Wilfredo E. De Jesus-Monge

The activity of Gli transcription factors is essential for Kras-induced pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here we find that transcriptional activation mediated by the Gli family of transcription factors, although dispensable for pancreatic development, is required for Kras-induced proliferation and survival in primary pancreatic epithelial cells in culture and for Kras-driven pancreatic intraepithelial neoplasia and PDAC formation in vivo. Further, ectopic Gli1 activation in the mouse pancreas accelerates Kras-driven tumor formation, underscoring the importance of Gli transcription factors in pancreatic tumorigenesis. Interestingly, we demonstrate Gli-regulated I-kappa-B kinase epsilon (IKBKE) and NF-κB activity in pancreatic cancer cells and show that this activity is a critical downstream mediator for Gli-dependent PDAC cell transformation and survival. Together, these studies demonstrate the requirement for Gli in Kras-dependent pancreatic epithelial transformation, suggest a mechanism of Gli-NF-κB oncogenic activation, and provide genetic evidence supporting the therapeutic targeting of Gli activity in pancreatic cancer.

Activated Wnt Signaling in Stroma Contributes to Development of Pancreatic Mucinous Cystic Neoplasms

BACKGROUND & AIMS Pancreatic mucinous cystic neoplasm (MCN), a cystic tumor of the pancreas that develops most frequently in women, is a potential precursor to pancreatic ductal adenocarcinoma. MCNs develop primarily in the body and tail of the pancreas and are characterized by the presence of a mucinous epithelium and ovarian-like subepithelial stroma. We investigated the involvement of Wnt signaling in KRAS-mediated pancreatic tumorigenesis and development of MCN in mice, and Wnt activation in human MCN samples. METHODS LSL-Kras(G12D), Ptf1a-cre mice were crossed with elastase-tva mice to allow for introduction of genes encoded by the replication-competent avian sarcoma-leukosis virus long-terminal repeat with splice acceptor viruses to pancreatic acinar cells and acinar cell progenitors, postnatally and sporadically. Repeat with splice acceptor viruses that expressed Wnt1 were delivered to the pancreatic epithelium of these mice; pancreatic lesions were analyzed by histopathology and immunohistochemical analyses. We analyzed levels of factors in Wnt signaling pathways in 19 MCN samples from patients. RESULTS Expression of Wnt1 in the pancreatic acinar cells and acinar cell progenitors of female mice led to development of unilocular or multilocular epithelial cysts in the pancreas body and tail, similar to MCN. The cystic lesions resembled the estrogen receptor- and progesterone receptor-positive ovarian-like stroma of MCN, but lacked the typical mucinous epithelium. Activated Wnt signaling, based on nuclear localization of β-catenin, was detected in the stroma but not cyst epithelium. Wnt signaling to β-catenin was found to be activated in MCN samples from patients, within the ovarian-like stroma, consistent with the findings in mice. CONCLUSIONS Based on studies of mice and pancreatic MCN samples from patients, the canonical Wnt signaling pathway becomes activated and promotes development of the ovarian-like stroma to contribute to formation of MCNs.

Abstract LB-105: Gli-IKBKE interactions in pancreatic tumorigenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oncogenic mutations in KRAS are detected in a majority of human pancreatic malignancies, and are considered an initiating event in pancreatic ductal adenocarcinoma (PDAC). The downstream mechanisms through which KRAS drives tumorigenesis remain elusive. While recent in vitro studies have suggested that Gli proteins, the transcriptional effectors of Hedgehog signaling, may mediate KRAS oncogenic activity in PDAC cell lines, a requirement for Gli activation in pancreatic tumorigenesis has not been demonstrated in vivo. Here, we use genetically modified mouse models, and human PDAC cell lines to establish a requirement for Gli activation in KRAS initiation of pancreatic tumorigenesis, and identify IKBKE as a novel oncogenic target gene of Gli in PDAC. We show for the first time that inhibition of Gli transcription in the pancreatic epithelium suppressed the ability of activated KRAS to initiate tumorigenesis in vivo. Also, we find that expression of Gli1 along with oncogenic KRAS led to a dramatic acceleration in the formation of pancreatic intraepithelial neoplasia (PanIN) lesions, and increased mortality. Importantly, we identify IKBKE as a novel transcriptional target of Gli in PDAC cells, and also in the pancreatic epithelium in vivo. We also demonstrate that IKBKE is required for the survival and tumorigenicity of PDAC cells, thus implicating IKBKE as a novel pancreatic cancer oncogene. We have found that IKBKE activates the NF-kB pathway, and also activates Akt by phosphorylation in PDAC cells as part of its tumorigenic activity. Our studies demonstrate for the first time a requirement for Gli transcriptional activity in KRAS induced pancreatic tumorigenesis in vivo, and establish IKBKE as a novel Gli target and pancreatic cancer oncogene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-105. doi:1538-7445.AM2012-LB-105

The Requirement of GLI Transcriptional Activity in KRAS-Induced Pancreatic Neoplasia

Background and Aim: Kras mutations are detected in 90% of human pancreatic ductal adenocarcinoma (PDAC) and are believed to be the critical oncogenic event during pancreatic tumorigenesis. However, the downstream transcriptional mechanisms by Kras in pancreatic cancer still remain poorly understood. Recent In Vitro studies suggested that Gli transcriptional factors, the intracellular mediator of Hedgehog signaling, are important for Kras activity in human PDAC cell lines. However, a requirement for Gli activation in pancreatic malignancy has not been demonstrated In Vivo. Here we carried out both loss-of-function and gain-offunction genetic analyses to establish the functional significance of Gli transcriptional activation in Kras-Induced pancreatic tumorigenesis using genetically modified mouse models. Methods: We utilized a conditional Rosa26 allele of Gli3T, R26-Gli3T. Gli3T is a dominant repressor of Gli3 that blocks Gli transcriptional activation. The R26-Gli3T mice were crossed to the Ptf1a-Cre;LDL-KrasG12D mice, a mouse model that allows pancreatic-specific Kras activation and tumor formation. A cohort of Ptf1a-Cre;LDL-KrasG12D;R26-Gli3T mice was generated and monitored for pancreatic tumor development. For Gli gain of function studies, we ectopically expressed Gli1 along with oncogenic Kras in mouse pancreas to determine whether Gli1 cooperates with Kras to promote tumorigenesis. Results: Our results showed that Gli3T expression caused a severe growth defect and loss of tumorigenicity in human PDAC cell lines. More importantly, the mice carrying Gli3T along with oncogenic Kras failed to develop PanIN lesions, in contrast to the mice with Kras activation alone. Mice expressing Gli1 along with activated Kras in pancreas also showed marked acceleration of tumor formation. Furthermore, our gene expression profiling identified a distinct transcriptional program mediated by Gli proteins in pancreatic tumor cells. Conclusions: Gli transcriptional activation not only accelerates pancreatic tumorigenesis in cooperation with Kras, but is also required for Kras-induced tumorigenesis In Vivo.

Electronic Clinical Challenges and Images in GIElectronic Clinical Challenges and Images in GI

r uestion: A previously healthy 31-year-old man was rought to the hospital complaining of dizziness, nausea, nd diffuse, intermittent, stabbing abdominal pain. The atient developed these symptoms before taking an airlane flight from a Caribbean island. This was his first pisode and he denied headache, chest pain, myalgia, rthralgia, diarrhea, gastrointestinal bleeding, and tremrs. He had a past medical history of cigarette smoking nd occasional alcohol intake. On physical examination, he man was alert, active, afebrile, hypertensive, oriented o person, time, and place, and complained of mild abominal pain. Bowel sounds were present, but his abdoen was diffusely tender to superficial and deep palpation ith voluntary guarding, no rebound, and no hepatoplenomegaly. Complete blood count, serum chemistry, urialysis, and coagulation panel were within normal limits. imilarly, urine toxicology was negative for amphetamines, arbiturates, benzodiazepines, marijuana, methadone, opites, and cocaine. An upright abdominal x-ray was done Figure A). What is the diagnosis? See the GASTROENTEROLOGY web site (www. astrojournal.org) for more information on submiting your favorite image to Clinical Challenges and Imges in GI. WILFREDO E. DE JESUS-MONGE, MD Department of Medicine University of Puerto Rico School of Medicine San Juan, Puerto Rico