

Gastric cancer (GC) is one of the leading causes of cancer associated deaths world-wide. Genomic aberration is the hallmark of cancers. Through copy number variation (CNV) study, recurrent amplification in 7q21.12-q22.3 region was observed in 22% of gastric cancer cell lines. TCGA and other primary gastric tumor samples’ CNV analysis also confirm the presence of this amplification in at-least 10% of patients. From the 159 genes present in 7q21.12-q22.3 region, many genes are expressed in amplification driven manner. A comprehensive analysis in both cell lines and tumor samples show 12 genes are over-expressed, consistently. Based on signature based pathway scoring and RNAi based in vitro pathway screening of these genes, we have identified 5 key genes to play key roles in gastric cancer. The identified genes found to have association with activated Src/Ras/Akt signaling cascade and suppressed p53 function in primary gastric tumors. A representative candidate gene was further investigated for its oncogenic features and reported to confer proliferation advantage through G2-M progression, micronuclei formation, inhibiting apoptosis, and conferring resistance to selected chemotherapeutic agents. Thus the key genes playing crucial role by 7q21.12-q22.3 amplification in GC and their cellular and molecular characteristics are defined for next generation gastric cancer therapeutics.

S3 FASL -844T>C promoter polymorphism in chronic myeloid leukemia

Prajitha E.M. ¹ , G. Manjula ¹ , K. Sailaja ¹ , Sugunakar V. ¹ , Anuradha C. ¹ , Santhoshi Rani N. ¹ , PhanniBhushann M. ¹ , Sarika J. ¹ , Sandhya A. ¹ , D. Raghunadha Rao ² , S. Vishnupriya ¹ . *

¹ Department of Genetics, Osmania University, Hyderabad; ² Homi Bhabha Cancer Hospital and Research Centre, Vizag, Andhra Pradesh. *Corresponding author: S. Vishnupriya.

Background: Bcr-Abl cells, despite the use of targeted drug, progresses into advanced phase through accumulation of Bcr-Abl mutations, activation of downstream pathways and failure of repair and apoptosis. Fas ligand (FASL/CD95L) initiates apoptosis by binding to its surface receptor, FAS.A T>C transition at −844, located in a binding motif for CEBP-β was found to be significantly associated withhigher basal expression of FASL. Increased expression of FasL has been detected in various cancers. Hence, the study was planned to find out the association of FASL -844T>C polymorphism with CML. Materials and Methods: 772 DNA samples, 386 CML cases (NIMS) and 386 age and gender matched controls (local population) were genotyped for FASL -844 T>C polymorphism by PCR-RFLP method and statistical analysis (SNPSTATs and SPSS) were performed. Results: The present studyrevealed a significant association of FASL CC genotype (OR-1.92, 95% CI, 1.38-2.66) and FASL CT genotype (OR-1.91, 95% CI, 1.22-3.00) (p-2e-04*) with CML. A borderline significant association (p-0.07) was observed between FasL -844TC and Partial Hematological Response (OR-1.04, 95% CI- 0.50-2.17). Survival analysis also showed an increase in mean EFS for FASL -844CC (33.489΁5.83) compared to other genotypes. Conclusion: The study suggest that FASL -844T>C polymorphism influence the development of CML.

S4 Changes in the methylation pattern of p53 gene promoter in the megaloblastic bone marrow disease

Manish K. Yadav ^#1 , Dr. Nandini N. Manoli ^# *, Insha Aman ^$ , Dr. SubbaRao V. Madhunapantula ^$ , Dr. Manjunath G.V. ^# , and Dr Suma M. Natraj <sup>$

#1</sup> Department of Pathology; ^$ Centre of Excellence in Molecular Biology and Regenerative Medicine (CEMR), Department of Biochemistry; Jagadguru Sri Shivarathreeshwara Medical College, Jagadguri Sri Shivarathreeshwara University, Mysore-570015, India. *Corresponding author: Dr. Nandini N. Manoli.

Megaloblastic anaemia, a disease originated by faulty DNA synthesis leading to abnormal maturation of haematopoietic cells, is characterized by the presence of abnormal, very large sized megaloblasts harboring a fine reticular nuclear structure. Prior studies have shown the deficiency of cobalamin (Vitamin B12 – VitB12) and Folic Acid (FA), which are essential for DNA biosynthesis, is the primary cause for megaloblastic bone marrow diseases. However, detailed mechanism(s) describing how these vitamin deficiencies transform a normoblast in to megaloblast are currently lacking. In addition, it is also unknown whether these vitamin deficiencies induce changes in promoter DNA methylation of genes, such as p53 (the guardian of the genome, involved in regulating cell cycle and apoptosis. Therefore, in this study we have estimated and compared the VitB12 and Folic Acid levels of megaloblastic bone marrow patients with that of control group and determined the changes in methylation pattern of P53 gene in bone marrow aspirate using methylation specific PCR. Preliminary findings identified low and very low levels of FA and VitB12 in megaloblastic anaemic patients compared to control individuals (FA >5.38 ng/ml, VitB12 – 211-911 pg/ml). The p53 promoter methylation and expression data will be presented at the time of presentation.

S5 Inhibition of pancreatic cancer cell line (MiaPaCa-2) by a novel approach: in situ allicin generation using targeted alliinase delivery

Sagar Chhabria ¹ , and Krutika Desai <sup>2

1</sup> Department of Biological Sciences; ² Department of Microbiology; SVKM’s Mithibai College, Vile Parle (W), Mumbai-400056, India. Email id: sagarchhabria7@gmail.com

Allicin (Diallyl Thiosulfinate), a highly active component in extracts of freshly crushed garlic, is the interaction product of non-protein amino acid alliin (S-allyl-L-cysteine sulfoxide) with the enzyme alliinase (Alliin lyase; EC 4.4.1.4). Allicin has been shown to have an anti-proliferative effect on cancer cells. This anti-proliferative effect of allicin was used to develop a novel approach to cancer treatment-based on site-directed generation of allicin (i.e. tumour targeted cytotoxicity). Alliinase purified from Cupriavidus necator was conjugated to a Monoclonal Antibody directed against a specific pancreatic cancer marker, CA19-9. This CA19-9 Antibody-Alliinase conjugate was bound to target pancreatic cancer cells. Addition of the substrate-alliin; resulted in the production of allicin which effectively killed CA19-9-expressing cells-MiaPaCa-2 cell line via. caspase dependent apoptosis by increasing ROS levels and up- regulating P21 ^Waf1/Cip1 expression. Moreover, using in-vitro cytotoxicity assays, we demonstrated for the first time, a high antitumor activity of allicin that was produced in situ by the conjugate, on alliin administration in vitro. This approach to cancer treatment, based on site-directed, tumour targeted generation of allicin, was developed and assessed by implementing IdMOC ^TM technology.

S6 Mdm2 gene (309 T/G) polymorphism in breast cancer

N. Santhoshi Rani ¹ , D. Surekha ¹ , Phanibhusan M. ¹ , Chiranjeevi Padala ¹ , T. Nageswara Rao ¹ , Anuradha C. ¹ , Sugunakar V. ¹ , Manjula G. ¹ , Prajitha E.M. ¹ , Sandhya A. ¹ , D. Raghunadha Rao ² , Satti Vishnupriya ^1, *

¹ Department of Genetics, Osmania University, Hyderabad; ² Nizam’s Institute of Medical Sciences, Hyderabad. *Corresponding author: Satti Vishnupriya; Presenting author email id: santhoshi.genetics@gmail.com

Background: MDM2 gene is negative regulator of the P53 gene, which plays significant role in cell cycle regulation/ DNA repair and apoptosis. MDM2 309 T/G polymorphism located in the promoter region was found to influence the expression of the gene. Hence, the present study was planned to determine the significance of MDM2-309 T/G polymorphism with Breast cancer development and to correlate with clinical parameters like receptor (ER, PR and HER2), lymph node status and stage of the cancer. Materials and Methods: The data consists of 301 Breast cancer samples collected from Nizam’s institute of Medical sciences, Hyderabad and 300 age matched healthy female controls from different rural and urban areas. Genomic DNA isolated by non-enzymatic/ salting out method was used for genotyping of MDM2309T>G polymorphism by ARMS-PCR. Statistical analysis was performed by using SNP stat and Medcalc software. Results: The TG, GG genotypes (p-0.0006) and G (p-0.001) allele were significantly associated with breast cancer and conferred risk by 1.66 fold [95% CI: 1.16-2.39], 1.71 fold [95% CI: 1.10-2.65] and 1.47 fold [95% CI: 1.16-1.86] respectively. The GG genotype was significantly association with tumor size >50nm [OR 1.90, 95% CI (1.05-3.43)], advanced stage [OR 1.91, 95% CI (1.02-3.57)] and with Her2 positive status [OR 1.82, 95%CI (1.35-2.14)]. Conclusions: The GG genotype of -309T>G polymorphism might influence breast cancer development and progression.

S7 Validation of altered expression of genes on 11q22 amplicon and its association with clinical outcome in OSCC patients

Priyanka Bhosale ¹ , Mickey Shah ¹ , Asawari Patil ² , Rajiv S. Desai ³ , Shubhada Kane ² , Manoj B. Mahimkar ^1, *

¹ Cancer Research Institute, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai; ² Tata Memorial Hospital, Parel, Mumbai; ³ Nair Dental College and Hospital, Mumbai Central, Mumbai. *Corresponding author: Dr. Manoj B. Mahimkar, Assistant Professor & Principal Investigator, Cancer Research Institute, ACTREC, Tata Memorial Centre, Navi Mumbai, India; Presenting author email id: pbhosale@actrec.gov.in

Background: Chromosomal instability is one of the key features in pathogenesis of OSCC, with gene gain/loss reflecting this genetic instability. We have demonstrated chromosomal gain of 11q22.1-q22.2 locus is associated with poor clinical outcome; this locus codes for cIAP1 and cIAP2, an important regulator of apoptosis. Overexpression of these proteins is shown to be associated with treatment resistance in several cancers. In this study, we have validated aCGH finding with Florescent In-Situ Hybridization (FISH) and analyzed overexpression of cIAP1/cIAP2 with outcome in OSCC patients. Methods: FISH analysis was performed to check alteration of 11q22.1-q22.2 locus. cIAP1/cIAP2 expression was analyzed by immunohistochemistry in normal, oral premalignant lesions (OPL) and OSCCs. Results: FISH analysis showed strong association of 11q22 amplification with nodal metastasis (p<0.001) and reduced survival (p=0.004). IHC analysis demonstrated differential localization as well as increased expression of cIAP1/cIAP2 in OSCC as compared to Normal and OPL. The correlation analysis for 11q22 amplification with expression of cIAP1/cIAP2 along with its effect on clinical outcome is on-going. Conclusion: Data implies that in presence of 11q22 amplification, patients fail to respond to treatment, enhancing covets for different treatment modalities that would improve patient outcome. Thus, treatment-response prediction may provide better treatment opportunities to OSCC patients.

S8 Deciphering active players in head and neck cancer: phospho EGFR, HIF-1α, CAIX and more…

Manishkumar Pandey ¹ , Tanuja A. Samant ¹ , Amit Joshi ² , Vanita Noronha ² , Poonam Gera ³ , Asawari Patil ⁴ , Shubhada V. Kane ⁴ , Kumar Prabhash ² , Manoj B. Mahimkar ^1, *

¹ Cancer Research Institute, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai; ² Department of Medical Oncology, Tata Memorial Hospital, Parel, Mumbai; ³ Biorepository, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai; ⁴ Department of Pathology, Tata Memorial Hospital, Parel, Mumbai. *Corresponding author: Dr. Manoj B. Mahimkar, Assistant Professor & Principal Investigator; Presenting author email id: manishp93@gmail.com

Background: Management of HNSCC has remained a challenge for decades and despite advancement in treatment modalities the patient survival has not improved significantly. Extensive molecular analysis on pathogenesis of HNSCC has uncovered various pathways involved; EGFR happens to be the only drugable target currently in clinics. Very few studies have correlated EGFR and hypoxia with treatment response. Additionally, association of HPV in HNSCC cannot be ignored and should be considered as stratification factor for treatment. Here we have comprehensively examined the protein expression of EGFR, phospho-EGFR, hypoxia markers (HIF-1α, CAIX) and HPV in HNSCC. Methods: Tumor tissues were analyzed for EGFR, pEGFR, HIF-1α, CAIX expression and p16 ^INK4a (surrogate-marker for HPV) by immunohistochemistry. Results: Immunohistochemistry analysis showed 4% tumors positive for HPV whereas EGFR overexpression was seen in 80% tumors, of which 45% expressed pEGFR. Similarly, HIF-1α was overexpressed in 90% tumors while only 30% concurrent cases expressed its downstream target (CAIX). Statistical analysis for these targets is ongoing. Conclusion: Preliminary data indicates that only fraction of tumors expressed pEGFR and CAIX compared to EGFR and HIF-1α, hence it’s important that the downstream targets and activated forms of these biomarkers along with HPV status should be considered while planning treatment strategy.

S9 Influence of anti-apoptotic BCL2 -938C>A polymorphism in breast cancer

Sarikajarjapu ¹ , PhanniBhushan Meka ¹ , Santhoshi Rani Nanchari ¹ , Gayathri H. ¹ , Surekha D. ¹ , Prajitha E.M. ¹ , Manjula Gorre ¹ , Anuradha Cingeetham ¹ , Sugunakar Vuree ¹ , Sandhya Annamaneni ¹ , Srinivasulu M. ² , Triveni B. ² , Raghunadharao D. ³ , Vishnupriya Satti ^1, *

¹ Department of Genetics, Osmania University, Hyderabad; ² MNJ Institute of Oncology & Regional Cancer Centre, Hyderabad; ³ Nizam’s Institute of Medical Sciences, Hyderabad, India

Introduction: Bcl-2-family proteins are known to play a central role in apoptosis and are capable of regulating diverse cell death mechanisms. Alterations in their expression and function contribute to the pathogenesis and progression of human cancers, including breast cancer.BCL2 gene polymorphism at -938 promoter region is found to affect the expression of the gene. Materials and Methods: The study was conducted on 110 BC patients and 204 age matched healthy controls. Genomic DNA was isolated from blood and tissue samples through phenol-chloroform method and used for SNP analysis by AS-PCR method. Immunohistochemistry was performed to evaluate the expression of BCL2. Further, cell proliferation was assessed by Ki67.Results were analyzed statistically using SPSS and SNPSTATs. Results: BCL2 -938AA genotype conferred risk in both co-dominant (OR-4.10 (95% CI-1.60-10.50) p-0.02*) and recessive (OR-2.86(95% CI- 1.10-7.41) p-0.06*) models. Comparison of clinicopathological characteristics revealed a significant association of -938AA with advanced stage (p=0.02), HER2 negative (p=0.02) and node negative patients (p<0.004). AA genotypes of this SNP were also found to be associated with Ki67 Index (p=0.005). Conclusion: The results revealed significant association of Bcl2 -938C>A promoter polymorphism with increased risk of breast cancer in individuals carrying -938AA genotype.

S10 Role of aberrant vimentin expression in early and late events of human oral cancer

Crismita Dmello, Sharda Sawant and Milind Vaidya*

Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 210, India. *Corresponding author: Dr. Milind Vaidya, mvaidya@actrec.gov.in; Presenting author email: crismitadmello@gmail.com

Vimentin is an intermediate filament protein predominantly expressed in mesenchymal cells. In our earlier study, we had seen the expression of vimentin in oral leukoplakia and submucous fibrosis which are known premalignant lesions for human oral cancer. Hence we overexpressed vimentin in human oral premalignant derived cell line to ask if vimentin is one of the causes or just a consequence of the process of neoplastic development. Critical EMT and stemness related molecular changes were observed upon vimentin overexpression. However no transformation related changes were observed hence carcinogenic stimulus was given using benzopyrene to both the clones till they showed an anchorage-independent state of growth. Remarkably vimentin expressing clones showed more number of soft agar colonies as compared to vector control clones. Vimentin is well reported in late events of cancer progression but the molecular pathway underlying it is unclear hence we downregulated vimentin in oral cancer derived cell line using shRNA technology. We found increased surface levels and decreased biological turnover of beta4 integrin in absence of vimentin. Another important proteins that we found altered upon vimentin knockdown, were the pair of K5/K14. Collectively this study provides insights into role of vimentin in the process of human oral oncogenesis.

S11 Role of PTEN in drug sensitivity of glioblastoma cells harboring wild type and mutant p53

Mansi Manchanda, Ratnakar Singh and Shyam S. Chauhan*

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India. *Corresponding author: Dr. Shyam S Chauhan, s_s_chauhan@hotmail.com; Presenting author email: mansimann16@gmail.com

p53 and PTEN are very frequently mutated/ deleted in glioblastoma which serve as tumor suppressors and as transcription factors regulating different genes involved in glioblastoma. We investigated the role of PTEN in relation to p53 status in influencing the sensitivity of the glioblastoma cells to temozolomide (TMZ), an alkylating drug used in chemotherapy of brain tumors. In the present study sensitivity of U87MG (p53 wild type, PTEN mutant) and U373MG (p53 mutant and PTEN mutant) cells which were stably transfected with the PTEN expression vector along with LN18 (p53 mutant and PTEN wild type) and LN229 (p53 wild type and PTEN wild type) to this drug was assessed. All the cells were treated with different concentration of TMZ for 48 hours followed by assessment of cell survival. LN229 cells (wild type PTEN and p53) along with U373MG and U87MG (wild type p53) cells were found to be most resistant to TMZ. PTEN expression in U373MG cells resulted in significant increase in their sensitivity to TMZ which was similar to LN18 cells where as U87MG cells exhibited a marginal increase. Expression levels of pro-apoptotic molecules involved in apoptotic pathway were found to be elevated and that of anti apoptotic proteins decreased on treatment with TMZ in LN18 and U373MG PTEN expressing cells as compared to p53 wild type U87MG and LN229 cells. Since LN18 cells has similar p53 and PTEN status to transfected U373MG cells differential TMZ sensitivity of these glioblastoma cells can be attributed to PTEN expression with drug being more effective in cells having mutant p53 and wild type PTEN.

S12 Sesamin: a novel agent for colorectal cancer chemoprevention

Shirly James, Manendra Babu L., J.S. Aparna, Sabira Mohammed and K.B. Harikumar*

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014. *Corresponding author e-mail: harikumar@rgcb.res.in; Presenting author e-mail: manendra@rgcb.res.in

Colorectal cancer (CRC) is the third most common cancer in men and second in women worldwide. Within Asia the incidence rate of CRC varies with different countries. However some recent studies on populations indicated a rising trend of CRC in Indian population. Several epidemiological evidences support the roles of diet, lifestyle, and medication in reducing the risk of colorectal cancer. There is a considerable increase in the pharmacological effects of nutraceuticals for cancer treatment and prevention. In this study we evaluated the possible role of Sesamin, a spice derived phytochemical as a chemopreventive agent in CRC. Our initial studies showed that this compound inhibited the growth of various colon cancer cells tested in vitro. We also found that Sesamin induces cell cycle arrest. The effect of Sesamin was also studied in mouse models of colitis induced by dextran sodium sulphate. We found that Sesamin protected the mouse colons from DSS induced colitis formation. We are presenting our preliminary findings indicating that Sesamin can be considered as a chemopreventive agent in colorectal cancer.

Acknowledgement: This work was funded by DBT Ramalingaswami Fellowship to KBH.

S13 Role of LCN2 gene promoter methylation in tumor microvessel density of breast cancer

Phannibhushann Meka ¹ , Santhoshi Rani Nanchari ¹ , Sarika Jarjapu ¹ , Gayatri ¹ , Prajitha EM ¹ , Manjula Gorre ¹ , Anuradha Cingeetham ¹ , Sugunakar Vuree ¹ , Sandhya Annamaneni ¹ , Srinivasulu M. ² , Triveni B. ² , Vishnupriya Satti ^1, *

¹ Osmania University, Hyderabad; ² MNJ Institute of Oncology & Regional Cancer Centre, Hyderabad, India. *Corresponding author: Dr. S. Vishnupriya, UGC Emeritus Fellow, e-mail: sattivishnupriya@gmail.com

Background: LCN2 (Lipocalin2) is a 25KD protein and reported to be a novel regulator of angiogenesis in human breast cancer.LCN2 gene expression was correlatedwith aberrant promoter methylation in breast cancer cell lines which reveals the possible relationship between epigenetic regulation and LCN2 gene expression in breast cancer. We studied the promoter methylation status of LCN2 gene and its influence on tumor microvessel density of breast cancer patients. Materials and methods: A total of 64 primary breast cancer tumors were collected from MNJ institute of oncology, Hyderabad, India.Genomic DNA was isolated by phenol chloroform method. Bisulfite treatment of genomic DNA was carried out for methylation-specific PCR (MSP) analysis.4΅m thick tissue sections were used for assessment of microvessel density (MVD) with monoclonal mouse anti human CD34. Results and Discussion: LCN2 promoter unmethylation was found in 43 of 64 (67.2%) breast cancer patients. Mean MVD was increased in unmethylated patients compared to methylated patients. This might be due to the activation of LCN2 gene through promoter DNA unmethylation which resulted in elevated expression of LCN2, which might promote angiogenesis through microvessel formation. Hence LCN2 promoter methylation might be a marker for predicting tumor angiogenesis in breast cancer.

S14 Expression of Caveolin-1 in oral squamous cell carcinoma tumor microenvironment of eastern India subpopulation

Samapika Routray ^1, *, Neeta Mohanty ¹ , Rupesh Das <sup>2

1</sup> Department of Oral Pathology & Microbiology, Institute of Dental Sciences, Sector-8, Kalinga Nagar, Ghatikia, Bhubaneswar, Odisha – 751003; ² Translational Research Wing, Gene Therapy for Cancer (RD Lab), Institute of Life Sciences, Bhubaneswar-23. *Corresponding author: Samapika Routray, drroutray.samapika@gmail.com

In Indian subcontinent, an upsurge in oral squamous cell carcinoma(OSCC) cases has been observed, predominantly due to various forms of industrialised tobacco usage, affecting younger generation more rampantly. Focus on the molecular characterization of these OSCC tumors will facilitate the development of more selective anticancer agents based on the targeted delivery of bioactive molecules to the tumor microenvironment. Caveolin-1(Cav-1), which plays an important role in the pathogenesis of oncogenic cell transformation, tumorigenesis, and metastasis, has confirmed its value as prognostic indicator in an array of tumors. Its been established that oxidative stress is the main cause for loss of stromal cav-1 via autophagy in the tumor microenvironment. Human studies encompassing the activity and expression of cav-1 with respect to progression of oral cancer from premalignancy to malignancy are largely lacking in India. In view of this background, the present study was designed to investigate the clinico-pathological correlation of OSCC with expression of Cav-1 in oral cancer patients of eastern India. We employed quantitative, real time polymerase chain reaction (qRT-PCR) to examine expression changes and further validated it by assessing its protein level by immunohistochemistry (IHC) analysis. Our results revealed up-regulation of cav-1 expression in qRT-PCR analysis in OSCC compared to normal controls. In tissue IHC analyses, Cav-1 exhibited increased protein expression in cancer compared to normal tissues.

S15 Study of inhibitory effect of C-Phycocyanin complex on human matrix metalloproteinases

Mugdha Kunte ¹ , Krutika Desai <sup>2

1</sup> School of Science, SVKM’s NMIMS, Vile Parle (W), Mumbai: ² Mithibai College, Vile Parle (W), Mumbai.

The MMPs are involved in homeostatic process such as tissue resorption, remodeling and repair. They are regulated by tissue inhibitors of matrix metalloproteases (TIMPs). Disruption of the MMP/inhibitor balance is observed during cancer. Inhibition of MMP activity has proven to be promising approach in controlling invasive and metastatic cancers. In the present study, attempt has been made to isolate and identify bioactive molecules as MMP inhibitors from a microalga Spirulina platensis. Growth conditions for microalga were optimised to obtain maximum concentration of C-phycocyanin (C-Pc) pigment-protein complex, a known antioxidant. C-Pc complex was extracted from cells and further purified using DEAE ion exchange chromatography. Their activity as MMP inhibitor was evaluated against serralysin; a bacterial metalloprotease; structurally and functionally similar to human MMP-2 & 9. Partially purified C-Pc complex was found to possess anti metalloprotease activity. However, purified pigment did not show any inhibitory activity against serralysins. Hence, inhibitory activity is probably a synergistic activity of proteins and C-Phycocyanin pigment present in the partially purified fraction. Partially purified C-Pc protein-pigment complex was eventually examined for inhibitory activity against human MMPs in HepG2 cell line.

S16 14-3-3 σ loss leads to an epithelial mesenchymal transition

Kumarkrishna Raychaudhuri ¹ , Mansa Gurjar ¹ , Sorab N. Dalal ^1, *

¹ KS215, Sorab Lab, Advanced Centre for Treatment Research & Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India. *Corresponding author: Sorab N. Dalal

The role of 14-3-3 proteins has been well established in regulation of cell cycle, DNA damage, regulation of gene expression, cytoskeletal dynamics, regulation of cell to cell adhesion, cancer progression etc. We observed that 14-3-3 σ loss leads to loss of epithelial markers e.g. E-cadherin, cytokeratin, plakoglobin and gain of mesenchymal markers e.g. N-cadherin and vimentin. Re-expression of 14-3-3σ in these cells leads to a reversal of these phenotypes. Loss of 14-3-3σ also leads to EMT specific phenotypic changes e.g. an increase in cell migration, decrease in cell to cell adhesion and decreased cell to ECM adhesion. Increased expression EMT specific transcription factors, Slug and Zeb1 was observed in 14-3-3σ null cells as compared to 14-3-3σ wild type cells. c- JUN is known to bind slug promoter and up-regulate Slug at transcriptional level. c JUN was found to be stabilized at protein level in 14-3-3 σ null cells, whereas, in 14-3-3σ wild type cells c JUN was targeted to proteasomal degradation. Our results show that 14-3-3 σ interacts with c JUN and this might lead to c JUN degradation. We report a novel mechanism through which c JUN-slug axis can promote EMT on loss of 14-3-3 σ.

S17 Role of miR-146a in regulating stemness of oral squamous cell carcinoma

Sangeeta Ghuwalewala, Dishari Ghatak and Susanta Roychoudhury*

Division of Cancer Biology and Inflammatory Disorder, CSIR – Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, India. *Corresponding author: Dr. Susanta Roychoudhury

Oral squamous cell carcinoma is a highly heterogeneous disease being driven by a small population of tumor-initiating cells called cancer stem cells. These cells may not be genetically distinct from the bulk of the tumor, and therefore must have some epigenetic mechanism that is reversible, highly dynamic in regulation of specific gene transcription and translation programs, thereby contributing to stem cell homeostasis. In the present study, we have used Magnetic activated cell sorting (Macs) based technique for the direct isolation of CD44+ CD24- population and characterized them as the CSC population, from various oral cancer cell lines. These cells showed enhanced expression of stem cell markers with greater sphere forming ability than the non-stem cancer cells. Amongst the development related signaling pathways implicated in stem cell dysregulation, we have found Notch/ Wnt pathways to be activated in these stem-like cells. Also we screened for epigenetic changes such as miRNA expression that may influence these cell signaling pathways. We found that mir-146a was over-expressed in cancer stem cell compared to the non-stem cell population of various oral cancer cell lines. This miRNA is already known to repress Numb that leads to Notch1/β-catenin stabilization which may impart stemness. We found that in OSCC, it also represses cancer stem cell marker, CD24, a novel target, which then leads to de-differentiation of cancer cells. Also the β-catenin is found to trans-activate mir-146a and maintain stemness by a positive feed-back loop. Hence we hypothesize that this mir-146a-CD24-β-catenin axis has an important contribution towards stem cell homeostasis. Our findings tend to elucidate the epigenetic mechanism behind cancer stem cell maintenance in tumor and how it contributes to oncogenesis.

S18 NPM1 is the most prevalent mutation followed by PML/RARα, AML1/ETO translocations and FLT3 mutation amongst the AML patients in Assam, India

Sukanta Nath ¹ , Jina Bhattacharyya ² , Kandarpa Kumar Saikia <sup>1

1</sup> Dept. of Bioengineering, Gauhati University, Guwahati; ² Dept. of Haematology, Gauhati Medical College & Hospital, Guwahati, Assam

Acute myeloid leukaemia (AML) is a type of cancer that affects the blood and bone marrow and one of the leading haematological malignancies in Assam. NPM1, FLT3, PML-RARα t(15;17)(q22;q21), AML1/ETO t(8;21)(q22;q22) and CBFβ/MYH11 t(16;16)(q22;p13) are reported to be the most frequent malignancies in AML with diagnostic and prognostic value. In this study we documented the prevalence of NPM1, FLT3 mutations and PML/RARα (bcr1 and bcr3 subtypes), AML1/ETO & CBFβ/MYH11 translocations with their co-relation with patient outcome. Blood/ bone marrow samples were collected from 90 AML patients with informed consent at the time of diagnosis. DNA & RNA were extracted to detect mutations by PCR. Out of 90 patients 50% were NPM1 positive, 17% were PML/RARα positive (bcr1 6% and bcr3 11%), AML1/ETO positive were 16% & FLT3-ITD positive were 7%. None of the patients were found to carry FLT3-D835 & CBFβ/MYH11. Our results conclude that NPM1 is the most prevalent mutation in the study population followed by PML/RARα (bcr3 subtype is more than bcr1 subtype), AML1/ETO translocations and FLT3-ITD mutation. Detection of mutations/translocations in the NPM1, PML-RARα, AML1/ETO associated with good prognosis whereas FLT3-ITD mutation showed poor prognosis. Our findings almost similar with global findings.

