Elena N. Fazio

Fibroblast Growth Factor 21 Reduces the Severity of Cerulein-Induced Pancreatitis in Mice

BACKGROUND & AIMS Fibroblast growth factor 21 (FGF21) acts as a hormonal regulator during fasting and is involved in lipid metabolism. Fgf21 gene expression is regulated by peroxisome proliferator-activated receptor (PPAR)-dependent pathways, which are enhanced during pancreatitis. Therefore, the aim of this study was to investigate FGF21s role in pancreatic injury. METHODS Fgf21 expression was quantified during cerulein-induced pancreatitis (CIP) or following mechanical or thapsigargin-induced stress through Northern blot analysis, in situ hybridization, and quantitative reverse transcription polymerase chain reaction. FGF21 protein was quantified by Western blot analysis. Isolated acinar cells or AR42J acinar cells were treated with recombinant FGF21 protein, and extracellular regulated kinase 1/2 activation was examined. The severity of CIP was compared between wild-type mice and mice overexpressing FGF21 (FGF21Tg) or harboring a targeted deletion of Fgf21 (Fgf21(-/-)). RESULTS Acinar cell Fgf21 expression markedly increased during CIP and following injury in vitro. Purified FGF21 activated the extracellular regulated kinase 1/2 pathway in pancreatic acinar cells. The severity of CIP is inversely correlated to FGF21 expression because FGF21Tg mice exhibited decreased serum amylase and decreased pancreatic stellate cell activation, whereas Fgf21(-/-) mice had increased serum amylase and tissue damage. The expression of Fgf21 was also inversely correlated to expression of Early growth response 1, a proinflammatory and profibrotic transcription factor. CONCLUSIONS These studies suggest a novel function for Fgf21 as an immediate response gene protecting pancreatic acini from overt damage.

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability.

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence.

Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice

BackgroundStanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo.ResultsSince PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2Tg) were examined. STC2Tgpancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2Tgmice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions.ConclusionsThis study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury.

Activating Transcription Factor 3 Promotes Loss of the Acinar Cell Phenotype in Response to Cerulein-Induced Pancreatitis in Mice

A novel role for ATF3 is identified in acinar-to–duct cell metaplasia during pancreatic injury. ATF3 targets transcriptional regulators that affect acinar cell fate and reduces the severity of pancreatic injury. However, by doing so, ATF3 may also increase the susceptibility for progression to pancreatic adenocarcinoma.

Abstract A47: The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse throughout the peritoneum via ascites to seed secondary lesions. We hypothesize that EOC spheroids are key mediators of metastasis, which use specific signaling pathways to alter cell metabolism for increased survival. Our lab discovered that AKT signaling is reduced during spheroid formation leading to cellular quiescence and autophagy. Given the induction of quiescence and autophagy in EOC spheroids, we are studying the 5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response and dormant phenotype of EOC spheroids. We demonstrate AMPK activity and its upstream kinase LKB1 are increased in quiescent EOC spheroids compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Targeted knockdown of STK11 , encoding LKB1, reduces cell viability in ovarian cancer cell line spheroids; PRKAA1 (AMPKα1) knockdown has little to no effect on EOC cell or spheroid viability. Combination of STK11 knockdown with carboplatin treatment leads to a synergistic enhancement in EOC spheroid cell death. In contrast, AICAR treatment of proliferating adherent ovarian cancer cell lines and primary EOC cells induces AMPK activity and causes either cytostasis or cell death. In addition, AICAR treatment of spheroids during reattachment decreases the dispersion capacity of migrating EOC cells. These results offer a glimpse of the potential important contributions of LKB1-AMPK pathway in stress signaling related to EOC cell survival during metastasis. In addition, downstream effectors of upregulated LKB1-AMPK signalling may provide additional mechanisms by which EOC evades chemotherapy. It is foreseeable that these findings will allow us to identify new therapeutic targets within this pathway critical to EOC progression for ultimate translation to the clinic. Citation Format: Teresa Peart, Elena Fazio, Yudith Ramos-Valdes, Monique Bertrand, Jacob McGee, Michel Prefontaine, Akira Sugimoto, Gabriel E. DiMattia, Trevor G. Shepherd. The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A47.