Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein.

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.

c-Jun N-terminal kinase regulates mGluR-dependent expression of post-synaptic FMRP target proteins.

Fragile X syndrome (FXS) is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of FMRP results in an elevated basal protein expression profile of FMRP targeted mRNAs, a loss of local metabotropic glutamate receptor (mGluR)-regulated protein synthesis, exaggerated long-term depression and corresponding learning and behavioral deficits. Evidence shows that blocking mGluR signaling in FXS models ameliorates these deficits. Therefore, understanding the signaling mechanisms downstream of mGluR stimulation may provide additional therapeutic targets for FXS. Kinase cascades are an integral mechanism regulating mGluR-dependent protein translation. The c-Jun N-terminal kinase (JNK) pathway has been shown to regulate mGluR-dependent nuclear transcription; however, the involvement of JNK in local, synaptic signaling has not been explored. Here, we show that JNK is both necessary and sufficient for mGluR-dependent expression of a subset of FMRP target proteins. In addition, JNK activity is basally elevated in fmr1 knockout mouse synapses, and blocking JNK activity reduces the over-expression of post-synaptic proteins in these mice. Together, these data suggest that JNK may be an important signaling mechanism downstream of mGluR stimulation, regulating FMRP-dependent protein synthesis. Furthermore, local, post-synaptic dysregulation of JNK activity may provide a viable target to ameliorate the deficits involved in FXS. Expression of many FMRP target proteins is enhanced in FXS. Here, we evaluated the role of JNKs in FXS. We found that JNK signaling is activated upon mGluR stimulation in wild-type neurons. Conversely, JNK activity is basally elevated in fmr1 knockout. Inhibiting JNK reduced the expression of FMRP target proteins and driving JNK activity increased the expression of these proteins.

Selenium and vitamin E for prostate cancer: post-SELECT (Selenium and Vitamin E Cancer Prevention Trial) status.

Various formulations of selenium and vitamin E, both essential human dietary components, have been shown to possess a therapeutic and preventive effect against prostate cancer. Fortuitous results of clinical trials also implied a risk-reduction effect of selenium and vitamin E supplements. The Selenium and Vitamin E Cancer Prevention Trial (SELECT), using oral selenium and vitamin E supplementation in disease-free volunteers, was designed to test a prostate cancer chemoprevention hypothesis. SELECT was terminated early because of both safety concerns and negative data for the formulations and doses given. Here, we review and discuss the studies done before and since the inception of SELECT, as well as the parameters of the trial itself. We believe that there is a lack of appropriate in vivo preclinical studies on selenium and vitamin E despite many promising in vitro studies on these agents. It seems that the most effective doses and formulations of these agents for prostate cancer chemoprevention have yet to be tested. Also, improved understanding of selenium and vitamin E biology may facilitate the discovery of these doses and formulations.

Melatonin, a novel Sirt1 inhibitor, imparts antiproliferative effects against prostate cancer in vitro in culture and in vivo in TRAMP model.

We recently demonstrated that Sirt1, a NAD(+) -dependent histone deacetylase, was overexpressed in prostate cancer (PCa) and its inhibition resulted in a significant antiproliferative response in human PCa cells. Studies have suggested a link between Sirt1 and circadian rhythms, the disruption of which has been linked to cancer. Interestingly, a decreased production of the pineal melatonin has been shown to deregulate the circadian rhythm machinery and increase cancer risk. Furthermore, disruption in melatonin production and circadian rhythmicity has been associated with aging. Here, we challenged our hypothesis that melatonin will impart antiproliferative response against PCa via inhibiting Sirt1. We demonstrated that melatonin significantly inhibited Sirt1 protein and activity in vitro in multiple human PCa cell lines, and melatonin-mediated Sirt1 inhibition was accompanied with a significant decrease in the proliferative potential of PCa cells, but not of normal cells. Forced overexpression of Sirt1 partially rescued the PCa cells from melatonin's antiproliferative effects, suggesting that Sirt1 is a direct target of melatonin. Employing transgenic adenocarcinoma of mouse prostate (TRAMP) mice, we also demonstrated that oral administration of melatonin, at human-achievable doses, significantly inhibited PCa tumorigenesis as shown by decreases in (i) prostate and genitourinary weight, (ii) serum insulin-like growth factor-1 (IGF-1)/IGF-binding protein-3 (IGFBP3) ratio, (iii) mRNA and protein levels of the proliferation markers (PCNA, Ki-67). This anti-PCa response was accompanied with a significant decrease in Sirt1 in TRAMP prostate. Our data identified melatonin as a novel inhibitor of Sirt1 and suggest that melatonin can inhibit PCa growth via Sirt1 inhibition.

Modulating polo-like kinase 1 as a means for cancer chemoprevention.

Naturally occurring agents have always been appreciated for their medicinal value for both their chemopreventive and therapeutic effects against cancer. In fact, the majority of the drugs we use today, including the anti-cancer agents, were originally derived from natural compounds, either in their native form or modified to enhance their bioavailability or specificity. It is believed that for maximum effectiveness, it will useful to design novel target-based agents for chemoprevention as well as the treatment of cancer. Recent studies have shown that the serine/threonine kinase polo-like kinase (Plk) 1 is widely overexpressed in a variety of cancers and is being increasingly appreciated as a target for cancer management. Additionally, several chemopreventive agents have been shown to inhibit Plk1 in cancer cells. In this review, we will discuss if Plk1 could also be a target for designing novel strategies for cancer chemoprevention.

