Overexpression Of Protein Kinase D1 Research Articles

Protein kinase D1 mitigation against etoposide induced DNA damage in prostate cancer is associated with increased α-Catenin.

The E-cadherin, α- and β-Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β-Catenin is dynamically linked to the cellular cytoskeleton through interaction with α-Catenin at amino acid positions threonine 120 (T120) to 151 of β-Catenin. Nuclear presence of α-Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α-Catenin and protein kinase D1 (PrKD1) in DNA damage response. Prostate cancer cells; LNCaP, LNCaP (Sh-PrKD1; silenced PrKD1), C4-2 and C4-2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound-10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4-2 and C4-2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound-10 by mice and generate preliminary data on efficacy of compound-10 in modulating the α-Catenin and PrKD1 expressions in inhibiting tumor progression. PrKD1, a novel serine threonine kinase, phosphorylates β-Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β- to α-Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β- and α-Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α-Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound-10, a selective PrKD inhibitor, demonstrating decreased total β- and α-Catenin protein levels, and β-Catenin T120 phosphorylation. Alteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α-Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

Abstract 3373: Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin

Abstract Interaction between E-cadherin and β-catenin at cell-cell adherens junctions is pivotal in cell adhesion, and dysregulation of these genes is associated with disease progression in various solid tumors, notably in prostate cancer. α-catenin acts as a physical linker between the cadherin/β-catenin complex and actin cytoskeleton, through its mechanosensory properties has potential to modify interactions between cadherins and cytoskeleton and results in sensitivity of cells to DNA damage and toxicity. Membranous β-catenin interacts with the cellular cytoskeleton through threonine 120 (T120) phosphorylation and its association with α-catenin. We want to investigate the role of protein Kinase D1 (PrKD1) in DNA damage response, using various prostate cancer cell lines; PrKD1-silenced cells (LNCaP sh-PrKD1) and overexpression of PrKD1 (C4-2 PrKD1). PrKD1 silence and overexpress cells were treated with PrKD1 inhibitor and Etoposide to observe the effect of DNA damage repair altering various genes in prostate cancer cells. Silencing α-Catenin increased p-γH2AX (ser-139) expression in C4-2 PrKD1 cells. Conversely silenced PrKD1 (LNCaP sh-PrKD1) cells represented decreased α-Catenin expression, and increased p-γH2AX (ser-139). Overexpression of PrKD1 in prostate cancer cells increased α-catenin levels and conferred resistance to etoposide-induced DNA damage. Silencing PrKD1 compromised DNA damage protective effect. Furthermore, we elucidate the role of Protein kinase D1 (PrKD1), a unique serine-threonine kinase that phosphorylates β-catenin at T120, in this interaction. We treated C4-2 and C4-2 PrKD1 cells with PrKD1 inhibitor and immuno-precipitated β-Catenin complex represented decrease in β-Catenin (T-120) and α-Catenin than untreated C4-2 PrKD1 cells. We observed crosstalk between α-catenin and PrKD1 in DNA damage repair. In-silico analysis affirmed that the loss of T120 phosphorylation disrupts β- and α-catenin interaction. PrKD1 inhibitor decreased cell viability in dose response fashion, and also decrease the p-γH2AX (ser-139) in C4-2 PrKD1 cells than C4-2 cells. In vitro findings were corroborated in a mouse prostate cancer PDX xenograft model by inhibiting PrKD1 kinase activity, resulting in reduced β-catenin T120 phosphorylation and α-catenin levels. In vivo experiments with PrKD1 inhibitor represented tolerability in mice and its efficacy in modulating α-catenin and PrKD1 expression to inhibit tumor progression. Study for the first time provides a mechanistic basis of the protective role of PrKD1 against DNA damage. Citation Format: Sanjeev Shukla, Samuel Serrano, Mohammed Al-Toubat, Teruko Osumi, K.C. Balaji. Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3373.

Protein Kinase D1 Correlates with Less Lymph Node Metastasis Risk, Enhanced 5-FU Sensitivity, and Better Prognosis in Colorectal Cancer.

