David R. Driscoll

Epidermal Growth Factor Receptor Kinase Domain Mutations in Esophageal and Pancreatic Adenocarcinomas

Purpose: Specific activating mutations within the epidermal growth factor receptor (EGFR) identify a subset of non–small cell lung cancers with dramatic sensitivity to the specific tyrosine kinase inhibitors (TKI), gefitinib and erlotinib. Despite the abundant expression of EGFR protein in a broad range of epithelial cancers, EGFR mutations have not been reported in a substantial fraction of other cancers. Given recent reports of TKI-responsive cases of esophageal and pancreatic cancer, this study was designed to determine the prevalence of EGFR mutations in these gastrointestinal cancers. Experimental Design: We sequenced exons 18 to 21 of EGFR from 21 cases of Barretts esophagus, 5 cases of high-grade esophageal dysplasia, 17 cases of esophageal adenocarcinoma, and 55 cases of pancreatic adenocarcinoma. Subsets of esophageal (n = 7) and pancreatic cancer cases (n = 5) were obtained from patients who were subsequently treated with gefitinib or erlotinib-capecitabine, respectively. Results: Mutations of EGFR were identified in two esophageal cancers (11.7%), three cases of Barretts esophagus (14.2%), and two pancreatic cancers (3.6%). The mutations consisted of the recurrent missense L858R and in-frame deletion delE746-A750, previously characterized as activating EGFR mutations in non–small cell lung cancer. We also identified the TKI drug resistance–associated EGFR T790M mutation in an untreated case of Barretts esophagus and the corresponding adenocarcinoma. Conclusion: The presence of activating mutations within EGFR in both esophageal and pancreatic adenocarcinomas defines a previously unrecognized subset of gastrointestinal tumors in which EGFR signaling may play an important biological role. EGFR mutations in premalignant lesions of Barretts esophagus also point to these as an early event in transformation of the esophageal epithelium. The role of genotype-directed TKI therapy should be tested in prospective clinical trials.

Functionally defective germline variants of sialic acid acetylesterase in autoimmunity

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

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.

Activation of WNT/β-Catenin Signaling Enhances Pancreatic Cancer Development and the Malignant Potential Via Up-regulation of Cyr61

Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of WNT signaling is also commonly observed in PDAC. To ascertain the impact of postnatal activation of WNT-stimulated signaling pathways in PDAC development, we combined the Elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and WNT1 stimulated the progression of premalignant pancreatic intraepithelial neoplasias (PanIN) and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A or RCAS-Wnt1 had reduced survival relative to RCAS-GFP-injected controls (P < .05). Ectopic expression of active β-catenin, or its DNA-binding partner TCF4, enhanced transformation associated phenotypes in PDAC cells. In contrast, these phenotypes were significantly impaired by the introduction of ICAT, an inhibitor of the β-catenin/TCF4 interaction. By gene expression profiling, we identified Cyr61 as a target molecule of the WNT/β-catenin signaling pathway in pancreatic cancer cells. Nuclear β-catenin and CYR61 expression were predominantly detected in moderately to poorly differentiated murine and human PDAC. Indeed, nuclear β-catenin- and CYR61-positive PDAC patients demonstrated poor prognosis (P < .01). Knockdown of CYR61 in a β-catenin-activated pancreatic cancer cell line reduced soft agar, migration and invasion activity. Together, these data suggest that the WNT/β-catenin signaling pathway enhances pancreatic cancer development and malignancy in part via up-regulation of CYR61.

mTORC2 signaling drives the development and progression of pancreatic cancer

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer. Cancer Res; 76(23); 6911-23. ©2016 AACR.

MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by resistance to currently employed chemotherapeutic approaches. Members of the mir-17~92 cluster of microRNAs (miRNAs) are upregulated in PDAC, but the precise roles of these miRNAs in PDAC are unknown. Using genetically engineered mouse models, we show that loss of mir-17~92 reduces ERK pathway activation downstream of mutant KRAS and promotes the regression of KRASG12D-driven precursor pancreatic intraepithelial neoplasias (PanINs) and their replacement by normal exocrine tissue. In a PDAC model driven by concomitant KRASG12D expression and Trp53 heterozygosity, mir-17~92 deficiency extended the survival of mice that lacked distant metastasis. Moreover, mir-17~92-deficient PDAC cell lines display reduced invasion activity in transwell assays, form fewer invadopodia rosettes than mir-17~92-competent cell lines and are less able to degrade extracellular matrix. Specific inhibition of miR-19 family miRNAs with antagomirs recapitulates these phenotypes, suggesting that miR-19 family miRNAs are important mediators of PDAC cell invasion. Together these data demonstrate an oncogenic role for mir-17~92 at multiple stages of pancreatic tumorigenesis and progression; specifically, they link this miRNA cluster to ERK pathway activation and precursor lesion maintenance in vivo and identify a novel role for miR-19 family miRNAs in promoting cancer cell invasion.

The p53R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates “prometastatic” gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

Abstract A08: Activation of Wnt/β-catenin in acinar cells accelerates Kras-induced PDAC, while activation of Wnt signaling pathways in stroma induces mucinous cystic neoplasm

Pancreatic ductal adenocarcinoma (PDAC) commonly develops following activating mutations in the KRAS oncogene. Activation of the Wnt signaling pathway is also commonly observed in PDAC, yet whether Wnt ligand induced signaling promotes pancreatic tumorigenesis in vivo remains unclear. To ascertain the impact of postnatal activation of the Wnt signaling pathways in PDAC development, we combined the elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-Kras G12D , Ptf1a-cre model. Delivery of RCAS viruses encoding β-catenin S37A and Wnt1 stimulated the progression of premalignant PanIN and PDAC development. Mice injected with RCAS-β-catenin S37A and Wnt1 had reduced survival relative to RCAS-GFP controls (log-rank test; p S37A nor RCAS-GFP, developed mucinous cystic neoplasm (MCN). These lesions displayed stereotypical ovarian-like stroma that was positive for estrogen receptor (ER) and progesterone receptor (PR), but they lacked the typical mucinous epithelium observed in human MCN. Analysis of tissue specimens confirmed of activation of both the Wnt/β-catenin and PCP signaling pathways in the stroma, but not the epithelium of MCN lesions. Analysis of human MCN cases identified activation of these Wnt signaling pathways in the ovarian-like stroma, but not the cyst epithelium. Together, these data suggest that the Wnt/β-catenin signaling pathway in acinar cells stimulates Kras-induced PDAC development, while activation of Wnt signaling pathways in the stroma stimulates the development of the ovarian-like stroma and contributes to MCN formation in vivo. Citation Format: Makoto Sano, David R. Driscoll, Wilfredo E. DeJesus-Monge, David S. Klimstra, Brian C. Lewis. Activation of Wnt/β-catenin in acinar cells accelerates Kras-induced PDAC, while activation of Wnt signaling pathways in stroma induces mucinous cystic neoplasm. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A08.

Abstract 2742: Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of the Wnt signaling pathway is also commonly observed in PDAC. To ascertain the impact of postnatal activation of the Wnt signaling pathways in PDAC development, we combined the elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and Wnt1 stimulated the progression of premalignant PanIN and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A and Wnt1 had reduced survival relative to RCAS-GFP controls (log-rank test; p < 0.05). Meanwhile, active β-catenin or its DNA-binding partner TCF4 enhanced PDAC cell proliferation, soft-agar colony formation, as well as migration and invasion activity. In contrast, these phenotypes were significantly blocked by the introduction of Icat, an inhibitor of the β-catenin/TCF4 interaction. Interestingly, Id2 (inhibitor of differentiation/DNA binding2) was significantly up-regulated by induction of β-catenin and TCF4, whereas Id2 expression was inhibited by Icat. Furthermore, nuclear β-catenin and Id2 were mainly observed in poorly differentiated PDACs and sarcomatoid tumors. Together, these data suggest that the Wnt/β-catenin signaling pathway stimulates pancreatic tumor development and progression through the activation of Id2. Citation Format: Makoto Sano, David R. Driscoll, Wilfredo E. DeJesus-Monge, David S. Klimstra, Brian C. Lewis. Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2013-2742