Andrew S. Liss

Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing “pancreatospheres” in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas.

Prognosis of invasive intraductal papillary mucinous neoplasm depends on histological and precursor epithelial subtypes

Objective Invasive cancers arising from intraductal papillary mucinous neoplasm (IPMN) are recognised as a morphologically and biologically heterogeneous group of neoplasms. Less is known about the epithelial subtypes of the precursor IPMN from which these lesions arise. The authors investigate the clinicopathological characteristics and the impact on survival of both the invasive component and its background IPMN. Design and patients The study cohort comprised 61 patients with invasive IPMN (study group) and 570 patients with pancreatic ductal adenocarcinoma (PDAC, control group) resected at a single institution. Multivariate analyses were performed using a stage-matched Cox proportional hazard model. Results The histology of invasive components of the IPMN cohort was tubular in 38 (62%), colloid in 16 (26%), and oncocytic in seven (12%). Compared with PDAC, invasive IPMNs were associated with a lower incidence of adverse pathological features and improved mortality by multivariate analysis (HR 0.58; 95% CI 0.39 to 0.86). In subtype analysis, this favourable outcome remained only for colloid and oncocytic carcinomas, while tubular adenocarcinoma was associated with worse overall survival, not significantly different from that of PDAC (HR 0.85; 95% CI 0.53 to 1.36). Colloid and oncocytic carcinomas arose only from intestinal- and oncocytic-type IPMNs, respectively, and were mostly of the main-duct type, whereas tubular adenocarcinomas primarily originated in the gastric background, which was often associated with branch-duct IPMN. Overall survival of patients with invasive adenocarcinomas arising from gastric-type IPMN was significantly worse than that of patients with non-gastric-type IPMN (p=0.016). Conclusions Tubular, colloid and oncocytic invasive IPMNs have varying prognosis, and arise from different epithelial subtypes. Colloid and oncocytic types have markedly improved biology, whereas the tubular type has a course that resembles PDAC. Analysis of these subtypes indicates that the background epithelium plays an equally, if not more, important role in defining the biology and prognosis of invasive IPMNs.

AP-1 Factors Play an Important Role in Transformation Induced by the v-rel Oncogene

ABSTRACT v-rel is the oncogenic member of the Rel/NF-κB family of transcription factors. The mechanism by which v-Rel induces transformation of avian lymphoid cells and fibroblasts is not precisely known. However, most models propose that v-rel disrupts the normal transcriptional regulatory network. In this study we evaluated the role of AP-1 family members in v-Rel-mediated transformation. The overexpression of v-Rel, c-Rel, and c-RelΔ resulted in a prolonged elevation of c-fos and c-jun expression and in a sustained repression of fra-2 at both the mRNA and protein levels in fibroblasts and lymphoid cells. Moreover, the transforming abilities of these Rel proteins correlated with their ability to alter the expression of these AP-1 factors. v-Rel exhibited the most pronounced effect, whereas c-Rel, with poor transforming ability, elicited only moderate changes in AP-1 levels. Furthermore, c-RelΔ, which exhibits enhanced transforming potential relative to c-Rel, induced intermediate changes in AP-1 expression. To directly evaluate the role of AP-1 family members in the v-Rel transformation process, a supjun-1 transdominant mutant was used. The supjun-1 mutant functions as a general inhibitor of AP-1 activity by inhibiting AP-1-mediated transactivation and by reducing AP-1 DNA-binding activity. Coinfection or sequential infection of fibroblasts or lymphoid cells with viruses carrying reloncogenes and supjun-1 resulted in a reduction of the transformation efficiency of the Rel proteins. The expression of supjun-1 inhibited the ability of v-Rel transformed lymphoid cells and fibroblasts to form colonies in soft agar by over 70%. Furthermore, the expression of supjun-1 strongly interfered with the ability of v-Rel to morphologically transform avian fibroblasts. This is the first report showing that v-Rel might execute its oncogenic potential through modulating the activity of early response genes.

Circulating tumor cells found in patients with localized and advanced pancreatic cancer

Objectives Isolation of circulating tumor cells (CTCs) holds the promise of diagnosing and molecular profiling cancers from a blood sample. Here, we test a simple new low-cost filtration device for CTC isolation in patients with pancreatic ductal adenocarcinoma (PDAC). Methods Peripheral blood samples drawn from healthy donors and PDAC patients were filtered using ScreenCell devices, designed to capture CTCs for cytologic and molecular analysis. Giemsa-stained specimens were evaluated by a pancreatic cytopathologist blinded to the histological diagnosis. Circulating tumor cell DNA was subjected to KRAS mutational analysis. Results Spiking experiments demonstrated a CTC capture efficiency as low as 2 cells/mL of blood. Circulating tumor cells were identified by either malignant cytology or presence of KRAS mutation in 73% of 11 patients (P = 0.001). Circulating tumor cells were identified in 3 of 4 patients with early (⩽American Joint Committee on Cancer stage IIB) and in 5 of 7 patients with advanced (≥ American Joint Committee on Cancer stage III) PDAC. No CTCs were detected in blood from 9 health donors. Conclusions Circulating tumor cells can be found in most patients with PDAC of any stage, whether localized, locally advanced, or metastatic. The ability to capture, cytologically identify, and genetically analyze CTCs suggests a possible tool for the diagnosis and characterization of genetic alterations of PDAC.