Protein kinase D1 (PKD1) controls tumor growth and invasion of gastrointestinal tract-related cancers, but its prognostic role in colorectal cancer (CRC) is not clear yet. Therefore, this research intended to assess the potential of PKD1 as a marker for CRC patients' management, also to evaluate its effect on 5-fluorouracil (5-FU) chemosensitivity in CRC cell lines. PKD1 protein and mRNA expressions were measured by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction assays in 214 CRC patients, respectively. The PKD1 overexpression plasmids and negative control (NC) plasmids were transfected into the HCT-116 and LoVo cell lines followed by 0-16 μM 5-FU treatment. PKD1 protein (P < 0.001) and mRNA expressions (P < 0.001) were both descended in tumor tissues compared to tumor-adjacent tissues. Meanwhile, tumor PKD1 protein and mRNA expressions were both negatively related to lymph node metastasis, N stage, and tumor-node-metastasis (TNM) stage (all P < 0.05). Prognostically, high expressions of PKD1 protein and mRNA were linked with prolonged disease-free survival (DFS) and overall survival (OS) (all P < 0.05). After adjustment by multivariate Cox analyses, PKD1 mRNA high expression independently forecasted longer DFS [hazard ratio (HR) = 0.199, P = 0.002] and OS (HR = 0.212, P = 0.022). In vitro experiments revealed that PKD1 overexpression decreased the half maximal inhibitory concentration value of 5-FU in the HCT-116 (P = 0.016) and LoVo (P = 0.007) cell lines. PKD1 expression links with less lymph node metastasis risk and satisfied prognosis in CRC patients, which promotes CRC cell chemosensitivity to 5-FU chemosensitivity as well.

Protein Kinase D1 (PKD1) Is a New Functional Non-Genomic Target of Bisphenol A in Breast Cancer Cells

Exposure to bisphenol A (BPA), one of the most widespread endocrine disruptors present in our environment, has been associated with the recent increased prevalence and severity of several diseases such as diabetes, obesity, autism, reproductive and neurological defects, oral diseases, and cancers such as breast tumors. BPA is suspected to act through genomic and non-genomic pathways. However, its precise molecular mechanisms are still largely unknown. Our goal was to identify and characterize a new molecular target of BPA in breast cancer cells in order to better understand how this compound may affect breast tumor growth and development. By using in vitro (MCF-7, T47D, Hs578t, and MDA-MB231 cell lines) and in vivo models, we demonstrated that PKD1 is a functional non-genomic target of BPA. PKD1 specifically mediates BPA-induced cell proliferation, clonogenicity, and anchorage-independent growth of breast tumor cells. Additionally, low-doses of BPA (≤10− 8 M) induced the phosphorylation of PKD1, a key signature of its activation state. Moreover, PKD1 overexpression increased the growth of BPA-exposed breast tumor xenografts in vivo in athymic female Swiss nude (Foxn1nu/nu) mice. These findings further our understanding of the molecular mechanisms of BPA. By defining PKD1 as a functional target of BPA in breast cancer cell proliferation and tumor development, they provide new insights into the pathogenesis related to the exposure to BPA and other endocrine disruptors acting similarly.

