Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. In order to analyze the effects of DNA methylation on prostate cancer, we established LNCaP-derived human prostate cancer cells that can pharmacologically induce global reactivation of hypermethylated genes by the methyl-CpG targeted transcriptional activation (MeTA) method. The MeTA suppressed the growth of LNCaP-derived cells and induced apoptosis. Microarray analysis indicated that PYCARD (PYD and CARD domain containing) encoding an apoptosis-inducing factor was upregulated by 65-fold or more after treatment with MeTA. We analyzed DNA methylation statuses using 50 microdissected primary prostate cancer tissues and found an extremely high frequency of tumor-specific promoter hypermethylation of PYCARD (90%, 45/50). Moreover, DNA methylation status was significantly associated with Gleason score ( P = 0.0063 ); the frequency of tumor-specific hypermethylation was 96% (44/46) in tumors with Gleason score ≥ 7 , whereas that in tumors with Gleason score 6 was 25% (1/4). Immunohistochemical analyses using these 50 cases indicated that only 8% (4/50) of cancerous tissues expressed PYCARD, whereas 80% (40/50) of corresponding normal prostate epithelial and/or basal cells expressed PYCARD. In addition, there was no relationship between PYCARD immunostaining and the Gleason score in cancerous tissue and surrounding normal tissue. Inducible expression of PYCARD inhibited cell proliferation by induction of apoptosis. These results suggest that aberrant methylation of PYCARD is a distinctive feature of prostate cancers with Gleason score ≥ 7 and may play an important role in escaping from apoptosis in prostatic tumorigenesis.

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. In order to analyze effects of DNA methylation in prostate cancer, we constructed LNCaP-derived human prostate cancer cells that can induce global reactivation of hypermethylated genes by the methyl-CpG targeted transcriptional activation (MeTA) method. In MeTA, a cassette construct of MBD2-derived methyl-CpG binding domain (MBD) with NFκB-derived transcriptional activation domain is transfected into cancer cells. Then expressed MBD domain specifically binds to the hypermethylated promoter regions, and NFκB transcriptional activation domain recruits p300/CREB-binding protein (CBP) and reactivates hypermethylation-mediated silenced genes. A cell proliferation assay indicated that MeTA suppressed the growth of LNCaP cells. Furthermore, both flow cytometry and TUNEL assays demonstrated that MeTA induced apoptosis. In order to search genes responsible for apoptosis, we performed gene expression microarray analysis of MeTA-uninduced and -induced LNCaP cells: Five genes encoding apoptosis-inducing factors upregulated two-fold or more in accordance with the induction of MeTA; PYCARD (PYD and CARD domain containing), TNFRSF25 (tumor necrosis factor receptor superfamily 25), HRK (harakiri, BCL2 interacting protein), BIK (BCL2 interacting killer), and CIDEA (cell death-inducing DFFA-like effector a). These genes also contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site. We focused on PYCARD and TNFRSF25 because these two genes upregulated 10-fold or more in accordance with the induction of MeTA. We found that both genes were hypermethylated and showed low expression levels in LNCaP prostate cancer cells, whereas only PYCARD was unmethylated in RWPE-1 normal prostate epithelial cells. We further analyzed primary prostate cancer tissues and found tumor-specific promoter hypermethylation of PYCARD in 53.8% (7/13; P < 0.01). These results suggest that methylation-mediated silencing of PYCARD contributes to escape from apoptosis in human prostate cancer, and MeTA may provide important clues to analyze changes in cancer cell phenotypes by DNA methylation alterations. Citation Format: Shinichi Fukushige, Toshiya Miyauchi, Teppei Okubo, Koji Mitsuzuka, Yoichi Arai, Akira Horii. Methylation-mediated silenced PYCARD plays a key role in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2017-4354