crt-0066101 and Pancreatic-Neoplasms

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.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Humans; Interleukin-8; Mice; Microvessels; Neoplasm Invasiveness; Neovascularization, Pathologic; Pancreatic Neoplasms; Protein Kinase C; Pyrimidines; Umbilical Veins; Vascular Endothelial Growth Factor A

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-kappaB activation; and abrogated the expression of NF-kappaB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 micromol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-kappaB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Humans; Male; Mice; Mice, Nude; Molecular Weight; Pancreatic Neoplasms; Protein Kinase C; Protein Kinase Inhibitors; Pyrimidines; Xenograft Model Antitumor Assays