Abstract 4367: Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1

Abstract Objective: Pancreatic cancer (PanCa) is the third most common cause of cancer-related deaths in the US. Protein Kinase D1 (PKD1) is a kinase molecule which is involved in various important cellular signaling processes. While PKD1 has been reported to play a role in PanCa progression, the molecular mechanisms involved have not been adequately studied. Herein, we investigate the underlying mechanisms which enable PKD1 in enhancing glucose metabolism and further contribute to PanCa aggressiveness. Methods: PanCa cells with high PKD1 expressing (Panc-1 and AsPC-1) and low PKD1 (HPAF-II and BXPC-3) were used in the study. Immunohistochemistry and confocal immunofluorescence were performed to analyze the expression of PKD1 in pancreatic cancer/normal tissues and cells, respectively. The effect of PKD1 gain/loss-in function was investigated on glucose uptake and lactate production in PanCa cells using commercially available kits. Western blotting and real-time PCR experiments were performed to analyze the expression of protein and mRNA levels. MTT assay was conducted to access the influence of PKD1 on cell proliferation. Boyden chamber migration assay and Matrigel invasion assays were performed to determine the migratory and invasive abilities of PanCa cells. The gene silencing was performed using specific siRNAs in the study. Results: Our results demonstrate that PKD1 upregulates glucose metabolism in PanCa cells as indicated by enhanced glucose consumption and lactate production. The invasive characteristics of PKD1 expressing cells are augmented in presence of L-Lactate and reduced in presence of 2DG. PKD1 overexpression demonstrated enhanced phosphorylation of mTOR (ser-2448), 4EBP1, s6kinase and AKT, suggesting the role of PKD1 in the activation of mTOR pathway. We further observed that the phosphorylation of mTOR (ser-2448) and ps6kinase was attenuated on silencing PKD1 or in presence of Rapamycin, suggesting the role of PKD1 in activation of mTORC1 complex, both being the main effectors of mTORC1. Additionally, the inhibition in the phosphorylation of mTOR (ser-2448) in presence of kinase dead PKD1 suggests that PKD1 phosphorylates/activates mTORC1 in PanCa. Decrease in glucose uptake and lactate production in presence of PKD1 overexpression was observed on silencing raptor but not rictor in the cells, further suggesting the involvement of mTORC1 in PKD1 induced metabolic reprogramming in PanCa. PKD1 induced enhanced phosphorylation of mTOR resulted in an activation of HIF-1α and Glut-1 proteins, involved in abberant glucose metabolism. Additionally, our results also demonstrate that altered glucose metabolism leads to chemoresistance and silencing of PKD1 expression sensitizes PanCa cells to gemcitabine. Conclusion: Our studies indicate that PKD1 acts as a key regulator of the glucose metabolism via mTOR activation and facilitates proliferation and invasion of PanCa cells. Citation Format: Sonam Kumari, Sheema Khan, Murali Yallapu, Subhash Chauhan, Meena Jaggi. Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4367.

Protein kinase D1 inhibition interferes with mitosis progression

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.

PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer.

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

Trypanosoma cruziextracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Protein kinase-D1 overexpression prevents lipid-induced cardiac insulin resistance

In the insulin resistant heart, energy fuel selection shifts away from glucose utilization towards almost complete dependence on long-chain fatty acids (LCFA). This shift results in excessive cardiac lipid accumulation and eventually heart failure. Lipid-induced cardiomyopathy may be averted by strategies that increase glucose uptake without elevating LCFA uptake. Protein kinase-D1 (PKD1) is involved in contraction-induced glucose, but not LCFA, uptake allowing to hypothesize that this kinase is an attractive target to treat lipid-induced cardiomyopathy. For this, cardiospecific constitutively active PKD1 overexpression (caPKD1)-mice were subjected to an insulin resistance-inducing high fat-diet for 20-weeks. Substrate utilization was assessed by microPET and cardiac function by echocardiography. Cardiomyocytes were isolated for measurement of substrate uptake, lipid accumulation and insulin sensitivity. Wild-type mice on a high fat-diet displayed increased basal myocellular LCFA uptake, increased lipid deposition, greatly impaired insulin signaling, and loss of insulin-stimulated glucose and LCFA uptake, which was associated with concentric hypertrophic remodeling. The caPKD1 mice on high-fat diet showed none of these characteristics, whereas on low-fat diet a shift towards cardiac glucose utilization in combination with hypertrophy and ventricular dilation was observed. In conclusion, these data suggest that PKD pathway activation may be an attractive therapeutic strategy to mitigate lipid accumulation, insulin resistance and maladaptive remodeling in the lipid-overloaded heart, but this requires further investigation.

Protein kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity.

Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells.

Functional role of protein kinase D1 in Aspergillums fumigates-induced activation of nuclear factor-κB signal pathway and transcription

