Expression of cancer/testis antigens in prostate cancer is associated with disease progression

Abstract

Background: The cancer/testis antigens (CTAs) are a unique group of proteins normally expressed in germ cells but aberrantly expressed in several types of cancers including prostate cancer (PCa). However, their role in PCa has not been fully explored.

Methods: CTA expression profiling in PCa samples and cell lines was done utilizing a custom microarray that contained probes for two-thirds of all CTAs. The data were validated by quantitative PCR (Q-PCR). Functional studies were carried out by silencing gene expression with siRNA. DNA methylation was determined by methylation-specific PCR.

Results: A majority of CTAs expressed in PCa are located on the X chromosome (CT-X antigens). Several CT-X antigens from the MAGEA/CSAG subfamilies are coordinately upregulated in castrate-resistant prostate cancer (CRPC) but not in primary PCa. In contrast, PAGE4 is highly upregulated in primary PCa but is virtually silent in CRPC. Further, there was good correlation between the extent of promoter DNA methylation and CTA expression. Finally, silencing the expression of MAGEA2 the most highly upregulated member, significantly impaired proliferation of prostate cancer cells while increasing their chemosensitivity.

Conclusions: Considered together, the remarkable stage-specific expression patterns of the CT-X antigens strongly suggests that these CTAs may serve as unique biomarkers that could potentially be used to distinguish men with aggressive disease who need treatment from men with indolent disease not requiring immediate intervention. The data also suggest that the CT-X antigens may be novel therapeutic targets for CRPC for which there are currently no effective therapeutics.

Fig. 1

MAGEA2 is predominantly expressed in castrate-resistant prostate cancer. Primary prostate cancer samples were obtained from radical prostatectomy while castrate-resistant prostate cancer were obtained from“rapid”autopsies and propagated as xenografts. Details of these samples are described in Supplemental Tables I and II. RNAsamples were run on awhole genome microarray as described in the Materials and Methods Section. Heat map shows the predominant upregulation of MAGEA genes from subclusters II–IV indicated in parenthesis as well as the CSAG genes. The color display is on a10-fold scale and red indicates overexpression relative to BPH. Green indicates downregulation.

Fig. 2

Q-PCRvalidationofCT-X antigen expression in prostate cancer specimens. A subset of samples from Figure 1 was used to determine expression by Q-OCR. Relative expression (normalized toTATA-binding protein, TBP) is shown for MAGEA12, CSAG2, MAGEA2, MAGEA6, and PAGE 4 (a–e), respectively.

Fig. 2

Q-PCRvalidationofCT-X antigen expression in prostate cancer specimens. A subset of samples from Figure 1 was used to determine expression by Q-OCR. Relative expression (normalized toTATA-binding protein, TBP) is shown for MAGEA12, CSAG2, MAGEA2, MAGEA6, and PAGE 4 (a–e), respectively.

Fig. 3

Epigenetic regulation of MAGEA2 expression in prostate cancer cells. a: Extent of methylation of cytosine residues in the androgen-responsive and androgen-independent prostate cancer cell lines. b: Relative expression of MAGEA2 normalized to TBP before and after 5AZA treatment. c: Fold-change of MAGEA2 expression before and after 5AZA treatment.

Fig. 4

MAGEA2 upregulation is associated with chemoresistance in prostate cancer cells. a: Effect of docetaxel treatment for 3 days on androgen-responsive LNCaP cells. b: Extent of apoptosis as detected by DNA fragmentation determined by flow cytometry.