INFLAMMATION AND CANCER: INTERACTION OF THE CYTOKINE, p53 AND MICRORNA PATHWAYS

Transcription

1 INFLAMMATION AND CANCER: INTERACTION OF THE CYTOKINE, p53 AND MICRORNA PATHWAYS Curtis Harris Laboratory of Human Carcinogenesis NCI Bethesda, MD

2 CHRONIC INFLAMMATION AND INFECTION CAN INCREASE CANCER RISK Inherited Disease Tumor Site Risk Hemochromatosis Liver 219 Crohn s Disease Colon 3 Ulcerative Colitis Colon 6 18% of human cancers, i.e., 1.6 million per year, are related to infection. - B. Stewart and P. Kleihues World Cancer Report, IARC Press, p. 57, 2003 Rheumatoid arthritis is an example of a chronic inflammatory disease without an increased cancer risk, e.g., joint sarcoma. Oncogenic human papilloma viruses are examples of cancer-prone chronic infections without inflammation. Acquired Disease Tumor Site Risk Viral Hepatitis B Liver 88 Hepatitis C Liver 30 Bacterial Helicobacter Pylori Gastric 11 PID Ovary 3 Parasitic S. hematobium Urinary Bladder 2-14 S. japonicum Colon 2-6 Liver Fluke Liver 14 Chemical/ Physical/Metabolic Acid reflux Esophagus Asbestos Lung pleural >10 Obesity Multiple sites *-CH

3 OBESITY IS A CHRONIC INFLAMMATORY DISEASE Weisberg et al. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest, 112: , Xu et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J. Clin. Invest, 112: , A-CH

4 CANCERS ASSOCIATED WITH OBESITY In Women Breast (postmenopausal) Endometrial Cervical Ovarian Colorectal Kidney Liver/ Gall Bladder Pancreatic Esophageal Hematopoietic Calle, E et al., NEJM 348: , 2003 In Men Prostate Colorectal Kidney Liver/Gall Bladder Pancreatic Esophageal Hematopoietic 3000-CH

5 FREE RADICALS AND INFLAMMATION ROS OH O2 - (Hydroxyl (Superoxide) radical) RNS NO ONOO - N 2 O 3 (Nitric Oxide) (Peroxynitrite) Protein Damage (DNA Repair eg Ogg1, Caspases eg 9) DNA Damage and Mutation Nitrosamines/Deamination 8--oxo-dG 8-nitroguanine Etheno Adducts M1G Adduct Base Excision Repair MDA (malondialdehyde) 4HNE (4-hydroxynonenal) Lipid Peroxidation Arachidonic Acid Cascade COX2 Eicosanoids Cell Proliferation 1760-CH

6 SF Kim et al., Science 310: 1966, CH

7 INFLAMMATION AND CARCINOGENESIS CM Ulrich et al., Nature Rev Cancer, 6: 131, CH

8 NITRIC OXIDE CAN ACTIVATE THE PROTECTIVE p53 STRESS RESPONSE PATHWAY AND CAUSE ONCOGENIC p53 MUTATIONS Cytokines Chronic Inflammation inos NO DNA damage p53 activation p53 mutations Anti-carcinogenic Effects Growth arrest DNA repair Apoptosis Anti-oxidant Pro-carcinogenic Effects Genomic instability Decrease cell cycle checkpoints, apoptosis and DNA repair Pro-oxidant 2507A*-CH

9 NITRIC OXIDE MODULATES TUMORIGENESIS IN A MURINE MODEL OF THE LI-FRAUMENI SYNDROME Normal NO levels delay spontaneous tumor development in p53-deficient C57B6 mice in a non-inflammatory microenvironment. Induced high NO levels accelerate spontaneous tumor development in p53- deficient C57B6 mice in an inflammatory microenvironment. Current Studies: Extend to murine models of colon, liver and lung cancer. 3793*-CH

10 LIVER CANCER: MOLECULAR SIGNATURES The poor prognosis of HCC is mainly due to: recurrence intrahepatic metastases HYPOTHESIS: The tumor and the liver marcoenvironment influence the metastatic propensity of HCC. Biomarkers Early Diagnosis Prognosis 3 molecular signatures mirnas Normal Cirrhosis HBV/HCV HCC 90% Majority of patients Cancer-associated gene expression Xin Wang and coworkers Metastasis tumor mrnas microenv t mrnas 4184-CH

11 A TUMOR METASTASIS mrna SIGNATURE PREDICTS HCC METASTASIS AND SURVIVAL A. Machine Learning Weighted Voting Value B. Kaplan Meier training set testing set M NM M CV (p<0.0005): 82% Sensitivity 67% Specificity NM M 153 genes Univariate: p= Multivariate: 2000 perm. Ye et al., Nature Medicine, 2003; Takafuji et al., Oncogene, CH

12 AN IMMUNE-RELATED HCC METASTASIS SIGNATURE IN NONTUMOR HEPATIC TISSUE Metastasis- Averse Microenvironment MAM MIM Metastasis- Inclined Microenvironment qrt-pcr IHC 17-Gene Refined Signature Th1-like cytokines Univariate T-test (p < 0.001); Multivariate test: 2000 random permutations. 454 significant genes with <10 FDR. Budhu et al., Cancer Cell, 2006 Th2-like cytokines 4186-CH

13 NONTUMOR CSF1 IS UPREGULATED IN HCC METASTASIS AND IS AN INDICATOR OF PATIENT PROGNOSIS qrt-pcr IHC Serum ELISA Kaplan Meier <165 pg/ml p= pg/ml 4187-CH

