Protein Kinase D1 Mediates Anchorage-dependent and -independent Growth of Tumor Cells via the Zinc Finger Transcription Factor Snail1

From the Department for Internal Medicine I, University Clinic Halle, Martin Luther University Halle-Wittenberg, Ernst-Grube Strasse 40, 06120 Halle (Saale), Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2012; 287(39):32367-80. DOI: 10.1074/jbc.M112.370999
Source: PubMed


We here identify protein kinase D1 (PKD1) as a major regulator of anchorage-dependent and -independent growth of cancer cells controlled via the transcription factor Snail1. Using FRET, we demonstrate that PKD1, but not PKD2, efficiently interacts with Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11. There was no change in the nucleocytoplasmic distribution of Snail1 using wild type Snail1 and Ser-11 phosphosite mutants in different tumor cells. Regardless of its phosphorylation status or following co-expression of constitutively active PKD, Snail1 was predominantly localized to cell nuclei. We also identify a novel mechanism of PKD1-mediated regulation of Snail1 transcriptional activity in tumor cells. The interaction of the co-repressors histone deacetylases 1 and 2 as well as lysyl oxidase-like protein 3 with Snail1 was impaired when Snail1 was not phosphorylated at Ser-11, which led to reduced Snail1-associated histone deacetylase activity. Additionally, lysyl oxidase-like protein 3 expression was up-regulated by ectopic PKD1 expression, implying a synergistic regulation of Snail1-driven transcription. Ectopic expression of PKD1 also up-regulated proliferation markers such as Cyclin D1 and Ajuba. Accordingly, Snail1 and its phosphorylation at Ser-11 were required and sufficient to control PKD1-mediated anchorage-independent growth and anchorage-dependent proliferation of different tumor cells. In conclusion, our data show that PKD1 is crucial to support growth of tumor cells via Snail1.

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    • "HeLa and Panc1 cells were seeded on glass coverslips, transfected as indicated, and processed as described in Eiseler et al. (2012). Samples were analyzed by a confocal laser scanning microscope, LSM710 (Zeiss, Jena, Germany) or TCS SP5 (Leica, Wetzlar, Germany ), equipped with respective 63× Plan Apo oil or 40× water immersion objective. "
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    ABSTRACT: Pancreatic cancer cell invasion, metastasis and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein Kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lower. We here report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in 3D-ECM cultures by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9), whereby MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in-vivo utilizing tumors growing on chorioallantois membranes (CAM). Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound VEGF-A, thereby increasing its bio-availability and angiogenesis. Interestingly, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and -2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in-vitro and in-vivo, addressing PKD isoform-specificity as a major factor for future therapeutic strategies.
    Molecular biology of the cell 12/2013; 25(3). DOI:10.1091/mbc.E13-06-0334 · 4.47 Impact Factor
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    • "Depending on the cell type and the activation mechanism, PKD enzymes are involved in many biological processes including cell adhesion, vesicle transport, cell survival and cell migration (reviewed in [35]). In prostate and breast tissue, PKD1 contributes to maintenance of the epithelial phenotype by inhibiting EMT and upregulating E-cadherin expression [2,23,36]. In addition, active PKD1 negatively impacts cell migration and invasion through inhibition of actin reorganization processes at the leading edge [6,7,9-11,37,38], as well as downregulation of expression of MMPs [12]. "
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    ABSTRACT: DNA methylation-induced silencing of genes encoding tumor suppressors is common in many types of cancer, but little is known about how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes protein kinase D1 (PKD1), a serine/threonine kinase that is expressed in cells of the normal mammary gland, where it maintains the epithelial phenotype by preventing epithelial-to-mesenchymal transition. The status of PRKD1 promoter methylation was analyzed by reduced representation bisulfite deep sequencing, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR in invasive and noninvasive breast cancer lines, as well as in humans in 34 cases of "normal" tissue, 22 cases of ductal carcinoma in situ, 22 cases of estrogen receptor positive, HER2-negative (ER+/HER2-) invasive lobular carcinoma, 43 cases of ER+/HER2- invasive ductal carcinoma (IDC), 93 cases of HER2+ IDC and 96 cases of triple-negative IDC. A reexpression strategy using the DNA methyltransferase inhibitor decitabine was used in vitro in MDA-MB-231 cells as well as in vivo in a tumor xenograft model and measured by RT-PCR, immunoblotting and immunohistochemistry. The effect of PKD1 reexpression on cell invasion was analyzed in vitro by transwell invasion assay. Tumor growth and metastasis were monitored in vivo using the IVIS Spectrum Pre-clinical In Vivo Imaging System. Herein we show that the gene promoter of PRKD1 is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, we show that reversion of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion. Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells.
    Breast cancer research: BCR 08/2013; 15(2):R66. DOI:10.1186/bcr3460 · 5.49 Impact Factor
  • H Zheng · Y Kang ·
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    ABSTRACT: Metastasis is the leading cause of cancer-associated death in most tumor types. Metastatic dissemination of cancer cells from the primary tumor is believed to be initiated by the reactivation of an embryonic development program referred to as epithelial-mesenchymal transition (EMT), whereby epithelial cells lose apicobasal polarity and cell-cell contacts, and gain mesenchymal phenotypes with increased migratory and invasive capabilities. EMT has also been implicated in the regulation of cancer stem cell property, immune suppression and cancer regression. Several transcription factors have been identified as master regulators of EMT, including the Snail, Zeb and Twist families, and their expression is tightly regulated at different steps of transcription, translation and protein stability control by a variety of cell-intrinsic pathways as well as extracellular cues. Here, we review the recent literature on the signaling pathways and mechanisms that control the expression of these master transcription factors during EMT and cancer progression.Oncogene advance online publication, 22 April 2013; doi:10.1038/onc.2013.128.
    Oncogene 04/2013; 33(14). DOI:10.1038/onc.2013.128 · 8.46 Impact Factor
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