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-κB activation; and abrogated the expression of NF-κB-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 μmol/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-κB-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. Mol Cancer Ther; 9(5); 1136-46. ©2010 AACR.

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRASG12C/G13D or BRAFV600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

No abstract

<div>Abstract<p>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-κB activation; and abrogated the expression of NF-κB-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 μmol/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 <i>in vivo</i>. CRT0066101 significantly reduced Ki-67–positive proliferation index (<i>P</i> < 0.01), increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive apoptotic cells (<i>P</i> < 0.05), and abrogated the expression of NF-κB–dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show <i>for the first time</i> that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth <i>in vivo</i> and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136–46. ©2010 AACR.</p></div>

Supplementary Data from A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth <i>In vitro</i> and <i>In vivo</i>

Supplementary Data from A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth <i>In vitro</i> and <i>In vivo</i>

No abstract

The invasive potential of carcinomas greatly contributes to their ability to metastasize, a process which is estimated to cause 90% of all human cancer deaths. The LIM kinase (LIMK) family of Ser/Thr kinases sit at a hub of signaling pathways downstream of the Rho family of GTPases. Functionally, LIMK is directly involved in regulating the activity of cofilin, a family of proteins which modulate cell movement through reorganization of the actin cytoskeleton network. LIMK is up-regulated in a number of invasive cancer cell lines and metastatic breast and prostate tumors, whilst an increase in LIMK activity has been shown to cause increased cellular invasion in multiple model systems. We demonstrate that LIMK inhibition by siRNA reduces the invasive capacity of MDA-MB-231-Luc breast cancer cells in an in vitro matrigel invasion assay and reduces fibroblast lead collective invasion in a co-culture organotypic model. Potent, selective, small molecule LIMK inhibitors have been identified that inhibit LIMK in vitro with sub-nanomolar activity in biochemical assays and low micromolar activity in cells as measured by cofilin phosphorylation. Treatment of MDA-MB-231-Luc cells or tumor associated fibroblasts with these small molecule inhibitors decreased cell invasion in vitro with minimal cellular toxicity. Collectively, these data further validate LIMK as an anti-invasive therapeutic target in tumor cells and demonstrate the potential utility of LIMK inhibitors in metastatic disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C210.