Störz, Peter

Pancreatic acinar-to-ductal metaplasia (ADM) is a reversible process that occurs after pancreatic injury, but becomes permanent and leads to pancreatic lesions in the presence of an oncogenic mutation in KRAS,. While inflammatory macrophage-secreted chemokines, growth factors that activate epidermal growth factor receptor (EGFR) and oncogenic KRAS have been implicated in the induction of ADM, it is currently unclear whether a common underlying signaling mechanism exists that drives this process. In this study, we show that different inducers of ADM increase levels of hydrogen peroxide, most likely generated at the mitochondria, and upregulate the expression of Protein Kinase D1 (PKD1), a kinase that can be activated by hydrogen peroxide. PKD1 expression in acinar cells affects their survival and mediates ADM, which is in part due to the PKD1 target NF-κB. Overall, our data implicate ROS-PKD1 signaling as a common feature of different inducers of pancreatic ADM.

The development of pancreatic cancer requires recruitment and activation of different macrophage populations. However, little is known about how macrophages are attracted to the pancreas after injury or an oncogenic event, and how they crosstalk with lesion cells or other cells of the lesion microenvironment. Here, we delineate the importance of CXCL10/CXCR3 signaling during the early phase of murine pancreatic cancer. We show that CXCL10 is produced by pancreatic precancerous lesion cells in response to IFNγ signaling and that inflammatory macrophages are recipients for this chemokine. CXCL10/CXCR3 signaling in macrophages mediates their chemoattraction to the pancreas, enhances their proliferation, and maintains their inflammatory identity. Blocking of CXCL10/CXCR3 signaling in vivo shifts macrophage populations to a tumor-promoting (Ym1 + , Fizz + , Arg1 + ) phenotype, increases fibrosis, and mediates progression of lesions, highlighting the importance of this pathway in PDA development. This is reversed when CXCL10 is overexpressed in PanIN cells.

Background: CD36 is a scavenger and antiangiogenic receptor that plays an important role in athero-thrombotic diseases, diabetes and cancer and contributes to obesity. Lysophosphatidic acid (LPA), a bioactive phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by down-regulating endothelial CD36 transcription via protein kinase PKD-1 signaling. However, the precise mechanism as to how angiogenic signaling is integrated to regulate endothelial specific CD36 transcription remain unknown. Hypothesis: LPA represses CD36 transcription through PKD-1-mediated formation of a nuclear transcriptional complex in endothelial cells. Methods: Microvascular endothelial cells expressing CD36 were used for studying signaling and CD36 transcription by real time RT-qPCR, Western blotting, co-immunoprecipitation or avidin-biotin-conjugated DNA-binding assay; angiogenesis gene array was used for angiogenic gene profiling in response to LPA exposure. Spheroid-based angiogenesis assay, in vivo Matrigel assay and tumor angiogenesis model in CD36 deficiency and wild type mice were established to elucidate mechanisms of angiogenic signaling. Results: CD36 transcriptional repression involved PKD-1 signaling mediated formation of FoxO1-HDAC7 complex in the nucleus of endothelial cells. Unexpectedly, turning off CD36 transcription initiated reprogramming MVECs to express ephrin B2, a critical “molecular signature” involved in angiogenesis and arteriogenesis, and increased phosphorylation of Erk1/2, the MAP kinase important in arterial differentiation. PKD-1 signaling was also shown in tumor endothelium of Lewis lung carcinomas, along with low CD36 expression or CD36 deficiency. Angiogenic branching morphogenesis and in vivo angiogenesis were dependent on PKD-1 signaling. Conclusion: LPA/PKD1-HDAC7-FoxO1 signaling axis regulates endothelial CD36 transcription and mediates silencing of the antiangiogenic switch, resulting in proarteriogenic reprogramming. Targeting this signaling cascade could be a novel approach for cancer, diabetes, athero-thrombotic diseases and obesity.