The suppression of SH3BGRL is important for v-Rel-mediated transformation

The v-rel oncogene is the most efficient transforming member of the Rel/NF-κB family of transcription factors. v-Rel induces avian and mammalian lymphoid cell tumors and transforms chicken embryo fibroblasts in culture by the aberrant regulation of genes under the control of Rel/NF-κB proteins. Here we report that the expression of SH3BGRL, a member of the SH3BGR (SH3 domain-binding glutamic acid-rich) family of proteins, is downregulated in v-Rel-expressing fibroblasts, lymphoid cells, and splenic tumor cells. Chromatin immunoprecipitation experiments demonstrated that v-Rel binds to the sh3bgrl promoter in transformed cells. Coexpression of SH3BGRL with v-Rel in primary splenic lymphocytes reduced the number of colonies formed by 76%. Mutations in the predicted SH3-binding domain of SH3BGRL abolished the suppressive effect on v-Rel transformation and resulted in colony numbers comparable to those formed by v-Rel alone. However, mutations in the predicted EVH1-binding domain of SH3BGRL only had a modest effect on suppression of v-Rel transformation. This study provides the first example of a gene that is downregulated in v-Rel-expressing cells that also plays a role in v-Rel transformation.

Circulating Epithelial Cells in Patients with Pancreatic Lesions: Clinical and Pathologic Findings.

BACKGROUND Circulating epithelial cell (CEC) isolation has provided diagnostic and prognostic information for a variety of cancers, previously supporting their identity as circulating tumor cells in the literature. However, we report CEC findings in patients with benign, premalignant, and malignant pancreatic lesions using a size-selective filtration device. STUDY DESIGN Peripheral blood samples were drawn from patients found to have pancreatic lesions on preoperative imaging at a surgical clinic. Blood was filtered using ScreenCell devices, which were evaluated microscopically by a pancreatic cytopathologist. Pathologic data and clinical outcomes of these patients were obtained from medical records during a 1-year follow-up period. RESULTS Nine healthy volunteers formed the control group and were found to be negative for CECs. There were 179 patients with pancreatic lesions that formed the study cohort. Circulating epithelial cells were morphologically similar in patients with a variety of pancreatic lesions. Specifically, CECs were identified in 51 of 105 pancreatic ductal adenocarcinomas (49%), 7 of 11 neuroendocrine tumors (64%), 13 of 21 intraductal papillary mucinous neoplasms (62%), and 6 of 13 patients with chronic pancreatitis. Rates of CEC identification were similar in patients with benign, premalignant, and malignant lesions (p = 0.41). In addition, CEC findings in pancreatic ductal adenocarcinoma patients were not associated with poor prognosis. CONCLUSIONS Although CECs were not identified in healthy volunteers, they were identified in patients with benign, premalignant, and malignant pancreatic lesions. The presence of CECs in patients presenting with pancreatic lesions is neither diagnostic of malignancy nor prognostic for patients with pancreatic ductal adenocarcinoma.

Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation

ABSTRACT Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS.

Pancreatic duct glands (PDGs) are a progenitor compartment responsible for pancreatic ductal epithelial repair.

Pancreatic duct glands (PDGs) have molecular features known to mark stem cell niches, but their function remains to be determined. To investigate the role of PDGs as a progenitor niche, PDGs were analyzed in both humans and mice. Cells were characterized by immunohistochemistry and microarray analysis. In vivo proliferative activity and migration of PDG cells were evaluated using a BrdU tag-and-chase strategy in a mouse model of pancreatitis. In vitro migration assays were used to determine the role of trefoil factor (TFF) -1 and 2 in cell migration. Proliferative activity in the pancreatic epithelium in response to inflammatory injury is identified principally within the PDG compartment. These proliferating cells then migrate out of the PDG compartment to populate the pancreatic duct. Most of the pancreatic epithelial migration occurs within 5days and relies, in part, on TFF-1 and -2. After migration, PDG cells lose their PDG-specific markers and gain a more mature pancreatic ductal phenotype. Expression analysis of the PDG epithelium reveals enrichment of embryonic and stem cell pathways. These results suggest that PDGs are an epithelial progenitor compartment that gives rise to mature differentiated progeny that migrate to the pancreatic duct. Thus PDGs are a progenitor niche important for pancreatic epithelial regeneration.

Sonic Hedgehog in pancreatic cancer: From bench to bedside, then back to the bench

Developmental genes are known to regulate cell proliferation, migration, and differentiation; thus, it comes as no surprise that the misregulation of developmental genes plays an important role in the biology of human cancers. One such pathway that has received an increasing amount of attention for its function in carcinogenesis is the Hedgehog (Hh) pathway. Initially the domain of developmental biologists, the Hh pathway and one of its ligands, Sonic Hedgehog (Shh), have been shown to play an important role in body planning and organ development, particularly in the foregut endoderm. Their importance in human disease became known to cancer biologists when germline mutations that resulted in the unregulated activity of the Hh pathway were found to cause basal cell carcinoma and medulloblastoma. Since then, misexpression of the Hh pathway has been shown to play an important role in many other cancers, including those of the pancreas. In many institutions, investigators are targeting misexpression of the Hh pathway in clinical trials, but there is still much fundamental knowledge to be gained about this pathway that can shape its clinical utility. This review will outline the evolution of our understanding of this pathway as it relates to the pancreas, as well as how the Hh pathway came to be a high-priority target for treatment.

Regulation of GLI underlies a role for BET bromodomains in pancreatic cancer growth and the tumor microenvironment

Purpose: The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. Experimental Design: In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. Results: In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. Conclusions: Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259–70. ©2016 AACR.