Störz, Peter

Objective— CD36 is a scavenger and antiangiogenic receptor that is important in atherothrombotic diseases, diabetes mellitus, cancer, and obesity. Lysophosphatidic acid, a phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by downregulating endothelial CD36 transcription via protein kinase D1 (PKD-1) signaling. We aimed to understand mechanisms by which lysophosphatidic acid–mediated angiogenic signaling is integrated to regulate CD36 transcription and endothelial cell function via a nuclear transcriptional complex. Approach and Results— Microvascular endothelial cells expressing CD36 were used for studying angiogenic signaling and CD36 transcription. Gene transfection and transduction, RT-qPCR, avidin–biotin-conjugated DNA-binding assay, chromatin immunoprecipitation assay, co-immunoprecipitation, proximal ligation assay, and immunofluorescence microscopy showed that lysophosphatidic acid–mediated CD36 transcriptional repression involved PKD-1 signaling mediated formation of forkhead box protein O1–histone deacetylase 7 complex in the nucleus. Unexpectedly, turning off CD36 transcription initiated reprogramming microvascular endothelial cells to express ephrin B2, a critical molecular signature involved in angiogenesis and arteriogenesis. Spheroid-based angiogenesis and in vivo Matrigel angiogenesis assays indicated that angiogenic branching morphogenesis and in vivo angiogenesis were dependent on PKD-1 signaling. A mouse tumor angiogenesis model revealed enhanced PKD-1 signaling and expression of ephrin B2 and smooth muscle actin in neovessels of Lewis Lung Carcinomas, along with low-CD36 expression or CD36 deficiency. Conclusions— Lysophosphatidic acid/PKD-1 signaling leads to nuclear accumulation of histone deacetylase 7, where it interacts with forkhead box protein O1 to suppress endothelial CD36 transcription and mediates silencing of antiangiogenic switch, resulting in proangiogenic and proarteriogenic reprogramming. Targeting this signaling cascade could be a novel approach for ischemic cardiovascular disease and cancer.

The Protein Kinase D (PKD) isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs) and diacylglycerol (DAG). PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT) and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development.

Abstract Invasive ductal carcinomas (IDC) of the breast are associated with altered expression of hormone receptors (HR), amplification or overexpression of HER2, or a triple-negative phenotype. The most aggressive cases of IDC are characterized by a high proliferation rate, a great propensity to metastasize, and their ability to resist to standard chemotherapy, hormone therapy, or HER2-targeted therapy. Using progression tissue microarrays, we here demonstrate that the serine/threonine kinase protein kinase D3 (PKD3) is highly upregulated in estrogen receptor (ER)–negative (ER−) tumors. We identify direct binding of the ER to the PRKD3 gene promoter as a mechanism of inhibition of PKD3 expression. Loss of ER results in upregulation of PKD3, leading to all hallmarks of aggressive IDC, including increased cell proliferation, migration, and invasion. This identifies ER− breast cancers as ideal for treatment with the PKD inhibitor CRT0066101. We show that similar to a knockdown of PKD3, treatment with this inhibitor targets all tumorigenic processes in vitro and decreases growth of primary tumors and metastasis in vivo. Our data strongly support the development of PKD inhibitors for clinical use for ER− breast cancers, including the triple-negative phenotype. Mol Cancer Ther; 14(6); 1306–16. ©2015 AACR.

Abstract Desmoplasia and an inflammatory environment are defining features of pancreatic cancer. Unclear is how pancreatic cells that undergo oncogenic transformation can cross-talk with immune cells and how this contributes to the development of pancreatic lesions. Here, we demonstrate that pancreatic acinar cells expressing mutant KRAS can expedite their transformation to a duct-like phenotype by inducing local inflammation. Specifically, we show that KRASG12D induces the expression of intercellular adhesion molecule-1 (ICAM-1), which serves as chemoattractant for macrophages. Infiltrating macrophages amplify the formation of KRASG12D-caused abnormal pancreatic structures by remodeling the extracellular matrix and providing cytokines such as TNF. Depletion of macrophages or treatment with a neutralizing antibody for ICAM-1 in mice expressing oncogenic Kras under an acinar cell–specific promoter resulted in both a decreased formation of abnormal structures and decreased progression of acinar-to-ductal metaplasia to pancreatic intraepithelial neoplastic lesions. Significance: We here show that oncogenic KRAS in pancreatic acinar cells upregulates the expression of ICAM-1 to attract macrophages. Hence, our results reveal a direct cooperative mechanism between oncogenic Kras mutations and the inflammatory environment to drive the initiation of pancreatic cancer. Cancer Discov; 5(1); 52–63. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1

