Search

Now entering its fourth decade, research on the biological function, small molecule inhibition, and disease relevance of the three known isoforms of protein kinase D, PKD1, PKD2, and PKD3, has entered a mature development stage. This mini-perspective focuses on the medicinal chemistry that provided a structurally diverse set of mainly active site inhibitors, which, for a brief time period, moved through preclinical development stages but have yet to be tested in clinical trials. In particular, between 2006 and 2012, a rapid expansion of synthetic efforts led to several moderately to highly PKD-selective chemotypes but did not yet achieve PKD subtype selectivity or resolve general toxicity and pharmacokinetic challenges. In addition to cancer, other unresolved medical needs in cardiovascular, inflammatory, and metabolic diseases would, however, benefit from a renewed focus on potent and selective PKD modulators.

The mitotic regulator Wee1 is a critical regulator of G2/M cell cycle checkpoint and DNA damage response pathways. Wee1-targeted agents are under active clinical investigation in multiple cancer types. However, Wee1 and its inhibitors have not been well studied in prostate cancer, particularly neuroendocrine prostate cancer (NEPC). In this study, we investigated the therapeutic potential of Wee1 inhibitor AZD1775 and its synthetic lethal combination with Chk1 inhibitor in the context of NEPC. Our data demonstrated increased Wee1 expression in NEPC cells and tumors. Treatment of NEPC cells with AZD1775 inhibited NEPC tumor cell survival with greater potency as compared to other prostate cancer cells, demonstrating increased vulnerability of NEPC cells to Wee1 inhibition. We further identified synthetic lethality between the inhibition of Wee1 and several protein kinases, particularly Chk1, another key G2/M checkpoint kinase. Using an autochthonous adenocarcinoma mouse prostate (TRAMP) model that gives rise to NEPC tumors, we assessed the efficacy of AZD1775 and a highly selective Chk1 inhibitor SRA737 as single agents and in combination on the progression, survival, and metastatic spread of NEPC tumors in vivo. AZD1775 and SRA737 when used as single agents significantly suppressed tumor growth, improved overall survival, and incidence of distant metastases of mice with NEPC tumors. The combination of AZD1775 and SRA737 on a sequential 2-wk treatment schedule was more efficacious in suppressing tumor growth and metastasis than each drug alone in castrated TRAMP NEPC tumors. Mechanistically, the combination of Chk1 and Wee1 inhibitors synergized to reduce the inhibitory phosphorylation of CDK1 at Y15 and Cdc25c at S216, resulting in enhanced activation of CDK1/cyclin B complex, abrogation of G2/M checkpoint, and consequent premature mitotic entry, which ultimately led to mitotic catastrophe and apoptosis. Our findings provide promising preclinical evidence supporting the therapeutic values of Wee1 and Chk1 inhibitors as single agents and in combination for the treatment of NEPC. Citation Format: Yapeng Chao, Yuzhou Chen, Yu Liu, Jaclyn A. Connelly, Hong Wang, Qiming Jane Wang. Wee1 inhibition and its synthetic lethal combination with Chk1 inhibition in mouse model of neuroendocrine prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5015.