To explore the functional role of protein kinase D1 (PKD1) in the activation of nuclear factor-κB (NF-κB) signal pathway and NF-κB transcription mediated by Aspergillus fumigatus. A549 cells and HEK293 cells were transfected with green fluorescence protein (GFP) or GFP-PKD1 followed by treatment with 1×10(5) CFU/ml Aspergillus fumigatus conidia for different time lengths. The phosphorylation levels of PKD1, IκB and p65 (pS276) in the transfected cells were measured by Western blotting. A549 cells were transfected with GFP-PKD1 or siRNA-PKD1, and the phosphorylation of IκB and p65 (pS276) was examined. Finally, NF-κB-luc and renilla luciferase reporter pRL-SV40 were cotransfected into GFP- or GFP-PKD1-transfected A549 cells before exposure of the cells to Aspergillus fumigatus conidia for 24 h, and NF-κB transcriptional activity in the cells was determined using dual-luciferase reporter assay. Overexpression of PKD1 significantly increased Aspergillus fumigatus conidia-stimulated phosphorylation of PKD1, IκB and p65 (pS276), whereas PKD1 knockdown by siRNA-PKD1 suppressed IκB and p65 (pS276) phosphorylation. Dual luciferase assay demonstrated that PKD1 overexpression markedly enhanced Aspergillus fumigatus-induced NF-κB transcription in A549 cells. PKD1 may contribute to the activation of NF-κB signal pathway and NF-κB transcription induced by Aspergillus fumigatus.

Abstract 2907: Protein Kinase D1 expression attenuates colon cancer progression

Abstract Objective: Protein Kinase D1 (PKD1) is an important modulator of several signal-transduction pathways in benign and malignant human diseases. Currently the role of PKD1 in colon cancer, which is the second-leading cause of cancer-related deaths, is not well established. We have found previously that advance stage prostate cancer has suppressed expression. This led us to analyze PKD1 expression patterns in various other cancer tissue samples including colon cancer. Materials and Methods: Tissue microarrays containing colon cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry. To determine PKD1 mediated effects on oncogenic β-catenin signaling in colon cancer, human colorectal adenocarcinoma cell line SW480. Stable transformants of SW480 were created with GFP and SW480-PKD1-GFP, PKD1 overexpression leads to attenuation of β-catenin/T cell factor (TCF) transcription activity measured by luciferase reporter assays. To determine the effect of PKD1 overexpression on other cancer associated genes, RT-PCR array (that had 84 cancer related genes) analysis was performed in SW480-GFP and SW480-PKD1-GFP cells. Results: Our expression analysis determined a significant decrease in PKD1 expression in advanced Dukes stage (II, III and IV) colon cancer samples as compared to non-neoplastic colon samples. We have also noticed downregulation and aberrant localization of β-catenin in colon cancer samples compared to non-neoplastic colon samples. Therefore, we also determined the function of PKD1 in oncogenic β-catenin signaling in colon cancer. Our confocal microscopy analyses demonstrates that PKD1 up-regulation caused translocation of β-catenin from the nucleus to the plasma membrane. PKD1 overexpression also reduced β-catenin/TCF interaction in the nucleus and suppressed the expression of its downstream signaling proteins. Phenotypic changes in the stable transformants were measured via proliferation assays, anchorage independent growth, anchorage dependent growth, cellular motility and invasion. The overexpression of PKD1 markedly suppressed cell proliferation in SW480 cells. The SW480-PKD1-GFP cells formed fewer colonies compared with SW480-GFP cells. The SW480-PKD-GFP cells showed very minimal or no motility and invasion compared to SW480-GFP cells. Our RT-PCR array data shows that PKD1 overexpression also modulates expression of tissue inhibitor of metalloproteinase 3 (TIMP3), interferon α, interferon β and tumor necrosis factor receptor (TNFR). Conclusion: In conclusion, PKD1 is down regulated in colon cancer and its downregulation is correlated with aberrant β-catenin subcellular localization. Our data suggest that the altered PKD1 expression may induce β-catenin dependent and independent signaling in colon cancer and suppression of PKD1 may play a critical role in colon carcinogenesis and colon cancer progression. 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 2907. doi:10.1158/1538-7445.AM2011-2907

