Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFß, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Low dosage combination treatment with metformin and simvastatin inhibits obesity-promoted pancreatic cancer development in male KrasG12D mice.

Pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with limited therapeutic options, may benefit from repurposing of FDA-approved drugs in preventive or interceptive strategies in high-risk populations. Previous animal studies demonstrated that the use of metformin and statins as single agents at relatively high doses restrained PDAC development. Here, four-week-old mice expressing KrasG12D in all pancreatic lineages (KC mice) and fed an obesogenic high fat, high calorie diet that promotes early PDAC development were randomized onto low dosage metformin, simvastatin, or both drugs in combination administered orally. Dual treatment attenuated weight gain, fibro-inflammation, and development of advanced PDAC precursor lesions (pancreatic intraepithelial neoplasia [PanIN]-3) in male KC mice, without significant effect in females or when administered individually. Dual-treated KC mice had reduced proliferation of PanIN cells and decreased transcriptional activity of the Hippo effectors, YAP and TAZ, which are important regulators of PDAC development. Metformin and simvastatin also synergistically inhibited colony formation of pancreatic cancer cells in vitro. Together, our data demonstrated that a combination of low doses of metformin and simvastatin inhibits PDAC development and imply that both drugs are promising agents for being tested in clinical trials for preventing pancreatic cancer progression.

Lipophilic statins inhibit YAP coactivator transcriptional activity in HCC cells through Rho-mediated modulation of actin cytoskeleton.

Hepatocellular carcinoma (HCC) is the third leading cause of liver-related death. Lipophilic statins have been associated with a decrease in HCC incidence, raising the possibility of their use as chemoprevention agents. The Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have emerged as an important pro-oncogenic mechanism in HCC. Statins modulate YAP/TAZ in other solid tumors, but few studies have assessed their mechanisms in HCC. We aimed to delineate how lipophilic statins regulate YAP protein localization by interrogating the mevalonate pathway in a stepwise manner using pharmacological and genetical approaches in HCC cells. Huh7 and Hep3B HCC cells were treated with the lipophilic statins cerivastatin and atorvastatin. YAP protein localization was determined using quantitative immunofluorescence (IF) imaging. The gene expression of CTGF and CYR61, known YAP/TEA-domain DNA-binding factor (TEAD)-regulated genes, was measured using quantitative real-time PCR. Rescue experiments were conducted using metabolites of the mevalonate pathway including mevalonic acid and geranylgeranyl pyrophosphate (GG-PP). The cellular cytoskeleton was assessed using F-actin IF staining. YAP protein was extruded from the nucleus to the cytoplasm with statin treatment. Consistently, CTGF and CYR61 mRNA expression significantly decreased with statins. Cytoskeletal structure was also compromised with statins. Gene expression, YAP protein localization, and cytoskeletal structure were all restored to baseline with exogenous GG-PP but not with other metabolites of the mevalonate pathway. Direct Rho GTPase inhibitor treatment mirrored the statin effects on YAP. YAP protein localization is regulated by lipophilic statins via Rho GTPases, causing cytoskeletal structural changes and is independent of cholesterol metabolites.NEW & NOTEWORTHY Statins are widely used for the treatment of cardiovascular diseases. Recently, their use has been associated with a decrease in the incidence of hepatocellular carcinoma (HCC); however, their mechanism(s) has remained elusive. In this study, we delineate the mechanism by which statins affect the Yes-associated protein (YAP), which has emerged as a key oncogenic pathway in HCC. We investigate each step of the mevalonate pathway and demonstrate that statins regulate YAP via Rho GTPases.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Statins inhibit protein kinase D (PKD) activation in intestinal cells and prevent PKD1-induced growth of murine enteroids.

We examined the impact of statins on protein kinase D (PKD) activation by G protein-coupled receptor (GPCR) agonists. Treatment of intestinal IEC-18 cells with cerivastatin inhibited PKD autophosphorylation at Ser916 induced by angiotensin II (ANG II) or vasopressin in a dose-dependent manner with half-maximal inhibition at 0.2 µM. Cerivastatin treatment inhibited PKD activation stimulated by these agonists for different times (5-60 min) and blunted HDAC5 phosphorylation, a substrate of PKD. Other lipophilic statins, including simvastatin, atorvastatin, and fluvastatin also prevented PKD activation in a dose-dependent manner. Using IEC-18 cell lines expressing PKD1 tagged with EGFP (enhanced green fluorescent protein), cerivastatin or simvastatin blocked GPCR-mediated PKD1-EGFP translocation to the plasma membrane and its subsequent nuclear accumulation. Similar results were obtained in IEC-18 cells expressing PKD3-EGFP. Mechanistically, statins inhibited agonist-dependent PKD activation rather than acting directly on PKD catalytic activity since exposure to cerivastatin or simvastatin did not impair PKD autophosphorylation or PKD1-EGFP membrane translocation in response to phorbol dibutyrate, which bypasses GPCRs and directly stimulates PKC and PKD. Furthermore, cerivastatin did not inhibit recombinant PKD activity determined via an in vitro kinase assay. Using enteroids generated from intestinal crypt-derived epithelial cells from PKD1 transgenic mice as a model of intestinal regeneration, we show that statins oppose PKD1-mediated increase in enteroid area, complexity (number of crypt-like buds), and DNA synthesis. Our results revealed a previously unappreciated inhibitory effect of statins on receptor-mediated PKD activation and in opposing the growth-promoting effects of PKD1 on intestinal epithelial cells.

Statins Inhibit Inflammatory Cytokine Production by Macrophages and Acinar-to-Ductal Metaplasia of Pancreatic Cells.

BACKGROUND AND AIMS: Animal data show that the presence of an oncogenic Kras mutation in pancreatic acinar cells leads to acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN), and pancreatic ductal adenocarcinoma (PDAC). Inflammatory macrophages play an important role in the formation of ADMs and transition to PanINs. Epidemiologically, statins are associated with a reduced risk of PDAC. We investigated whether statins inhibit inflammatory cytokine production in macrophages and whether this leads to reduced ADM formation. METHODS: The efficacy of statins on inflammatory cytokine production in 2 macrophage cell lines was measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of macrophage-conditioned medium on ADM in primary pancreatic acinar cells was investigated. Mouse pancreatic tissue samples were analyzed for macrophage numbers, cytokine levels, and neoplastic/dysplastic area. RESULTS: Lipophilic statins prevented inflammatory cytokine production in Raw264.7 and J774A.1 cells stimulated by lipopolysaccharide. The inhibitory effect of statins was mediated by inhibition of mevalonate and geranylgeranyl pyrophosphate synthesis and disruption of the actin cytoskeleton but not by a reduction in intracellular cholesterol. Treatment of macrophages with lipophilic statins also blocked ADM formation of primary pancreatic acinar cells. Furthermore, oral administration of simvastatin was associated with a reduction in the number of intrapancreatic macrophages, decreased inflammatory cytokine levels in the pancreas, and attenuated ADM/PanIN formation in mice. CONCLUSION: Our data support the hypothesis that statins oppose early PDAC development by their effects on macrophages and ADM formation. The inhibitory actions of statins on macrophages may collaborate with direct inhibitory effects on transformed pancreatic epithelial cells, which cumulatively may reduce early PDAC development and progression.

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Opposite Effects of Src Family Kinases on YAP and ERK Activation in Pancreatic Cancer Cells: Implications for Targeted Therapy.

Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive disease that is expected to become the second cause of cancer fatalities during the next decade. As therapeutic options are limited, novel targets, and agents for therapeutic intervention are urgently needed. Previously, we identified potent positive crosstalk between insulin/IGF-1 receptors and G protein-coupled (GPCR) signaling systems leading to mitogenic signaling in PDAC cells. Here, we show that a combination of insulin and the GPCR agonist neurotensin induced rapid activation of Src family of tyrosine kinases (SFK) within PANC-1 cells, as shown by FAK phosphorylation at Tyr576/577 and Tyr861, sensitive biomarkers of SFK activity within intact cells and Src416 autophosphorylation. Crucially, SFKs promoted YAP nuclear localization and phosphorylation at Tyr357, as shown by using the SFK inhibitors dasatinib, saracatinib, the preferential YES1 inhibitor CH6953755, siRNA-mediated knockdown of YES1, and transfection of epitogue-tagged YAP mutants in PANC-1 and Mia PaCa-2 cancer cells, models of the aggressive squamous subtype of PDAC. Surprisingly, our results also demonstrate that exposure to SFK inhibitors, including dasatinib or knockdown of YES and Src induces ERK overactivation in PDAC cells. Dasatinib-induced ERK activation was completely abolished by exposure to the FDA-approved MEK inhibitor trametinib. A combination of dasatinib and trametinib potently and synergistically inhibited colony formation by PDAC cells and suppressed the growth of Mia PaCa-2 cells xenografted into the flank of nude mice. The results provide rationale for considering a combination(s) of FDA-approved SFK (dasatinib) and MEK (e.g., trametinib) inhibitors in prospective clinical trials for the treatment of PDAC.

Obesity and Pancreatic Cancer: Insight into Mechanisms.

The prevalence of obesity in adults and children has dramatically increased over the past decades. Obesity has been declared a chronic progressive disease and is a risk factor for a number of metabolic, inflammatory, and neoplastic diseases. There is clear epidemiologic and preclinical evidence that obesity is a risk factor for pancreatic cancer. Among various potential mechanisms linking obesity with pancreatic cancer, the adipose tissue and obesity-associated adipose tissue inflammation play a central role. The current review discusses selected topics and mechanisms that attracted recent interest and that may underlie the promoting effects of obesity in pancreatic cancer. These topics include the impact of obesity on KRAS activity, the role of visceral adipose tissue, intrapancreatic fat, adipose tissue inflammation, and adipokines on pancreatic cancer development. Current research on lipocalin-2, fibroblast growth factor 21, and Wnt5a is discussed. Furthermore, the significance of obesity-associated insulin resistance with hyperinsulinemia and obesity-induced gut dysbiosis with metabolic endotoxemia is reviewed. Given the central role that is occupied by the adipose tissue in obesity-promoted pancreatic cancer development, preventive and interceptive strategies should be aimed at attenuating obesity-associated adipose tissue inflammation and/or at targeting specific molecules that mechanistically link adipose tissue with pancreatic cancer in obese patients.

Crosstalk between KRAS, SRC and YAP Signaling in Pancreatic Cancer: Interactions Leading to Aggressive Disease and Drug Resistance.

Pancreatic ductal adenocarcinoma (PDAC), the predominant form of pancreatic cancer, remains a devastating disease. The purpose of this review is to highlight recent literature on mechanistic and translational developments that advance our understanding of a complex crosstalk between KRAS, YAP and Src tyrosine kinase family (SFK) in PDAC development and maintenance. We discuss recent studies indicating the importance of RAS dimerization in signal transduction and new findings showing that the potent pro-oncogenic members of the SFK phosphorylate and inhibit RAS function. These surprising findings imply that RAS may not play a crucial role in maintaining certain subtypes of PDAC. In support of this interpretation, current evidence indicates that the survival of the basal-like subtype of PDAC is less dependent on RAS but relies, at least in part, on the activity of YAP/TAZ. Based on current evidence, we propose that SFK propels PDAC cells to a state of high metastasis, epithelial-mesenchymal transition (EMT) and reduced dependence on KRAS signaling, salient features of the aggressive basal-like/squamous subtype of PDAC. Strategies for PDAC treatment should consider the opposite effects of tyrosine phosphorylation on KRAS and SFK/YAP in the design of drug combinations that target these novel crosstalk mechanisms and overcome drug resistance.

Metformin: review of epidemiology and mechanisms of action in pancreatic cancer.

Pancreatic ductal adenocarcinoma continues to be a lethal disease, for which efficient treatment options are very limited. Increasing efforts have been taken to understand how to prevent or intercept this disease at an early stage. There is convincing evidence from epidemiologic and preclinical studies that the antidiabetic drug metformin possesses beneficial effects in pancreatic cancer, including reducing the risk of developing the disease and improving survival in patients with early-stage disease. This review will summarize the current literature about the epidemiological data on metformin and pancreatic cancer as well as describe the preclinical evidence illustrating the anticancer effects of metformin in pancreatic cancer. Underlying mechanisms and targets of metformin will also be discussed. These include direct effects on transformed pancreatic epithelial cells and indirect, systemic effects on extra-pancreatic tissues.

Ligation of HLA Class I Molecules Induces YAP Activation through Src in Human Endothelial Cells.

Ab cross-linking of HLA class I (HLA I) molecules on the surface of endothelial cells (EC) triggers proliferative and prosurvival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. Despite the importance of Ab-mediated rejection in transplantation, the mechanisms involved remain incompletely understood. In this study, we examined the regulation of yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in human ECs challenged with Abs that bind HLA I. In unstimulated ECs, YAP localized mainly in the cytoplasm. Stimulation of these cells with Ab W6/32 induced marked translocation of YAP to the nucleus. The nuclear import of YAP was associated with a rapid decrease in YAP phosphorylation at Ser127 and Ser397, sites targeted by LATS1/2 and with the expression of YAP-regulated genes, including connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Transfection of small interfering RNAs targeting YAP/TAZ blocked the migration of ECs stimulated by ligation of HLA I, indicating that YAP mediates the increase in EC migration induced by HLA I ligation. Treatment of intact ECs with Src family inhibitors induced cytoplasmic localization of YAP in unstimulated ECs and, strikingly, blocked the nuclear import of YAP induced by Ab-induced HLA I activation in these cells and the increase in the expression of the YAP-regulated genes CTGF and CYR61 induced by HLA I stimulation. Our results identify the Src/YAP axis as a key player in promoting the proliferation and migration of ECs that are critical in the pathogenesis of transplant vasculopathy.

Metformin inhibition of colorectal cancer cell migration is associated with rebuilt adherens junctions and FAK downregulation.

E-cadherin, a central component of the adherens junction (AJ), is a single-pass transmembrane protein that mediates cell-cell adhesion. The loss of E-cadherin surface expression, and therefore cell-cell adhesion, leads to increased cell migration and invasion. Treatment of colorectal cancer (CRC)-derived cells (SW-480 and HT-29) with 2.0 mM metformin promoted a redistribution of cytosolic E-cadherin to de novo formed puncta along the length of the contacting membranes of these cells. Metformin also promoted translocation from the cytosol to the plasma membrane of p120-catenin, another core component of the AJs. Furthermore, E-cadherin and p120-catenin colocalized with ß-catenin at cell-cell contacts. Western blot analysis of lysates of CRC-derived cells revealed a substantial metformin-induced increase in the level of p120-catenin as well as E-cadherin phosphorylation on Ser838/840 , a modification associated with ß-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and ß-catenin localization suggest that metformin induces rebuilding of AJs in CRC-derived cells. Those modifications were accompanied by the inhibition of focal adhesion kinase (FAK), as revealed by a significant decrease in the phosphorylation of FAK at Tyr397 and paxillin at Tyr118 . These changes were associated with a reduction in the numbers, but an increase in the size, of focal adhesions and by the inhibition of cell migration. Overall, these observations indicate that metformin targets multiple pathways associated with CRC development and progression.

Metformin alters the duodenal microbiome and decreases the incidence of pancreatic ductal adenocarcinoma promoted by diet-induced obesity.

Pancreatic ductal adenocarcinoma (PDAC)'s growing incidence has been linked to the rise in obesity and type 2 diabetes mellitus. In previous work, we have shown that metformin can prevent the increased incidence of PDAC in a KrasG12D mouse model subjected to a diet high in fat and calories (HFCD). One potential way that metformin can affect the host is through alterations in the gut microbiome. Therefore, we investigated microbial associations with PDAC development and metformin use in the same mouse model. Lox-Stop-Lox Kras G12D/+ (LSL-Kras G12D/+); p48-Cre (KC) mice were given control diet, HFCD, or HFCD with 5 mg/mL metformin in drinking water for 3 mo. At the end of the 3 mo, 16S rRNA sequencing was performed to characterize microbiome composition of duodenal mucosal, duodenal luminal, and cecal luminal samples. KC mice on an HFCD demonstrated depletion of intact acini and formation of advanced pancreatic intraepithelial neoplasia. This effect was completely abrogated by metformin treatment. HFCD was associated with significant changes in microbial composition and diversity in the duodenal mucosa and lumen, much of which was prevented by metformin. In particular, Clostridium sensu stricto was negatively correlated with percent intact acini and seemed to be inhibited by the addition of metformin while on an HFCD. Administration of metformin eliminated PDAC formation in KC mice. This change was associated with significant microbial changes in both the mucosal and luminal microbiome of the duodenum. This suggests that the microbiome may be a potential mediator of the chemopreventive effects of metformin.NEW & NOTEWORTHY Pancreatic ductal adenocarcinoma (PDAC)'s growing incidence has been linked to the rise in obesity and type 2 diabetes mellitus. Administration of metformin eliminated PDAC formation in KC mice with diet-induced obesity. This change was associated with significant microbial changes in both the mucosal and luminal microbiome of the duodenum. This suggests that the microbiome may be a potential mediator of the chemopreventive effects of metformin.

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 inhibits ß-catenin phosphorylation on Ser-552 through an AMPK/PI3K/Akt pathway in colorectal cancer cells.

Several epidemiologic studies have revealed strong inverse associations between metformin use and risk of colorectal cancer development. Nevertheless, the underlying mechanisms are still uncertain. The Wnt/ß-catenin pathway, which plays a central role in intestinal homeostasis and sporadic colorectal cancer development, is regulated by phosphorylation cascades that are dependent and independent of Wnt. Here we report that a non-canonical Ser552 phosphorylation in ß-catenin, which promotes its nuclear accumulation and transcriptional activity, is blocked by metformin via AMPK-mediated PI3K/Akt signaling inhibition.

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.

Protein kinase D1 inhibition interferes with mitosis progression.

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.

KRAS, YAP, and obesity in pancreatic cancer: A signaling network with multiple loops.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a lethal disease with no efficacious treatment modalities. The incidence of PDAC is expected to increase, at least partially because of the obesity epidemic. Increased efforts to prevent or intercept this disease are clearly needed. Mutations in KRAS are initiating events in pancreatic carcinogenesis supported by genetically engineered mouse models of the disease. However, oncogenic KRAS is not entirely sufficient for the development of fully invasive PDAC. Additional genetic mutations and/or environmental, nutritional, and metabolic stressors, e.g. inflammation and obesity, are required for efficient PDAC formation with activation of KRAS downstream effectors. Multiple factors "upstream" of KRAS associated with obesity, including insulin resistance, inflammation, changes in gut microbiota and GI peptides, can enhance/modulate downstream signals. Multiple signaling networks and feedback loops "downstream" of KRAS have been described that respond to obesogenic diets. We propose that KRAS mutations potentiate a signaling network that is promoted by environmental factors. Specifically, we envisage that KRAS mutations increase the intensity and duration of the growth-promoting signaling network. As the transcriptional activator YAP plays a critical role in the network, we conclude that the rationale for targeting the network (at different points), e.g. with FDA approved drugs such as statins and metformin, is therefore compelling.