Christopher J. Halbrook

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.

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.

Abstract A32: GFAT: The crossroads of glucose and glutamine metabolism in pancreatic cancer

Due to a lack of early detection methods and effective treatment strategies, pancreatic cancer is slated to become the second leading cause of cancer related death in the United States. Pancreatic ductal adenocarcinoma (PDA), the most common type of pancreatic cancer, is initiated almost universally by mutations in the Kras oncogene. Kras mutations rewire metabolism in the cancer cells by enhancing nutrient capture and facilitating metabolic pathways that support survival and proliferation in the austere tumor microenvironment. Among these pathways, we have previously demonstrated that mutant Kras drives glucose flux into the hexosamine biosynthetic pathway (HBP) through upregulation of the rate limiting enzyme, glutamine fructose-6-phosphate transaminase (GFAT). The HBP plays a well-established role in the generation of the precursor substrates in glycosylation. In addition to this, GFAT has a less well appreciated facet, as it requires input from both glucose and glutamine (Gln). Specifically, GFAT deamidates the terminal nitrogen on Gln and releases glutamate, which can serve as an anaplerotic carbon source for the TCA cycle. Accordingly, GFAT sits at a nexus in the metabolism of the two canonical nutrients used by proliferating cells. Previous work from our group demonstrated that GFAT1 knockdown in mouse PDA cells results in the inhibition of colony formation and decreased growth of subcutaneous xenografts in mice. These results combined with the nearly ubiquitous nature of Kras mutations in pancreatic cancer, strongly indicate that targeting the HBP by inhibition of GFAT could offer a powerful new avenue of treatment for PDA. Utilizing a Cas9/CRISPR approach to modulate GFAT expression in human PDA cells, we have generated cell lines which require the addition of downstream HBP metabolites to maintain cell viability. We have also found several cell lines in which the loss of GFAT expression appears to have no impact on cell viability, suggesting novel mechanisms of glucose and Gln metabolism in these cells. In addition to validating GFAT as a therapeutic option in PDAC, we have also explored the utility of the GFAT reaction as an imaging tool. To accomplish this, we have developed a method to monitor the conversion of Gln to glutamate by magnetic resonance spectroscopy using hyperpolarized glutamine. We further assessed the importance of GFAT in this conversion utilizing our GFAT-/- cell lines. Taken together, these results highlight the benefit of working to understand the complex nature of metabolic rewiring in pancreatic cancer cells, where a further understanding of these pathways may lead to more effective early detection and therapeutic options. Citation Format: Christopher J. Halbrook, Daniel M. Kremer, Lucia Salamanca-Cardona, Kayvan R. Keshari, Costas A. Lyssioitis.{Authors}. GFAT: The crossroads of glucose and glutamine metabolism in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A32.

Abstract A27: Redefining the role of Notch in acinar to ductal metaplasia

The EGFR and Notch signaling pathways have been widely implicated in relation to both chronic pancreatitis and pancreatic ductal adenocarcinoma in humans, however the link between the two pathways has remained obscure. Acinar to ductal metaplasia (ADM,) a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells, has been linked to both the Notch and EGFR signaling pathways. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasia, a precursor lesion to pancreatic cancer. Recently we and others have shown that genetic ablation of EGFR in the pancreas effectively prevents acinar to ductal metaplasia in cerulein model of pancreatitis in mice, and prevents tumorigenesis driven by oncogenic Kras. Additionally, we have found that acinar cells harvested from EGFRf/f;Ptf1aCre/+ mice (EKO) mice are resistant to ADM in vitro upon cerulein treatment, or by the expression of oncogenic Kras. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has previously been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has also been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EKO mice. Interestingly, these acinar cells fail to transdifferentiate in vitro upon transduction of N2ICD in 3D culture; however the transdifferentiation event can be rescued by co-expression of oncogenic Kras with N2ICD in acinar cells lacking EGFR. These results indicate that EGFR is important in Notch induced ADM, however there are other pathways that possibly can be utilized by acinar cells expressing oncogenic Kras to drive ADM. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Redefining the role of Notch in acinar to ductal metaplasia. [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 A27.

Abstract A77: Investigating the role of ADAM10 in pancreatic tumor differentiation

Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer related deaths in the United States and has a dismal 5-year survival rate of 6%. This prognosis is attributed the disease’s fast and asymptomatic progression to metastasis, leading to late diagnosis upon which time there are few treatment options available. The A Disintegrin and Metalloproteinase (ADAM) family are sheddase proteins known to regulate cell adhesion and function. This regulation can allow ADAMs to influence the ability of tumor cells to disseminate from the primary tumor and metastasize to distant organs. ADAM10, which is seen to be up-regulated in both chronic pancreatitis and PDAC, cleaves a variety of substrates including several cell adhesion molecules such as L1-CAM, N-Cadherin, and E-Cadherin. Using a conditional knockout for ADAM10 in the Kras+/G12D;Ptf1a+/Cre (KC) mouse model of pancreatic cancer, we have observed an absence of metastasis when ADAM10 is genetically ablated. To investigate the potential role that ADAM10 plays in this, we used a shRNA approach to knock-down ADAM10 expression in various human pancreatic cancer cells lines. Analysis of these cell lines suggests a partial restoration of a less aggressive, differentiated phenotype, consistent with the increased longevity of the ADAM10 knockout KC mice. Citation Format: Louise V. Peverley, Christopher J. Halbrook, Jason C. Hall, Christine M. Ardito, Howard C. Crawford. Investigating the role of ADAM10 in pancreatic tumor differentiation. [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 A77.

