Protein Kinases D2 and D3 Are Novel Growth Regulators in HCC1806 Triple-negative Breast Cancer Cells
Department of Biochemistry, The University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, Texas 75708, U.S.A. . Anticancer research
(Impact Factor: 1.83).
Aim: The role of protein kinase D (PKD) in the context of breast cancer cell biology is not clear.
The expression of PKD isoforms was assessed in various breast cancer cell lines and PKD isoform-specific siRNAs and selective inhibitors were used to study the role of PKD in breast cancer cell growth.
PKD2 and PKD3 were two major isoforms expressed at the highest levels in tumorgenic HCC1806 triple-negative breast cancer cells. Silencing PKD2 or PKD3 significantly inhibited HCC1806 cell proliferation, and PKD3 silencing had a higher inhibitory effect than PKD2 silencing on cell growth and PKD-mediated signaling. HCC1806 breast cancer cells were highly responsive to PKD inhibitors but not to a general protein kinase C (PKC) inhibitor.
We have identified PKD2 and PKD3, especially PKD3, as novel cell growth regulators in HCC1806 triple-negative breast cancer cells. Targeting PKD instead of all PKCs effectively inhibited cell proliferation in a number of breast cancer cell lines.
Available from: Raymond Mckenzie
- "We have shown that PKD1 regulates the production of proinflammatory cytokines by vascular endothelial growth factor in endothelial cells  and that PKD2 is pivotal for angiogenesis . We also found that both PKD2 and PKD3 were novel growth regulators in triple-negative breast cancer cells . Moreover, it has been shown that PKD1 is a key modulator of macrophage activation by toll-like receptors (TLRs)  and that PKD inhibition suppresses microbial Ag-induced hypersensitivity pneumonitis in mice . "
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.
Available from: Peter Storz
- "This confirms previously described data showing that these two isoforms may have tumor-promoting functions. For example, both have been shown to contribute to cell proliferation and growth of triple-negative breast cancer cells
. It may be speculated that to become aggressive, breast cancer cells undergo an isoform switch in PKD proteins. "
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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.
Available from: Peter Storz
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