KRAS-induced Pancreatic Tumorigenesis Research Articles

Tumor Cell-Intrinsic SETD2 Deficiency Reprograms Neutrophils to Foster Immune Escape in Pancreatic Tumorigenesis.

Genetic and epigenetic alterations play central roles in shaping the immunosuppressive tumor microenvironment (TME) to evade immune surveillance. The previous study shows that SETD2-H3K36me3 loss promotes KRAS-induced pancreatic tumorigenesis. However, little is known about its role in remodeling the TME and immune evasion. Here, it is shown that SETD2 deficiency can reprogram neutrophils to an immunosuppressive phenotype, thereby promoting immune escape during pancreatic tumor progression. By comprehensive profiling of the intratumoral immune cells, neutrophils are identified as the subset with the most significant changes upon Setd2 loss. Setd2-deficient pancreatic tumor cells directly enhance neutrophil recruitment and reprogramming, thereby inhibiting the cytotoxicity of CD8+ T cells to foster tumor progression. Mechanistically, it is revealed that Setd2-H3K36me3 loss leads to ectopic gain of H3K27me3 to downregulate Cxadr expression, which boosts the PI3K-AKT pathway and excessive expression of CXCL1 and GM-CSF, thereby promoting neutrophil recruitment and reprogramming toward an immunosuppressive phenotype. The study provides mechanistic insights into how tumor cell-intrinsic Setd2 deficiency strengthens the immune escape during pancreatic tumorigenesis, which may offer potential therapeutic implications for pancreatic cancer patients with SETD2 deficiency.

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2

Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

SETDB1 Inhibits p53-Mediated Apoptosis and Is Required for Formation of Pancreatic Ductal Adenocarcinomas in Mice

SETDB1, a histone methyltransferase that trimethylates histone H3 on lysine 9, promotes development of several tumor types. We investigated whether SETDB1 contributes to development of pancreatic ductal adenocarcinoma (PDAC). We performed studies with Ptf1aCre; KrasG12D; Setdb1f/f, Ptf1aCre; KrasG12D; Trp53f/+; Setdb1f/f, and Ptf1aCre; KrasG12D; Trp53f/f; Setdb1f/f mice to investigate the effects of disruption of Setdb1 in mice with activated KRAS-induced pancreatic tumorigenesis, with heterozygous or homozygous disruption of Trp53. We performed microarray analyses of whole-pancreas tissues from Ptf1aCre; KrasG12D; Setdb1f/f, and Ptf1aCre; KrasG12D mice and compared their gene expression patterns. Chromatin immunoprecipitation assays were performed using acinar cells isolated from pancreata with and without disruption of Setdb1. We used human PDAC cells for SETDB1 knockdown and inhibitor experiments. Loss of SETDB1 from pancreas accelerated formation of premalignant lesions in mice with pancreata that express activated KRAS. Microarray analysis revealed up-regulated expression of genes in the apoptotic pathway and genes regulated by p53 in SETDB1-deficient pancreata. Deletion of Setdb1 from pancreas prevented formation of PDACs, concomitant with increased apoptosis and up-regulated expression of Trp53 in mice heterozygous for disruption of Trp53. In contrast, pancreata of mice with homozygous disruption of Trp53 had no increased apoptosis, and PDACs developed. Chromatin immunoprecipitation revealed that SETDB1 bound to the Trp53 promoter to regulate its expression. Expression of an inactivated form of SETDB1 in human PDAC cells with wild-type TP53 resulted in TP53-induced apoptosis. We found that the histone methyltransferase SETDB1 is required for development of PDACs, induced by activated KRAS, in mice. SETDB1 inhibits apoptosis by regulating expression of p53. SETDB1 might be a therapeutic target for PDACs that retain p53 function.

Transgenic expression of Sag/Rbx2 E3 causes early stage tumor promotion, late stage cytogenesis and acinar loss in the Kras–PDAC model

SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our previous studies showed that Sag/Rbx2 co-operated with Kras or Pten loss to promote tumorigenesis in the lung and prostate, respectively, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. The role of SAG in KRAS-induced pancreatic tumorigenesis is unknown. In this study, we mined a cancer database and found that SAG is overexpressed in pancreatic cancer tissues and correlates with decreased patient survival. Whether Sag overexpression plays a causal role in pancreatic tumorigenesis is unknown. Here, we reported the generation of Sag transgenic mouse model alone (CS), or in combination with KrasG12D, driven by p48-Cre (KCS mice) for pancreatic specific Sag expression. Sag transgenic expression alone has no phenotypical abnormality, but in combination with KrasG12D promotes ADM (acinar-to-ductal metaplasia) conversion in vitro and mPanIN1 formation in vivo at the early stage, and impairs pancreatic functions at the late stage, as evidenced by poor glucose tolerance and significantly reduced α-Amylase activity, and induction of cytogenesis and acinar cell loss, eventually leading to atrophic pancreata and shortened mouse life-span. Mechanistically, Sag transgenic expression altered several key signaling pathways, particularly inactivation of mTORC1 signaling due to Deptor accumulation, and activation of the antioxidant Nrf2-Nqo1 axis. Thus, Sag plays a stage dependent promotion (early) and fate-changing (late) role during Kras-pancreatic tumorigenesis, likely via regulating its key substrates, which control growth-related signal transduction pathways.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

ATDC is required for the initiation of KRAS-induced pancreatic tumorigenesis.