S19 MiR-22 inhibits the growth of human colorectal cancer cells and Wnt signaling

Chetlangia Neha*, Srinivasa Rao Rao** and Devarajan Karunagaran*

*Department of Biotechnology, Indian Institute of Technology Madras, Chennai – 600036, India; **Present address: Nuffield Dept. of Surgical Sciences, University of Oxford, Oxfordshire, OX3 7LD, United Kingdom. Corresponding author: Devarajan Karunagaran.

Deregulated expression of microRNAs is frequently observed in different cancers. MicroRNA-22 expression is reported to be significantly lower in colon cancer tissues compared to normal adjacent mucosa. Bioinformatics prediction tools enlisted several Wnt signaling intermediates as potential targets of miR-22. Aberrant activation of Wnt signaling has been implicated in various cancers especially of breast and colon. Therefore we investigated the role of miR-22 in colon cancer cells having constitutively active Wnt Signaling. We observed that miR-22 overexpression decreased TOPFlash luciferase activity in SW480, SW620 and HCT116 (human colon cancer) cells. It was also noticed that miR-22 inhibited LiCl-induced activation of Wnt signaling in HEK293 (human embryonic kidney) cells. miR-22 repressed Wnt signaling target genes CyclinD1, c-Myc and TCF7L2, at both mRNA and protein levels. It also decreased the mesenchymal markers (Snail and Vimentin) and increased epithelial marker (E-cadherin) at the protein level. miR-22 was found to target BCL9L, as demonstrated by luciferase reporter assay. Over-expression of miR-22 significantly reduced cell viability, although cell cycle profile showed no observable difference. Cell migration was inhibited by miR-22. Thus our study shows that miR-22 exerts its growth suppressive effects in colon cancer cells potentially by targeting the Wnt coactivator BCL9L.

S20 Polymeric black tea polyphenols (PBPs) modulate tobacco carcinogen induced cellular responses in A/J mice lung carcinogenesis model

Rasika Hudlikar ¹ , Varadha Balaji Venkadakrishnan ¹ , Rajeev Kumar Kaushal ² , Rahul Thorat ³ , Arvind Ingle ³ , Saral Desai ² , Girish Maru ¹ , Manoj Mahimkar <sup>1,#

1</sup> Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai-410210; ² Tata Memorial Hospital, Tata Memorial Centre, Parel, Mumbai-400012; ³ Laboratory Animal Facility, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai-410210. ^# Corresponding author: Dr. Manoj B. Mahimkar, Assistant Professor & Principal Investigator; Presenting author email: rasikahudlikar@gmail.com

Lung cancer is one of the leading causes of deaths in smokers. Although tobacco cessation is important for lung cancer prevention, ex-smokers still exhibit higher risk. Chemoprevention with phytochemicals is evolving and promising approach for management of lung cancer. We studied the chemopreventive efficacy of polymeric black tea polyphenols (PBPs), abundantly found in black tea, to inhibit Benzo(a)pyrene [B(a)P] and 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) induced lung adenoma in A/J mice. In present study, administration of PBPs throughout the treatment period significantly decreased the multiplicity of surface tumors as well as microscopic lung lesions, including adenomas. Although, tumor incidence and latency period remains unaffected, histopathological evaluation of lung at 4,6,10 and 18 weeks post carcinogen treatment period showed decrease in tumor multiplicity which was also correlated with different molecular markers. PBP rich fraction down-regulated the B (a) P and NNK induced cell proliferation (diminished PCNA and Bcl-2 expression) and enhanced apoptosis (increased Bax expression) through phosphorylation of p38 and Akt. Anti-inflammatory action of PBP was demonstrated by reduced Cox-2 expression. In conclusion, the protective effects of PBP fractions could be attributed through inhibition of cellular proliferation and induction of apoptosis. Some of these biomarkers are likely to be helpful in monitoring clinical chemoprevention trials.

S21 Study of RASSF1A and Hippo signaling molecules in urothelial carcinoma of bladder

Madhuram Khandelwal ¹ , Vivek Anand ¹ , Sandeep Appunni ¹ , Amlesh Seth ² , Sandeep Mathur ³ , Alpana Sharma ^1, *

¹ Department of Biochemistry, AIIMS, New Delhi; ² Department of Urology, AIIMS, New Delhi – 110029; ³ Department of Pathology, AIIMS, New Delhi. *Corresponding author: Dr. Alpana Sharma, Email: dralpanasharma@gmail.com

Bladder cancer is 9th most common cancer globally. Silencing of tumor suppressor genes by epigenetic modification is an important mechanism involved in pathogenesis of cancer. RASSF1A (Ras association domain family 1A) is well studied tumour suppressor gene in cancer till date and its link with Hippo pathway has been reported. Aberrant expression of Hippo pathway molecules, that normally regulates cell cycle and growth, causes carcinogenesis. Thus aim of study was to observe methylation status of RASSF1A and expression of RASSF1A and Hippo pathway molecules (MST, YAP) in Urothelial carcinoma of bladder (UCB). Expression of RASSF1A and Hippo pathway molecules (MST, YAP) at mRNA level by Q-PCR, and methylation status of RASSF1A by MS-PCR were observed. Expression of RASSF1A was significantly (p<0.05) lower in tumor tissue of patients (n=20) versus adjacent normal tissue and found to be correlated with methylation of RASSF1A gene. Yap showed higher expression in tumor tissue compared to normal tissue while MST showed no significant difference at mRNA level. This study indicate significant alteration in expression of RASSF1A and YAP in UCB. Study of downstream signalling molecules may validate the useful link of Hippo pathway with RASSF1A in bladder cancer and its future potential in novel therapeutics.

S22 Exploring the role of versican as a potential diagnostic marker in multiple myeloma

Nidhi Gupta ¹ , Rehan Khan ¹ , Raman Kumar ¹ , Lalit Kumar ² , Alpana Sharma ^1, *

¹ Department of Biochemistry, All India Institute of Medical Sciences, New Delhi – 110029, India; ² Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India. *Corresponding author: Dr. Alpana Sharma, Additional Professor, Email: dralpanasharma@gmail.com

Multiple myeloma (MM) is malignancy of B-cells characterized by proliferation of malignant plasma cells in the bone marrow (BM). Versican (VCAN), an extracellular matrix (ECM) protein emerged to be involved in pathophysiology of several cancers. Identifying optimum diagnostic markers and delineating its association with disease severity might be important for controlling MM. We have investigated expression of VCAN and its associated molecules (β-catenin, β1 integrin & FAK) in 60 subjects to evaluate their usefulness as diagnostic markers. Circulatory & molecular levels of above molecules were analyzed in their BM and Blood using ELISA, Q-PCR and western blotting along with ROC curve analysis. Pearson correlation was determined to observe interrelationship between molecules. Circulatory levels of VCAN, β-catenin and FAK were significantly higher and also show a trend with disease severity. β-catenin and FAK intracellular levels were significantly elevated. mRNA and protein levels of all molecules were also significantly higher in patients. Significant elevation of VCAN and its associated molecules imply their role in MM. ROC curve analysis of VCAN in serum showed absolute combination of sensitivity and specificity to utilize its importance as potential diagnostic marker for active disease which can be further validated in large patient cohort.

S23 Expression of small leucine rich proteoglycans (Decorin, Biglycan and Lumican) in urothelial carcinoma of bladder

Sandeep Appunni ¹ , Vivek Anand ¹ , Madhuram Khandelwal ¹ , Amlesh Seth ² , Sandeep Mathur ³ , Alpana Sharma ^1, *

¹ Department of Biochemistry, AIIMS, New Delhi – 110029. *Corresponding author: Dr. Alpana Sharma, Additional Professor, email: dralpanasharma@gmail.com

Bladder cancer is 9th most common cancer worldwide. Small Leucine Rich Proteoglycans (SLRP) are ubiquitously present in extracellular matrix having well-established role in regulating cell growth and proliferation. Three SLRPs decorin, biglycan and lumican are implicated in cancer with limited study in urothelial carcinoma of bladder. Decorin acts as a tyrosine kinase inhibitor and upregulates p21 which prevents cell proliferation. Biglycan and lumican are other two SLRPs which regulates cell proliferation. Thirty patients of confirmed untreated bladder cancer and 30 healthy controls were enrolled. Blood was collected from all subjects and tumour/adjacent normal tissue was taken from patients. Circulatory levels were estimated by ELISA, relative mRNA expression by qPCR and protein expression by immunohistochemistry. The circulatory levels of biglycan (p=0.0038) and lumican (p<0.0001) were significantly elevated and decorin (p<0.0001) was reduced in patients as compared to controls. Relative mRNA expression (tissue specimen) for biglycan (p=0.0033) was significantly increased and for decorin (p=0.0003) was decreased in patients. Protein expression (IHC) of biglycan (p=0.03) was significantly higher and lower for decorin (p<0.0001) in patients. Significant alteration of matrix SLRPs in urothelial carcinoma of bladder was observed and their diagnostic/prognostic potential can be further validated in large patient cohort.

S24 Identification of small molecules with potential anticancer activity and elucidation of their biological target

Silpa G ¹ , Rayala SK ¹ , and Kesavan V ^2, *

¹ Molecular Oncology Lab, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, IIT-Madras, Chennai, India; ² Chemical Biology Lab, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, IIT Madras, Chennai, India. *Corresponding author: Kesavan V.

Novel small molecule inhibitors against cancer can be designed from chemical starting points obtained by methods like peptidomimetic approaches, screening of natural products or synthetic libraries or virtual screening/structure based design or by slight minor modifications to the existing drugs to improve their efficacy. However, many novel small molecule inhibitors designed are being discarded due to lack of understanding of its mechanistic action (which leads to undesirable off-target effects), lack of specificity to cancer cells and acquired drug resistance. In this present study, novel small molecules are designed following physicochemical and biological properties necessary for potent drugs (Alpha Methylene Gamma Lactone derivatives, HDAC Inhibitor analogs, Tubulin Inhibitor analogs, Isatin derivatives) and are screened against different tumor cell lines respective to cancers highly prevailing world wide (Breast, Brain and Lung). Range of dose concentration and time points are analysed using cell based cytotoxicity assay. Lead drugs from the preliminary screen are meticulously tested on cancer type matched normal cells to find out their toxicity.

S25 Identifying mechanisms regulating tumor progression upon plakophilin3 loss

Srikanta Basu, Rahul Thorat and Sorab N. Dalal*

KS215, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India. *Corresponding author: Sorab N. Dalal.

Desmosomes are adherens like junctions present in epithelial cells that anchor intermediate filaments at membrane associated plaques in adjoining cells allowing the formation of an intercellular network that promotes tissue organization and rigidity. The desmosomal plaque protein plakophilin3 binds to a broad repertoire of proteins such as the desmosomal cadherins, desmoglein 1-3, desmocollins 1 and 3; cytokeratin 18; desmoplakin and plakoglobin. Previous results from our laboratory demonstrate that plakophilin3 loss leads to alterations in the desmosome size, a decrease in cell-cell adhesion, increased cell migration, anchorage independent growth, growth to high density in culture, accelerated tumor formation in nude mice and increased metastasis to lung. Plakophilin3 expression is known to be lost in high grade poorly differentiated oropharyngeal cancer, colon cancer, gastric cancer and bladder cancers. The mechanism underlying tumor progression in plakophilin3 knockdown clones remained unclear. Gene expression analysis demonstrated that plakophilin3 loss leads to an increase in the expression of Lipocalin2 (LCN2). Knockdown of LCN2 in HCT116 derived plakophilin3 knock-down clone leads to an increase in cell-cell adhesion, a decrease in migration and abolishes the ability of cells to form soft agar colonies. LCN2 knockdown also inhibits tumor formation in nude mice. Luciferase reporter assays using different LCN2 promoter fragments driving firefly luciferase gene confirmed that LCN2 expression is regulated at the transcriptional level. The mechanisms by which the loss of plakophilin3 leads to an increase in LCN2 levels is still being elucidated and results addressing this question will be presented at the meeting.

S26 In vitro efficacy evaluation of diallyl disulfide (DADS) for the treatment of breast and colorectal carcinomas

Rathod Venkatesh ^1,# , Sujatha P ¹ , Pramod N. Jadhav ¹ , Shrisiri J. Prakash ¹ , Muslima Muhusin ¹ , Shruthi A. Nagarajarao ¹ , Siddarth V. Talluri ^2,# , Gowthamarajan Kuppuswamy ² , and SubbaRao V. Madhunapantula ^1, *

¹ Center of Excellence in Molecular Biology and Regenerative Medicine (CEMR), Department of Biochemistry, JSS Medical College, JSS University, Mysore, Karnataka, 570015; ² Department of Pharmaceutics, JSS College of Pharmacy, Ooty, Tamilanadu; #Contributed equally to the work; *Corresponding author: madhunapantulas@yahoo.com

Objective: The objective of this study is to determine the therapeutic efficacy of oil-soluble diallyl disulfide (DADS) for treating cell lines representing carcinomas of breast, colon and rectum. Materials and Methods: DADS was procured and purity tested using HPLC. The 85% pure DADS was structurally characterized using LC-MS. IC50 of DADS for cell lines representing carcinomas of breast (MCF-7 and MDA-MB-231) and colon and rectum (HCT-116 and HT-29) was determined by sulforhodamine-B (SRB) assay. The mechanistic basis for cytotoxicity was elucidated using Western blotting. Results: Cytotoxicity assays using 85% pure DADS demonstrated a time and dose dependent increase in the efficacy against colorectal carcinoma cell lines HT29 and HCT116, and breast cancer cell lines MDA-MB-231 and MCF-7. For example at 24 h the IC50 of DADS for HCT116 was ~400 ΅M, the IC50 decreased to 125 ΅M at 48 h. Similar trend was also observed with other cell lines. Detailed molecular mechanisms responsible for DADS induced cytotoxicity will be presented at the time of presentation. Conclusion: Naturally occurring DADS appears to be good therapeutic agent for treating colon and breast cancers. Further studies are required to determine the efficacy of DADS in animal models of colon and breast cancers.

S27 Randomness and preserved patterns in the Breast Cancer Network

Aparna Rai ¹ , Pramod Shinde ¹ , Alok Yadav ² , Camellia Sarkar ¹ , Sanjiv K Dwivedi ² and Sarika Jalan ^1,2, *

¹ Centre for Biosciences and Bio-Medical Engineering, Indian Institute of Technology Indore, Khandwa Road, Indore-452017; ² Complex Systems Lab, Discipline of Physics, Indian Institute of Technology Indore, Khandwa Road, Indore-452017. *Corresponding author: Sarika Jalan.

Breast cancer has been reported to account for the maximum cases among all female cancers till date. In order to gain a deeper insight into the complexities of the disease, we analyze the breast cancer network and its normal counterpart at the proteomic level through a novel mathematical tool, random matrix theory. This theory has previously been used to understand the complexities of various physical systems ranging from quantum chaos to galaxy. The random matrix analysis of the protein-protein interaction networks for both the cases reveal insightful structural patterns involving functionally important proteins and also provide an evidence towards the importance of random connections in the underlying networks. The analysis being time and cost proficient provides a benchmark for designing novel drugs and therapeutic targets which involves a subgraph instead of individual proteins.

