Central role of Yes-associated protein and WW-domain-containing transcriptional co-activator with PDZ-binding motif in pancreatic cancer development.

Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC. Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network. Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators

Cooperation of Gq, Gi, and G12/13 in Protein Kinase D Activation and Phosphorylation Induced by Lysophosphatidic Acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha q/11 (Galpha q/11 KO cells) or Galpha 12/13 (Galpha 12/13 KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha 12/13 KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha 12/13 KO cells in autophosphorylation activity) achieved at 3 µM. In contrast, LPA did not induce any significant increase in PKD activity in Galpha q/11 KO cells. However, LPA induced a significantly increased PKD activity when Galpha q/11 KO cells were transfected with Galpha q. LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha 12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Galpha 12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi, and G12 and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways

Yes-associated protein (YAP) in pancreatic cancer: at the epicenter of a targetable signaling network associated with patient survival.

Pancreatic ductal adenocarcinoma (PDAC) is generally a fatal disease with no efficacious treatment modalities. Elucidation of signaling mechanisms that will lead to the identification of novel targets for therapy and chemoprevention is urgently needed. Here, we review the role of Yes-associated protein (YAP) and WW-domain-containing Transcriptional co-Activator with a PDZ-binding motif (TAZ) in the development of PDAC. These oncogenic proteins are at the center of a signaling network that involves multiple upstream signals and downstream YAP-regulated genes. We also discuss the clinical significance of the YAP signaling network in PDAC using a recently published interactive open-access database (www.proteinatlas.org/pathology) that allows genome-wide exploration of the impact of individual proteins on survival outcomes. Multiple YAP/TEAD-regulated genes, including AJUBA, ANLN, AREG, ARHGAP29, AURKA, BUB1, CCND1, CDK6, CXCL5, EDN2, DKK1, FOSL1,FOXM1, HBEGF, IGFBP2, JAG1, NOTCH2, RHAMM, RRM2, SERP1, and ZWILCH, are associated with unfavorable survival of PDAC patients. Similarly, components of AP-1 that synergize with YAP (FOSL1), growth factors (TGFα, EPEG, and HBEGF), a specific integrin (ITGA2), heptahelical receptors (P2Y2R, GPR87) and an inhibitor of the Hippo pathway (MUC1), all of which stimulate YAP activity, are associated with unfavorable survival of PDAC patients. By contrast, YAP inhibitory pathways (STRAD/LKB-1/AMPK, PKA/LATS, and TSC/mTORC1) indicate a favorable prognosis. These associations emphasize that the YAP signaling network correlates with poor survival of pancreatic cancer patients. We conclude that the YAP pathway is a major determinant of clinical aggressiveness in PDAC patients and a target for therapeutic and preventive strategies in this disease

Lipophilic statins inhibit YAP nuclear localization, co-activator activity and colony formation in pancreatic cancer cells and prevent the initial stages of pancreatic ductal adenocarcinoma in KrasG12D mice.

We examined the impact of statins on Yes-associated Protein (YAP) localization, phosphorylation and transcriptional activity in human and mouse pancreatic ductal adenocarcinoma (PDAC) cells. Exposure of sparse cultures of PANC-1 and MiaPaCa-2 cells to cerivastatin or simvastatin induced a striking re-localization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEAD-regulated genes Connective Tissue Growth Factor (CTGF) and Cysteine-rich angiogenic inducer 61 (CYR61). Statins also prevented YAP nuclear import and expression of CTGF and CYR61 stimulated by the mitogenic combination of insulin and neurotensin in dense culture of these PDAC cells. Cerivastatin, simvastatin, atorvastatin and fluvastatin also inhibited colony formation by PANC-1 and MiaPaCa-2 cells in a dose-dependent manner. In contrast, the hydrophilic statin pravastatin did not exert any inhibitory effect even at a high concentration (10 μM). Mechanistically, cerivastatin did not alter the phosphorylation of YAP at Ser127 in either PANC-1 or MiaPaCa-2 cells incubated without or with neurotensin and insulin but blunted the assembly of actin stress fiber in these cells. We extended these findings with human PDAC cells using primary KC and KPC cells, (expressing KrasG12D or both KrasG12D and mutant p53, respectively) isolated from KC or KPC mice. Using cultures of these murine cells, we show that lipophilic statins induced striking YAP translocation from the nucleus to the cytoplasm, inhibited the expression of Ctgf, Cyr61 and Birc5 and profoundly inhibited colony formation of these cells. Administration of simvastatin to KC mice subjected to diet-induced obesity prevented early pancreatic acini depletion and PanIN formation. Collectively, our results show that lipophilic statins restrain YAP activity and proliferation in pancreatic cancer cell models in vitro and attenuates early lesions leading to PDAC in vivo

Metformin Decreases the Incidence of Pancreatic Ductal Adenocarcinoma Promoted by Diet-induced Obesity in the Conditional KrasG12D Mouse Model.

Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly disease. Chronic conditions, including obesity and type-2 diabetes are risk factors, thus making PDAC amenable to preventive strategies. We aimed to characterize the chemo-preventive effects of metformin, a widely used anti-diabetic drug, on PDAC development using the KrasG12D mouse model subjected to a diet high in fats and calories (HFCD). LSL-KrasG12D/+;p48-Cre (KC) mice were given control diet (CD), HFCD, or HFCD with 5 mg/ml metformin in drinking water for 3 or 9 months. After 3 months, metformin prevented HFCD-induced weight gain, hepatic steatosis, depletion of intact acini, formation of advanced PanIN lesions, and stimulation of ERK and mTORC1 in pancreas. In addition to reversing hepatic and pancreatic histopathology, metformin normalized HFCD-induced hyperinsulinemia and hyperleptinemia among the 9-month cohort. Importantly, the HFCD-increased PDAC incidence was completely abrogated by metformin (p < 0.01). The obesogenic diet also induced a marked increase in the expression of TAZ in pancreas, an effect abrogated by metformin. In conclusion, administration of metformin improved the metabolic profile and eliminated the promoting effects of diet-induced obesity on PDAC formation in KC mice. Given the established safety profile of metformin, our findings have a strong translational potential for novel chemo-preventive strategies for PDAC

Kir4.1 Potassium Channel Subunit Is Crucial for Oligodendrocyte Development and In Vivo Myelination

To understand the cellular and in vivo functions of specific K^+ channels in glia, we have studied mice with a null mutation in the weakly inwardly rectifying K^+ channel subunit Kir4.1. Kir4.1−/− mice display marked motor impairment, and the cellular basis is hypomyelination in the spinal cord, accompanied by severe spongiform vacuolation, axonal swellings, and degeneration. Immunostaining in the spinal cord of wild-type mice up to postnatal day 18 reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes, but probably not in neurons or glial fibrillary acidic protein-positive (GFAP-positive) astrocytes. Cultured oligodendrocytes from developing spinal cord of Kir4.1−/− mice lack most of the wild-type K^+ conductance, have depolarized membrane potentials, and display immature morphology. By contrast, cultured neurons from spinal cord of Kir4.1−/− mice have normal physiological characteristics. We conclude that Kir4.1 forms the major K^+ conductance of oligodendrocytes and is therefore crucial for myelination. The Kir4.1 knock-out mouse is one of the few CNS dysmyelinating or demyelinating phenotypes that does not involve a gene directly involved in the structure, synthesis, degradation, or immune response to myelin. Therefore, this mouse shows how an ion channel mutation could contribute to the polygenic demyelinating diseases

Down-regulation of pancreatic and duodenal homeobox-1 by somatostatin receptor subtype 5: a novel mechanism for inhibition of cellular proliferation and insulin secretion by somatostatin

Somatostatin (SST) is a regulatory peptide and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells. SST's actions are mediated by a family of seven transmembrane domain G protein-coupled receptors that comprise five distinct subtypes (SSTR1-5). SSTR5 is one of the major SSTRs in the islets of Langerhans. Homeodomain-containing transcription factor pancreatic and duodenal homeobox-1 (PDX-1) is essential for pancreatic development, β cell differentiation, maintenance of normal β cell functions in adults and tumorigenesis. Recent studies show that SSTR5 acts as a negative regulator for PDX-1 expression and that SSTR5 mediates somatostatin's inhibitory effect on cell proliferation and insulin expression/excretion through down-regulating PDX-1 expression. SSTR5 exerts its inhibitory effect on PDX-1 expression at both the transcriptional level by down-regulating PDX-1 mRNA and the post-translational level by enhancing PDX-1 ubiquitination. Identification of PDX-1 as a transcriptional target for SSTR5 may help in guiding the choice of therapeutic cancer treatments

Identification of caspase 3 motifs and critical aspartate residues in human Phospholipase D1b and Phopsholipase D2a

Stimulation of mammalian cells frequently initiates phospholipase D-catalysed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change and movement. A number of studies have highlighted that PLD-based signalling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase-3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis