Kathleen E. DelGiorno

PI3K Regulation of RAC1 Is Required for KRAS-Induced Pancreatic Tumorigenesis in Mice

BACKGROUND & AIMS New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110β (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Stromal reengineering to treat pancreas cancer

Pancreatic ductal adenocarcinoma co-opts multiple cellular and extracellular mechanisms to create a complex cancer organ with an unusual proclivity for metastasis and resistance to therapy. Cell-autonomous events are essential for the initiation and maintenance of pancreatic ductal adenocarcinoma, but recent studies have implicated critical non-cell autonomous processes within the robust desmoplastic stroma that promote disease pathogenesis and resistance. Thus, non-malignant cells and associated factors are culprits in tumor growth, immunosuppression and invasion. However, even this increasing awareness of non-cell autonomous contributions to disease progression is tempered by the conflicting roles stromal elements can play. A greater understanding of stromal complexity and complicity has been aided in part by studies in highly faithful genetically engineered mouse models of pancreatic ductal adenocarcinoma. Insights gleaned from such studies are spurring the development of therapies designed to reengineer the pancreas cancer stroma and render it permissive to agents targeting cell-autonomous events or to reinstate immunosurveillance. Integrating conventional and immunological treatments in the context of stromal targeting may provide the key to a durable clinical impact on this formidable disease.

Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions

The development of pancreatic cancer requires the acquisition of oncogenic KRas mutations and upregulation of growth factor signaling, but the relationship between these is not well established. Here, we show that mutant KRas alters mitochondrial metabolism in pancreatic acinar cells, resulting in increased generation of mitochondrial reactive oxygen species (mROS). Mitochondrial ROS then drives the dedifferentiation of acinar cells to a duct-like progenitor phenotype and progression to PanIN. This is mediated via the ROS-receptive kinase protein kinase D1 and the transcription factors NF-κB1 and NF-κB2, which upregulate expression of the epidermal growth factor, its ligands, and their sheddase ADAM17. In vivo, interception of KRas-mediated generation of mROS reduced the formation of pre-neoplastic lesions. Hence, our data provide insight into how oncogenic KRas interacts with growth factor signaling to induce the formation of pancreatic cancer.

Identification and Manipulation of Biliary Metaplasia in Pancreatic Tumors

BACKGROUND & AIMS Metaplasias often have characteristics of developmentally related tissues. Pancreatic metaplastic ducts are usually associated with pancreatitis and pancreatic ductal adenocarcinoma. The tuft cell is a chemosensory cell that responds to signals in the extracellular environment via effector molecules. Commonly found in the biliary tract, tuft cells are absent from normal murine pancreas. Using the aberrant appearance of tuft cells as an indicator, we tested if pancreatic metaplasia represents transdifferentiation to a biliary phenotype and what effect this has on pancreatic tumorigenesis. METHODS We analyzed pancreatic tissue and tumors that developed in mice that express an activated form of Kras (Kras(LSL-G12D/+);Ptf1a(Cre/+) mice). Normal bile duct, pancreatic duct, and tumor-associated metaplasias from the mice were analyzed for tuft cell and biliary progenitor markers, including SOX17, a transcription factor that regulates biliary development. We also analyzed pancreatic tissues from mice expressing transgenic SOX17 alone (ROSA(tTa/+);Ptf1(CreERTM/+);tetO-SOX17) or along with activated Kras (ROSAtT(a/+);Ptf1a(CreERTM/+);tetO-SOX17;Kras(LSL-G12D;+)). RESULTS Tuft cells were frequently found in areas of pancreatic metaplasia, decreased throughout tumor progression, and absent from invasive tumors. Analysis of the pancreatobiliary ductal systems of mice revealed tuft cells in the biliary tract but not the normal pancreatic duct. Analysis for biliary markers revealed expression of SOX17 in pancreatic metaplasia and tumors. Pancreas-specific overexpression of SOX17 led to ductal metaplasia along with inflammation and collagen deposition. Mice that overexpressed SOX17 along with Kras(G12D) had a greater degree of transformed tissue compared with mice expressing only Kras(G12D). Immunofluorescence analysis of human pancreatic tissue arrays revealed the presence of tuft cells in metaplasia and early-stage tumors, along with SOX17 expression, consistent with a biliary phenotype. CONCLUSIONS Expression of Kras(G12D) and SOX17 in mice induces development of metaplasias with a biliary phenotype containing tuft cells. Tuft cells express a number of tumorigenic factors that can alter the microenvironment. Expression of SOX17 induces pancreatitis and promotes Kras(G12D)-induced tumorigenesis in mice.

Response to Chauhan et al.: Interstitial Pressure and Vascular Collapse in Pancreas Cancer—Fluids and Solids, Measurement and Meaning

Chauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond.

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b+ F4/80+, CD11b+ Ly6Cint Ly6G+ and CD11b+ Ly6Chi Ly6G− cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.

Interstitial pressure and vascular collapse in pancreas cancer:fluids and solids, measurement and meaning

Chauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond.

Abstract IA13: Acinar cell transdifferentiation sets the stage for early tumor heterogeneity

Prior to PanIN formation, acinar cells harboring oncogenic Kras gradually lose their acinar character and take on a ductal phenotype in a process known as acinar-to-ductal metaplasia. Rather than mimicking a normal pancreatic duct, metaplastic ducts take on a proliferative biliary progenitor phenotype, marked by the expression of SOX17 and PDX1 and the presence of numerous tuft cells, recently identified as a PanIN initiating cells. Manipulation of SOX17 and PDX1 in vivo reveal them to be a transdifferentiation promoter and suppressor, respectively, both greatly affecting tuft cell genesis and tumor formation. These opposing roles of developmental transcription factors during tumorigenesis implicate the usurpation of a differentiation program that significantly contributes to cellular heterogeneity within early pre-cancerous lesions of the pancreas. Citation Format: Kenneth K. Takeuchi, Kathleen E. Delgiorno, Christopher J. Halbrook, Howard C. Crawford. Acinar cell transdifferentiation sets the stage for early tumor heterogeneity. [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 IA13.

Abstract B05: Assessing and removing biophysical barriers to treatment

Pancreatic ductal adenocarcinoma (PDA) is a confounding cancer with a penchant for metastasis and resistance to therapy. Cell autonomous events, such as mutations in the Kras proto-oncogene, are essential for the initiation and maintenance of PDA; however non-malignant cells as well as non-cellular components have been shown to contribute to tumor growth, immunosuppression, and chemotherapeutic resistance. The stromal compartment can comprise more than 80% of tumor content and is characterized by a dynamic and deregulated extracellular matrix (ECM). In addition to a role in tissue structure, the ECM provides biophysical and biochemical cues that determine cell responses. PDA presents its own characteristic ECM signature including large deposits of the negatively charged glycosaminoglycan (GAG) hyaluronan (HA), as well as fibrillary collagens which increases matrix stiffness. We have previously demonstrated that high levels of HA in PDA contribute to extraordinary interstitial fluid pressures (IFP) and vascular collapse. We show here that IFP is comprised of both freely mobile and immobile fluid phases. Due to its highly charged nature, HA binds large amounts of water to create an immobile-fluid phase with a significant swelling pressure. This pressure is not detected by conventional methods that can measure only free fluid pressure. The swelling pressure stresses abundant collagen fibrils which contract through cellular efforts to maintain homeostasis, further contributing to IFP. Targeting HA through systemic administration of pegylated hyaluronidase (PEGPH20) liberates the immobile fluid phase and dramatically reduces IFP, increasing vessel patency and bioavailability of systemically delivered agents. We present results on the effects of targeting additional ECM components and signaling pathways to help remove biophysical barriers to chemotherapeutic access. We also compare the abilities of different methodologies to measure interstitial pressures associated with the distinct fluid phases in a variety of experimental contexts. Citation Format: Kathleen E. DelGiorno, Markus Carlson, Paolo P. Provenzano, Scott Brockenbough, Sunil R. Hingorani. Assessing and removing biophysical barriers to treatment. [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 B05.

Abstract A92: Murine clinical trials program

Enormous advances in the biomedical sciences have raised tenable hope of a new era of personalized medicine. Significant hurdles to realizing this goal include the inordinately high and evolving degree of genetic and biologic diversity in carcinomas, even within categories of defined organ and cell-type. In addition, the tissue culture and xenograft preclinical model systems that have been instrumental in elucidating fundamental principles of cancer biology do not recapitulate the essential geometry or tissue and stromal interactions encountered in the native disease. As a result, the ability to kill cancer cells in these contexts has had limited impact on treating the diseases in patients. Genetically engineered mouse models of pancreas cancer appear to encompass the biological diversity and complexity seen in patients. In addition to revealing important aspects of disease pathobiology, these model systems form the basis of a preclinical platform, the Murine Clinical Trials Program (MCTP), designed to accelerate the development and evaluation of early detection, chemoprevention and therapeutic strategies most likely to impact patients in the clinic. By screening these strategies in highly faithful animal surrogates for the human disease, we hope to rapidly identify and translate the most effective approaches to the clinic, while concomitantly defining the appropriate disease subtype-specific context in which to employ each application. Attempting to accomplish these aims by performing initial validation and optimization experiments directly in patients can slow progress by many years, if not preclude it altogether. Treatment strategies are advanced through the MCTP in a systematic manner with defined endpoints and criteria (“Go/No-Go”) for each step. If these criteria are met in phase A (Pilot Study), they advance to Phase B (Full Survival Study). Strict enrollment criteria ensure the presence of invasive PDA (with or without metastatic disease) while also allowing sufficient time on investigational therapy to reveal potential resistance mechanisms, unanticipated secondary or off-target effects and delayed toxicities. Each of these aspects is described in detail in our presentation. Each new investigational agent in both the pilot and survival studies is analyzed in three arms: 1) agent alone; 2) agent + standard cytotoxic agent(s); 3) agent + cytotoxics + anti-stromal agent. In our standard approach, pilot studies enroll n=4-5 animals per agent. A 2-5 cm mass measurable by high-resolution ultrasound is required for enrollment. The standard 3 weeks ‘on’/1 week ‘off’ treatment regimen used routinely in patients is recapitulated in mice, together with a dose and route designed to be as similar as possible to that anticipated for human patients. Primary endpoints for pilot studies (1-2 cycles of treatment) include objective response and cellular effects including tumor cell apoptosis and proliferation, immune cell responses, changes in content and character of ECM components, vessel density and cross-sectional diameter, and so on. Results from pilot studies are used to determine whether a full survival study is warranted. Examples of recent MCTP pilot and survival studies will be presented. Citation Format: J. Scott Brockenbrough, Libing Feng, Ingunn M. Stromnes, Kathleen E. DelGiorno, Martin C. Whittle, Carlos Cuevas, Ashley M. Dotson, Joseph S. Ryan, Shelley M. Thorsen, Sunil R. Hingorani. Murine clinical trials program. [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 A92.