14 Predicted Probabilities Probability Prediction Analysis of Microarray (PAM) NM M Training (20/20=100%) Sample THE 17-GENE NONTUMOR SIGNATURE CAN PREDICT METASTASIS AND IS ASSOCIATED WITH SURVIVAL p=4.2e-11 Probability Kaplan Meier NM p= Censored NM M Survival days Recurrence days M NM M 0.2 Test (87/95=92%) Cox Regression Survival Sample Univariate Analysis Multivariate Analysis Hazard ratio Hazard ratio Clinical variable (95% CI) p value (95% CI) p value Mim/MAM predictor 9.2( )< ( ) Age (>50 vs. <50 yrs) 1.0( ) n.a. Sex (male vs. female) 3.3( ) n.a. HBV (AVR-CC vs. CC) 0.9( ) ( ) AFP (>300 vs. <300ng/ml) 2.0( ) n.a. ALT (>50 vs. <50 U/l) 1.0( ) ( ) Albumin (>0.15 vs. 0.15g/l) 0.3( ) n.a CH

15 CONCLUSIONS HCC tumors exhibit a gene expression profile that is related to metastasis and predictive of outcome The non-tumor macroenvironment influences the metastatic propensity of HCC, predominantly due to a global shift in the inflammatory cytokine response of non-cancerous hepatic tissues. This unique immune-responsive 17-gene signature is predictive of HCC metastasis/recurrence CH

16 CLONAL AND PARALLEL MODELS OF TUMOR EVOLUTION TO METASTASIS Clonal evolution Normal Cell Dual proclivity Proliferate lesion Primary tumor The tendency to metastasize can be acquired by both tumor and it s non-tumor macroenvironment early during breast, prostate, liver and lung tumorigenesis (Ye et al 2003; Ramaswamy et al, 2003; van de Vijver et al, 2002; Yanaihara et al, 2006). Colon and esophagus, unpublished data Metastasis Figure modified from Joe Gray, Cancer Cell CH

17 MICRORNA AND HUMAN CANCER MicroRNA Small non-coding RNAs that are evolutionarily conserved and regulate gene expression. Mature micrornas, nucleotides are generated by sequential processing of primary transcripts (pri-micrornas) mediated by two RNAse III enzymes, Drosha and Dicer. Mature micrornas negatively regulate protein expression of specific mrna by either translational inhibition or mrnas degradation. Human Cancer MicroRNAs are differentially expressed in human cancers. Genes coding micrornas are frequently chromosomally located at fragile sites, regions of amplification, or deletion. MicroRNA signatures can predict survival of cancer patients. 3690*-CH

18 THE BIOGENESIS OF microrna AND sirna AND THEIR POSTTRANSCRIPTIONAL REGULATION OF mrna Common Processing enzymes, Drosha and Dicer Different Target recognition micrornas- multiple mrna targets sirna- single mrna target He l and Hannon GJ. Nat Rev Genet, 5: 522, CH

19 mirnas CAN HAVE MULTIPLE REGULATORS AND TARGETS IN DIFFERENT CELLULAR PROCESSES Examples: Gene Amplification AAAAAAAA Examples: Cell Cycle Checkpoints Gene Deletion AAAAAAAA Bioenergy Cellular Stress mir-xxx AAAAAAAA Cell Death Epigenetic Mechanisms AAAAAAAA DNA Repair Cytokines AAAAAAAA Senescence 3887-CH

20 EXPERIMENTAL OVERVIEW mirna Expression Profiling Microarray Quantification of Specific mirnas Real-time PCR Functional Analysis mirna and mirna Inhibitor Delivery Identification of Target Coding Gene of Specific mirnas Computational Analysis _AGCUUAU CU.... AAGCUUAUUUUUCCU 3257-CH

21 HYPOTHESES: 1: mirnas are differentially expressed in and diagnostic of colon tumors. 2: mirna profiles are predictive of colon cancer prognosis. Strategy Use microrna microarrays to profile microrna expression patterns in tumors and paired non-tumor tissue from the Maryland test cohort. Validate the association of microrna expression using qrt-pcr in the Hong Kong Validation cohort 1-AS

22 MicroRNAs ARE DIFFERENTIALLY EXPRESSED IN TUMORS FROM THE MARYLAND TEST COHORT 37 Independent micrornas are differentially expressed in tumors (p<0.001; FDR < 0.5%) 26 are higher in tumors 11 are reduced in tumors 4190-CH

23 HIGH mirna EXPRESSION IN TUMORS PREDICTS POOR SURVIVAL IN THE VALIDATION COHORT 4200-CH

24 CONCLUSIONS mirna expression in colon tumors is altered in systematic ways. mirna expression is increased in adenomas. More advanced tumors express higher levels of specific mirnas. Multivariate analysis indicates that mirna expression in tumors is an independent prognostic biomarker of two different cohorts. If causal in colon carcinogenesis, specific mirnas may be novel therapeutic and chemopreventive targets CH

25 CLINICAL IMPLICATIONS OF MicroRNA AND CYTOKINE EXPRESSION IN HUMAN CANCER Diagnosis of cancer. Prediction of prognosis, including stage 1. Insight into signaling pathways involved in carcinogenesis and tumor progression. Prediction of therapeutic response. Identification of molecular targets. Add to other classes of biomarkers to formulate a more robust panel-algorithm 3952-CH

26 COLLABORATORS Anuradha Budhu Aaron Schetter Carlo Croce Suet Y. Leung Jane Sohn Xin Wang 1470-CH