Most cancer patients die because of tumor metastasis not due to tumors at the primary site. Prostate tumors in advance stages frequently metastasize to the bone, which is the main cause of death for the disease. The family of protein kinase D (PKD) has been implicated in prostate cancer development, however, its role in prostate cancer metastasis has not been investigated. In this study, we examined the contribution of PKD to the metastatic potential of prostate cancer cells and the impact of PKD inhibition on prostate cancer bone metastasisin vivo. Our data showed that depletion of PKDs by siRNA or inhibition of PKD by an inhibitor CRT0066101 in a highly invasive prostate cancer PC3-ML cells potently inhibited colony formation and cell migration. Furthermore, depletion or inhibition of PKD significantly blocked invasion of PC3-ML cells and suppressed expression of genes related to bone metastasis. The reduced invasive activity resulted from PKD depletion was in part mediated through the transcription factor Runx2 as its silencing decreased PKD-mediated metastatic gene expression. Mechanistically, our data indicate that PKD modulated Runx2 target gene expression through the MEK/ERK1/2 signaling axis. Additionally, we examined whether PKD inhibitor CRT0066101 could prevent prostate cancer bone metastasis in a mouse model of metastasis, where intracardiac injection of PC3-ML cells led to metastasis of cells to the bone. We found that CRT0066101 potently decreased the frequency of micrometastases in mouse bone. These results indicate that PKDs play an important role in bone metastasis of prostate cancer cells and its inhibition may be beneficial for treatment of advanced stages of cancer.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. The regulatory mechanisms of BCSC interactions with the vascular niche within the tumor microenvironment (TME) and their self-renewal are currently under extensive investigation. We have demonstrated the existence of an arteriolar niche in the TME of human BC tissues. Intriguingly, BCSCs tend to be enriched within the arteriolar niche in human estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the lysophosphatidic acid (LPA)/protein kinase D (PKD-1) signaling pathway, which promotes both arteriolar differentiation of ECs and self-renewal of CSCs likely via differential regulation of CD36 transcription. This study indicates that CSCs may enjoy blood perfusion to maintain their stemness features. Targeting the LPA/PKD-1 -CD36 signaling pathway may have therapeutic potential to curb tumor progression by disrupting the arteriolar niche and effectively eliminating CSCs., Jiang, Guo, Hao et al. investigate the role of the tumour microenvironment in breast cancer stem cell regulation and identify an arteriolar niche in which BCSCs tended to be enriched in human estrogen receptor positive breast cancer. Here, BCSCs are found to interact with arteriolar endothelial cells through LPA/PKD-1 signalling-mediated arteriolar differentiation of ECs and self-renewal of BCSCs.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. The regulatory mechanisms of BCSC interactions with the vascular niche within the tumor microenvironment (TME) and their self-renewal are currently under extensive investigation. We have demonstrated the existence of an arteriolar niche in the TME of human BC tissues. Intriguingly, BCSCs tend to be enriched within arteriolar niche in human estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the lysophosphatidic acid (LPA)/protein kinase D (PKD-1) signaling pathway, which promotes both arteriolar differentiation of ECs and self-renewal of CSCs. This study indicates that CSCs may enjoy blood perfusion to maintain their stemness features. Targeting the LPA/PKD-1 signaling pathway in combination with inhibition of CD36 function may have therapeutic potential to curb tumor progression by disrupting the arteriolar niche and eliminating CSCs.

Protein kinase D (PKD) is a family of serine/threonine protein kinases operating in the signaling network of the second messenger diacylglycerol. The three family members, PKD1, PKD2, and PKD3, are activated by a variety of extracellular stimuli and transduce cell signals affecting many aspects of basic cell functions including secretion, migration, proliferation, survival, angiogenesis, and immune response. Dysregulation of PKD in expression and activity has been detected in many human diseases. Further loss- or gain-of-function studies at cellular levels and in animal models provide strong support for crucial roles of PKD in many pathological conditions, including cancer, metabolic disorders, cardiac diseases, central nervous system disorders, inflammatory diseases, and immune dysregulation. Complexity in enzymatic regulation and function is evident as PKD isoforms may act differently in different biological systems and disease models, and understanding the molecular mechanisms underlying these differences and their biological significance in vivo is essential for the development of safer and more effective PKD-targeted therapies. In this review, to provide a global understanding of PKD function, we present an overview of the PKD family in several major human diseases with more focus on cancer-associated biological processes.