Abstract A04: PI3K regulation of RAC1 is required for Kras-induced pancreatic tumorigenesis

Nearly all pancreatic ductal adenocarcinomas are caused by oncogenic mutations in the KRAS gene. Pharmacological inhibition of mutant KRAS has thus far been unsuccessful in the clinical setting, precipitating a need to understand the pathways downstream of KRAS which may prove more easily targeted with small molecule inhibitors. Here we show that PI3K p110α is absolutely required for pancreatic tumorigenesis while p110β is dispensable for this process. Surprisingly, ablation of p110α does not impair the ability of KRAS to activate AKT, demonstrating that AKT activation is not sufficient for transformation. Instead we find that p110α is required for robust activation of RAC1, a small GTPase required for pancreatic metaplasia. Consistent with this, our data show that p110α is necessary for regulating epithelial expression and activation of RAC-GEFs including Vav1, Tiam1 and Ect2. Ultimately, these results define the mechanistic role of p110α in pancreatic tumorigenesis and suggest selective inhibition of this PI3K isoform as a promising therapeutic approach to treating patients with pancreas cancer. Citation Format: Kenneth K. Takeuchi, Eileen Carpenter, Claire Wu, Christopher J. Halbrook, Richard Z. Lin, Howard C. Crawford. PI3K regulation of RAC1 is required for Kras-induced pancreatic tumorigenesis. [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 A04.

Abstract A09: Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia

Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

Abstract 3030: Regulation of EGFR in pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC EGFR and EGFR ligand upregulation has been widely seen in both human chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) for many years; however the role of EGFR in PDA was poorly understood. Recently we and others have shown that genetic ablation of EGFR effectively prevents both spontaneous and cerulein induced tumorigenesis in the pancreas of the LSL-KrasG12D/+;Ptf1aCre/+ (KC) mouse model of pancreatic tumorigenesis. Pockets of EGFR upregulation localized in single acini of KC mice appear to precede pancreatic intraepithelial neoplasia (PanIN) formation in spontaneous tumorigenesis, coupled with a distinct spatially localized immune response. Ubiquitous EGFR upregulation is observed throughout the pancreas upon induction of pancreatitis in wild-type (WT) and KC mice, returning to normal levels in WT mice but continuing to increase along with the widespread tumorigenesis in cerulein treated KC mice. Comparison of cerulein treated KC mice with the EGFRf/f;LSL-KrasG12D/+;Ptf1aCre/+ mice (EKC), which are largely resistant to cerulein induced pancreatitis, offers an opportunity to identify signals in the KC mouse which could be responsible for the upregulation of EGFR and subsequent tumorigenesis. Citation Format: Christopher J. Halbrook, Kenneth K. Takeuchi, Howard C. Crawford. Regulation of EGFR in 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 3030. doi:10.1158/1538-7445.AM2013-3030

Abstract B73: Tumor-associated pancreatic metaplasia assumes a biliary duct-like phenotype.

Pancreatic ductal adenocarcinoma (PDA) manifests as a highly lethal disease due to lack of early detection and effective therapeutics, making the study of initiating events invaluable. An elementary aberrant epithelial structure consistently associated with PDA development is the metaplastic duct, known to be dedifferentiated and hypothesized to be preneoplastic. Through lineage tracing and immunohistochemical analysis we have determined that pancreatic ductal metaplasia derives in part from acinar cells but assumes characteristics of the developmentally related biliary “duct gland.” Previously shown to be a source of progenitor/stem cells for the liver, bile duct, and pancreas, as well as a potential source of pancreatic disease, we find that duct glands are characteristic of the biliary tract but are absent in the normal murine pancreatic duct. Consistent with the assumption of a biliary phenotype, pancreatic metaplasia expresses PDX1+/Sox17+ reminiscent of the pancreatobiliary progenitor cell. In addition, pancreatic metaplasia is consistently associated with the presence of tuft cells, another characteristic of normal bile duct glands, but not of the normal murine pancreas. Found throughout the hollow organs of the digestive and respiratory tracts, tuft cells are solitary chemosensory cells (SCC) that make up the diffuse chemosensory system (DCS), expressing taste cell signaling components thought to sample the environment and respond with effectors. Tuft cells express differentiation markers of both inflammatory cells and neuronal cells, as well as stem cell markers DCLK1 and LGR5, suggesting a possible role as multipotent progenitor cells. In addition, tuft cells represent an abundant source of proinflammatory prostaglandins, as well as endorphins, with the potential to exacerbate, if not drive, the disease state. Tuft cells, in addition to a number of developmental factors, represent transdifferentiation of the diseased pancreatic epithelium to a glandular, biliary duct-like phenotype rather than direct derivation from the pancreatic duct, giving new insight into tumorigenesis and PDA development. Citation Format: Kathleen E. DelGiorno, Kenneth Takeuchi, Christopher Halbrook, Howard C. Crawford. Tumor-associated pancreatic metaplasia assumes a biliary duct-like phenotype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B73.