Epigenetic regulation of inhibin alpha-subunit gene in prostate cancer cell lines

Abstract

Inhibin was first identified as a gonad-derived regulator of pituitary FSH; however, it has subsequently been shown to be a tumour suppressor in the gonad and adrenal glands. Whereas non-malignant regions of human primary prostate carcinomas express inhibin -subunit (INHA), malignant tissues lack INHA transcript and protein, which is consistent with epigenetic regulation of the inhibin -subunit gene (INHA) promoter. This study investigated whether methylation of the INHA promoter was responsible for inactivation of INHA transcription and translation in the prostate cancer cell lines, LNCaP, DU145 and PC3. Methylation of the promoter was revealed by bisulphite genomic sequencing and use of inhibitors of methylation and histone deacetylation resulted in reactivation of the INHA transcription and translation. Significant (P<0·05) downregulation of a luciferase reporter gene downstream from a methylated INHA promoter compared with unmethylated INHA promoter occurred in vitro. The data demonstrate that promoter methylation is associated with downregulation of the INHA gene in prostate cancer cell lines, which is consistent with its tumour suppressive role. Therefore INHA has a significant role in prostate tumorigenesis.

Elevated level of inhibin-α subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer

“…Studies from our laboratory on metastatic PCa epithelial cell lines demonstrated that high concentrations (400 ng ml À1 ) of recombinant inhibin A inhibited growth of androgen-responsive LNCaP cells, but not androgen-independent DU145 cells, suggesting that the effect of inhibin may be dependent on cell phenotypes related to specific stages of PCa (McPherson et al, 1997). Furthermore, we have shown loss of heterozygosity or epigenetically regulated loss or down-regulation of INHa in PCa patient samples and LNCaP, DU145 and PC3 cell lines (Mellor et al, 1998;Schmitt et al, 2002;Balanathan et al, 2004). Collectively, these data suggest a tumour suppressive function for INHa in the prostate.…”

“…Stable transfection of LNCaP and PC3 cell lines LNCaP and PC3 were obtained from American Type Culture Collection (Rockville, MD, USA) and routinely cultured as described previously (Balanathan et al, 2004). Expression vectors pcDNA3.1 (empty vector, EV) and human INHa cDNA subcloned into pcDNA3.1 (pcDNA3.1 (INHa)) were purchased from Invitrogen (Mount Waverley, Victoria, Australia) and prepared for transfection according to the manufacturer's instructions.…”

“…Reverse transcription (RT) was performed as previously described (Balanathan et al, 2004). b 2 -microglobulin (b 2 mg) was used as a housekeeping gene for block PCR.…”

“…Monoclonal human mitochondria antibody (1 : 100; Chemicon, Temecula, CA, USA) was used to determine the presence of human cells in the tumours as previously described (McCulloch et al, 2005). The monoclonal R1 antibody (7.5 mg ml À1 ), kindly provided by Dr Nigel Groome, was used to determine INHa expression in tumours as previously described (Balanathan et al, 2004).…”

“…Lymphatic vessels were counted using stereological methods as previously described (Balanathan et al, 2004). Lymphatic vessels were counted within tissue sections (of randomly selected INHApositive prostate tumours, n ¼ 15 and EV tumours, n ¼ 11; using n ¼ 2 randomly selected sections per tumour) to assess the LVD within the tumour (intra-tumoural) region, the region in contact with both the tumour and the stroma (peritumoural) and the region away from tumour.…”