S28 The hepatitis B virus ‘X’ oncoprotein associates with nucleophosmin to promote rDNA transcription and cellular transformation

Richa Ahuja, Neetu Rohit Kapoor and Vijay Kumar*

Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India. *Corresponding author: Vijay Kumar, PhD, Group Leader, e-mail: vijay@icgeb.res.in; Presenting author email: rahuja@icgeb.res.in

Chronic infection of hepatitis B virus (HBV) is considered as a major risk factor for the development and progression of hepatocellular carcinoma (HCC) which is the third leading cause of cancer related deaths worldwide. The HBV-encoded HBx oncoprotein is widely regarded as a key etiological factor for HCC. The pleiotropic HBx oncoprotein induces the expression of ribosomal RNAs and several host proteins that supports the oncogenic transformations. While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates with HCC progression, its regulation by viral HBx and consequent perturbations in the nucleolar functions remain enigmatic. The present study shows that HBx regulates NPM levels and hijacks its functions to promote cellular proliferation. We found that HBx expression stabilizes NPM by promoting its phosphorylation at Thr ¹⁹⁹ by cyclin-dependent kinase 2. Further, HBx directly interacted with NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery on the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving NPM acetylation and subsequent depletion of histones from the rDNA promoter. Apparently, the enhanced rRNA expression along with NPM accumulation under the HBx microenvironment promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our findings strongly implicate NPM as a mediator of the oncogenic effects of HBx.

S29 A novel inhibitor of nonhomologous DNA end joining, SCR7 brings down the effective dose of radiation during cancer therapy

Divya Lakshmanan, Mrinal Srivastava, Supriya Vartak and Sathees C. Raghavan*

Department of Biochemistry, Indian Institute of Science, Bangalore – 560012, India. *Corresponding author: Sathees C. Raghavan, sathees@biochem.iisc.ernet.in Presenting author email: divyalmangalath@gmail.com

DNA double-strand breaks (DSBs) are one of the most lethal type of DNA damages within cells. In higher eukaryotes, homologous recombination (HR) and nonhomologous end joining (NHEJ) are the major DSB repair pathways. NHEJ plays a major role in providing resistance to cancer cells against radio and chemotherapeutic agents. DNA ligase-IV is one of the initial enzymes involved in NHEJ. Recently SCR7, an inhibitor against NHEJ, targeted against ligase-IV was shown to impede tumour progression. SCR7 also has the potential to improve radio and chemotherapy. In the present study, we show that SCR7 sensitises cancer cell lines several fold, when treated with a single dose of 0.25 Gy gamma radiation, which was equivalent to a dose of 2 Gy. When SCR7 was co-administered in mouse solid tumour models with a dose of 1.5 Gy radiation, the observed tumor regression was equivalent to that of 6 Gy of radiation, suggesting efficient reduction in radiation dose. The combination treatment led to CASPASE3 activation followed by cleavage of XRCC4 (p35) indicating apoptotic cell death. Further, immunohistochemical examination and TUNEL assay confirmed DNA fragmentation leading to apoptosis. Comet assay and gamma-H2AX staining showed that SCR7 in conjunction with 0.25 Gy radiation led to significant increase in unrepaired DSBs within the cells in comparison to individual doses. Thus, our results demonstrate that SCR7 could bring down the effective dose of radiation significantly in both cell lines and mouse tumor models. Hence, SCR7 could improve the clinical side effects of radiotherapy by lowering the radiation dose.

S30 Evaluating the binding properties of NHEJ proteins: sequence preferences of DNA ligase IV and KU70/80

Monica Pandey ¹ and Sathees C. Raghavan ^1, *

¹ Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India. *Corresponding author: Dr. Sathees C. Raghavan, sathees@biochem.iisc.ernet.in; Presenting author email: monicapandey808@gmail.com

Repair of DNA breaks is essential for maintaining genomic integrity. Double-strand breaks (DSBs) are repaired mainly by Homologous recombination and Nonhomologous end joining (NHEJ) pathway. NHEJ is the predominant DSB repair pathway in eukaryotic cells. During NHEJ, KU70/KU80 heterodimer binds to the dsDNA ends and recruits DNA-PKcs, Artemis and Pol ΅ or λ to the repair site, resulting in end-processing followed by Ligase IV, XRCC4 and XLF complex-mediated ligation. The DNA sequence preference of these proteins has not been determined yet. Ligase IV has a multidomain architecture with DNA Binding domain (DBD) at N-terminus and two adjacent BRCT motifs at C-terminus, that aid in interaction with XRCC4. In the present study, using radioactively labeled single stranded or double stranded oligomeric DNA substrates mimicking in vivo DNA breaks, we show that DNA Binding Domain (DBD) of Ligase IV alone fails to bind to dsDNA. Interestingly, DBD Ligase IV and KU70/80 heterodimer bind much firmly to ssDNA than dsDNA substrates. This study would help in defining the sequence preference of NHEJ proteins. We further characterised two genetic polymorphisms (A3V and T9I) identified within DBD of ligase IV in Lig4 syndrome patients. The extensive structural and functional analyses of these mutations would facilitate designing potential novel therapeutics, targeting NHEJ in cancer cells.

S31 Mechanism of immune dysfunction mediated by HLA-DR ^_/low CD33 ⁺ CD11b ⁺ myeloid cells in oral cancer patients

Asif Dar ¹ , Trupti Pradhan ¹ , Anil D’Cruz ² , Devendra Chaukar ² , Shubhada Chiplunkar ^1, *

¹ Chiplunkar Lab, ACTREC, Tata Memorial Centre, Navi Mumbai-410210, India; ² Tata Memorial Hospital, Tata Memorial Centre, Mumbai-400012, India. *Corresponding author: Dr. Shubhada Chiplunkar, schiplunkar@actrec.gov.in

Immune dysfunction contributes to tumour progression in oral cancer (OC) patients. Suppression of T cell activation and function by myeloid-derived suppressor cells (MDSCs) leads to impairment in the anti tumour immune response and contributes to immune dysfunction. The frequency and function of a new population of MDSCs denoted as HLA-DR ^-/low CD33 ⁺ CD11b ⁺ were investigated in OC patients. MDSCs were characterized for phenotype and function from peripheral blood (n=65) and tumour (n=19) of OC patients. The frequency of MDSCs in OC patients was significantly higher than healthy individuals (HI). Non-classical MDSCs (CD14 ⁺⁺ CD16 ⁺ CD15 ⁺ ) were prevalent in periphery while granulocytic MDSCs (CD14 ^– CD16 ⁺⁺ CD15 ⁺ ) dominated the tumour compartment. High levels of MDSCs correlated with cancer stage. MDSCs suppressed autologous T-cell proliferation; decreased CD3-ζ chain expression and IFN-γ production. MDSCs expressed higher levels of COX-2, arginase, pSTAT3 and IL-10. IL-10 may be facilitating the crosstalk between MDSCs and Tregs. Tregs (CD4 ⁺ CD25 ⁺ CD127 ^– Foxp3 ⁺ ) were higher in periphery and tumor of OC patients and correlated with stage of the tumour. Tregs of OC patients expressed higher levels of CCR6 and CXCR4 and may aid their migration to the tumor site. Understanding the mechanism of action of MDSC and Tregs in OC patients is important in designing effective immunotherapeutic protocol for OC patients.

S32 Clinical significance of preoperative neutrophil: lymphocyte and platelet: lymphocyte ratios in oral squamous cell carcinoma

Dr. Swetha Acharya ¹ , Dr. Pragati Rai ¹ , Dr. Kaveri Hallikeri ¹ , Dr. Ventakesh Anehousur ² , Dr. Jyoti Kale <sup>1

1</sup> Department of Oral Pathology and Microbiology; ² Department of Oral and Maxillofacial Surgery; SDM College of Dental Sciences and Hospital, Dharwad- 580009, India. Corresponding author: Dr. Swetha Acharya, Reader.

Systemic inflammatory response is deemed to reflect both disease activity as well as the host’s innate response towards the tumor. This can be easily and reproducibly quantified in patients using a number of indices such as the neutrophil to lymphocyte (NLR) and platelet to lymphocyte ratio (PLR), both derived from inflammation-induced derangements in the full blood count. Therefore the aim of the study was to assess whether the NLR and PLR before complete surgical staging provide information on lymph node metastasis in oral squamous cell carcinomas (OSCC). Sixty eight patients with OSCC who underwent surgical treatment at our institution from 2011-14 were identified retrospectively from databases. The clinicopathological data and preoperative complete blood investigation details were obtained from medical records and departmental database. Receiver operating characteristics curve analysis was used to evaluate cut – off, sensitivity and specificity values for predictive NLR and PLR to predict lymph node metastasis. Univariate analysis was applied to analyse the categorical variables. The best cut-off values for predicting lymph node metastasis was 128.5 for the PLR, with 75% sensitivity and 70.45% specificity. The rate of lymph node involvement was higher in the PLR> 128.5 group than in the PLR≤ 128.5 [p=0.00]. There was an association between PLR cut-off and tumor stage. There was no significant difference between means of NLR between the lymph node positive and negative groups. Preoperative PLR is directly associated with nodal involvement status of OSCC.

S33 Study of clinicopathological parameters in assessing the behavior pattern of tongue carcinoma

Kaveri Hallikeri ^1, *, Swetha Acharya ¹ , Shilpa S Chatni ² , Darshika Gandhi <sup>1

1</sup> Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences and Hospital, Dharwad-580009; ² Department of Oncosurgery, Karnataka Cancer Therapy and Research Centre, Navnagar, Hubli. Corresponding author: Dr. Kaveri Hallikeri, Professor and Head.

The clinicopathological parameters play an undisputable role in determining the prognosis of squamous cell carcinoma. The aim of this study was to assess the correlation between tumor thickness, depth of invasion and involvement of anatomical structures with clinicopathological parameters. A retrospective study was conducted on 56 cases of radically dissected squamous cell carcinoma of tongue from year 2008-2014 in SDM Dental College and Hospital, Dharwad and Cancer Hospital, Navnagar. Parameters such as age, gender, habits, site, size, staging-grading, involvement of anatomical structures, entire tumor thickness, depth of invasion, nature of invasive front, eosinophils, resected margin, lymphovascular and perineural invasion and their relation to the development of nodal metastases was determined by assessing the H&E stained sections under light microscope by two observers. Carcinoma was observed predominantly among male more than 40 years of age having tobacco chewing habit, belonging to T1-T2 size (63%) and stage III and IV (79%). Statistics was done using univariant analysis, showed significant association between nature of invasive front and involvement of anatomical structures (p=0.001), also observed as the tumor thickness increased, tumor cells were discohesive. Eosinophils were found to be associated with tumor thickness (p=0.044) as well as depth of invasion (p=0.047). 71% cases showed more than 5mm depth of invasion and 77% showed more than 4mm tumor thickness. Eosinophils, margins, lymphovascular and perineural invasion showed no statistical significance with nodal metastasis. To conclude, the nature of invasive front and tumor thickness can be a prognosticator in squamous cell carcinoma and presence of eosinophils implicate a role in the progression of disease.

S34 Regulation of centrosome duplication by 14-3-3 proteins

Arunabha Bose ¹ , Amitabha Mukhopadhyay ¹ , Lalit Sehgal ¹ , Renu Balyan ² and Sorab N. Dalal ^1, *

¹ ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India; ² National Institute of Immunology, JNU complex, New Delhi, India. Corresponding author: Sorab N. Dalal.

The DNA replication cycle has to be synchronized with the centrosome duplication cycle as an inability to do so, leads to genomic instability and tumor progression. Previous work from our laboratory has demonstrated that loss of 14-3-3γ in HCT116 cells leads to an abrogation of the S and G2/M checkpoint pathways. 14-3-3γ knockdown cells display a higher mitotic index and an increase in centrosome number. In this study, we focus on the regulation of the centrosome cycle by 14-3-3 proteins. In order to study the interaction between 14-3-3γ and the centrosome, we have identified centrosomal proteins that form a complex with 14-3-3γ. Experiments are currently underway to map the 14-3-3γ binding sites in these proteins and determine whether this interaction is required for the regulation of centrosome duplication.

S35 Ultradian rhythms of redox oscillations in cancer cells

Uma K., Karunagaran D., Suraishkumar G.K.*

Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai-600036. *Corresponding author email: gk@iitm.ac.in

Circadian and ultradian rhythms are fundamental characteristics of eukaryotic cells and are manifested in cellular processes such as ROS signaling. ROS homeostasis is regulated by a number of feedback loops, among which those of the antioxidant enzymes, namely catalases and superoxide dismutases, are crucial. Analyses of the basal and altered rhythms of ROS and antioxidant enzymes are expected to facilitate an understanding towards stress response and ROS homeostasis in cancer treatment. In this study the effect of a single pulse treatment of various chemicals, one at a time (viz. hydrogen peroxide, menadione and curcumin), which alter ROS homeostasis have been analyzed in the cervical cancer cell line SiHa. The specific intracellular levels of superoxide anions and hydroxyl radicals were measured periodically in intact cells using membrane permeable fluorescent dyes and the intracellular catalase and superoxide dismutase levels were determined correspondingly from cell lysates. A reset of the endogenous rhythm with a reduction in period of oscillation was observed in reactive species and superoxide dismutase while no appreciable change in pattern was observed for catalase. This reset was dependent upon the concentration of the chemicals used. Continuing investigations are expected to provide insights into cancer therapeutic strategies that alter ROS homeostasis.

S36 Centrifuged liquid based cytology (CLBC): A diagnostic augmentation

Nikhil Yadav ¹ , Veda Hegde ¹ , Shwetha Nambiar ¹ , Kaveri Hallikeri <sup>1

1</sup> Department of Oral Pathology and Microbiology, SDM College of Dental Sciences, Dharwad, India. *Corresponding author: Dr. Nikhil Yadav, dr.nikhily@gmail.com

Oral squamous cell carcinoma (OSCC) is one of the most common cancer arising in the oral cavity from potentially malignant disorders. Detecting oral cancer in early stage enhances survival rate. Centrifuged liquid based cytology (CLBC) offers high quality smears compared to the conventional cytology and helping in enhanced initial evaluation and diagnosis of oral lesion. In this prospective comparative study, we are evaluating the efficacy of CLBC smears over conventional cytology smears in normal oral mucosa and in patients with OSCC. Smears were obtained from normal mucosa and lesions of OSCC with a soft tooth brush after obtaining prior written consent. One smear was made using the conventional technique and another with the centrifuged liquid based cytology technique. Both the smears were stained by Papanicolaou method and values obtained were subjected to chi-square test. CLBC technique presented smears with clear background, adequate cellularity and improved cell morphology compared to the conventional technique. Hence, CLBC technique offered better prospect of slide observation and can be implemented for routine diagnostic purposes over conventional cytology technique.

