Regulation of the ITGA2 gene in prostate cancer

Prostate cancer is the most commonly diagnosed cancer in males and is the second leading cause of cancer deaths in men after skin cancer. It is still associated with significant mortality and morbidity and statistics indicate that 80% of prostate cancer deaths have metastatic bony lesions. Previous work from our group has identified integrin alpha 2 (ITGA2) as a putative prostate cancer susceptibility gene through a familial genetic study. There is an increasing body of evidence suggesting it is involved in prostate cancer progression, particularly contributing to the preferential metastasis of prostate cancer cells to the bone. Integrins are surface receptors which play important roles in cell migration, invasion, survival and angiogenesis. Altered expression of integrins has been found to mediate tumour cell invasion and metastasis in a range of cancers including prostate cancer. Recently, it has become evident that epigenetic mechanisms play important roles in the progression of prostate cancer, affecting a large number of genes associated with the disease, which leads to the hypothesis that deregulation of ITGA2 expression by epigenetic alterations may be associated with prostate tumour progression and metastasis. To examine this hypothesis, regulation of the ITGA2 gene was investigated in a panel of prostate cell lines which represents different aspects of prostate cancer biology. The ITGA2 promoter is associated with a large CpG island and reduced methylation at the ITGA2 promoter was observed in the bone metastatic cell line, PC3 when compared to the non-tumorigenic cell line, LNCaP and the benign prostate cell line, PWR-1E. Reduced methylation correlated with increased ITGA2 expression levels in these cell lines. Chromatin accessibility and histone acetylation at the ITGA2 promoter was found to be higher in the more highly expressing PC3 cell line. A lower percentage of nucleosome occupancy at the transcription start site also correlated with the higher expression of ITGA2 in 22Rv1 as compared to LNCaP cells. In addition, inhibition of DNA methylation and histone acetylation in combination increased ITGA2 expression in LNCaP cells. These data are consistent with DNA methylation, nucleosome occupancy and histone acetylation contributing to ITGA2 regulation. Higher ITGA2 gene expression levels were also found to correlate with higher cell migration capacity. This was consistent with LNCaP cells displaying an epithelial-like phenotype while the highly expressing PC3 cells displayed a mesenchymal-like phenotype. Knockdown of ITGA2 in the metastatic prostate cancer cell line, PC3, resulted in reduced cell migration, without affecting epithelial to mesenchymal transition (EMT). However, selection of cells with increased ITGA2 expression by serial passaging of cells on collagen matrix correlated with altered expression of transcription factors known to modulate EMT. Higher ITGA2 expression correlated with increased Twist and decreased Snail expression. Further examination of the regulation of ITGA2 gene by transcription factors suggests that the Sp1 transcription factor activates ITGA2 expression. However, Sp1 is expressed at equivalent levels in both LNCaP and PC3 cells and thus, can not account for the differential ITGA2 expression observed. While Twist showed inconsistent activity, androgen treatment and Snail overexpression repressed ITGA2 promoter activity. Since Snail is more highly expressed in LNCaP as compared to PC3 cells, this could explain the lower ITGA2 expression observed in LNCaP compared to PC3 cells. Overall, ITGA2 has been shown by others to be involved in the selective metastasis of prostate cancer to the bone, which is the major cause of prostate cancer related death. ITGA2 may therefore represent a potential therapeutic target in metastatic prostate cancer. This study has shown that both epigenetic factors involving DNA methylation, histone acetylation and nucleosome occupancy and the transcription factors, Sp1, Snail and androgen receptor cooperate to regulate ITGA2 gene expression. Further, the data presented suggest that alterations to epigenetic factors and/ or the EMT transcription factor Snail may contribute to aberrant ITGA2 expression during prostate cancer metastasis.