TARGETING PROTEIN KINASE D BY NOVEL SMALL MOLECULE INHIBITORS AND RNA INTERFERENCE IN PROSTATE CANCER

Protein kinase D (PKD) has been implicated in a variety of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD-mediated cellular responses remains difficult due to lack of a potent and selective inhibitor. Here, we report the discovery of a novel class of pan-PKD inhibitors, CID755673 and its analogs. Subsequently, we use these inhibitors in conjunction with RNA interference technology to show that PKD regulates prostate cancer cell growth and motility. CID755673 was discovered in collaboration with our colleagues at the University of Pittsburgh as a compound demonstrating nanomolar potency and high selectivity for PKD inhibition. To enhance its selectivity and potency for potential in vivo application, several analogs of CID755673 were generated. After initial activity screening, 5 analogs having equal or greater in vitro potencies as CID755673 were chosen for further analysis. Our data showed that modifications to the aromatic core structure significantly increased potency while retaining high specificity for PKD. In prostate cancer cells, all compounds inhibited phorbol 12-myristate 13-acetate (PMA)-induced autophosphorylation of PKD1, with kb-NB142-70 being most active. Importantly, these inhibitors caused a dramatic arrest in cancer cell proliferation. Migration and invasion were also inhibited by this class of compounds, with varying potencies that correlated to their cellular activity, suggesting an active role for PKD in these processes. To confirm PKD involvement in prostate cancer biology, we used short hairpin RNA (shRNA)- and small interfering RNA (siRNA)-mediated knockdown of specific PKD isoforms, demonstrating that knockdown of PKD2 and/or PKD3 significantly reduces proliferation, migration, and invasion in metastatic PC3 prostate cancer cells. We also found that inhibition of PKD expression or activity decreases secretion of several key tumor-promoting factors including matrix metalloproteinase (MMP)-9, interleukin (IL)-6, IL-8, and growth-regulated oncogene α (GROα). Finally, we demonstrated that inducible knockdown of PKD3 in both subcutaneous and orthotopic xenograft models leads to reduced prostate tumor growth. Taken together, these data provide much-needed pharmacological tools for the study of PKD function, validate PKD as a promising therapeutic target in prostate cancer treatment, and broaden our understanding of the molecular mechanisms of PKD function in prostate cancer progression

Novel protein kinase D inhibitors cause potent arrest in prostate cancer cell growth and motility

<p>Abstract</p> <p>Background</p> <p>Protein kinase D (PKD) has been implicated in a wide range of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD-mediated cellular responses remains difficult due to lack of a potent and selective inhibitor. Previously, we identified a novel pan-PKD inhibitor, CID755673, with potency in the upper nanomolar range and high selectivity for PKD. In an effort to further enhance its selectivity and potency for potential <it>in vivo </it>application, small molecule analogs of CID755673 were generated by modifying both the core structure and side-chains.</p> <p>Results</p> <p>After initial activity screening, five analogs with equal or greater potencies as CID755673 were chosen for further analysis: kb-NB142-70, kb-NB165-09, kb-NB165-31, kb-NB165-92, and kb-NB184-02. Our data showed that modifications to the aromatic core structure in particular significantly increased potency while retaining high specificity for PKD. When tested in prostate cancer cells, all compounds inhibited PMA-induced autophosphorylation of PKD1, with kb-NB142-70 being most active. Importantly, these analogs caused a dramatic arrest in cell proliferation accompanying elevated cytotoxicity when applied to prostate cancer cells. Cell migration and invasion were also inhibited by these analogs with varying potencies that correlated to their cellular activity.</p> <p>Conclusions</p> <p>Throughout the battery of experiments, the compounds kb-NB142-70 and kb-NB165-09 emerged as the most potent and specific analogs <it>in vitro </it>and in cells. These compounds are undergoing further testing for their effectiveness as pharmacological tools for dissecting PKD function and as potential anti-cancer agents in the treatment of prostate cancer.</p

Analysis of oncogenic activities of protein kinase D1 in head and neck squamous cell carcinoma.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated.MethodsThe expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and&nbsp;subcutaneous xenograft mouse model was established by implantation of&nbsp;the stable doxycycline (Dox)-inducible PKD1 expression cell lines&nbsp;for analysis of tumorigenic activity in vivo.ResultsPKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells.ConclusionsOur study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC

Analysis of oncogenic activities of protein kinase D1 in head and neck squamous cell carcinoma

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. Methods The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. Results PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. Conclusions Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC

Big data analytics in healthcare: promise and potential

Objective To describe the promise and potential of big data analytics in healthcare. Methods The paper describes the nascent field of big data analytics in healthcare, discusses the benefits, outlines an architectural framework and methodology, describes examples reported in the literature, briefly discusses the challenges, and offers conclusions. Results The paper provides a broad overview of big data analytics for healthcare researchers and practitioners. Conclusions Big data analytics in healthcare is evolving into a promising field for providing insight from very large data sets and improving outcomes while reducing costs. Its potential is great; however there remain challenges to overcome

Design, Synthesis, and Biological Evaluation of PKD Inhibitors

Protein kinase D (PKD) belongs to a family of serine/threonine kinases that play an important role in basic cellular processes and are implicated in the pathogenesis of several diseases. Progress in our understanding of the biological functions of PKD has been limited due to the lack of a PKD-specific inhibitor. The benzoxoloazepinolone CID755673 was recently reported as the first potent and kinase-selective inhibitor for this enzyme. For structure-activity analysis purposes, a series of analogs was prepared and their in vitro inhibitory potency evaluated