Over the last 25 years roles have been established for vitamin D receptor (VDR) in influencing cell proliferation and differentiation. For example, murine knock-out approaches have revealed a role for the VDR in controlling mammary gland growth and function. These actions appear widespread, as the enzymes responsible for 1α,25-dihydroxycholecalciferol generation and degradation, and the VDR itself, are all functionally present in a wide range of epithelial and haematopoietic cell types. These findings, combined with epidemiological and functional data, support the concept that local, autocrine and paracrine VDR signalling exerts control over cell-fate decisions in multiple cell types. Furthermore, the recent identification of bile acid lithocholic acid as a VDR ligand underscores the environmental sensing role for the VDR.In vitroandin vivodissection of VDR signalling in cancers (e.g. breast, prostate and colon) supports a role for targeting the VDR in either chemoprevention or chemotherapy settings. As with other potential therapeutics, it has become clear that cancer cells displayde novoand acquired genetic and epigenetic mechanisms of resistance to these actions. Consequently, a range of experimental and clinical options are being developed to bring about more targeted actions, overcome resistance and enhance the efficacy of VDR-centred therapeutics.

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21 ^{( waf1/cip1 )} ). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter . The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G ₁ and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21 ^{( waf1/cip1 )} activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21 ^{( waf1/cip1 )} expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21 ^{( waf1/cip1 )} expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.

Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity. MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions. In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis. Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)₂D₃ responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)₂D₃ partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21^(waf1/cip1)) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)₂D₃ treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)₂D₃-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)α/γ may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARα/γ target genes, for example CDKN1A [encodes p21 ^(waf1/cip1) ]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A . Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARα/γ ligands in prostate cancer cell lines. Pursuing PPARα/γ signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARα/γ activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARα/γ gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1 . Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARα/γ to regulate key target genes ( CDKN1A and TGFBRAP1 ). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1–PPARα/γ and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARα/γ selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.