Courtney R. LaValle

Potent and Selective Disruption of Protein Kinase D Functionality by a Benzoxoloazepinolone

Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of PKD in these processes is severely hampered by the lack of a PKD-specific inhibitor that can be readily applied to cells and in animal models. We now report the discovery of the first potent and selective cell-active small molecule inhibitor for PKD, benzoxoloazepinolone (CID755673). This inhibitor was identified from the National Institutes of Health small molecule repository library of 196,173 compounds using a human PKD1 (PKCμ)-based fluorescence polarization high throughput screening assay. CID755673 suppressed half of the PKD1 enzyme activity at 182 nm and exhibited selective PKD1 inhibition when compared with AKT, polo-like kinase 1 (PLK1), CDK activating kinase (CAK), CAMKIIα, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cell-based assays, CID755673 blocked phorbol ester-induced endogenous PKD1 activation in LNCaP cells in a concentration-dependent manner. Functionally, CID755673 inhibited the known biological actions of PKD1 including phorbol ester-induced class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis virus glycoprotein transport from the Golgi to the plasma membrane, and the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 inhibited prostate cancer cell proliferation, cell migration, and invasion. In summary, our findings indicate that CID755673 is a potent and selective PKD1 inhibitor with valuable pharmacological and cell biological potential.

Protein kinase D as a potential new target for cancer therapy.

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.

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

Background 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 in vivo application, small molecule analogs of CID755673 were generated by modifying both the core structure and side-chains. Results 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. Conclusions Throughout the battery of experiments, the compounds kb-NB142-70 and kb-NB165-09 emerged as the most potent and specific analogs in vitro 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.

Tumor Epidermal Growth Factor Receptor and EGFR PY1068 Are Independent Prognostic Indicators for Head and Neck Squamous Cell Carcinoma

Purpose: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC). Patients and Methods: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR. Results: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26–6.00 and HR = 3.29; 95% CI = 1.34–8.14, respectively]. Conclusions: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR. Clin Cancer Res; 18(8); 2278–89. ©2012 AACR.

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.

Inducible Silencing of Protein Kinase D3 Inhibits Secretion of Tumor-Promoting Factors in Prostate Cancer

Protein kinase D (PKD) acts as a major mediator of several signaling pathways related to cancer development. Aberrant PKD expression and activity have been shown in multiple cancers, and novel PKD inhibitors show promising anticancer activities. Despite these advances, the mechanisms through which PKD contributes to the pathogenesis of cancer remain unknown. Here, we establish a novel role for PKD3, the least studied member of the PKD family, in the regulation of prostate cancer cell growth and motility through modulation of secreted tumor-promoting factors. Using both a stable inducible knockdown cell model and a transient knockdown system using multiple siRNAs, we show that silencing of endogenous PKD3 significantly reduces prostate cancer cell proliferation, migration, and invasion. In addition, conditioned medium from PKD3-knockdown cells exhibits less migratory potential compared with that from control cells. Further analysis indicated that depletion of PKD3 blocks secretion of multiple key tumor-promoting factors including matrix metalloproteinase (MMP)-9, interleukin (IL)-6, IL-8, and GROα but does not alter mRNA transcript levels for these factors, implying impairment of the secretory pathway. More significantly, inducible depletion of PKD3 in a subcutaneous xenograft model suppresses tumor growth and decreases levels of intratumoral GROα in mice. These data validate PKD3 as a promising therapeutic target in prostate cancer and shed light on the role of secreted tumor-promoting factors in prostate cancer progression. Mol Cancer Ther; 11(7); 1389–99. ©2012 AACR.

Androgen suppresses protein kinase D1 expression through fibroblast growth factor receptor substrate 2 in prostate cancer cells

In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment.

Abstract 3829: Inducible silencing of PKD3 reveals a critical role for PKD3 in prostate cancer growth and metastasis

Protein kinase D is a family of novel serine/threonine kinases that act as major mediators of several signaling pathways related to cancer development. Increasing evidence has demonstrated that specific PKD isoforms are deregulated in human cancers and that PKD plays an active role in cancer cell proliferation, migration, and invasion. Here, we present evidence on the development of a PC3 tetracycline-inducible PKD3 knockdown prostate cancer cell line and its application in both the study of PKD3 function in cells and the validation of PKD3 as a therapeutic target in vivo. We show that tetracycline treatment results in >50% knockdown of endogenous PKD3 expression in two distinct clones expressing PKD3 shRNA (shPKD3-C7 and shPKD3-C26), but has no effect on cells expressing scrambled shRNA (shSCR). Furthermore, endogenous PKD1 or PKD2 expression levels are not altered in PKD3 knockdown cells treated with tetracycline, supporting the specific targeting of the shRNA to the PKD3 isoform. Phenotypic analysis revealed that, similar to studies utilizing PKD siRNAs or novel PKD inhibitors, tetracycline-induced silencing of PKD3 causes potent arrest in cell growth and motility. Additionally, we found that conditioned media collected from tetracycline-treated shPKD3-C7 cells causes a reduction in PC3 cell migration, and we observed increased secretion of the tissue inhibitor of metalloproteinases-2 (TIMP-2) protein, which has important anti-angiogenic functions, from tetracycline-treated shPKD3-C7 cells. Western blotting revealed that both siRNA-mediated transient knockdown of PKD2 or PKD3 or treatment with novel PKD inhibitors led to decreased levels of the matrix metalloproteinases MMP-9, MMP-11, and MMP-14 in PC3 cells. Finally, we found that reduction of PKD3 expression by tetracycline-inducible shRNA significantly retarded both tumor growth in a subcutaneous xenograft model as well as tumor growth and metastasis in an orthotopic prostate cancer xenograft model. Taken together, these data suggest that PKD is a critical signaling protein that modulates prostate cancer cell growth and motility through a pathway involving multiple secreted factors, and that PKD3 is a potential therapeutic target in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2011-3829