S37 Reconstruction and characteristisation of in-vitro three dimensional human oral organotypic co-culture model

Archana Kumari Singh ¹ , Daniela Costea ² , Shriya Joshi ¹ , Harsh Dongre ¹ , Rahul Mhatre ¹ , Milind Vaidya ¹ , Sharada S. Sawant ^1, *

¹ Vaidya Lab, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, India; ² Gade Laboratory, Pathology, Department of Clinical Medicine, Haukeland University Hospital, University of Bergen, N-5021 Bergen, Norway. *Corresponding author: Sharada S. Sawant

Most in vitro studies in experimental biology have been done in 2-dimensional (2D) monocultures. Accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells. These interactions may have an important role in neoplastic transformation and progression of the tumour. Hence, we have reconstructed a 3-D co-culture model system from normal, dysplasia and tumour tissues of human oral mucosa to study the cell-cell and cell-ECM interactions during the major stages of oral tumour development. Primary Keratinocytes and fibroblasts are isolated from normal and tumour tissues using direct explant method and characterized for their epithelial and fibroblast purity. Keratinocytes were seeded on collagen matrix and air liquid interface maintained for 12 days. Cultures were maintained in organotypic media, DMEM + Ham’s F12 (3:1), containing special growth factors. At 12 ^th day cultures harvested, fixed and processed for histopathology and immunohistochemical studies. IHC performed for different functional molecules specific for Keratins (epithelia), Vimentin (connective tissues), B4 integrins (basal lamina), PCNA (proliferative cells) and Involucrin (differentiating cells). We found that in-vitro reconstructed tissue maintained its architecture and molecular characterstics mimicking to in-vivo tissue. In future, we like to use this model for anticancer drugs testing.

S38 Determination of plasma homocysteine level in oral submucous fibrosis & oral squamous cell carcinoma using high power liquid chromatography (HPLC)

Dr. Shravya Shetty ¹ , Dr. Kaveri Hallikeri ^1, *, Dr. S.V. Hiremath <sup>2

1</sup> Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences & Hospital, Dharwad, India; ² Department of Biotechnology and Microbiology, K.L.E Society, P.C. Jabin Science College, Hubli, India. *Corresponding author: Dr. Kaveri Hallikeri.

Nutritional deficiencies occur primarily or secondary to disease processes. The folate deficiency developing secondary to that of iron causes hyperhomocysteinemia which induces oxidative DNA damage and altered metabolic states initiating carcinogenesis. Hyperhomocystenemia has been recorded in potentially malignant disorders like Oral submucous fibrosis (OSF) and Oral squamous cell carcinoma (OSCC). The study is aimed to evaluate plasma homocysteine levels in healthy controls, OSF and OSCC patients and to determine it’s plausibility as a potential biomarker. A study sample comprising of healthy controls (n=7), OSF (n=15) and OSCC (n=9) patients were selected. The plasma homocysteine levels were evaluated using HPLC analysis. Statistical analysis was carried out using Non-parametric tests. Plasma homocysteine level was increased in OSF as compared to healthy controls and in contrast, the levels were decreased in OSCC. The plasma levels of Homocysteine were correlated amongst the three groups along with age, gender, duration, frequency and types of habits, which did not show any statistical significance. Nutrition is required to maintain the health and integrity of the mucosa and deficiency leads to increased susceptibility to carcinogens from the betel quid and areca nut. Since hypofolatemia leads to hyperhomocystenemia, the folate (Vit B12) supplementation can arrest disease progression in OSF and play a major role in the chemoprevention and treatment of OSCC.

S39 Role of IL17 producing gdT cells (Tgd17) in gall bladder cancer pathogenesis

¹ Rushikesh Sudam Patil, ¹ Sagar Umesh Shah, ² Shailesh V. Shrikhande, ² Mahesh Goel, ² Rajesh Dikshit, and ^1, *Shubhada Vivek Chiplunkar

¹ Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India, ² Tata Memorial Hospital, Parel, Mumbai-400012, India. *Corresponding author: Shubhada Vivek Chiplunkar, schiplunkar@actrec.gov.in; Presenting author email: rushikesh.patil17@gmail.com

The tumor environment complements chronic inflammation and modulates immune surveillance. 70-80% of Gallbladder cancer (GBC) patients are associated with inflammatory condition of cholelithiasis. The present study aims to understand the dynamics and functions of proinflammatory (Th17, Tγδ17, Tc17) and anti-inflammatory (Treg) immune cells which might be contributing to GBC. Multicolor flowcytometry has revealed that the levels of Tγδ17, Th17 and Tc17 cells increased in peripheral blood lymphocytes (PBLs) and tumor infiltrated lymphocytes (TILs) of GBC patients compared to healthy individuals (HI). CD4 ⁺ CD25 ⁺ CD127 ^low/- Tregs were decreased in PBLs however, the expression of FOXP3 in Tregs and their suppressive potential were comparable in HI and GBC patients. The ratios of Th17/Treg, Tγδ17/Treg and Tc17/Treg increased in GBC patients while γδ⁺ IFNγ⁺ and CD8 ⁺ IFNγ⁺ cells decreased in TILs. Cytokines (IL6, IL23, IL1β) required for differentiation of IL17 producing cells were elevated in patients’ sera. γδT cells migrated towards tumor bed through CXCL9/CXCL10-CXCR3 axis, and skewed towards Tγδ17 phenotype. The Tγδ17 cells induced VEGF production and angiogenesis related proteins in GBC cells through IL17 production. Tγδ17 cells were associated with poor survival of GBC patients as studied by Kaplan-Meier analysis. Our data unravelled novel protumorigenic functions of Tγδ17 cells in GBC, opening new avenues for targeted therapies.

S40 Aberrant cell kinetics: as evidenced by c-Jun expression in normal buccal mucosa, oral submucous fibrosis, severe epithelial dysplasia and well differentiated squamous cell carcinoma

Dr. Shraddha K.S. ^1, *, Dr. Niranjan K.C. ¹ , Dr. Kaveri Hallikeri <sup>1

1</sup> Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences & Hospital, Dharwad, India. *Corresponding author: Dr. Shraddha K.S.

Background and Objective: c-Jun is a component of the transcription factor complex AP-1, which has a role in cell proliferation, differentiation, apoptosis and oncogenic transformation. Over-expression of c-Jun has been implicated in the pathogenesis of several types of cancer including oral cancer. The aim of this study was to correlate the expression of c-Jun in Normal buccal mucosa (NM), Oral submucous fibrosis (OSF), severe epithelial dysplasia (ED) and well differentiated squamous cell carcinoma (WDSCC). Methods: Quantitative and qualitative assessment of c-Jun expression was evaluated in a total of 60 cases which included 15 each of NM, OSF, ED and WDSCC. The obtained results were subjected for statistical evaluation. Results: The average c-Jun nuclear expression in NM, OSF, ED and WDSCC was 35.02%, 35.61%, 89.09% and 83.31% respectively. A significant difference was found quantitatively between NM and ED, NM and WDSCC, OSF and ED, OSF and WDSCC & ED and WDSCC. Qualitatively, statistical significance was seen in intense staining of c-Jun expression between OSF and ED, OSF and WDSCC & ED and WDSCC. Conclusion: The over expression of c-Jun in ED and WDSCC demonstrates its role in early carcinogenesis as evidenced in our study. Therefore, c-Jun might act in different mechanisms and pathways that lead to malignant transformation in oral lesions. Keywords: AP-1, Transcription factor, c-Jun, Oral submucous fibrosis, Severe epithelial dysplasia, Well differentiated squamous cell carcinoma.

S41 Methyl-β-cyclodextrin potentiates doxorubicin mediated apoptosis in breast and hepatocellular carcinoma cells through p53 activation and Fas

Naoshad Mohammad ¹ , Shivendra Vikram Singh ¹ , Dipti Athavale ¹ , Parmanand Malvi, Balkrishna Chaube ¹ and Manoj Kumar Bhat1,*

¹ National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune- 411007, India. *Corresponding author: Manoj Kumar Bhat

Doxorubicin-based chemotherapy is commonly used in treating breast and liver cancers. However, its clinical application is limited due to severe side effects as well as development of multidrug resistance phenotype. Number of studies has attempted to find efficient approaches with which to overcome these. Methyl-β-cyclodextrin (MCD), a potent cholesterol depleting agent has been reported to enhance the cytotoxic effects of chemotherapeutic drugs like carboplatin, 5-flurouracil and tamoxifen in various cancer. In the present study, we explored MCD as a tool to sensitize MCF-7 and Hepa1-6 cells to DOX and also investigated the mechanism underlying this combination regimen. Combination treatment with MCD and the marginal dose of DOX reduces the cell viability and promotes apoptosis through induction of pro-apoptotic protein, Bax and down regulation of anti-apoptotic protein, Bcl-2 in the cells. Mechanistically, sensitization to DOX by MCD was due to induction of FasR/FasL death pathway because of p53 activation. Furthermore, inhibition of p53 by using inhibitor (PFT α) or siRNA attenuated the combination effect of MCD and DOX. Additionally, in vivo experiments were performed by using C57BL/6J mouse isografted (Hepa1-6) mode. In mice treated by MCD in-combination with DOX, tumor grows very slowly as compared to control, MCD and DOX alone treated mice. Taken together, our results suggested that the use of MCD in-combination with DOX could be a very efficient way to achieve enhanced therapeutic efficacy against breast and liver cancer.

S42 Quantitative assessment of eosinophils in metastatic and non-metastatic oral squamous cell carcinomas

Wasim Raja Mumtaz ^1, *, Veda Hegde <sup>1

1</sup> Department of Oral Pathology and Microbiology, SDM College of Dental Sciences, Dharwad-580009, India. *Corresponding author: Dr. Wasim Raja Mumtaz, wasimmumtaz786@gmail.com

Oral squamous cell carcinoma (OSCC) is the most common cancer in India. Therefore, any innovation that facilitates early detection of this neoplasm has the potential to improve survival. Tumor associated tissue eosinophils (TATE) are believed to play a significant role in the biological behavior of carcinoma. The present study is aimed to evaluate the efficacy of Congo red staining to discern eosinophils in the inflammatory infiltrate in OSCC and whether it has a role as prognostic indicator or stromal invasion in metastatic and non-metastatic OSCC. This retrospective study includes a total of 30 paraffin embedded tissue blocks of previously diagnosed cases of metastatic and non-metastatic OSCC. TATE was quantified using H&E and Congo red stains in a systematic manner in ten high power field. TATE was compared statistically with age, sex, habit, site & TNM staging of the patient. Statistical analysis were made using t test and statistical significant difference was noted in eosinphil count between metastatic & non-metastatic OSCC and also between non-metastatic OSCC and age group of the patient. So, it is concluded that eosinophils have an important role to play in OSCC and quantitative assessment of eosinophils should become a part of the routine histopathological diagnosis for OSCC.

S43 Absence of galectin-3 contributes to immune dysfunction favoring lung metastasis of B16F10 melanoma in mice

Aparna Chaudhari ¹ , Rajiv Gude, Rajiv Kalraiya, Shubhada V. Chiplunkar ^1, *

¹ Chiplunkar Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India. *Corresponding author: Shubhada V. Chiplunkar, schiplunkar@actrec.gov.in; Presenting author email: achaudhari@actrec.gov.in

Galectin-3, a β-galactoside-binding lectin, is involved in cancer progression and metastasis. Galectin-3 is also an important regulator of immune responses. The present study was aimed at analyzing how Galectin-3 regulates tumor-specific immunity and lung metastasis in B16F10 murine melanoma model. 6-8 weeks old female Gal-3 ^+/+ (wild-type), Gal-3 ^+/- (hemizygous) and Gal-3 ^-/- (null) mice were used. B16F10 cells were injected intravenously to induce lung metastasis in these mice. Gal-3 ^-/- non-tumor-bearing mice exhibited decreased levels of cytotoxic cells and lower NK cytotoxicity against tumor targets, compared to Gal-3 ^+/+ and Gal-3 ^+/- mice. On stimulation, Gal-3 ^+/- and Gal-3 ^-/- mice splenocytes showed increased T cell proliferation and intracellular calcium flux, but lower intracellular ROS levels than Gal-3 ^+/+ mice. Surprisingly, the ability of B16F10 melanoma cells to form lung metastatic colonies in Gal-3 ^+/+ and Gal-3 ^-/- mice remained comparable. Serum Th1/Th2 cytokine balance was disturbed in Gal-3 ^-/- mice, skewing to inflammation induced immunosuppression. Serum IFN-γ, IFN-gR1 receptor, STAT1 and phospho-STAT1(Tyr701) expression in splenocytes were reduced, whereas SOCS1 and SOCS3 increased in Gal-3 ^-/- mice. Our data indicates that absence of Galectin-3 facilitates lung metastasis of B16F10 cells in mice, which may be due to immune dysfunction resulting from decreased NK cytotoxicity, disturbed serum cytokine balance and attenuated IFN-γ signaling.

S44 Glucose dependent alterations in PCSK9 in hepatocellular carcinoma (HCC) cells

Dipti Athavale, Vimal Pandey and Manoj Kumar Bhat*

National Centre for Cell Science (NCCS), Savitribai Phule Pune University Campus, Ganeshkhind, Pune-411007, India. *Corresponding author: Dr. Manoj Kumar Bhat.

PCSK9 (proprotein convertase subtilisin like kexin 9) also known as NARC1 (neural apoptosis regulated convertase) is a member of serine protease. It is secreted mainly by liver cells. PCSK9 binds the LDL receptor (LDLR) on hepatocytes, stimulates its internalization, promotes LDLR lysosomal degradation and prevents recycling of LDLR to the cell surface. Cancer cells use glucose aggressively compared to normal cells. To check the effect of increased glucose flux on molecules involved in lipid metabolism, we have screened a panel of human and murine origin hepatocellular carcinoma cell lines. Only in HepG2 cells, PCSK9 levels were affected by glucose concentration [high (25 mM) and low (5 mM) glucose]. This phenomenon was further confirmed by RT PCR, time dependent protein expression levels and media replenishment studies. Inhibition of glucose uptake significantly reduces the levels of PCSK9 in high glucose conditions. Since LDLR is target of PCSK9, We further checked status of LDLR under 5 mM and 25 mM glucose by confocal microscopy and surprisingly HepG2 cells cultured under 5 mM glucose showed more staining for LDLR suggesting role of glucose in lipid metabolism in HCC cells. This work might be clinically relevant in the group of HCC patients exhibiting hypercholesterolemia.