Protein kinase D1 promotes anchorage‐independent growth, invasion, and angiogenesis by human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases. Novel molecularly targeted therapies are urgently needed. Here, we extended our studies on the role of protein kinase D1 (PKD1) in PDAC cell lines. Given that Panc-1 express moderate levels of PKD1, we used retroviral-mediated gene transfer to create a Panc-1 derivative that stably over-expresses PKD1 (Panc-1-PKD1). Reciprocally, we used shRNA targeting PKD1 in Panc-28 to produce a PKD1 under-expressing Panc-28 derivative (Panc-28-shPKD1). Our results demonstrate that Panc-1-PKD1 cells exhibit significantly increased anchorage-independent growth in soft agar and increased in vitro invasion compared with Panc-1-mock. Reciprocally, Panc-28-shPKD1 cells show a significant decrease in anchorage-independent growth and invasiveness, as compared with Panc-28-mock cells. The selective PKD family inhibitor CRT0066101 markedly decreased colony-forming ability and invasiveness by either Panc-1-PKD1 or Panc-28-mock cells. Secretion of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and CXC chemokines (CXCL8) was significantly elevated by PKD1 over-expression in Panc-1 cells and reduced either by depletion of PKD1 via shRNA in Panc-28 cells or by addition of CRT0066101 to either Panc-1-PKD1 or Panc-28-mock cells. Furthermore, human umbilical vein endothelial cell (HUVEC) tube formation was significantly enhanced by co-culture with Panc-1-PKD1 compared with Panc-1-mock in an angiogenesis assay in vitro. Conversely, PKD1 depletion in Panc-28 cells decreased their ability to induce endotube formation by HUVECs. PDAC-induced angiogenesis in vitro and in vivo was markedly inhibited by CRT0066101. Our results lend further support to the hypothesis that PKD family members provide a novel target for PDAC therapy.

M1970 Knock Down of Signal Tranducer and Avtivator of Transcripton3 (STAT3) Enhance Gemcitabine Mediated Apoptosis in Chemoresistant Pancreatic Cancer Cells In Vitro and In Vivo

Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family, separate from the previously identified PKCs. PKD family members are rapidly activated by G protein-coupled receptor (GPCR) agonists which are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors, including pancreatic ductal adenocarcinoma, a devastating disease with overall 5 year survival of only 3-5%. A variety of GPCR agonists, including neurotensin (NT), stimulate DNA synthesis in human pancreatic adenocarcinoma cell lines. We used inducible stable expression of wild-type (WT) and kinase-dead (K618N) forms of PKD1 in PANC-1 cells to elucidate its function in the regulation of NT-induced MAP kinases, DNA synthesis and cell proliferation. Results: Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun Nterminal Kinase (JNK) and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser63 phosphorylation (indicative of JNK activity) in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser63 phosphorylation either at the level of or upstream of MKK4. This is the first demonstration that PKD1 suppresses GPCR-induced JNK/cJun activation in any cell type. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation and stimulated DNA synthesis and proliferation of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]coated dishes to eliminate cell adhesion (anchorage-independent growth). Conclusion: The reciprocal influence of PKD1 signaling on pro-mitogenic ERK and pro-apoptotic JNK/c-Jun pathways provides a mechanism by which PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. These results suggest that PKDs could be a novel target for the development of therapeutic drugs that restrict the proliferation of pancreatic cancer cells.

M1969 Induced Overexpression of Protein Kinase D1 Stimulates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells

M1969 Induced Overexpression of Protein Kinase D1 Stimulates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells

Abstract 997: PKD1-β3-integrin complex increases E-cadherin shedding through MMP-2 and −9 secretion in prostate cancer