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of β-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.

Pancreatic DCLK1+ cells originate distinctly from PDX1+ progenitors and contribute to the initiation of intraductal papillary mucinous neoplasm in mice

PanINs and IPMNs are the two most common precursor lesions that can progress to invasive pancreatic ductal adenocarcinoma (PDA). DCLK1 has been identified as a biomarker of progenitor cells in PDA progressed from PanINs. To explore the potential role of DCLK1-expressing cells in the genesis of IPMNs, we compared the incidence of DCLK1-positive cells in pancreatic tissue samples from genetically-engineered mouse models (GEMMs) for IPMNs, PanINs, and acinar to ductal metaplasia by immunohistochemistry and immunofluorescence. Mouse lineage tracing experiments in the IPMN GEMM showed that DCLK1+ cells originated from a cell lineage distinct from PDX1+ progenitors. The DCLK1+ cells shared the features of tuft cells but were devoid of IPMN tumor biomarkers. The DCLK1+ cells were detected in the earliest proliferative acinar clusters prior to the formation of metaplastic ductal cells, and were enriched in the “IPMN niches”. In summary, DCLK1 labels a unique pancreatic cellular lineage in the IPMN GEMM. The clustering of DCLK1+ cells is an early event in Kras-induced pancreatic tumorigenesis and may contribute to IPMN initiation.

An exploratory open-label, investigator-initiated study to evaluate the efficacy and safety of combination sonidegib and buparlisib for advanced basal cell carcinomas

To the Editor: Although smoothened inhibitors (SIs) are Food and Drug Administration approved for advanced basal cell carcinomas (aBCCs), over 50% of patients do not respond. 1 Sekulic A. Migden M.R. Basset-Seguin N. et al. Long-term safety and efficacy of vismodegib in patients with advanced basal cell carcinoma: final update of the pivotal ERIVANCE BCC study. BMC Cancer. 2017; 17: 332 Crossref PubMed Scopus (222) Google Scholar , 2 Lear J.T. Migden M.R. Lewis K.D. et al. Long-term efficacy and safety of sonidegib in patients with locally advanced and metastatic basal cell carcinoma: 30-month analysis of the randomized phase 2 BOLT study. J Eur Acad Dermatol Venereol. August, 28 2017; ([Epub ahead of print])https://doi.org/10.1111/jdv.14542 Crossref Scopus (121) Google Scholar Preclinical work in models of hedgehog-dependent tumors has revealed the importance of phosphoinositide-3-kinase (PI3K) in promoting SI resistance, 3 Gonnissen A. Isebaert S. Haustermans K. Targeting the hedgehog signaling pathway in cancer: beyond smoothened. Oncotarget. 2015; 6: 13899-13913 Crossref PubMed Scopus (149) Google Scholar , 4 Buonamici S. Williams J. Morrissey M. et al. Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med. 2010; 2: 51ra70 Crossref PubMed Scopus (388) Google Scholar , 5 Rajurkar M. De Jesus-Monge W.E. Driscoll D.R. et al. The activity of GLI transcription factors is essential for KRAS-induced pancreatic tumorigenesis. Proc Natl Acad Sci U S A. 2012; 109: E1038-E1047 Crossref PubMed Scopus (105) Google Scholar , 6 Zhou J. Zhu G. Huang J. et al. Non-canonical GLI1/2 activation by PI3K/AKT signaling in renal cell carcinoma: a novel potential therapeutic target. Cancer Lett. 2016; 370: 313-323 Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar with concurrent smoothened and PI3K inhibition delaying SI resistance in medulloblastoma 4 Buonamici S. Williams J. Morrissey M. et al. Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med. 2010; 2: 51ra70 Crossref PubMed Scopus (388) Google Scholar (Supplemental Fig 1; available at http://www.jaad.org). 1 Sekulic A. Migden M.R. Basset-Seguin N. et al. Long-term safety and efficacy of vismodegib in patients with advanced basal cell carcinoma: final update of the pivotal ERIVANCE BCC study. BMC Cancer. 2017; 17: 332 Crossref PubMed Scopus (222) Google Scholar , 3 Gonnissen A. Isebaert S. Haustermans K. Targeting the hedgehog signaling pathway in cancer: beyond smoothened. Oncotarget. 2015; 6: 13899-13913 Crossref PubMed Scopus (149) Google Scholar , 4 Buonamici S. Williams J. Morrissey M. et al. Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med. 2010; 2: 51ra70 Crossref PubMed Scopus (388) Google Scholar , 5 Rajurkar M. De Jesus-Monge W.E. Driscoll D.R. et al. The activity of GLI transcription factors is essential for KRAS-induced pancreatic tumorigenesis. Proc Natl Acad Sci U S A. 2012; 109: E1038-E1047 Crossref PubMed Scopus (105) Google Scholar , 6 Zhou J. Zhu G. Huang J. et al. Non-canonical GLI1/2 activation by PI3K/AKT signaling in renal cell carcinoma: a novel potential therapeutic target. Cancer Lett. 2016; 370: 313-323 Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar , 7 Chang A.L.S. Oro A.E. Initial assessment of tumor regrowth after vismodegib in advanced basal cell carcinoma. Arch Dermatol. 2012; 148: 1324-1325 Crossref PubMed Scopus (150) Google Scholar Due to the lack of good treatment options available to SI-resistant aBCC patients, this open-label, single-arm, proof-of-concept study of combined hedgehog and PI3K inhibition on aBCCs was explored using sonidegib, an SI, and buparlisib, a pan-PI3K inhibitor.

