Janelle N. Ruiz Assignment 11

Vocabulary

1. Heterogeneous: consisting of elements that are not of the same kind or nature http://www.google.com/search?rlz=1C1SNNT_enUS360US361&sourceid=chrome&ie=UTF-8&q=define%3A+heterogeneous
2. Prostate-specific antigen (PSA) is a protein produced by cells of the prostate gland. The PSA test measures the level of PSA in the blood. The higher a man’s PSA level, the more likely it is that cancer is present, but there are other possible reasons for an elevated PSA level http://www.cancer.gov/cancertopics/factsheet/Detection/PSA
3. Indolent: sluggish, slow to develop www.sfaf.org/custom/glossary.aspx
4. Stroma - The supportive framework of an organ (or gland or other structure), usually composed of connective tissue http://www.google.com/search?rlz=1C1SNNT_enUS360US361&sourceid=chrome&ie=UTF-8&q=define%3Astromal
5. Dissemination - the property of being diffused or dispersed http://www.google.com/search?hl=en&rlz=1C1SNNT_enUS360US361&q=define%3Adisseminate.&btnG=Search
6. Fibroblast: - a cell from which connective tissue develop http://www.google.com/search?hl=en&rlz=1C1SNNT_enUS360US361&q=define%3Afibroblasts&btnG=Search
7. Metastasis - the spreading of a disease (especially cancer) to another part of the body wordnetweb.princeton.edu/perl/webwn
8. Prostatectomy: surgical removal of part or all of the prostate gland wordnetweb.princeton.edu/perl/webwn
9. Transduction: the transformation of sensory stimulus energy into a cellular signal, such as a receptor potential www.macalester.edu/psychology/whathap/ubnrp/smell/definitions.html
10. Immunohistochemistry - an assay that shows specific antigens in tissues by the use of markers that are either fluorescent dyes or enzymes (such as horseradish peroxidase) wordnetweb.princeton.edu/perl/webwn

Outline

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

Introduction

• Prostate cancer is the most common cancer in the United States affecting over 198,000 individuals in 2001
• Recently, increase in proportion of patients diagnosed with tumors confined to the prostate gland alone

1. Results of increased public awareness and early detection and measurement of PSA levels in blood
2. Early disease is genetically heterogeneous because many patients have a slow-moving cancer (more likely to survive)

• Once metastatic disease develops, majority of patients die
• Prostate cancer now considered in a series of stages of disease: metastasized and localized tumors

1. Understanding biological basis of differences in stages is crucial to assess prognosis, therapy, and treatment.

• Tumor metastasis = most clinically significant (i.e. deadly) event in prostate cancer development

1. Requires that cancer cell participate in specific interactions between itself and host
2. Cells from primary tumors must detach, invade stromal tissue, and penetrate vessels in order to spread themselves throughout the body
3. Must survive this circulation step in order to reach next site in which they stop or bind to tissue
4. To form tumors outside prostate, cancer cells must multiply in the new tissue and obtain a blood supply
5. The tumor cells that are selected for those which are most "fit" -- this process involves many genes and transcriptional programs

• Purpose of study: Identification of genes, gene expression profiles, and biological pathways which contribute to metastasis

1. This is important to improve tumor identification and therapy
2. To do this, authors performed genome-wide expression analysis and identified differences in gene expression between primary and metastatic prostate cancers

Materials and Methods

• Samples:

1. Tissues from

3 non-cancerous prostates (controls) 23 primary prostate cancers 9 metastatic prostate cancers Each tissue examined histologically using H&E-stained cryostat sections

• Gene Expression Analysis:

1. RNA was extracted from tissues
2. Complimentary DNA synthesized from total RNA labeled with biotinylated nucleotides
3. Gene expression analysis carried out using Affymetrix U95 gene arrays with 63,175 features for individual gene/EST clusters

Five distinct microarrays, each containing probes for ~12,000 unique genes/EST transcripts Two response measures: Average Difference (primary measure of expression levels) and Absolute Call (secondary measure of expression level) extracted fro each gene on every sample Expression values on each array scaled to have avg expression of 2500

• Data Analysis:

1. Scanned image files analyzed using Microarray Suite v4.0
2. Differential expression (DE) filtered to include only those with mean expression values that differed at least 3-fold between groups. DE evaluated using:

