Role of TGF-β and Wnt antagonist gene sFRP4 in predicting overall survival and clinic-pathological outcomes in lung cancer patients treated with platinum based doublet chemotherapy A Thesis Submitted in the partial fulfillment of the requirement for the award of the degree of MASTER OF TECHNOLOGY IN BIOTECHNOLOGY Under the supervision of: Submitted by: Dr. Siddharth Sharma Sheetal Vats Assistant Professor Roll No. 601504009 DEPARTMENT OF BIOTECHNOLOGY THAPAR UNIVERSITY, PATIALA -147004 Scanned by CamScanner Scanned by CamScanner Scanned by CamScanner ABSTARCT Title: Role of TGF beta and Wnt antagonists gene sFRP4 in predicting overall survival and clinic-pathological outcomes in lung cancer patients treated with platinum based doublet chemotherapy. TGF-β1 gene is located at chromosome no. 19q13.1-13.39. TGF-β1 maintains balance between cell renewal and cell differentiation by inhibiting cell cycle progression through G1 arrest. Depending upon the tumor type and stage, TGF-β has been reported for tumor suppression as well as for tumor activation properties. sFRP4 is 346 amino acid long sequence, 10.99 Kb long and located on 7p14.1. They are extracellular glycoproteins which act as Wnt antagonists as they bind with Wnt proteins and block Wnt signaling pathways. Objectives: To investigate the role of TGF-β1 polymorphism in modulating the survival and clinical outcomes of lung cancer patients and to examine the role of sFRP4 polymorphisms in affecting the overall survival and prognosis of lung cancer patients. Materials and methods: A total of 186 and 340 cases were genotyped in case of TGF beta and sFRP4 respectively using PCR-RFLP. The overall survival was done using Kaplan-Meier and Cox regression analysis. Results: Subjects with CA and CA+AA genotype for sFRP4 rs1802073 and ECOG (0-1) showed significant association with overall survival when analyzed by using univariate analysis (HR=1.97; 95%CI=1.06-3.66; P=0.004 and HR=1.93; 95%CI=1.04-3.56; P=0.005), whereas analysis by Cox regression showed significant association in genotypes AA (HR=0.42; 95%CI=0.24-0.72; P=0.001), CA (HR=0.43; 95%CI=0.20-0.92; P=0.03) and CA+AA (HR=0.46; 95%CI=0.27-0.79; P=0.005). Subjects with ECOG (2-4) sFRP4 rs1802073 with Cox regression model showed significant for mutant AA genotype (HR=2.54; 95%CI=1.33- 4.86; P=0.004). Conclusion: Our study demonstrated that TGF-β1 rs1800469 C/T polymorphism is not an important factor contributing to survival and clinical outcomes of lung cancer patients in north Indian population. On the other hand sFRP4 rs1802073 (Pro320Thr) is significantly contributing in increasing survival and clinical outcomes of lung cancer. iv TABLE OF CONTENTS S. No. Title Page No. Declaration i Certificate ii Acknowledgement iii Abstract iv Table of Contents v-vi List of Tables vii-viii List of Figures ix-x Abbreviations xi-xii 1. Introduction 1-4 2. Review of Literature 5-30 2.1 Overview of Lung Cancer 5-10 2.2 Transforming Growth Factor-β1 10-12 2.3 Signaling of Transforming Growth Factor beta 1 gene 12-16 2.4 TGF-β signaling pathway alterations and lung cancer 17-18 2.5 TGF-β1 rs1800469 polymorphism mechanism and splicing 18-19 2.6 Wnt protein secretion 19-22 2.7 Functions of Wnt protein 23-24 2.8 Wnt signaling and cancer 24-25 2.9 Wnt signaling and lung cancer 25-27 2.10 sFRP (Secreted Frizzled related receptor) and signaling pathway 27-28 2.11 Secreted Frizzled related receptor 4 (sFRP4) and genetic variants 28-30 3. Aim of Study 31 4. Materials and Methods 32-42 4.1 Sample collection 32 4.2 DNA extraction from peripheral blood 32-34 4.3 Quantitative estimation of DNA samples 34-35 4.4 Agarose gel method qualitative estimation of DNA 35-37 4.5 PCR (Polymerase Chain Reaction) 37 4.6 PCR amplification of TGF β1 rs1800469 C/T 37-38 4.7 PCR amplification of sFRP4 rs1802073 C/A 38-40 4.8 Restriction Digestion of TGF β1 rs1800469 C/T 40-41 4.9 Restriction Digestion of sFRP4 rs1802073 C/A 41-42 4.10 Statistical Analysis 42 5. Results 43-81 5.1 DNA isolation 43 5.2 Polymerase Chain Reaction of TGF β1 rs1800469 C/T 43 5.3 Polymerase Chain Reaction of sFRP4 rs1802073 C/A 44 5.4 Restriction digestion of TGF β1 rs1800469 C/T with enzyme Bsu361 44-45 5.5 Restriction digestion of sFRP4 rs1802073 C/A with restriction enzyme 45 Hinf1 5.6 Demographic distribution of lung cancer patients for TGF β1 gene 45-47 5.7 Association of TGF-β1 rs1800469 C/T on the overall survival of lung 47-50 cancer patients v 5.8 Genotypic distribution and association of TGF-β1 rs1800469 C/T with 50-55 overall survival of lung cancer patients on the basis of histological Subtypes 5.9 Genotypic distribution and association of TGF-β1 rs1800469 C/T with 56 overall survival of lung cancer patients on the basis of gender 5.10 Association of TGF-β1 