1. 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
Department of Biology
Loyola Marymount University
April 11, 2010

2. Microarray analysis to determine differential gene expression
Outline
The importance of investigating differences in gene expression profiles between progressive and non-progressive tumors
Microarray analysis to determine differential gene expression
Differentially expressed gene between metastatic and primary tumors
Cell Cycle Regulation
Mitosis
Signaling
DNA Replication
Metastatic tumors had higher proliferation index than primary tumors
Both previously-identified and novel differentially expressed genes were identified

3. 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
Development of metastatic disease leads to death.
Improving tumor classification and therapy can be achieved by identifying
Genes,
Gene expression profiles
Biological pathways
Metastisized = progressive
Localized = non-progressive

4. 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
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

5. Differences in Clinical and Pathological Features Between Primary and Metastatic Tumors

6. Gene Expression Clusters 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

7. Genes Involved in Cell Cycle Regulation, DNA Replication and Repair, and Mitosis Were Highly Differentially Expressed in Metastatic Tumors

8. Genes Involved in Signaling and Signal Transduction Were Differentially Expressed in Metastatic Tumors

9. Genes Involved in Transcriptional Regulation and Cell Adhesion Were Highly Differentially Expressed in Metastatic Tumors

10. Metastatic Tumors Present 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.

11. Metastatic Tumors Present 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.

12. 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

13. 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

14. 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
Further studies would determine function of unknown genes 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

15. 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)