S45 Notch signaling augments the TCR driven effector functions of human gd T cells through IL2 signaling pathway

Sajad Bhat and Shubhada Vivek Chiplunkar*

Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai – 410210, India. *Corresponding author: Shubhada Vivek Chiplunkar, schiplunkar@actrec.gov.in; Presenting author email: sajadbhat386@gmail.com

gd T cells have been implicated in innate immune responses against various tumors and pathogens. Earlier we have shown that notch signaling regulates cytotoxic potential and IFN-g production of gd T cells. gd T cells depend on IL2 for proliferation and survival. The objective of the present study was to investigate if notch regulates IL-2 signaling pathway and influences the TCR driven effector functions of gd T cells. It was observed that abrogation of notch signaling by gamma secretase inhibitor (GSI) decreases the ability of gd T cells to lyse tumor targets and decreased expression of effector molecules (perforin and granzyme) and master transcription regulators (Eomes and Tbet) in gd T cells. Inhibition of notch signaling by GSI decreased the expression of IL-2 receptor subunits (i.e. CD25, CD122 and CD132) and downstream signaling molecules pAKT and NF-kB. Down regulation of IL-2 signaling through notch signaling caused gd T cells to be arrested in G0/G1 phase of cell cycle which was due to increase in expression of p53 and decrease in MDM2 expression. In conclusion, the studies demonstrate that notch regulates proliferation and survival of gd T cells through IL2 signaling pathway and has important implications in designing gd T cell based therapy for cancer.

S46 Incidence of Aspergillus in oral squamous cell carcinoma and oral submucous fibrosis

Dr. Lalit S. Kanthale, Dr. Madhuri Gawande

Department of Oral Pathology and Microbiology, Sharad Pawar Dental College, Sawangi Meghe, Wardha, India

The carcinogenic, immunotoxic properties of arecanuts are well documented. The nuts may contain aflotoxin, but these have not been adequately quantified in products. The variable nut consumption pattern among chewers makes estimation of exposure to aflotoxin different. This is of consequences as varied concentrations of aflotoxin may enhance the cacinogenic effect of these nuts on human tissue. Oral submucous fibrosis (OSMF) is a chronic, incidious, disabling condition of the oral mucosa affecting any part of the mouth and rarely the pharynx, larynx and oesophagus. Altough a number of contributory factors in the causation of OSMF have been reported, it is generally regarded that areca nut usage either alone or as an ingreidient of the betel nut is the main etiological factor. The precancerous nature of OSMF has been well established with a frequency of malignant transformation rate of 3-6%. Aspergillus is a group of around 200 moulds (fungi) found worldwide. The presence of arecoline as a constituent of arecanut promotes the formation of Aspergillus. Thus, consumption of the nut, in the oral cavity may cause an increase in incidence of Aspergillosis. Eventually, the fungus grows and enters the oral cavity and invades the mucous membrane. Further studies will be needed on the evaluation of role of aspergillus as an causative agent in oral submucous fibrosis.

S47 Analysis of collagen in invasive front of oral squamous cell carcinoma using Picro-Sirius red and polarizing microscope

Dr. Devendra Alrani ^1, *, Dr. Niranjan KC ² , Dr. Swetha Acharya ³ , Dr. Kaveri Hallikeri ⁴

Department of Oral Pathology, SDM College of Dental Science and Hospital, Dharwad, India. *Corresponding author: Dr. Devendra Alrani, dalrani@yahoo.in, dalranialrani86@gmail.com

Evidence for the potent influence of stromal organization and function on invasion and metastasis of oral cancer is ever growing. There are limited studies in the literature on methods to detect, quantify and analyze the collagen in the tumor invasive front of oral squamous cell carcinoma (OSCC). Hence, the aim of the present study is to assess the nature of collagen (thickness, organization, hue, density of packing, orientation) at the invasive front (IF) of OSCC and its clinicopathological correlation. Tissue sections of 30 OSCC cases with IF were stained with hematoxylin, eosin and picrosirius red staining for evaluation of the nature of collagen under polarizing microscope. Results of statistical analysis are awaited. The interim observations suggest that the collagen in most of the discohesive invasive tumor front were thin, disorganized, poorly packed, yellowish – green in hue with moderate-weak birefringence under polarizing microscope.

S48 Differential gene expression profiling in MCF7 vs. MDA-MB-231 cell lines and identifying the molecular markers and drug targets for Triple Negative Breast Cancer (TNBC)

Madheswaran Suresh, Venil N. Sumantran, Natarajan Sudhakar*

Department of Biotechnology, Dr. M.G.R. Educational and Research Institute (University), Maduravoyal, Chennai-600095. *Corresponding author: nsudha79@gmail.com

Background: Breast cancer is one of the most common cancers amongst women and the disease is heterogeneous. There are effective treatment options available for hormone receptor positive breast cancers. Whereas, hormone receptor negative or triple negative breast cancer (TNBC) is an aggressive breast cancer type with lack of targeted therapies. Aim: To study the differential gene expression profiling between MCF7 and MDA-MB-231 cell lines to identify the molecular markers and drug targets for TNBC. Method: BRB-array tool was used for analysis of microarray data. Six different data sets were selected from Gene Expression Omnibus (GEO) repository. Univariate t-test algorithm was used with false discovery rate 0.01 and 1000 random permutation to compare genes that are statistically significant between two classes MCF7 and MDA MB231. Results: We selected 18 well characterized genes which are differentially expressed >10 fold in MDA-MB-231 compared to MCF7. Out of these genes, 12 genes were significantly up regulated and 6 genes were significantly down regulated in MDA-MB-231. The up regulated genes that can act as a gene expression signature in TNBC were TGFBR2, AXL, GAS6, ADORA2B, IGFBP7, PLAU/PLAUR, S100A4, ETS1, DUSP4, DUSP6, GPR116, XAGE1A/1B. The down regulated genes were ESR1, PGR, ERBB3, CDH1, FOXA1 and TG2. Among the up regulated genes, TGFBR2, GPR116, DUSP4 and DUSP6 genes are playing a vital role in invasion and metastasis and thus act as potential targets for TNBC.

S49 Comparative assessment of the tumoricidal properties of Annona squamosa and Annona muricata fruits in-vitro

Shashanka K. Prasad ¹ , Anand Prakash ² , Revathy Baskaran ² , Suma M. Natraj ¹ , SubbaRao V. Madhunapantula ^1, *

¹ Center of Excellence in Molecular Biology and Regenerative Medicine (CEMR), Department of Biochemistry, Jagadguru Sri Shivarathreeshwara Medical College, Jagadguru Sri Shivarathreeshwara University, Mysore – 570015, India; ² Department of Fruits and Vegetables Technology, CSIR-Central Food Technological Research Institute (CSIR-CFTRI), Mysore-570020, India. *Corresponding author: SubbaRao V. Madhunapantula, email: madhunapantulas@yahoo.com

Accumulating evidences from experimental and observational studies now, support the hypothesis that consumption of fruits of Annona squamosa (Custard Apple) and Annona muricata (Soursop/ Lakshmanaphala), helps to fight against cancer. However, it is clearly unknown about the key constituents of these fruits responsible for exhibiting the anti-cancer activity. Therefore we have made an attempt to fractionate and identify the potential anti-carcinogenic phytochemicals from these fruits. Pulp is the edible portion of A. squamosa and A. muricata. Hence, first, we have separated the pulp and freeze dried to obtain a dry powder. Next, the dried pulp-powder was extracted with 70% aqueous ethanol to separate free phenolic compounds. The residue remained is further extracted with 10% sodium hydroxide to harvest bound phenolics. In a separate experiment, the total phenols were extracted from the dried pulp by incubating with 10% sodium hydroxide. All these fractions have been analyzed for (a) total phenol content; (b) reducing sugar levels; (c) antioxidant activity and (d) anti-cancer activity against breast, prostate and colorectal carcinomas. The data demonstrated a dose dependent anti-cancer activity with both free and bound phenols with former being more potent compared to latter. Detailed results will be presented at the time of meeting.

S50 Elucidation of drug resistance mechanism in a lung cancer model

Sajitha I.S., Srinivas K.P., Santhik S.L., T.R. Santhosh Kumar and M. Radhakrishna Pillai

Cancer Research Program – 9, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India. Corresponding author: M. Radhakrishna Pillai, email: mrpillai@rgcb.res.in

Non-small cell lung cancer accounts for 80-85% of lung cancer and multi drug resistance contributes to chemotherapeutic failure and death in these cases. A better understanding of drug resistance mechanisms may help in the development of better treatment options. In the present study, A549 cell line and its podophyllotoxin resistant variant was used to elucidate the mechanism of drug resistance using in vitro and in vivo models. Drug resistant cells were developed by exposure of parental cells to lethal dose of the drug. The emerging clones of cells after a short stay in a senescent phase were expanded and used as drug resistant cells. Both parental and drug resistant cells were injected in SCID mice and differences in the biology of tumour growth was studied. The drug resistant cells formed highly invasive and malignant tumors when compared to the parental cells. Parental cells invaded nearby lymph node and to the lungs, whereas drug resistant cells metastasized to multiple organs including lungs, liver, stomach, intestine spleen and pancreas. Molecular evaluation showed differential expression of drug transporter proteins, proteins associated with cell proliferation, apoptosis and DNA repair suggesting that multiple mechanisms are involved in the development of multi drug resistance in lung cancer.

S51 A prototype COX-2 inhibitor NS-398 enhances the cytotoxicity of BCNU under varying oxygen concentrations

Akansha Jalota ^1,2 , Mukesh Kumar ³ , Bhudev C. Das ⁴ , Ajay K. Yadav ² , Kunzang Chosdol ¹ and Subrata Sinha ^3, *

¹ Department of Biochemistry, All India Institute of Medical Sciences, New Delhi-110002; ² Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007; ³ National Brain Research Centre, Manesar, Gurgaon-122051; 4Amity Institute of Molecular Medicine and Stem Cell Research, Amity University, Noida-201313. *Corresponding author: Subrata Sinha.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumors which is characterized by high degree of intra-tumor hypoxia. Hypoxia regulates expression of many genes including COX-2 which results in increased cell proliferation, angiogenesis and chemoresistance. Rational combinations have the possibility of improving therapeutic response. NS-398, a COX-2 inhibitor is known to suppress tumor growth. Carmustine (BCNU) is often used after surgical removal of glioma but does not significantly prolong survival due to chemoresistance. Since COX-2 is over-expressed in subset of GBM, a COX-2 inhibitor in combination with BCNU may prove to be an effective rational chemotherapy. We have, here, demonstrated that when NS-398 was combined with BCNU, the cytotoxicity was synergistically enhanced under normoxia as well as hypoxia in COX-2 expressing cell lines (U87MG and LN229). Further elucidation of the mechanism of apoptosis showed high levels of caspase3/7 activity as well as increased expression of pro-apoptotic (Bax, caspase 3 and cytochrome c) and decreased expression of anti-apoptotic markers (Bcl2). The cell population remaining after the combination treatment showed lower expression of EMT markers (vimentin and N-cadherin). The in-vitro study showed the potential of the combination for reducing chemoresistance and metastasis under hypoxia in glioma cells.

S52 Metastatic Tumor Antigen 1, coregulator transcriptionally represses DNA methyl transferase 3a in cancer cells

Deivendran S. ¹ , Hezlin Marzook, T.R. Santhosh Kumar, Rakesh Kumar* and M. Radhakrishna Pillai*

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India. *Corresponding authors: M. Radhakrishna Pillai, Rakesh Kumar

Metastatic Tumor Antigen 1 (MTA1) is a major overexpressed oncogene in human cancer. MTA1 is a chromatin modifier that regulates transcription by virtue of its dual functional activity both as a corepressor and coactivator. MTA1 contains a BAH domain associated with methylation of DNA, histone and non-histone proteins. We postulate a role of MTA1 or MTA1-containing complexes in target gene methylation and consequently, expression of genes that might be mechanistically involved in the action of MTA1. Through datamining, we found that mRNA levels of DNMT3a and MTA1 to be inversely correlated in the existing microarray databases. This was experimentally validated by MTA1 overexpression which resulted in a significant reduction in the level of DNMT3a mRNA as well as protein; while MTA1 knockdown resulted in an increased expression of DNMT3a in breast cancer cells. These observations suggest that MTA1 may regulate DNMT3a at the transcriptional level as we also found preliminary evidence of MTA1 recruitment onto DNMT3a promoter. Further, our co-immunoprecipitation results showed presence of DNMT3a in MTA1-pulled complex. These studies elucidate a potential role of MTA1 in the regulation of DNMT3a and resulting functions, in addition to bringing DNMT3a into the regulation of gene expression by MTA1 in cancer cells.

S53 Decoding the regulatory functions of MTA ¹ , a stress responsive protein under hypoxic conditions

Hezlin Marzook, S. Deivendran, T.R. Santhosh Kumar, Rakesh Kumar* and M. Radhakrishna Pillai*

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India. *Corresponding Authors: M. Radhakrishna Pillai (mrpillai@rgcb.res.in), Rakesh Kumar (rakeshkumar@rgcb.res.in),

Solid tumors often utilize microenvironmental factors such as hypoxia to exert resistance to chemotherapy and radiotherapy while gaining a tendency to predispose for metastases. This work focus on a master chromatin modifier, Metastasis-Associated Tumor Antigen 1 (MTA1), the founding member of MTA family, which are found to play an important role in epigenetic regulation favoring tumor progression. Experiments conducted in breast cancer cell lines provided a proof-of-concept about the alterations in the sub-cellular distribution of MTA1 during serum starvation and/or hypoxia. It is revealed that nutrient deprivation or hypoxia could re-set the nuclear functions of MTA1, largely due to its reduced levels in the nucleus. MTA1 targets downstream effector SGK1, at both transcriptional and translational levels. We suspect that SGK1 might be a new target of MTA1 and stimulated due to a loss of MTA1’s co-repressive activity in the nucleus. It is possible that MTA1 may have a protective role against stress, presumably, due to its ability to regulate a sub-set of relevant genes in the nuclear compartment. Our findings are revealing how MTA1 modulates hypoxic survival of cancer cells independent of HIF1α regulation and to sustain growth and proliferation under oxygen and nutrient deprived conditions.

S54 Evaluation of anticancer and antioxidant properties of kumari-lota against breast cancer cell lines

Monoj Kr. Das, Anindita Dutta, Pitambar Baishya, Jonita Chongtham and Anand Ramteke*

Cancer Genetics and Chemoprevention Research Group, Dept. of Molecular Biology and Biotechnology, Tezpur University, Tezpur-28, Assam, India. Email: anand@tezu.ernet.in

Phyto-chemicals are increasingly being used in the treatment of cancer because of their availability, potential anti-cancer activity with less adverse effects when compared with chemotherapy. The plant extract was studied for its effects on growth of the malignant cell lines including human MCF-7 and MDA-MB435S breast cancer cell lines using assays for cell viability (MTT) and cell death (EtBr/AO and Hoechst DNA staining). The extract exhibited decreased cell viability, inhibited cell proliferation, and induced cell death in a dose dependent manner. The antioxidant properties were evaluated by measuring DPPH, ABTS, hydroxyl and superoxide anion radical scavenging/inhibitory potentials, total reducing activity and Fe ²⁺ chelation. The hydroalcoholic extract scavenge/chelate in significant concentration dependent pattern. Further studies are necessary for chemical characterization of the active principles and more extensive biological evaluations.

Acknowledgement: The authors MKD and AD are thankful to UGC and DBT, Govt. of India respectively for fellowship.

