Faculty Bibliography

Pancreatic ductal adenocarcinomas comprise a hierarchy of tumor cells that develop around a population of cancer stem cells. The cancer stem cells promote tumor growth and progression through a number of mechanisms, including differentiation into bulk tumor cells, metastasis, alteration of adjacent stromal cells, and evasion of conventional therapies. As with other cancer stem cells, pancreatic cancer stem cells (PCSCs) can be distinguished from bulk tumor cells based on their expression of unique surface markers, abilities to form spheres under nonadherent conditions and tumors in mice, and self-renewal and differentiation capacities. We review the markers used to identify PCSCs, the signaling pathways that regulate PCSC functions, the complex interactions between PCSCs and stromal cells, and approaches to therapeutically target PCSCs and improve treatment of patients with pancreatic cancer.

Transforming growth factor beta (TGFbeta) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFbeta signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFbeta activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFbeta-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFbeta-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.

Protein kinases represent the most effective class of therapeutic targets in cancer; therefore, determination of kinase aberrations is a major focus of cancer genomic studies. Here, we analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types, and defined distinct "outlier kinases" in individual breast and pancreatic cancer samples, based on highest levels of absolute and differential expression. Frequent outlier kinases in breast cancer included therapeutic targets like ERBB2 and FGFR4, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines, as tested by siRNA knockdown and/or pharmacologic inhibition. Outlier expression of polo-like kinases was observed in a subset of KRAS-dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI-6727. Our results suggest that outlier kinases represent effective precision therapeutic targets that are readily identifiable through RNA sequencing of tumors.

OBJECTIVE: Endoscopic ultrasound-guided fine-needle aspiration and bile duct brushings are utilized in the cytologic evaluation of solid and cystic pancreaticobiliary tract lesions. We sought to determine the diagnostic accuracy of cytology. METHODS: Five hundred seventy-nine pancreatic resections with 727 corresponding cytology specimens were identified from 1997 to 2012. Histologic diagnoses included benign, carcinoma, pancreatic endocrine neoplasm (PEN), nonepithelial neoplasms, cystic neoplasms, and ampullary adenomas. Standard interpretative categories-nondiagnostic, negative, atypical, suspicious, and positive--were utilized for preoperative cytology specimens. RESULTS: For solid masses, the sensitivity and specificity of positive fine-needle aspiration (FNA) cytology for detecting carcinoma were 74 and 100 %, respectively. FNAs performed better than brushings (sensitivity, 40 %; specificity, 98 %) in detecting carcinomas. Similar findings were seen for PENs and nonepithelial neoplasms. For cystic lesions, the sensitivity of FNA for predicting malignancy was lower (24 %) with a specificity of 97 %. Sequentially combining suspicious and atypical categories with the positive category resulted in increases in sensitivity and decreases in specificity for all cases except for cystic lesions. CONCLUSIONS: Cytology adds to the assessment of solid masses, but its utility in cystic lesions is less clear. Consideration of a suspicious cytologic interpretation as a positive diagnosis for triaging patients to surgery is supported by our study.

BACKGROUND: Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma. METHODS: We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing. RESULTS: Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue. CONCLUSIONS: Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.

In a pilot study, multimodal optical spectroscopy coupled with quantitative tissue-optics models distinguished intraductal papillary mucinous neoplasm (IPMN), a common precursor to pancreatic cancer, from normal tissues in freshly excised human pancreas. A photon-tissue interaction (PTI) model extracted parameters associated with cellular nuclear size and refractive index (from reflectance spectra) and extracellular collagen content (from fluorescence spectra). The results suggest that tissue optical spectroscopy has the potential to characterize pre-cancerous neoplasms in human pancreatic tissues.

PURPOSE: Local failure in unresectable pancreatic cancer may contribute to death. We hypothesized that intensification of local therapy would improve local control and survival. The objectives were to determine the maximum tolerated radiation dose delivered by intensity modulated radiation with fixed-dose rate gemcitabine (FDR-G), freedom from local progression (FFLP), and overall survival (OS). METHODS AND MATERIALS: Eligibility included pathologic confirmation of adenocarcinoma, radiographically unresectable, performance status of 0-2, absolute neutrophil count of >/= 1,500/mm(3), platelets >/= 100,000/mm(3), creatinine <2 mg/dL, bilirubin <3 mg/dL, and alanine aminotransferase/aspartate aminotransferase /= 3, neutropenic fever, or deterioration in performance status to >/= 3 between day 1 and 126. Dose level was assigned using TITE-CRM (Time-to-Event Continual Reassessment Method) with the target dose-limiting toxicity (DLT) rate set to 0.25. RESULTS: Fifty patients were accrued. DLTs were observed in 11 patients: G3/4 anorexia, nausea, vomiting, and/or dehydration (7); duodenal bleed (3); duodenal perforation (1). The recommended dose is 55 Gy, producing a probability of DLT of 0.24. The 2-year FFLP is 59% (95% confidence interval [CI]: 32-79). Median and 2-year overall survival are 14.8 months (95% CI: 12.6-22.2) and 30% (95% CI 17-45). Twelve patients underwent resection (10 R0, 2 R1) and survived a median of 32 months. CONCLUSIONS: High-dose radiation therapy with concurrent FDR-G can be delivered safely. The encouraging efficacy data suggest that outcome may be improved in unresectable patients through intensification of local therapy.

PURPOSE: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy. EXPERIMENTAL DESIGN: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays. RESULTS: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition. CONCLUSIONS: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling.