T-LAK cell-originated protein kinase (TOPK) enhances androgen receptor splice variant (ARv7) and drives androgen-independent growth in prostate cancer.

Despite impressive advances in the treatment of prostate cancer with various efficacious inhibitors along the androgen/androgen receptor axis, eventual development of incurable metastatic Castration-Resistant Prostate Cancer (mCRPC) is inevitable and remains a major clinical challenge. Constitutively active androgen receptor (AR) spliced variants have emerged as primary means of resistance to anti-androgens and androgen synthesis inhibitors. The alternatively spliced AR variant, ARv7, has attracted significant interest due to its constitutively active status in CRPC that drives androgen-independence. Factors that are involved in regulating ARv7 levels in CRPC are not clearly known. We recently demonstrated that a protein kinase, T-LAK cell-originated protein kinase (TOPK) level correlates with the aggressiveness of prostate cancer and its invasive behavior. In this study, we investigated whether TOPK plays a role in driving androgen-independence in prostate cancer cells. Our data demonstrate that TOPK overexpression in androgen-dependent LNCaP and VCaP induces ARv7 and drives androgen-independent growth. On the other hand, pharmacological inhibition of TOPK in androgen-independent LNCaP95 and 22Rv1 represses AR transactivation, and AR stability. In summary, this study illustrates a direct role of TOPK in regulating ARv7 and driving androgen-independence in prostate cancer cells.

A naturally derived small molecule disrupts ligand-dependent and ligand-independent androgen receptor signaling in human prostate cancer cells.

Continued reliance on androgen receptor (AR) signaling is a hallmark of prostate cancer, including the development of castration-resistant prostate cancer (CRPC), making it an attractive therapeutic target for prostate cancer treatment. Mahanine is a novel carbazole alkaloid derived from the leaves of Murraya koenigii, commonly known as the curry leaf plant, which grows widely across East-Asia. We show here that mahanine possesses the ability to inhibit ligand-dependent and -independent AR transactivation, leading to a prominent decline in AR target gene expression. Mahanine treatment causes a time- and dose-dependent decline in AR protein levels, including truncated AR splice variants, in a panel of androgen-responsive and -independent prostate cancer cells. The decrease in AR levels induced by mahanine occurs posttranslationally by proteasomal degradation, without any change in the AR gene expression. Mahanine treatment induces an outward movement of the AR from the nucleus to the cytoplasm, leading to an initial increase in cytoplasmic AR levels, followed by a gradual decline in the AR levels in both cellular compartments. Ligand-induced AR phosphorylation at Ser-81, a phospho-site associated with prostate cancer cell growth and AR transactivity, is greatly diminished in the presence of mahanine. The decline in AR phosphorylation at Ser-81 by mahanine occurs via the inactivation of mitotic kinase CDK1. Collectively, our data demonstrate that mahanine strongly disrupts AR signaling and inhibits the growth of androgen-dependent and -independent prostate cancer cells, thereby implicating a therapeutic role of mahanine in prostate cancer treatment.

Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

BACKGROUND: Hypermethylation of the promoter of the tumor suppressor gene RASSF1A silences its expression and has been found to be associated with advanced grade prostatic tumors. The DNA methyltransferase (DNMT) family of enzymes are known to be involved in the epigenetic silencing of gene expression, including RASSF1A, and are often overexpressed in prostate cancer. The present study demonstrates how mahanine, a plant-derived carbazole alkaloid, restores RASSF1A expression by down-regulating specific members of the DNMT family of proteins in prostate cancer cells. RESULTS: Using methylation-specific PCR we establish that mahanine restores the expression of RASSF1A by inducing the demethylation of its promoter in prostate cancer cells. Furthermore, we show that mahanine treatment induces the degradation of DNMT1 and DNMT3B, but not DNMT3A, via the ubiquitin-proteasome pathway; an effect which is rescued in the presence of a proteasome inhibitor, MG132. The inactivation of Akt by wortmannin, a PI3K inhibitor, results in a similar down-regulation in the levels DNMT1 and DNMT3B. Mahanine treatment results in a decline in phospho-Akt levels and a disruption in the interaction of Akt with DNMT1 and DNMT3B. Conversely, the exogenous expression of constitutively active Akt inhibits the ability of mahanine to down-regulate these DNMTs, suggesting that the degradation of DNMT1 and DNMT3B by mahanine occurs via Akt inactivation. CONCLUSIONS: Taken together, we show that mahanine treatment induces the proteasomal degradation of DNMT1 and DNMT3B via the inactivation of Akt, which facilitates the demethylation of the RASSF1A promoter and restores its expression in prostate cancer cells. Therefore, mahanine could be a potential therapeutic agent for advanced prostate cancer in men when RASSF1A expression is silenced.

The cellular functions of RASSF1A and its inactivation in prostate cancer.

Epigenetic events significantly impact the transcriptome of cells and often contribute to the onset and progression of human cancers. RASSF1A (Ras-association domain family 1 isoform A), a well-known tumor suppressor gene, is frequently silenced by epigenetic mechanisms such as promoter hypermethylation in a wide range of cancers. In the past decade a vast body of literature has emerged describing the silencing of RASSF1A expression in various cancers and demonstrating its ability to reverse the cancerous phenotype when re-expressed in cancer cells. However, the mechanisms by which RASSF1A exerts its tumor suppressive properties have not been entirely defined. RASSF1A appears to mediate three important cellular processes: microtubule stability, cell cycle progression, and the induction of apoptosis through specific molecular interactions with key factors involved in these processes. Loss of function of RASSF1A leads to accelerated cell cycle progression and resistance to apoptotic signals, resulting in increased cell proliferation. In this review, we attempt to summarize the current understanding of the biological functions of RASSF1A and provide insight that the development of targeted drugs to restore RASSF1A function holds promise for the treatment of prostate cancer.

UVB upregulates the bax promoter in immortalized human keratinocytes via ROS induction of Id3.

Id3 belongs to the inhibitor of differentiation family of helix-loop-helix transcription factors, important in proliferation, differentiation and apoptosis. We showed that Id3, but not Id2 or Id1, mediates the UVB-sensitization of immortalized keratinocytes by inducing caspase 9-dependent apoptosis. In this study, quantitative PCR analysis revealed a time-dependent increase in Id3 mRNA induced by UVB, dependent on reactive oxygen species. UVB upregulated promoter activity of Id3, but not Id2, at early time points, as shown by reporter assays and also stabilized Id3 mRNA, increasing its half-life from 10 to approximately 60 min. We next examined downstream events related to UVB-induced Id3 upregulation and investigated the effects of UVB or ectopic expression of Id3 on bax promoter activity. Regulatory elements in the bax promoter that mediate transcriptional activation by UVB and Id3, in the absence of p53, were identified. Bax promoter deletion analysis revealed that transcriptional activation by UVB involves a 738-bp region upstream from the transcription start site of bax. Mimicking the effects of UVB, ectopic expression of Id3 also upregulated bax mRNA and activated this 738-bp fragment. Mutational analysis of the transcription binding sites further showed that point mutations of the E-box region found in the 738-bp fragment, but not in a 174-bp fragment, completely abolished Id3- and UVB-inducible bax promoter activity, thus confirming the importance of Id3 and UVB-mediated Id3 upregulation in activating the bax promoter. These results suggest a mechanism whereby reactive oxygen species upregulation of Id3 relieves repression of bax via E-box-binding factors.