Loyola Marymount University Comprehensive Gene Expression Analysis of Prostate Cancer Reveals Distinct Transcriptional Programs Associated With Metastatic Disease Kevin Paiz-Ramirez Janelle N. Ruiz Biology 398.01 Department of Biology Loyola Marymount University April 11, 2010

Outline Classification of prostate cancer tumors and importance of investigating differences in gene expression profiles between progressive and non-progressive tumors Microarray analysis to determine differential gene expression Results: Genes involved in functions such as cell cycle regulation, mitosis, signaling, and DNA replication and repair were highly differentially expressed between metastatic and primary tumors Metastatic tumors had higher proliferation index than primary tumors Both previously-identified and novel differentially expressed genes were identified

Importance of investigating gene expression profiles of prostate cancer tumors Carcinoma of the prostate is the most common cancer in the United States Tumors can be classified as being either metastasized or localized Once metastatic disease develops, majority of patients die Identifying the genes, gene expression profiles, and biological pathways that lead to metastasis will be important to improve tumor classification and therapy Metastisized = progressive Localized = non-progressive

Genome wide expression analysis of primary and metastatic prostate cancers Tissues samples were taken from: 3 Non cancerous Patients 23 Primary Prostate Cancer Patients 9 Metastatic Prostate cancer Patients Collected as biopsies from 1993-1999 Analysis was preformed with Affymetrix chips Expression values on each array were multiplicatively scaled with an average expression of 2500 across the central 96% of all genes on the array Expression dataset was filtered to include mean expression values that differed by 3 fold between two groups Absolute base 10 Log of the ratio of the means

Differences in clinical and pathological features b/w patients with primary and metastatic tumors

Gene expression clusters were differentially expressed between primary and metastatic tumors Figure 1: Representative gene expression clusters enriched for genes differentially expressed between primary (blue boxes) and metastatic (orange boxes) prostate carcinomas

Genes involved in cell cycle regulation, DNA replication and repair, and mitosis were highly differentially expressed in metastatic tumors

Genes involved in signaling and signal transduction were highly differentially expressed in metastatic tumors

Genes involved in transcriptional regulation and cell adhesion were highly differentially expressed in metastatic tumors

Metastatic tumors had higher proliferation index than primary tumors Proliferation indices for prostate carcinoma samples A: Dendrogram showing overall similarity of gene expression profiles. Blue boxes represent primary tumors and orange boxes represent metastatic tumors B: Bar graph representing proliferation indices based on Ki67 immunohistochemistry C: Section of multitissue block of primary prostate carcinomas immunostained for Ki67 , section of multitissue block of primary prostate carcinomas immunostained for Ki67. Inset, high-power magnification of representative core tissue section. D, section of multitissue block of metastatic prostate carcinomas immunostained for Ki67. Inset, high-power magnification of representative core tissue section.

Metastatic tumors had higher proliferation index than primary tumors Proliferation indices for prostate carcinoma samples A: Dendrogram showing overall similarity of gene expression profiles. Blue boxes represent primary tumors and orange boxes represent metastatic tumors B: Bar graph representing proliferation indices based on Ki67 immunohistochemistry C: Section of multitissue block of primary prostate carcinomas immunostained for Ki67 , section of multitissue block of primary prostate carcinomas immunostained for Ki67. Inset, high-power magnification of representative core tissue section. D, section of multitissue block of metastatic prostate carcinomas immunostained for Ki67. Inset, high-power magnification of representative core tissue section.

Quantitative RT-PCR confirms relative expression values observed from microarray Figure 3: Comparison of relative expression values for selected differentially expressed genes based on Q-RT-PCR and microarray analysis

Microarray data reveals both previously-identified and novel differentially expressed genes between primary and metastatic tumors Previous studies have revealed similar differentially expressed gene expression levels Differentially expressed genes reflect biological distinctions and functional pathways previously implicated in aggressive disease Analysis reveled hundreds of poorly characterized gene clusters that likely represent novel genes of unknown function Biological activity of these genes can be inferred from other known genes with shared expression patterns Few prior studies have used high-throughput gene expression analysis to study prostate cancer metastasis and differences in gene expression between non-progressive and progressive tumors Why? Well-preserved surgical samples are rare -- limiting availability Previous studies with high-throughput analysis have revealed similar differentially expressed gene expression levels: Partial agreement despite different methodology= encouraging and helps validate results One study (comparing fewer samples): 5 of 9 genes found to be commonly differentially expressed agreed with data here Another study: two of genes over-expressed in metastatic cancer as compared with primary prostate cancer agreed with data presented here Another study: two of five genes identified as differentially over-expressed in aggressive cancer agreed with findings here Predicted function of differentially expressed genes gives insight into biology of prostate cancer progression Differentially expressed genes reflect biological distinctions and functional pathways previously implicated in aggressive disease Some of genes differentially expressed may identity critical functional pathways: Ex: MYBL2 -- over expressed in many metastatic tumors -- activate CDC2 gene expression in proliferating fibroblasts --> catalytic subunit of protein kinase complex that induces entry into mitosis (cyclin E modulates functional activity of these genes) This pathway may be critical component of cell cycle regulation in metastatic prostate cancer because all elements of pathway over-expressed in metastatic cells This may serve as therapeutic target Analysis reveled hundreds of poorly characterized EST clusters that likely represent novel genes of unknown function Biological activity of these genes can be inferred from other known genes with shared expression patterns Many are likely to play important roles to those predicted for known gene products (described above) Next Steps: determine function of unknown genes will provide new insights into biology of prostate cancer  

Determining function of unknown genes may identify potential therapeutic targets Few prior studies have used high-throughput gene expression analysis to study prostate cancer metastasis Some of genes differentially expressed may identity critical functional pathways: Example: MYBL2 may signify critical component of cell cycle regulation in metastatic cancer Next steps for research: determining function of unknown genes to gain new insights into biology of prostate cancer and identify potential therapeutic targets for treatment Took out resutls slide (can discuss with figures – this info not so relevant to presentation of reuslts) Expression of genes corresponding to 5992 probe sets were reliably detected in all 23 primary prostate carcinomas 22665 did not detect expression in any of the samples 9 of 23 primary sample patients experienced a recurrence. 132 were over-expressed in primary tumors and 360 in metastasis

References LaTulippe E, Satagopan J, Smith A, Scher H, Scardino P, Reuter V, and Gerald L. Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease. Cancer Res 2002 Aug 1; 62(15) 4499-506.