Protein kinase D (PKD) is a novel family of serine/threonine kinases and diacylglycerol (DAG) receptors and has been documented in a variety of cellular processes. To get high purity catalytic domain of PKD1 (PKD1-cat) for crystallography study, the GST-tagged PKD1-cat gene was cloned into a baculovirus transfer vector pFastBac1 (donor plasmid). When the recombinant plasmid was transformed into DH10Bac competent Escherichia coli, which contains a baculovirus shuttle vector (bacmid), transposition occurs to generate a recombinant bacmid containing the gene of interest (GST-PKD1-cat). The recombinant bacmid DNA was transfected into Spodoptera frugiperda Sf9 insect cells to generate recombinant baculovirus, which was then amplified through multiple rounds of infection in Sf9 cells. After that, Trichoplusia ni insect cells in suspension culture were infected with baculoviral stock at a multiplicity of infection (MOI) of 5 PFU/cell. SDS-PAGE and Western blotting analysis confirmed the detection of a 68 kDa protein by the glutathione S-transferase (GST) monoclonal antibody. The recombination protein was purified by Glutathione sepharose affinity chromatography and cleaved by PreScission Protease to remove GST tag, and a highly pure 42 kDa protein which was consistent with the molecular weight of the expected PKD1-cat protein was detected on SDS-PAGE. The activity of purified PKD1-cat protein was determined by in vitro PKD kinase assay. Our data showed that the kinase activity increased with the concentration of purified PKD1-cat protein. These results showed that the truncated recombinant PKD1-cat protein was highly active and pure, and could potentially be used for solving 3D structure of this protein by Nuclear Magnetic Resonance (NMR) or crystallography.

The catalytic domain of overexpressed protein kinase C (PKC)-δ mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-δ/εV5 and -ε/δV5, by swapping the V5 domains of PKC-δ and -ε. PKC-δ/εV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-δ's functionality. The other chimera, PKC-ε/δV5, endowed inactive PKC-ε with nearly all PKC-δ's apoptotic ability, confirming the importance of PKC-δ in this function. Green fluorescent protein (GFP)-tagged PKC-δV5 and -ε/δV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-ε and -δ/εV5 showed significantly less, indicating that the V5 region of PKC-δ contains determinants critical to its nuclear distribution. PKC-ε/δV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-ε, implicating the PKC-εV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-δ and -ε.

The catalytic domain of overexpressed protein kinase C (PKC)-delta mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-delta/epsilonV5 and -epsilon/deltaV5, by swapping the V5 domains of PKC-delta and -epsilon. PKC-delta/epsilonV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-delta's functionality. The other chimera, PKC-epsilon/deltaV5, endowed inactive PKC-epsilon with nearly all PKC-delta's apoptotic ability, confirming the importance of PKC-delta in this function. Green fluorescent protein (GFP)-tagged PKC-deltaV5 and -epsilon/deltaV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-epsilon and -delta/epsilonV5 showed significantly less, indicating that the V5 region of PKC-delta contains determinants critical to its nuclear distribution. PKC-epsilon/deltaV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-epsilon, implicating the PKC-epsilonV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-delta and -epsilon.

Phorbol esters, analogs of diacylglycerol, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. Although the apoptotic pathways induced by PMA are well characterized, little is known of PMA-regulated pro-survival pathways such as ERK1/2 and Akt. We now provide experimental evidence to indicate that PKD1 promotes survival signals in LNCaP cells in response to PMA treatment. In turn, PMA induces dose- and time-dependent downregulation of PKD1 to facilitate apoptosis. This effect can be blocked by PKC inhibitor Ro 31-8220, indicating that PKC activation is required for PKD1 downregulation. Depletion of endogenous PKCδ and PKCε reveals that PKD1 activation and subsequent downregulation are mainly controlled by PKCδ. Knockdown of endogenous PKD1 and PKD2 decreases ERK1/2 and Akt activities and potentiates PMA-induced apoptosis as measured by cleaved PARP and sub-G0/G1 population, whereas overexpression of wild-type and constitutive-active PKD1 enhances ERK1/2 and Akt activities and suppresses PMA-induced apoptosis. Further analysis shows that the downregulation of PKD1 is mediated by ubiquitin/proteasome degradation pathway. In summary, our data indicate that PKD1 plays a pro-survival role in PMA-induced apoptosis by modulating downstream ERK1/2 and Akt activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3127.