S55 Antioxidants, cell viability and apoptosis are the targeting node for screening of anticancer efficacy of ‘karphool’ against breast cancer

Monoj Kr. Das, Anindita Dutta, Jonita Chongtham, Pitambar Baishya and Anand Ramteke*

Cancer Genetics and Chemoprevention Research Group, Dept. of Molecular Biology and Biotechnology, Tezpur University, Tezpur-28, Assam, India. Email: anand@tezu.er net.in

Breast cancer is the most leading sites of cancer in female and it account for 19-34 % of all cancer cases among women in India. The current study investigated the in-vitro antioxidant and free radical scavenging activities determined by measuring the scavenging/reducing/inhibitory potentials of ABTS, total reducing activity and Fe ²⁺ chelation, DPPH, hydroxyl and superoxide radicals and compared with BHA/Ascorbic acid/EDTA/Gallic acid. The crude extract of karpool scavenge/inhibit the ABTS, DPPH, Hydroxyl, superoxide radicals and chealate Fe ²⁺ in significant concentration dependent pattern. Present study also evaluated cell viability and apoptotic effect of crude extract of karphool on human breast cancer cells (MCF7, MDA-MB435S). The MTT based assay exhibited dose dependent decreases of cell viability. The extract treated cells revealed hallmark properties of apoptosis such as membrane blebbing, nuclear condensation, and DNA fragmentation. Further the present findings evaluated the cytotoxic effect of karphool on the human lymphocytes, suggest non cytotoxic in the normal cells. Thus the present study revealed potential anti-cancer activities of the crude extract of karphool against human breast cancer cells.

Acknowledgement: The author MKD is thankful to UGC, Govt. of India for fellowship.

S56 Assessment of Jilmil for its cytotoxicity, anticancer efficacy and in vitro antioxidant along with drug metabolizing enzyme profile

Pitambar Baishya, Monoj Kr. Das, Jagrity Choudhury and Anand Ramteke*

Cancer Genetics and Chemoprevention Research Group, Dept. of Molecular Biology and Biotechnology, Tezpur University, Tezpur-28, Assam, India. Email: anand@tezu.ernet.in

Despite major advances in research, cancer remains one of the main causes of mortality worldwide. As a result, there is a great need for the development and implementation of novel, effective, and affordable chemopreventive strategies that can reach a diverse global population. Aiming at the exploration of traditional use of herbal medicine, Jilmil, a commonly used medicinal plant was screened for their cytotoxicity, anticancer efficacy and in vitro antioxidant along with drug metabolizing enzyme profile. The present findings suggest the viability of human PBMC cells treatment of crude extract, using MTT based method. The membrane protective activity was also determined by measuring the haemolysis of human erythrocytes. Dose dependent decrease in cell viability of breast cancer cell lines (MCF7, MDA-MB435S) suggest the anticancer efficacy of the plant extract. The in vitro antioxidant and radical scavenging potential of the crude extract was determined by measuring the scavenging/reducing/inhibitory potentials compared to standard. Antioxidant enzyme profile of hepatic HepG2 cells on treatment of Jilmil was assessed, underlining the antioxidant activity of Jilmil.

Acknowledgement: The authors MKD and PB are thankful to UGC and TU, Govt. of India respectively for fellowship.

S57 Correlation of antioxidant, xenobiotic metabolism and anti-proliferation efficacy of bok phool against breast cancer

Monoj Kr. Das, Jonita Chongtham, Pitambar Baishya, Anindita Dutta and Anand Ramteke*

Cancer Genetics and Chemoprevention Research Group, Dept. of Molecular Biology and Biotechnology, Tezpur University, Tezpur-28, Assam, India. Email: anand@tezu.ernet.in

In the present we have studied the anti-proliferative efficacy of the herbal extract against breast cancer cell lines and have correlated with the antioxidant and its related enzyme profiles. The cytotoxicity was tested on lymphocytes (MTT based assay) and anti-proliferative activity of the plant extract was assessed in breast cancer cell lines (MCF-7 and MDA-MB435S). Apoptosis was studied by Hoechst staining and AO/EtBr staining and COMET assay to study the amount of DNA damage. Present findings showed anti-proliferative activity via induction of apoptosis in the cancer cells in dose dependent manner; however the extract did not show any cytotoxic effects in the human lymphocytes.

Acknowledgement: The authors MKD and JC are thankful to UGC and DBT, Govt. of India respectively for fellowship.

S58 A comprehensive study of the role of H2A isoforms in carcinogenesis

Saikat Bhattacharya, Divya Reddy and Sanjay Gupta*

Gupta Lab, Epigenetics and Chromatin Biology Group, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai- 410210, India. *Corresponding author: Sanjay Gupta; Presenting author email: sbhattacharya@actrec.gov.in

It is now well appreciated that epigenetics contribute immensely in the process of carcinogenesis. It is increasingly being recognised that the histone isoforms are members of the repertoire of molecules by which the epigenetic controls are enforced. Earlier studies from our lab has shown the differential expression of two H2A isoforms H2A.1 and H2A.2, both in hepatocellular carcinoma and liver development in rats. However, the non-redundant roles of the H2A isoforms at the molecular level is not understood. We present here the findings of the study which show non-redundant roles of H2A.1 and H2A.2 isoforms. Our in vitro studies suggest that H2A.2/H2B dimer is more stable compared to H2A.1/H2B dimer this differential stability is also reflected in the stability of nucleosomes with H2A.1 nucleosome being more stable than H2A.2 containing ones. Ectopic overexpression of these variants in cell lines suggest that H2A.1 overexpression leads to higher proliferation, however, it did not affect the migratory potential of cells. In accordance with their role in proliferation the relative level of H2A.1 was found to increase during regeneration of liver following partial hepactectomy. This is the first in depth investigation of the structural and functional roles of histone isoforms in epigenetic regulation.

S59 Xenobiotic drug metabolizing enzymes, antioxidant and cytotoxic potential of ‘Gohona bon’

Monoj Kr. Das, Jagrity Choudhury, Pitambar Baishya and Anand Ramteke*

Cancer Genetics and Chemoprevention Research Group, Dept. of Molecular Biology and Biotechnology, Tezpur University, Tezpur-28, Assam, India. Email: anand@tezu.ernet.in

Free radicals, in the form of reactive oxygen and nitrogen species, are an integral part of normal physiology. An over-production of these reactive species due to oxidative stress can cause damages to a large number of biomolecules resulting in increased risk of cancer. Dietary antioxidants can augment cellular defences and help to prevent oxidative damage to cellular components. Aiming at the exploration of traditional use of herbal medicine, ‘Gohona Bon’ a commonly used medicinal plant was investigated for the xenobiotic drug metabolizing enzyme activity, in-vitro antioxidant and free radical scavenging activity. It also investigates the cytotoxic potential in breast cancer cell lines ( MCF7 and MDA-MB435S). Xenobiotic drug metabolizing enzymes were studied by evaluating both the enzymatic and non enzymatic antioxidant enzymes. MTT assay was carried out to test the cytotoxicity on lymphocytes. The anti proliferative activity of the plant extract was studied on Breast cancer cell lines (MCF-7 and MDA-MB435S) by MTT based assay and Apoptosis by AO/EtBr staining and COMET assay to study the amount of DNA damage. Significant dose dependent anti-proliferative activity was observed on the cancer cells via DNA damage but no cytotoxic effect were observed in the normal cells.

Acknowledgement: The authors MKD and JC are thankful to UGC and DBT, Govt. of India respectively for fellowship.

S60 O-linked β-N-acetylglucosamine (O-GlcNAcylation) and phosphorylation crosstalk on keratin 18 regulates its stability and filament organization

Poonam Kakade ¹ , Srikanth Budnar ² , Rajiv Kalraiya ^1, *

¹ Kalraiya Lab, Tata Memorial Centre (TMC), Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, 410210, India; ² Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Australia. *Corresponding author: Rajiv Kalraiya

Addition of a single N-acetyl-glucosamine (GlcNAc) on Ser/Thr residues, referred to as O-GlcNAcylation, is a novel type of glycosylation seen on nuclear and cytoplasmic proteins. The enzymes for its cycling: O-GlcNActransferase (OGT) and O-GlcNAcase (OGA) respectively reside in nucleus/ cytoplasm, and O-GlcNAcylation like phosphorylation is a dynamic modification. Cytokeratin 8 and 18 (K8/18) pair is dynamically modified with O-GlcNAcylation and phosphorylation at multiple sites. Both the modifications are important functional determinants of K8/18. They regulate solubility, stability and filament organization of keratins. Role of phosphorylation in regulation of K8/18 functions in site specific manner is well studied. Presence of multiple sites of glycosylation adjacent to site of phosphorylation on K8/18 prompted us to investigate the possible crosstalk by generating site specific glycol- and phospho-mutants. K18 glycosylation sites (Ser29/30/48) are adjacent with major phosphorylation sites (Ser33/52). Triple mutant and S30A mutation on K18 resulted in decreased phosphorylation on S33 and rescuing S30 also increased S33 phosphorylation. S30A mutation resulted in significantly altered keratin filament architecture and stability similar to that observed on mutation of S33A. We for the first time demonstrate that as opposed to Ying Yang regulation, O-GlcNAcylation on S30 positively regulates phosphorylation on S33 and thus the filament architecture and stability.

S61 Role of an oncoprotein – Gankyrin, in promoting alteration of EGFR gene expression

Indrajit Sahu, Minal Mane, Burhan Ud Din Farooqee and Prasanna Venkatraman*

Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India.

Gankyrin (PSMD10), a non-ATPase subunit of proteasome, an oncoprotein associated with many cancers and modulates several critical signalling pathways. Our initial microarray data indicated that gankyrin overexpression in HEK293 cells, results in alteration of ErBb signalling pathway. In cancer ErBb-1 (EGFR) gene undergoes different alterations (Deletions/point-mutations) or gene amplification which facilitates tumor growth and progression. We hypothesized that gankyrin may promote EGFR gene alteration in cancer cells. To address this question we established gankyrin overexpressed stable cell lines in HEK293, MCF10A and A431. By semi-qPCR and RT-qPCR we found that amplification around 8 ^th -exon of EGFR significantly decreased, but there was no change in amplification around tyrosine kinase domain. Amplification of entire extracellular domain of EGFR showed an alteration in amplicon size upon gankyrin overexpression. Furthermore we have showed that hnRNPA1, an mRNA splicing protein, as a novel interacting partner of gankyrin in mammalian cells. To find out the possible involvement of hnRNPA1 interaction with gankyrin in EGFR gene alteration, we performed immunofluorescence and proximal ligation assay. Results suggested a significant nuclear interaction between gankyrin and hnRNPA1. Since nucleus is the primary locus of gene alteration, we speculate the involvement of this interaction in EGFR gene alteration. The above preliminary data suggested there may be an involvement of gankyrin in splicing of EGFR around its 8 ^th -exon which might be facilitating the tumour growth and progression.

S62 Role of keratin 8 phosphorylation in neoplastic progression of squamous cell carcinoma

Richa Tiwari, Hunain Alam, Milind M Vaidya*

Advanced Centre for Treatment, Research and Education in Cancer, Tata memorial Centre, Kharghar, Navi Mumbai – 410210, India.

Keratins were thought to be mere structural proteins but current progress in the field of keratin biology has indicated their key regulatory functions. We have shown role of K8/18 in malignant transformation in stratified epithelial cells. Recent data from our lab suggests that K8/18 promotes tumor progression by deregulating β4 integrin signaling in OSCC. K8/18 undergoes several post translational modifications including phosphorylation which regulates various cellular processes; however its significance in neoplastic progression is still immerging. Our mutational studies showed that loss of K8 phosphorylation leads to increased migration and tumorigenicity in OSCC cells. Next we wanted to investigate role of K8 phosphorylation in skin epidermoid carcinoma cell line (A431) at phenotypic and molecular levels. To address this question K8 was stably knocked down in A431 cells which led to decreased tumorigenic and migratory potential of these cells. Further in K8 null background shRNA resistant K8 wild type, phospho-dead and phospho-mimetic mutants were stably over expressed. To our surprise phospho-dead clones showed significant decrease in cell migratory and invasive phenotype in comparison to wild type clones whereas phospho-mimetic clones showed significantly more migratory and invasive behaviour compared to phospho-dead clones and is more towards wild type. We are also investigating the molecular basis for these changes by iTRAQ analysis. These studies would help us to better understand the role of K8 phosphorylation in tumor progression of SCC.

S63 p38MAPK/ MSK1 pathway mediated increase in histone H3Ser10 phosphorylation leads to poor prognosis in gastric cancer

S.A. Khan ¹ , R. Amnekar ¹ , B. Khade ¹ , S.G. Barreto ^2,4 , M. Ramadwar ³ , S.V. Shrikhande ² , S. Gupta ^1, *

¹ Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai-410210; ² Gastrointestinal and Hepato-Pancreato-Biliary Service, Department of Surgical Oncology, ³ Department of Pathology, Tata Memorial Hospital, Mumbai-400012; ⁴ Medanta Institute of Hepatobiliary & Digestive Sciences, Gurgaon-122001, India. Email: sakbiotech@gmail.com, sgupta@gmail.com

Global level of histone post-translational modifications have demonstrated their diagnostic and prognostic utility in multiple cancers. However, comparative studies of histone marks between tumor and surgical resection margin tissues are minimal. Here, in human gastric cancer, status of several acetylation, methylation and phosphorylation marks of histone H3 and H4 were compared between paired tumor and resection margin tissues and phosphorylation of histone H3 Serine 10 (H3S10P) was found to be most consistent and significantly (p=0.0001) higher in tumor tissues. Immunohistochemical studies showed a significant correlation of H3S10P with tumor grade (p=0.0001), depth of invasion (p=0.0211), lymph node positivity (p=0.008) and also found to be a predictor of survival (p<0.01). In addition, the resection margins, both proximal and distal with the distance of < 4 cm showed higher level of H3S10P compared to > 4cm (p<0.01) helped in predicting survival and defining the true negative resection margin. Increase in the levels of immediate early (IE) genes c-jun, c-fos and also in mitogen and stress activated kinase 1 (MSK1) and phos-MSK1 in tumor tissues further corroborated with the increase of H3S10P in gastric cancer and suggested MAPK pathway mediated regulation of H3S10P. Further investigation of MAPK pathway both in tissues and cell lines (AGS and KATOIII) confirmed p38 MAPK/MSK1 mediated regulation of H3S10P in gastric cancer. Moreover, higher level of H3S10P and IE genes in transformed cell lines of different tissue origins (skin, liver, colon and breast) compared to their untransformed counterparts indicate the possibility of H3S10P as a tumor associated universal histone mark.