Abstract In response to inflammation, pancreatic acinar cells can undergo acinar-to-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a duct-like phenotype and in the context of additional oncogenic stimulation, contributes to development of pancreatic cancer. The signalling mechanisms underlying pancreatitis-inducing ADM are largely undefined. Our results provide evidence that macrophages infiltrating the pancreas drive this transdifferentiation process. We identify the macrophage-secreted inflammatory cytokines RANTES and TNFα as mediators of such signaling. Both RANTES and TNFα induce ADM through activation of nuclear factor κ-B and its target genes involved in regulating survival, proliferation and degradation of extracellular matrix. In particular we identify matrix metalloproteinases (MMPs) as targets that drive ADM, and provide in vivo data suggesting that MMP inhibitors may be efficiently applied to block pancreatitis-induced ADM in therapy. Citation Format: Geou-yarh Liou, Heike Doeppler, Peter Storz. Macrophage-secreted cytokines drive acinar-to-ductal metaplasia of the pancreas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-342. doi:10.1158/1538-7445.AM2013-LB-342

Abstract CD36 is an endothelial cell (EC) receptor that mediates angiostatic activity and oxidative stress, which are important in ischemic disease and tumor progression. CD36 interacts with TSR domain proteins including thrombospondin-1. This results in a signal that is likely to initiate an antiangiogenic “switch," subsequently converting a growth factor-mediated proangiogenic response to an antiangiogenic response. However, in pathologic settings including malignant tumors, robust angiogenesis occurs despite the abundance of TSR-containing proteins in the microenvironment. This suggests that there is a mechanism by which TSR-mediated antiangiogenesis could be blunted via the localized downregulation of EC CD36 transcription. We previously reported that lysophosphatidic acid (LPA), a bioactive signaling phospholipid mediator, down-regulates microvascular EC (MVEC) CD36 transcription by protein kinase PKD-1 signaling via G protein coupled receptor LPA1,3. However, further elucidating the CD36 transcriptional mechanisms is critical to understanding the antiangiogenic switch. To this end, we recently discovered that obesity-derived LPA inhibited CD36 expression in tumor-associated ECs (TAECs). Similarly, inducing the constitutively active PKD-1 or PKD-CA expression also resulted in CD36 downregulation. Mechanistically, LPA treatment led to nuclear accumulation of PKD-1, HDAC7 and FoxO1. Furthermore, LPA-mediated PKD-1 signaling enhanced FoxO1-HDAC7 interaction in the nucleus. This interaction was attenuated with PKD-1 silencing. Intriguingly, doxycycline induced PKD-CA expression increased ephrin B2 expression and ERK activation, which are two critical “molecular signatures” involved in arteriogenesis. Functionally, FGF-2 induced a potent proangiogenic response even in the presence of TSP-1 in ECs pre-treated with LPA. Doxycycline induced PKD-CA expression stimulated robust arterial EC branching in a 3D spheroid assay. This angiogenic response was inhibited by a novel PKD-1 specific inhibitor. Moreover, in a mouse Lewis lung carcinoma model, we detected a minimal level of endothelial CD36 expression with robust tumor angiogenesis. The role of PKD-1 signaling in CD36 transcriptional repression and tumor arteriogenesis is a critical yet underexplored and underappreciated field. We propose that the LPA-PKD-FoxO1 signaling axis is essential for CD36 transcriptional repression and arteriogenic signaling and responses. The PKD-1 stimulated nuclear signaling network is critical to epigenetic suppression of CD36 transcription and silencing of CD36 antiangiogenic switch, subsequently turning on the angiogenic switch and stimulating the arteriogenic responses. Targeting the LPA-PKD1-FoxO1 signaling axis could have therapeutic potential in the ischemic disease and malignant tumors. Citation Format: Jacob D. Kohlenberg, Yiliang Chen, Brad Best, Peter Storz, Randall T. Peterson, Roy Silverstein, Bin Ren. A novel LPA-PKD1-FoxO1 pathway in endothelial cells provides an angiogenic switch via down-regulation of CD36 transcription and induction of arteriogenic responses . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-338. doi:10.1158/1538-7445.AM2013-LB-338