Regulation of mitosis via mitotic kinases: new opportunities for cancer management.

Mitosis, a critical and highly orchestrated event in the cell cycle, decides how cells divide and transmit genetic information from one cell generation to the next. Errors in the choreography of these events may lead to uncontrolled proliferation, aneuploidy, and genetic instability culminating in cancer development. Considering the central role of phosphorylation in mitotic checkpoints, spindle function, and chromosome segregation, it is not surprising that several mitotic kinases have been implicated in tumorigenesis. These kinases play pivotal roles throughout cellular division. From DNA damage and spindle assembly checkpoints before entering mitosis, to kinetochore and centrosome maturation and separation, to regulating the timing of entrance and exit of mitosis, mitotic kinases are essential for cellular integrity. Therefore, targeting the mitotic kinases that control the fidelity of chromosome transmission seems to be a promising avenue in the management of cancer. This review provides an insight into the mechanism of mitotic signaling, especially the role of critical mitotic kinases. We have also discussed the possibilities of the use of mitotic kinases in crafting novel strategies in cancer management.

Numb regulates stability and localization of the mitotic kinase PLK1 and is required for transit through mitosis.

Numb functions in progenitor cell fate determination and early development, but it is also expressed in postdevelopmental tissues and cancers where its role is unclear. In this study, we report that a targeted knockdown of Numb expression causes a G(2)-M arrest and reduced cell growth in human melanoma cells. Co-immunoprecipitation and colocalization studies showed that Numb interacts with the serine/threonine polo-like kinase Plk1 and Numb cycles in a cell-cycle-dependent fashion along with this mitotic regulator. Interestingly, Numb expression was required for Plk1 protein stability and localization to the spindle poles during mitosis. Reduction in Numb expression resulted in mislocalization of Plk1 at both metaphase and anaphase, leading to disorganized γ-tubulin recruitment in centrosomes. Together, our findings present a novel function for Numb during symmetric cell division. We suggest that dysregulation of Numb expression results in mislocalized Plk1 and poor centrosomal γ-tubulin recruitment, potentially contributing to mitotic errors, aneuploidy, and cancer development.

Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells.

Melanoma, one of the most lethal forms of skin cancer, remains resistant to currently available treatments. Therefore, additional target-based approaches are needed for the management of this neoplasm. Polo-like kinase 1 (Plk1) has been shown to be a crucial regulator of mitotic entry, progression, and exit. Elevated Plk1 level has been associated with aggressiveness of several cancer types and with poor disease prognosis. However, the role of Plk1 in melanoma is not well established. Here, we show that Plk1 is overexpressed in both clinical tissue specimens and cultured human melanoma cells (WM115, A375, and HS294T) when compared with normal skin tissues and cultured normal melanocytes, respectively. Furthermore, Plk1 gene knockdown through Plk1-specific shRNA or its activity inhibition by a small-molecule inhibitor resulted in a significant decrease in the viability and growth of melanoma cells without affecting normal human melanocytes. In addition, Plk1 inhibition resulted in a significant (i) decrease in clonogenic survival, (ii) multiple mitotic errors, (iii) G(2)/M cell-cycle arrest, and (iv) apoptosis of melanoma cells. This study suggests that Plk1 may have a functional relevance toward melanoma development and/or progression. We suggest that the targeting of Plk1 may be a viable approach for the treatment of melanoma.

Polo-like kinase 1 (Plk1) in non-melanoma skin cancers.

Polo-like kinase 1 (Plk1) is becoming an increasingly attractive target for cancer management. Plk1 has been shown to be overexpressed in a variety of cancers; however its role in skin cancers is not well-understood. We recently demonstrated that Plk1 is overexpressed in human melanoma and gene-knockdown as well as chemical-inhibition of Plk1 resulted in a significant decrease in melanoma cell viability and growth without affecting the growth of the normal human epidermal melanocytes (NHEMs). Further, the observed anti-proliferative response of Plk1 was found to be accompanied with a significant G(2)/M cell cycle arrest, mitotic catastrophe and induction of apoptosis in melanoma cells. In this study, we determined the expression profile of Plk1 in non-melanoma skin cancers viz. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Our data demonstrated that like melanoma, Plk1 is significantly overexpressed in BCC and SCC samples. Further, we also found that compared to normal human epidermal keratinocytes (NHEKs), Plk1 was overexpressed at both the protein and mRNA levels in squamous A253 and A431 cells. In addition, a similar protein expression pattern was found for the downstream targets of Plk1, viz. Cdk1, Cyclin B1 and Cdc25C. We believe that the expression pattern of Plk1 in the various skin cancers, the observed insusceptibility of normal cells to Plk1 inhibition and the easy accessibility for topical applications lends the skin as an attractive tissue for Plk1 based cancer chemoprevention and chemotherapeutic applications.