S64 Epigenetic landscape of cancer: role of H2A isoforms and H3 variants

Divya Reddy*, Saikat Bhattacharya and Sanjay Gupta

Gupta Lab, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai- 410210, India; Presenting author email: divyavelga@gmail.com

The sequence divergent form of the core histones, the histone variants and the histone isoforms, have been reported to be aberrantly expressed in a variety of physiological conditions. The functional and structural role of few histone variants has been established, example being the increase in expression of H2A.Z in bteast cancer. The correlation between H2A.Z and cancer is so strong that now it is being veiwed as a target for therapy. Although the relation between variants and cancer has been established but the functinal importance of isoforms has been not fully understood yet. But recent reports have highlighted the non-redundant functional importance of H2A isoforms. Studies from our lab has shown that to H2A isoforms, H2A.1 and H2A.2, which are differentially exressed during hepatocellular carcinoma (HCC) progression and liver development impart differential stability to nucleosomes and H2A.1 promotes proliferation (unpublished data). Interestingly, we present here the findings that the H3 variants are also differentially expressed in HCC with upregulation of H3 variant H3.2 and downregulation of H3.3. The H3.2 chaperone CAF-1 was also found to be overexpressed. Additionally, in normal tissue uH2A.1 levels are higher and there is loss of H2A ubiquitination mediated gene repression in cancer without marked alteration in the level of H2A ubiquitinating/deubiquitinating enzymes. Ectopic overexpression of H2A could decrease the level of chromatin bound uH2A suggesting that transcriptional regulation of the H2A isoforms might be critical in determining uH2A mediated repression. In this regard we have identified some very interesting differences in the cis-acting transcription regulatory elements of H2A.1 and H2A.2. Further, although the ratio of H2A.1/H2A.2 increased during liver regeneration H3.2/H3.3 ratio was not altered. In summary, our results suggest that the levels of H2A isoforms and H3 variants together may bring about activation/repression of different subset of genes important in governing the epigenetic landscape of cancer.

S65 Galectin-3 mediated cellular spreading and movement utilizes distinct molecular mechanism as compared to those used on fibronectin

Shyam K. More, Rajiv D. Kalraiya*

Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai-410210, India. *Corresponding author: Rajiv D. Kalraiya, rkalraiya@actrec.gov.in

Galectin-3 is secreted in a non-classical manner which often gets incorporated on the surface of neighboring cells, extra-cellular-matrix and basement membrane. Such immobilized galectin-3 is utilized by cancer cells for their spreading and movement for metastatic dissemination. Here we explore the molecular mechanism by which galectin-3 brings about these effects and elucidate how it is different from the classical components of the matrix like fibronectin. Galectin-3 was found to induce parallel actin bundles at the lamellipodial region resulting in unique morphological features, from the early time point as compared to stress fibers throughout the cell body, in case of fibronectin, as observed by fixed and live cell imaging. Actin turnover in the lamellipodial region was much higher on galectin-3 as compared to that on fibronectin, as analyzed by FRAP. Regulation of AKT and Rac1 was found to be inversely regulated on galectin-3 and fibronectin whereas regulation of ERK remained same. Inhibition of AKT and ERK inhibited spreading and motility of cells on galectin-3 while these inhibitors did not affect the same on fibronectin. These results demonstrate that secreted galectin-3 may serve as a useful extracellular component for tumor cell dissemination that utilizes multiple signaling mechanisms possibly via different glycoprotein carriers.

S66 Transcription regulation of histone H2A.1 and H2A.2 variants

Monica Tyagi*, Shafqat Ali Khan, and Sanjay Gupta

Cancer Research Institute, Advanced Centre for Treatment, Education and Research in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India. *Email: mtyagi@actrec.gov.in

Chromatin is highly organized structure that acts as regulatory switch for genetic information and the possible mechanisms so far demonstrated involves PTMS, DNA methylation and histone variants. Recent studies report number of histone variants that account for specialized cellular function under various patho-physiological conditions. On the basis of previous lab report on differential expression of H2A.1/H2A.2 variant during sequential development of HCC, in the present study we demonstrate a strong correlation of H2A.1 expression with undifferentiated hepatocytes, overlapping signatures of histone modifications and chromatin architecture. The plausible reason hypothesized for such observation is the transcriptional regulatory mechanism of H2A.1 and H2A.2 variant gene. Therefore, in the current study we have delineated mechanism of H2A.1 and H2A.2 transcription regulation by step wise identification of TSS, 5’UTR and conserved stem-loop with 62bp and 88bp long 3’UTR, suggesting their replication-dependent expression. Further, both H2A.1 and H2A.2 are TATA-driven with conserved CAAT sequence with different cis-acting elements in their promoters signifying their different regulatory mechanisms. The binding of transcription factors, p53 and Sox-9 have been identified, in vitro and in vivo, as negative transcriptional regulators of H2A.1 and H2A.2 gene promoters, respectively. The, H2A.1 and H2A.2 variants might play a potential role in defining highly specialized chromatin organization during different biological processes, differentiation and/or de-differentiation suggesting their role in epigenetic reprogramming of genome.

S67 Evaluation of different isolation techniques for cancer stem cells in cervical cancer

Anuka Sharma ¹ , Ajay Kumar ¹ , Shalmoli Bhattacharyya* ¹

Department of Biophysics, PGIMER, Chandigarh, India. *Correspondence author: Dr. Shalmoli Bhattacharyya, PhD, FISBT, Associate Professor. E-mail: shalmoli2007@yahoo.co.in; Presenting author email: anuka.sharma2@gmail.com

Cervical cancer can be treated successfully if detected in early stages; however the advanced stage, recurrent or metastatic disease is still incurable. Recent evidences implicate a small pool of self-renewing malignant progenitors cells i.e. cancer stem cells (CSCs) for the generation of metastasis and resistance to therapy. So, it is important to identify and isolate these cells from bulk tumor population. No established marker for CSCs has been identified in cervical cancer. In present study, cervical cancer cell line (s) were screened for the presence of CSCs using already known methods of CSC isolation in other types of cancer like; CD-44 expression (99.59΁0.24%), CD-133expression (2΁0.53%), Aldehyde dehydrogenase (ALDH) activity (4.33΁0.43%) and Side population (SP) analysis (~2%). Based on the preliminary results, SP cells identified on the basis of Hoechst exclusion method were selected for further studies. SP cells were observed to show distinct CSCs like features namely high sphere formation ability in non-adherent conditions. These cells also showed higher expression of drug resistance and pluripotency genes in comparison to non-SP cells. So, targeting SP cells may represent a novel strategy for designing drugs to prevent relapse due to drug resistance in cervical cancer.

S68 Presence of epithelial to mesenchymal transition (EMT) a metastatic process promotes tumor invasion in renal cell carcinoma

Mamta Singla ¹ , Amanjit Bal ² , Arup K. Mandal ³ , Shrawan K. Singh ³ and Shalmoli Bhattacharyya* <sup>,1

1</sup> Dept. of Biophysics; ² Dept. of Histopathology; ³ Dept. of Urology; Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. *Corresponding author: Dr. Shalmoli Bhattacharyya, Associate Professor, Department of Biophysics, Research Block-B, PGIMER, Chandigarh – 160012, India, Email: shalmoli2007@yahoo.co.in; Presenting author: mamta17pgi@gmail.com

The transition from epithelial to mesenchymal phenotype (EMT) plays an essential role in tumor progression and metastasis in various solid tumors. EMT converts the polarized epithelial cells to a migratory, sarcomatoid phenotype,is regarded as an important event during malignant progression and metastases.However, the role of EMT in tumor metastasis has not been demonstrated in all tumors. In this study, we explored the relative expression of EMT associated genes in patients of renal cell carcinoma. Surgical specimens of tumor and adjoining normal looking parenchyma were collected from patients undergoing nephrectomy for kidney tumor with their written consent.Histological diagnosis was done for confirmation of RCC. Expression of E-cadherin, Vimentin and Snail were analyzed by real time PCR. Gene expression analysis in patients showed 0.5 fold decrease in expression of E-Cadherin in tumor tissue as compared to its adjoining normal kidney tissue (p<0.0048). Vimentin was found to be upregulated by 5.9 folds (p<0.0012) while Snail expression increased by 3.8 fold (p <0.06) in tumor tissue as compared to adjoining normal kidney tissue. EMT phenotype in tissue samples were also confirmed in Western immunoblot.. EMT was also confirmed in in vitro culture of cells from normal looking parenchyma and tumor tissue of RCC patient. Immunohistochemical analysis was done for EMT signature proteins which showed an increase in cells from the tumor tissue and the tumor cells showed increased invasiveness. This shows the process of EMT is involved in the metastatic spread of RCC and may be targeted during designing the anti-metastatic therapy.

S69 Human papillomavirus antibody patterns in the Indian 2- versus 3- dose quadrivalent HPV vaccination trial

¹ Priya R. Prabhu, ² Michael Pawlita, ¹ M. Radhakrishna Pillai, ³ Neerja Bhatla and ⁴ R. Sankaranarayanan for Indian Multi Centre HPV Vaccination Study Group

1Rajiv Gandhi Centre for Biotechnology, Trivandrum, India, 2German Cancer Research Centre, Heidelberg, Germany, 3AIIMS, New Delhi, India, 4International Agency for Research on Cancer, Lyon, France. Corresponding author: Dr. R. Sankaranarayanan, Head and Senior Scientist, Screening Group, Early Detection and Prevention (EDP), International Agency for Research on Cancer, 150 cours Albert Thomas, 69008 Lyon, France. Email: SankarR@iarc.fr

Vaccination against human papillomavirus (HPV) types 16 and 18 is safe and effectively prevents infection and disease induced by these types and also showed some cross-protection against related HPV types. A multi-centre vaccination study that permits comparing the effectiveness of 2 versus the standard 3 doses is on-going in India since 2009.In this study we compare immunogenicity of 2 versus 3 doses of quadrivalent HPV vaccine in India in the first 3 years post vaccination. The trial at 9 Indian study sites enrolled 17,729 girls aged 9 to 18 years to receive 2 (months 0 and 6) or 3 (months 0, 2 and 6) doses of quadrivalent HPV vaccine (Gardasil;). Plasma samples (n=10900) collected before and at different time points after vaccination were analyzed for HPV capsid protein L1-binding antibodies by bead-based multiplex serology (Waterboer, ClinChem 2005) and for HPV neutralizing antibodies (n=498) by pseudovirion-based neutralization assays (Sehr, PLOSOne 2013). The geometric mean titer of L1-binding antibodies to vaccine types in the 2-dose per protocol recipients at months 7, 18, 24 and 36 months after first dose was non-inferior to 3-dose recipients. Similarly, neutralizing antibody titers measured at months 18 and 24 did not differ significantly. Preliminary data analysis suggests that prevalence of cross-neutralizing antibodies against non-vaccine HPV types 31, 33 and 45 may be reduced by 20%. Antibody response to vaccine-targeted HPV types after the 2-dose quadrivalent HPV vaccine schedule was non-inferior to the standard 3-dose schedule.

S70 Interaction of triplex forming oligonucleotide with the promoter region of high mobility group box 1 (hmgb1) and its inhibitory effect on HMGB1 expression in HepG2 human hepatocellular carcinoma cell line

Neelam Lohani and Moganty R. Rajeswari*

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi – 110029, India. *Corresponding author e-mail: rajeswari3011@hotmail.com

HMGB1 (high mobility group box-1), is a chromatin associated architectural protein with extracellular and intracellular roles. Intracellularly it plays important role in transcription, VDJ recombination, chromatin remodeling, DNA repair etc. Extracellular HMGB1 act as a cytokine to mediate inflammatory reaction through a variety of receptors including toll like receptor and receptor for advance glycation end product etc. Overexpression of HMGB1 has been reported in the development and progression of various cancers and suggested as tumour marker. Thus HMGB1 appers to be a promising therapeutic target. The present study is a comparative in vitro binding study of triplex forming oligonucleotide (TFO) to the promoter region of hmgb1 and its effect on the expression of HMGB1 in HepG2. The potential triplex forming oligonucleotide target sequences (TTS) was screened in hmgb1 gene promoter region using TTS Mapping software. The formation of stable triplex was studied using spectrophotometric and calorimetric technique including UV melting, circular dichroic technique, Fourier transform infra red spectroscopy, and isothermal titration calorimetry. Binding of TFO with the promoter region of hmgb1 revealed that the binding of TFO is much higher than that of potential anticancer drug. Treatment of HepG2 cell with hmgb1 TFOs significantly downregulated HMGB1 expression at the level of mRNA, protein and inhibited cell proliferation as investigated by RT-PCR, Western blot and propidium iodide staining. Interestingly, we found that the combination of TFO and anticancer drug showed a synergistic effect on HMGB1 expression. All these results taken together suggest that TFO based antigene strategy to inhibit HMGB1 expression could be an effective way to treat various cancer in which it is implicated to play a central role.

S71 To determine the incidence of tobacco chewing in families of patients suffering from oral squamous cell carcinoma

Rashmi Nigudkar, Dr. Minal Chaudhary

Sharad Pawar Dental College, Sawangi Meghe, Wardha

Background: Use of smokeless tobacco is major cause of oral squamous cell carcinoma. Use of this tobacco has social sanction and faces few impediments in widespread use. In communities and families it has been found that this habit passes from one generation to another. Aim: To determine the incidence of tobacco chewing in families of patients suffering from tobacco associated oral squamous cell carcinoma. Material and Method: The study is being carried out at Sharad Pawar Dental College, Sawangi, Wardha. The families of 30 known cases of oral squamous cell carcinoma are taken into consideration. The family members have been asked to fill the questionnaire regarding tobacco use, its duration and frequency. Analysis and results of proforma will be evaluated statistically.

S72 Secretory phospholipase A2 IIA/ enhancing factor over expression shows disruption in epidermal homeostasis and stem cell depletion

Rahul Sarate, Gopal Chovatiya, Vagisha Ravi, Nirmala Mansukhani, Sanjeev K Waghmare*

Stem Cell Biology Group, Waghmare Lab, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, India; *Email: swaghmare@actrec.gov.in

Epidermis is maintained throughout the adult life by stem cells that self-renew and also generate progeny that undergo terminal differentiation. Enhancing factor (EF), a growth factor modulator, is the mouse homologue of human secretory group II phospholipase A2 (sPLA2 IIA). While it is involved in various cancers, its role in epidermal homeostasis is obscure. This present study showed the role of EF in epidermal stem cell regulation and maintenance. In K14-sPLA2 IIA mice, we attempt to study the hair follicle stem cell behaviour during the first hair cycle at various postnatal ages by performing histological analysis, tail whole mount and immunofluorescence staining of hair follicle stem cell markers as well as proliferation and differentiation markers. Further, hair follicle stem cell pool was assessed by Flow Cytometry and subsequently performed BrdU Label Retaining Cells (LRCs) study in the skin tissue in the K14-sPLA2 IIA mice as compared to wild type littermate. To study functional characterisation, colony forming assays was performed. K14-sPLA2 IIA mice showed epidermal hyperplasia, loss of ortho-parakeratotic organization and increase in differentiation. The data showed gradual decrease in the hair follicle stem cell pool, and also there is a loss of slow cycling BrdU Label Retaining Cells (LRCs). The result showed that there is a decrease in colony forming ability in the K14-sPLA2 IIA mice. Over expression of Secretory phospholipase A2 IIA/ enhancing factor in mice epidermis resulted in the depletion of the hair follicle stem cell pool and disruption of epidermal homeostasis.