Abstract Introduction: We have previously reported that Protein Kinase D1 (PKD1), a serine threonine kinase, is down regulated in advanced prostate cancer (PC) and known complex with cell adhesion protein β3-integrin to influence cell motility. Cell motility in facilitated by degradation of extracellular matrix, which is accomplished through secretion of proteins such as Matrix Metalloproteinases (MMPs), which are neutral proteinases that catalyze degradation of various transmembrane and extracellular matrix proteins including E-cadherin at cell surface. Here, we demonstrate the presence of PKD1-β3-integrin complex in PC cells that increases MMP-2 and −9 secretion and consequently enhances E-cadherin shedding. Methods: Protein expression, phosphorylation and secretion studies using PC cell lines were done by western blot analysis or gelatin zymography. Cell proliferation was measured by MTS assay. In silico analysis was done to validate correlation between PKD1 and MMPs expression in human PC. Results: To find out the complex formation among PKD1 and β3-integrin in PC cell, we immunoprecipitated the PKD1 protein and immunoblotted with β3-integrin antibody and observed that the PKD1 and β3-integrin proteins were in same protein complex. Then we found that overexpression of PKD1 also colocalized in PC3 cells and that was suppressed with RGD peptide, a specific β3-integrin inhibitor, treated same cells. Additionally, we found that PKD1 overexpressed cell increased in secretion of MMP-2 and −9 as well as activation of Erk and Akt those were suppressed in RGD peptide treated cells. In contrast, we observed that downregulation of PKD1 by shRNA could suppress the phosphorylation of Erk and Akt as well as MMP-2 and MMP-9 secretion in DU145 cells. Interestingly, PKD1 enhanced the expression of E-cadherin and secretion of extracellular part of E-cadherin (sE-cadherin) in conditioned media of DU145 cells, which was suppressed by treatment by MMP-2 and −9 inhibitors. In addition, suppression of cell proliferation by PKD1 was augmented by recombinant MMP-2 and −9 proteins and rescued by MMP-2 and MMP-9 inhibitor in DU145 cells, suggesting a surprising antiproliferative role for MMP-2 and −9 in PC cells. In silico analysis of publicly available DNA microarray data set derived from human PC confirmed that expression of PKD1 and MMP-2 correlated directly in metastatic PC compared to benign prostate tissue. Conclusions: Our study has identified a novel role of PKD1-β3-integrin complex for secretion of MMP-2 and −9 in prostate cancer, which is associated with E-cadherin shedding. Our data also suggested that the proteins complex limits PC cell proliferation by modulation MMP-2 and −9 secretion. 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 997.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC‐1 cells

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

Protein Kinase D1–Mediated Phosphorylation and Subcellular Localization of β-Catenin

beta-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells and also acts as a key cofactor for transcription activity. We previously showed that protein kinase D1 (PKD1), founding member of the PKD family of signal transduction proteins, is down-regulated in advanced prostate cancer and interacts with E-cadherin. This study provides evidence that PKD1 interacts with and phosphorylates beta-catenin at Thr(112) and Thr(120) residues in vitro and in vivo; mutation of Thr(112) and Thr(120) results in increased nuclear localization of beta-catenin and is associated with altered beta-catenin-mediated transcription activity. It is known that mutation of Thr(120) residue abolishes binding of beta-catenin to alpha-catenin, which links to cytoskeleton, suggesting that PKD1 phosphorylation of Thr(120) could be critical for cell-cell adhesion. Overexpression of PKD1 represses beta-catenin-mediated transcriptional activity and cell proliferation. Epistatic studies suggest that PKD1 and E-cadherin are within the same signaling pathway. Understanding the molecular basis of PKD1-beta-catenin interaction provides a novel strategy to target beta-catenin function in cells including prostate cancer.

Protein kinase D1 mediates NF-kappaB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells.

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

β‐catenin mediates alteration in cell proliferation, motility and invasion of prostate cancer cells by differential expression of E‐cadherin and protein kinase D1

We have previously demonstrated that Protein Kinase D1 (PKD1) interacts with E-cadherin and is associated with altered cell aggregation and motility in prostate cancer (PC). Because both PKD1 and E-cadherin are known to be dysregulated in PC, in this study we investigated the functional consequences of combined dysregulation of PKD1 and E-cadherin using a panel of human PC cell lines. Gain and loss of function studies were carried out by either transfecting PC cells with full-length E-cadherin and/or PKD1 cDNA or by protein silencing by siRNAs, respectively. We studied major malignant phenotypic characteristics including cell proliferation, motility, and invasion at the cellular level, which were corroborated with appropriate changes in representative molecular markers. Down regulation or ectopic expression of either E-cadherin or PKD1 significantly increased or decreased cell proliferation, motility, and invasion, respectively, and combined down regulation cumulatively influenced the effects. Loss of PKD1 or E-cadherin expression was associated with increased expression of the pro-survival molecular markers survivin, beta-catenin, cyclin-D, and c-myc, whereas overexpression of PKD1 and/or E-cadherin resulted in an increase of caspases. The inhibitory effect of PKD1 and E-cadherin on cell proliferation was rescued by coexpression with beta-catenin, suggesting that beta-catenin mediates the effect of proliferation by PKD1 and E-cadherin. This study establishes the functional significance of combined dysregulation of PKD1 and E-cadherin in PC and that their effect on cell growth is mediated by beta-catenin.