Lysophosphatidic acid (LPA), a bioactive signaling phospholipid, down-regulates CD36 expression in microvascular endothelial cells (MVECs) via protein kinase PKD-1 signaling, thereby abolishing endothelial cell responses to its antiangiogenic ligand thrombospondin-1. However, little is known regarding mechanisms by which MVEC-specific CD36 transcription is regulated. We describe that in MVECs LPA represses CD36 transcription by activating a PKD-1 signaling that induces formation of a HDAC7/NCoR1/FoxO1 complex in the nucleus. Promoter analysis first identified FoxO1 as a transcription factor responsible for the CD36 transcription, which was confirmed by a chromatin-immunoprecipitation assay. Using a combination of PKD-1 gene transduction with co-immmunoprecipitation assay, we showed an increased interaction of HDAC7/NCoR1 with FoxO1 in response to LPA. However, HDAC7 and FoxO1 interaction was attenuated with PKD-1 silencing. Furthermore, based on results from an angiogenesis profiling with real time qPCR, doxycycline inducible constitutively active PKD-1 plasmids were transduced into tumor associated endothelial cells using a Lentiviral system to induce the PKD-1 expression. The results showed that turning off CD36 transcription reprograms by PKD-1 signaling was accompanied by an induced expression of ephrin B2 and activation of MAPK/ERK1/2 signaling, which are two critical “molecular signatures” involved in arteriogenesis. Moreover, three dimensional spheroid assay, a modified Boyden Chamber assay and in vivo Matrigel assay revealed that turning off CD36 transcription promoted angiogenesis in vitro and in vivo in a PKD-1-dependent manner. Immunofluorescence microscopy also showed the presence of this signaling pathway in the vasculature of Lewis lung carcinomas grown in cd36 deficient mice. In summary, our data suggest that a LPA-PKD-1-HDAC7/NCoR1-FoxO1 signaling axis is critical for transcriptional regulation of CD36 and mediates silencing of this antiangiogenic switch. This subsequently results in MVEC reprogramming for proangiogenic and arteriogenic responses. Therefore, targeting this signaling cascade could be a novel approach for malignant tumors, cardiovascular ischemia and other thrombotic diseases.

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-2 to control vasculogenesis and angiogenesis. Dysregulation of these physiological processes contributes to the pathologies of heart disease, stroke, and cancer. Protein kinase D (PKD) plays a crucial role in the regulation of angiogenesis by modulating endothelial cell proliferation and migration. In human umbilical vein endothelial cells (HUVEC) and human blood outgrowth endothelial cells (BOEC), knockdown of PKD-1 or PKD-2 downregulates VEGFR-2 and significantly inhibits VEGF-induced endothelial cell proliferation and migration. We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, activating enhancer binding protein 2 (AP2) binding sites exist within the VEGFR-2 promoter. Thus, we hypothesized PKD may downregulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. Indeed, AP2β binds the VEGFR-2 promoter upon PKD knockdown in HUVEC as evident by chromatin immunoprecipitation assay. Luciferase reporter assays using serial deletions of AP2β binding sites within the VEGFR-2 promoter revealed transcriptional activity negatively correlated with the number of AP2β binding sites, thus confirming negative regulation of VEGFR-2 transcription by AP2β. Next, using siRNA, we demonstrated that upregulation of AP2β decreased VEGFR-2 expression and loss of AP2β enhanced VEGFR-2 expression. In vivo studies confirmed this finding as we observed increased VEGFR-2 immunostaining in the dorsal horn of the spinal cord of embryonic day 13 AP2β knockout mice. We hypothesize that PKD directly regulates AP2β function by serine phosphorylation and ongoing studies are being conducted to determine phosphorylation sites in AP2β directly regulated by PKD. Taken together, we demonstrate AP2β negatively regulates VEGFR-2 transcription and VEGFR-2 is a major downstream target of PKD. Our findings describing how PKD regulates angiogenesis may contribute to the development of therapies to improve the clinical outcome of patients afflicted by heart disease, stroke, and cancer.

Summary Activating mutations of the K‐ras proto‐oncogene are detected in over 95% of pancreatic adenocarcinoma (PDA) and its precursor lesions. One mechanism by which activated K‐ras can drive the development of PDA is activation of the transcription factor nuclear factor κ‐B (NF‐κB), which has been shown to regulate genes involved in all aspects of tumor formation and progression. We here describe the PRKD1 gene promoter as a target for K‐ras‐NF‐κB signaling. We identify the binding sites for NF‐κB in the PRKD1 promoter and show that K‐ras‐driven upregulation of expression of PRKD1 and its gene product protein kinase D1 (PKD1) is linked to increased cell survival and oncogenic signaling in PDA. Conclusion Our data provide a functional link between oncogenic K‐ras, NF‐κB and PKD1, a targetable kinase in this cancer. Grant Funding Source : Supported by the NCI R01 grant CA140182

Protein kinase D3 (PKD3) is upregulated in triple‐negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro‐oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA‐MB‐231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF‐H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF‐H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC.