Hsin-Yuan Su

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.

The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells

Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5–2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.

Insulin promotes proliferation and fibrosing responses in activated pancreatic stellate cells.

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.

The Combination of Alcohol and Cigarette Smoke Induces Endoplasmic Reticulum Stress and Cell Death in Pancreatic Acinar Cells

BACKGROUND & AIMS Smoking, an independent risk factor for pancreatitis, accelerates the development of alcoholic pancreatitis. Alcohol feeding of mice induces up-regulation of spliced X-box binding protein 1 (XBP1s), which regulates the endoplasmic reticulum (ER) unfolded protein response and promotes cell survival upon ER stress. We examined whether smoking affects the adaptive mechanisms induced by alcohol and accelerates disorders of the ER in pancreatic acinar cells. METHODS We studied the combined effects of ethanol (EtOH) and cigarette smoke extract (CSE) on ER stress and cell death responses in mouse and human primary acini and the acinar cell line AR42J. Cells were incubated with EtOH (50 mmol/L), CSE (20-40 μg/mL), or both (CSE+EtOH), and analyzed by immunoblotting, quantitative reverse-transcription polymerase chain reaction, and cell death assays. Some cells were incubated with MKC-3946, an inhibitor of endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1) that blocks XBP1s formation. Male Sprague-Dawley rats were fed isocaloric amounts of an EtOH-containing (Lieber-DeCarli) or control diet for 11 weeks and exposed to cigarette smoke or room air in an exposure chamber for 2 hours each day. During the last 3 weeks, a subset of rats received intravenous injections of lipopolysaccharide (LPS, 3 mg/kg per week) to induce pancreatitis or saline (control). Pancreatic tissues were collected and analyzed by histology and immunostaining techniques. RESULTS In AR42J and primary acini, CSE+EtOH induced cell death (necrosis and apoptosis), but neither agent alone had this effect. Cell death was associated with a significant decrease in expression of XBP1s. CSE+EtOH, but neither agent alone, slightly decreased adenosine triphosphate levels in AR42J cells, but induced oxidative stress and sustained activation (phosphorylation) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, also called PERK) and increased protein levels of DNA damage inducible transcript 3 (DDIT3, also called CHOP). CHOP regulates transcription to promote apoptosis. Incubation of AR42J or primary mouse or human acinar cells with MKC-3946 reduced expression of XBP1s, increased levels of CHOP, and induced cell death. In rats fed an EtOH diet, exposure to cigarette smoke increased ER stress in acinar cells and sensitized the pancreas to LPS-induced pathology. CONCLUSIONS Cigarette smoke promotes cell death and features of pancreatitis in EtOH-sensitized acinar cells by suppressing the adaptive unfolded protein response signaling pathway. It also activates ER stress pathways that promote acinar cell death.

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model

Background & Aims Heavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins. Methods Wild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations. Results Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice. Conclusions Results documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

The Differential Role of Human Cationic Trypsinogen (PRSS1) p.R122H Mutation in Hereditary and Nonhereditary Chronic Pancreatitis: A Systematic Review and Meta-Analysis

Background Environmental factors and genetic mutations have been increasingly recognized as risk factors for chronic pancreatitis (CP). The PRSS1 p.R122H mutation was the first discovered to affect hereditary CP, with 80% penetrance. We performed here a systematic review and meta-analysis to evaluate the associations of PRSS1 p.R122H mutation with CP of diverse etiology. Methods The PubMed, EMBASE, and MEDLINE database were reviewed. The pooled odds ratio (OR) with 95% confidence intervals was used to evaluate the association of p.R122H mutation with CP. Initial analysis was conducted with all etiologies of CP, followed by a subgroup analysis for hereditary and nonhereditary CP, including alcoholic or idiopathic CP. Results A total of eight case-control studies (1733 cases and 2415 controls) were identified and included. Overall, PRSS1 p.R122H mutation was significantly associated with an increased risk of CP (OR = 4.78[1.13–20.20]). Further analysis showed p.R122H mutation strongly associated with the increased risk of hereditary CP (OR = 65.52[9.09–472.48]) but not with nonhereditary CP, both alcoholic and idiopathic CP. Conclusions Our study showing the differential role of p.R122H mutation in various etiologies of CP indicates that this complex disorder is likely influenced by multiple genetic factors as well as environmental factors.

Abstract 1769: Rottlerin induces ER stress-mediated cell death in pancreatic stellate cells

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate under 5%. Whereas activated pancreatic stellate cells (PaSC) are important players in PDAC stroma, simultaneously targeting both cancer and stromal cells can be beneficial for cancer therapy. We previously reported that the phytochemical rottlerin dose-dependently reduces PaSC viability through dysregulation of autophagy and induction of apoptosis, and attenuates tumor progression in PDAC experimental models. Interestingly, both rottlerin and metformin have been shown to affect mitochondrial function, and metformin has been demonstrated to induce AMPK-dependent, CHOP-mediated cell death. Here we further studied the mechanisms underlying cell death and autophagy in rottlerin-treated mouse PaSC. Rottlerin rapidly depolarized mitochondrial membrane, increased oxygen consumption and decreased cellular ATP levels and the expression of the mitochondrial transmembrane protein TOM20, suggesting mitochondrial damage. Rottlerin dysregulated autophagy as indicated by rapid and prolonged increasing in LC3I-LC3II conversion and p62/SQSTM1 accumulation. However, rottlerin-induced LC3II did not further change in the presence of inhibitors of autophagic flux, suggesting that rottlerin blocks autophagy progression in PaSC. In addition, rottlerin induced marked AMPK activation and inhibited mTOR signaling. In contrast to metformin, AMPKα1/2 knockdown had no effect on rottlerin-induced inhibition of mTOR signaling and cell viability. Furthermore, rottlerin induced ER stress as manifested by persistent activation of the PERK pathway and upregulation of proapoptotic transcription factor CHOP. Importantly, rottlerin-induced apoptosis was greatly reduced in PaSC isolated from CHOP null mice. In summary, these data indicate that rottlerin reduces PaSC viability through impairing mitochondrial function, inhibiting mTOR signaling and inducing prolonged ER stress. Specifically, CHOP induction plays an important role in determining cell-fate in mitochondria-targeting agents, such as rottlerin and metformin. Citation Format: Hsin-Yuan Su, Richard Waldron, Raymond Gong, Stephen Pandol, Aurelia Lugea. Rottlerin induces ER stress-mediated cell death in pancreatic stellate cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2015-1769