Döppler, Heike

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 Epigenetic silencing of tumor suppressing genes by promoter-specific DNA methylation is common in many types of cancer. As an early event, this process has been well shown to promote tumor initiation and progression; however little is known how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes Protein Kinase D1 (PKD1), a serine/threonine kinase expressed in epithelial cells of the normal mammary gland that maintains the epithelial phenotype of normal breast cells and prevents epithelial-to-mesenchymal transition (EMT). PKD1 is also a critical suppressor of tumor cell invasion and is silenced in expression and activity during breast tumor progression. Here, we show that aberrant methylation of PRKD1 promoter region is not only correlated with the silencing of its expression but is also associated with invasiveness of breast cancer cell lines and with aggressiveness of breast tumors. Using the highly invasive MDA-MB-231 cells, we show that the inhibition of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and significantly decreases their invasive abilities in vitro. More importantly, in a tumor xenograft model it dramatically 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. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-24.

In response to inflammation, pancreatic acinar cells can undergo acinar-to-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a ductlike phenotype and, in the context of additional oncogenic stimulation, contributes to development of pancreatic cancer. The signaling 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 tumor necrosis factor α (TNF) as mediators of such signaling. Both RANTES and TNF induce ADM through activation of nuclear factor κB 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.

PAKs (p21-activated kinases) are effectors of RhoGTPases. PAK4 contributes to regulation of cofilin at the leading edge of migrating cells through activation of LIMK (Lin-11/Isl-1/Mec-3 kinase). PAK4 activity is regulated by an autoinhibitory domain that is released upon RhoGTPase binding as well as phosphorylation at Ser474 in the activation loop of the kinase domain. In the present study, we add another level of complexity to PAK4 regulation by showing that phosphorylation at Ser99 is required for its targeting to the leading edge. This phosphorylation is mediated by PKD1 (protein kinase D1). Phosphorylation of PAK4 at Ser99 also mediates binding to 14-3-3 protein, and is required for the formation of a PAK4–LIMK–PKD1 complex that regulates cofilin activity and directed cell migration.

Increases in reactive oxygen species (ROS) have been implicated in age-related diseases, including cancer. The serine/threonine kinase protein kinase D1 (PKD1) is a stress-responsive kinase and sensor for reactive oxygen species, which can initiate cell survival through NF-κB signaling. We have previously shown that in response to ROS, PKD1 is activated at the mitochondria. However, the initial signaling events leading to localization of PKD1 to the mitochondria are unknown. Here, we show that formation of mitochondrial diacylglycerol (DAG) and its binding to PKD1 is the means by which PKD1 is localized to the mitochondria in response to ROS. Interestingly, DAG to which PKD1 is recruited in this pathway is formed downstream of phospholipase D1 (PLD1) and a lipase-inactive PLD1 or inhibition of PLD1 by pharmacological inhibitors blocked PKD1 activation under oxidative stress. To date it has been viewed that monosaturated and saturated DAG formed via PLD1 have no signaling function. However, our data describe a role for PLD1-induced DAG as a competent second messenger at the mitochondria that relays ROS to PKD1-mediated mitochondria-to-nucleus signaling.