rs1800469 C/T with overall survival of lung 56-57 cancer patients on the basis of their smoking status 5.11 Genotypic distribution and association of TGF-β1 rs1800469 C/T with 57-58 overall survival of lung cancer patients on the basis of regimen 5.12 Genotypic distribution and association of TGF-β1 rs1800469 C/T with 59-60 overall survival of lung cancer patients on the basis of performance status after receiving chemotherapy 5.13 Genotypic distribution and association of TGF β1 rs1800469 C/T with 61 clinic-pathological parameters 5.14 Genotypic distribution and Association of TGF β1 rs1800469 C/T with 62 lung cancer on the basis of chemotherapy response on the basis of gender and metastasis 5.15 Genotypic distribution and Association of TGF β1 rs1800469 C/T with 62-63 lung cancer on the basis of chemotherapy response 5.16 Demographic distribution of lung cancer patients for sFRP4 gene 63-65 5.16 Association of sFRP4 rs1802073 C/A on the overall survival of lung 65-67 cancer patients 5.17 Genotypic distribution and association of sFRP4 rs1802073 C/A with 67-73 overall survival of lung cancer patients on the basis of histological Subtypes 5.18 Genotypic distribution and association of sFRP4 rs1802073 C/A with 73-74 overall survival of lung cancer patients on the basis of gender 5.19 Association of sFRP4 rs1802073 C/A with overall survival of lung 74-75 cancer patients on the basis of their smoking status 5.20 Genotypic distribution and association of sFRP4 rs1802073 C/A with 75-76 overall survival of lung cancer patients on the basis of regimen 5.21 Genotypic distribution and association of sFRP4 rs1802073 C/A with 76-79 overall survival of lung cancer patients on the basis of performance status after receiving chemotherapy 5.22 Genotypic distribution and Association of sFRP4 rs1802073 C/A with 80 lung cancer on the basis of chemotherapy response 5.23 Genotypic distribution and association of sFRP4 rs1802073 C/A with 80-81 clinicpathological parameters 6. Discussion 82-86 7. Conclusion 87 8. References 88-97 9. Appendix 98 vi List of Tables S. No. Title Page No. 2.1 Various lung cancer causing agents in humans and their exposure types 6-7 2.2 Polymorphism of gene TGF-β1 rs1800469 19 2.3 Wnt pathway alterations in NSCLC 26 4.1 Representing requirements for preparation of washing buffer 33 4.2 Representing requirements for preparation of lysis buffer 33 4.3 Representing requirements for preparation of TE buffer 33 4.4 Requirements for Polymerase Chain Reaction (PCR) for TGF β gene 38 4.5 Steps of PCR along with their specified temperature for TGF β gene 38 4.6 Requirements for Polymerase Chain Reaction (PCR) for sFRP4 gene 39 4.7 Steps of primary PCR along with their specified temperature (sFRP4 40 gene) 4.8 Steps of secondary PCR along with their specified temperature 40 (sFRP4 gene) 5.1 Distribution of demographic characteristics for TGF β rs 1800469 C/T 46-47 of LC cases 5.2 Relationship of TGF-β genotype with overall survival of lung cancer 48 Patients 5.3 Relationship of TGF-β genotype with overall survival of lung cancer 50-51 patients on the basis of histology 5.4 Relationship of TGF-β genotype with overall survival of lung cancer 56 patients on the basis of gender 5.5 Relationship of TGF-β genotype with OS of lung cancer patients on 57 the basis of smoking status 5.6 Relationship of TGF-β genotype with overall survival of lung cancer 58 patients on the basis of regimen 5.7 Relationship of TGF-β genotype with overall survival of lung cancer 60 patients on the basis of performance status 5.8 Relationship of different genotypes with the clinic-pathological 61 Parameters 5.9 Relationship of different genotypes with the clinic-pathological 62 Parameters 5.10 Relationship of different genotypes with the chemotherapy response 63 5.11 Distribution of demographic characteristics for sFRP4 rs1802073 C/A 64-65 of LC cases 5.12 Relationship of sFRP4 genotype with overall survival of lung cancer 65 vii Patients 5.13 Relationship of sFRP4 genotype with overall survival of lung cancer 67-68 patients on the basis of histology 5.14 Relationship of sFRP4 genotype with overall survival of lung cancer 74 patients on the basis of gender 5.15 Relationship of sFRP4 genotype with overall survival of lung cancer 74-75 patients on the basis of smoking status 5.16 Relationship of sFRP4 genotype with overall survival of lung cancer 75-76 patients on the basis of regimen 5.17 Representing relationship of sFRP4 genotype with overall survival of 76-78 lung cancer patients on the basis of performance status (KPS & ECOG) 5.18 Relationship of different genotypes of sFRP4 with the chemotherapy 80 Response 5.19 Relationship of different genotypes of sFRP4 with the clinic- 81 pathological parameters viii List of Figures S.