First probed were ranked based on magnitude of difference b/w means of two sample sets Second, datasets normalized by standardizing each gene to mean = 0 and variance = 1 Clustering and result display performed using Cluster and TreeView software Specific genes corresponding to Unigene clusters identified using GenBank of the clone used to produce probe set

• Immunohistochemistry:

1. Tissue prepared using tissue arrayer

Immunohistochemical detection carried out using standard methods

• Quantitative Reverse Transcriptase-PCR:

Q RT-PCR: Utilized SYBR Green system, Measured mRNA expression levels quantitatively

Results

• Gene Expression Analysis Using Oligonucleotide Arrays:

1. Analyzed gene expression using hybridization of RNA target to oligonucleotide microarrays with 63,175 features for gene/EST clusters
2. Highest proportion of expressed genes represented by probe sets on U95A microarray corresponding to genes with near full-length cDNAs

• Identification of Differentially Expressed Genes:

1. Compared 14 tumors from patients that did not recur with 9 metastatic prostate cancer to see differences

Expression data first filtered to include only probe sets detecting genes with mean expression levels that differed by at least three-fold between two groups -- a total of 3,436 probe sets were found to contain differences of this magnitude

1. Probes then ranked based on relative magnitude of different (t test) between the means
2. Of the probe sets for full-length genes, 132 were over-expressed in primary tumors and 360 in metastatic tumors (100 most highly ranked tumor genes based on t test listed in Table 2)

• Functional Attributes of Differentially Expressed Genes:

1. Based on review of literature, assigned general molecular or biological function to each gene (Table 2)
2. These reflect biological differences between primary and metastatic tumors
3. 26 of 100 high ranked genes play some role in cell cycle regulation, DNA replication and repair, or mitosis

Examples: RFC5, TOP2A, etc - known to be up-regulated in highly proliferative cells

1. 15 of 100 corresponded to genes known to be involved in signaling an signal transduction
2. 9 of 100 may contribute to cell adhesion, migration, or extracellular matrix

Ex: HMMR -- encodes extracellular matrix binding protein which pays a role in cell motility through RAS-ERK signaling pathway

1. Large proportion of 100 believed to be involved in regulation of gene expression and gene product function

13 encode trxn factors, components of transcriptional complex, or other protein contributing to regulation of trxn 3 encode products which participate in RNA splicing or metabolism 3 encode products which participate in chromatin modifications - may also impact trxn regulation 5 contribute to post-trxn regulation of protein function Findings suggest that progression of metastatic prostate cancer associated with changes in gene expression levels related to cell proliferation, interaction with micro-environment, cell motility, activated signal transduction pathways, and regulation of gene product synthesis and function

• Validation:

1. Gene expression values validated by:

Some gene transcripts represented by more than one probe set (each detected similar levels of expression) Cancer Genome Anatoy Project verified expression of many genes Q-RT-PCR -- some of 100 genes selected and measured for transcript levels using Q-RT-PCR --results in agreement with relative levels of microarray gene expression Immunohisotchemical analysis of tumor samples: established protein expression levels of select 100 correlated with mRNA levels from microarray Several genes identified have already been previously shown to be differentially expressed in metastatic prostate cancer

• Table 1: Clinical and Pathological Features of Prostate Carcinomas

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

• Table 2: 100 highest ranked differentially expressed genes and functional classification
• Figure 2: Proliferation indices for prostate carcinoma samples

1. A: Dendrogram showing overall similarity of gene expression profiles. Blue boxes represent primary tumors and orange boxes represent metastatic tumors
2. B: Bar graph representing proliferation indices based on Ki67 immunohistochemistry
3. C: Section of multitissue block of primary prostate carcinomas immunostained for Ki67

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

Discussion

• 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

1. Why?
2. 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

1. One study (comparing fewer samples): 5 of 9 genes found to be commonly differentially expressed agreed with data here
2. Another study: two of genes over-expressed in metastatic cancer as compared with primary prostate cancer agreed with data presented here
3. 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:

1. 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)
2. This pathway may be critical component of cell cycle regulation in metastatic prostate cancer because all elements of pathway over-expressed in metastatic cells
3. This may serve as therapeutic target

• Analysis reveled hundreds of poorly characterized EST clusters that likely represent novel genes of unknown function

1. Biological activity of these genes can be inferred from other known genes with shared expression patterns
2. 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

Presentation

File:Prostatecancerpres.ppt

Janelle N. Ruiz

Class Links

BIOL 398.01/Spring 2010

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