Kenneth K. Takeuchi

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.

Plectin-1 as a biomarker of malignant progression in intraductal papillary mucinous neoplasms a multicenter study

Objective This study aimed to evaluate Plectin-1 expression as a biomarker of malignant risk for intraductal papillary mucinous neoplasms (IPMNs). Methods Plectin-1 immunohistochemistry (IHC) was performed retrospectively on surgical (n = 71) and cytological (n = 33) specimens from Mayo Clinic Jacksonville and UCLA Medical Center, including IPMNs with low-grade dysplasia, high-grade dysplasia (HGD), or an associated invasive adenocarcinoma. Results Plectin-1 expression was increased in invasive adenocarcinoma compared with adjacent in situ IPMN (P = 0.005), as well as the in situ HGD component of IPMNs with invasive cancer compared with HGD of IPMNs without invasive cancer (P = 0.02). Plectin IHC discriminated IPMNs with invasive adenocarcinoma from noninvasive IPMN (area under the curve [AUC] of 0.79, 75% sensitivity, and 85% specificity) but was insufficient for discriminating HGD IPMN from low-grade dysplasia IPMNs in surgical resections (AUC of 0.67, 56% sensitivity, and 64% specificity) or fine-needle aspiration specimens (AUC of 0.45). Conclusions Although Plectin-1 IHC has insufficient accuracy to be used as a definitive biomarker for malignant risk in the evaluation of IPMN biopsy or cytological specimens, increased Plectin-1 expression observed in both invasive cancer and in situ HGD of malignant IPMNs suggests that it might be successfully leveraged as a cyst fluid biomarker or molecular imaging target.

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 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 B58: EGFR and IR/IGFR activation sustains ERK1/2 and AKT signaling independently of mutant Kras in pancreatic cancer cells lines

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Roughly 45,000 patients are diagnosed with the disease each year and of these patients 94% will not survive past 5 years. The most common genetic alteration associated with PDAC is an oncogenic mutation in the KRAS gene, which is believed to initiate the disease. Mutant Kras is generally thought to signal constitutively to its downstream effector proteins, such as Raf/MEK/Erk and PI3K/AKT. However, recently we and others have demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling upstream of mutant Kras is necessary for unlocking its oncogenic potential in the pancreas, distinct from Kras driven tumorigenesis in the lung or colon. Unfortunately, while EGFR inhibition is effective in blocking pancreatic tumorigenesis in mice, it has limited benefit in treating human PDAC patients. Furthermore, it has been shown that wild-type Nras and Hras have indispensable signaling roles in cells expressing oncogenic Kras, and that both the mutant and WT Ras signals are required for cell growth. Here we show that pancreatic cancer cell lines containing oncogenic Kras still require upstream signaling to maintain high levels of both active Erk and AKT although these signals are not necessarily EGFR derived. We have identified a subset of pancreatic cancer cell lines that rely upon IR/IGFR signaling to maintain the high levels of Erk and AKT signaling required for continued cell growth. Additionally, this signaling appears to be independent of oncogenic Kras and instead correlates with the levels of active wild-type Nras and Hras. This data indicates that patients may benefit from the simultaneous inhibition of both the EGFR and IR/IGFR signaling pathways. Citation Format: Kenneth K. Takeuchi, Howard C. Crawford. EGFR and IR/IGFR activation sustains ERK1/2 and AKT signaling independently of mutant Kras in pancreatic cancer cells lines. [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 B58. doi: 10.1158/1557-3125.RASONC14-B58

Use of a preclinical model of pancreas cancer to identify potential candidates for rapalogue therapy

The oft-repeated statistics are dismal: pancreatic ductal adenocarcinoma (PDA) has only a 6% 5-year survival rate that has not changed significantly in 30 years and the vast majority of PDA patients perish within 6 months of diagnosis. Several possible reasons contribute to these grim results. PDA is difficult to detect, it is recalcitrant to most targeted treatments and conventional therapies only have a modest effect on survival. Compared with other cancers, PDA was revealed to be remarkably genetically homogenous with 95% or more of PDA harbouring oncogenic mutations in the Kras gene,1 generating hope that finding effective ways of interrupting Kras signalling would increase survival for PDA patients. However, Kras-targeted therapies have proven elusive thus far, leaving its downstream effectors as the most obvious potential targets. The true genetic heterogeneity of PDA has just begun to be appreciated2 ,3 within a background of the almost universal presence of Kras mutation and the statistically predictable identities of many of the subsequent tumour suppressor mutations that propel its progression and metastasis. Of course, …

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.

Abstract B84: EGFR activation by the ADAM17 sheddase is required for Kras-induced pancreatic tumorigenesis.

Nearly 45% of early PanIN lesions and 90% of pancreatic ductal adenocarcinoma (PDA) harbor a Kras mutation which inhibits Kras9 intrinsic GTP-ase function, resulting in a constitutively active Kras. The canonical Ras-Raf-Erk pathway suggests that constitutively active Kras would also result in constant Erk activation, independent of upstream components. In this study we demonstrate that genetic ablation of either EGFR or ADAM17, the primary EGFR ligand sheddase, effectively prevents tumorigenesis in the LSL-Kras G12D/+ ; Ptf1a Cre/+ (KC) mouse model of pancreatic tumorigenesis. Both mice are also protected from cerulein-induced chronic pancreatitis. Surprisingly, both knockouts fail to show robust activation of ERK, even with the expression of oncogenic Kras. This observation can be reproduced in human pancreatic cancer cell lines harboring a mutant Kras, where inhibition of EGFR with erlotinib resulted in a significant decrease in Erk activity. Conversely, stimulation of EGFR by either addition of exogenous TGF-α or stimulation of ADAM 17 with the phorbol 12-myristate 13-acetate (PMA) resulted in an increase in Erk activity. These data demonstrate a clear link between EGFR signaling and Erk activity despite of the presence of a mutant Kras. That EGFR signaling is also required for the chronic pancreatitis phenotype suggests EGFR inhibition could be a potential method of preventative therapy for patients at risk of developing pancreatitis or cancer derived from it. Citation Format: Christopher J. Halbrook, Kenneth K. Takeuchi, Christine M. Ardito, Barbara M. Gruener, Jens T. Siveke, Howard C. Crawford. EGFR activation by the ADAM17 sheddase is required for Kras-induced pancreatic tumorigenesis. [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 B84.