Hypoexpression and epigenetic regulation of candidate tumor suppressor gene CADM-2 in human prostate cancer.
OBJECTIVE
Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer.


METHODS
The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR.


RESULTS
We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression.


CONCLUSIONS
CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.
Department of Urology, University of Pittsburgh, 5200 Centre Avenue, Pittsburgh, PA 15232, USA.
Guimin
G
Chang
Shuping
S
Xu
Rajiv
R
Dhir
Uma
U
Chandran
Denise S
DS
O'Keefe
Norman M
NM
Greenberg
Jeffrey R
JR
Gingrich
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics*; D049109:Cell Proliferation; D003001:Cloning, Molecular; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D055815:Young Adult
C556123:CADM2 protein, human; D015815:Cell Adhesion Molecules; D012333:RNA, Messenger; D011994:Recombinant Proteins