IKBKE Is Required during KRAS-Induced Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies lacking effective therapeutic strategies. Here, we show that the noncanonical IκB-related kinase, IKBKE, is a critical oncogenic effector during KRAS-induced pancreatic transformation. Loss of IKBKE inhibits the initiation and progression of pancreatic tumors in mice carrying pancreatic-specific KRAS activation. Mechanistically, we demonstrate that this protumoral effect of IKBKE involves the activation of GLI1 and AKT signaling and is independent of the levels of activity of the NF-κB pathway. Further analysis reveals that IKBKE regulates GLI1 nuclear translocation and promotes the reactivation of AKT post-inhibition of mTOR in PDAC cells. Interestingly, combined inhibition of IKBKE and mTOR synergistically blocks pancreatic tumor growth. Together, our findings highlight the functional importance of IKBKE in pancreatic cancer, support the evaluation of IKBKE as a therapeutic target in PDAC, and suggest IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic. Cancer Res; 77(2); 320-9. ©2017 AACR.

Abstract A65: Initiation of pancreatic metaplasia requires ADAM17-mediated autocrine signaling, not paracrine signaling derived from macrophages

Abstract Acinar-to-ductal metaplasia (ADM) has been frequently seen in the damaged pancreatic tissues. It is also associated with PanIN lesions in PDAC samples derived from human and mouse and has been shown to act as a preneoplastic lesion in mice. Therefore, understanding the mechanisms of lesion formation at the earliest phase would provide needed information for studying pancreatic tumorigenesis. Previously, we have shown that EGFR activity is required for metaplastic duct formation and KRas-induced pancreatic tumorigenesis. In addition, a prominent macrophage infiltration has been shown to be important in the early phase of lesion formation. To investigate the origin of EGFR ligands for EGFR activation during ADM formation in vivo, we examined the metaplasia formation by administration of cerulein in the mice with the conditional knockout of a key EGFR ligand sheddase ADAM17 in the pancreas (ADAM17fl/fl;Ptf1aCre/+) or in the myeloid cells (ADAM17fl/fl;LysM-Cre). With 2 weeks cerulein treatment, wild type and ADAM17fl/fl;LysM-Cre mice showed a similar degree of metaplastic and fibrotic response. However, the mice with ADAM17-null pancreas displayed significant protection from chronic pancreatitis induced by cerulein, including a lack of significant macrophage infiltration, making the analysis of any additional contribution from macrophage derived EGFR ligands. To remedy this, we conditionally knocked out HB-EGF (Heparin-binding EGF-like growth factor) one of the most robustly expressed EGFR ligands in macrophages using LysM-Cre. We examined cerulein-induced metaplasia in HB-EGFfl/fl;LysM-Cre mice. Compared to wild type mice, HB-EGFfl/fl;LysM-Cre mice showed a similar degree of metaplastic and fibrotic response and a decrease in cell proliferation; moreover, myeloid cell-specific ablation of HB-EGF dramatically delayed the healing and the restoration of normal pancreatic epithelium after cessation of cerulein treatment. These results indicate that an ADAM17/EGFR ligand/EGFR autocrine loop derived from pancreatic parenchymal cells is required for metaplastic duct formation, and macrophage-derived HBEGF acting in a paracrine fashion contributes to cell survival and tissue remodeling. Citation Format: Hui-Ju Wen, Howard C. Crawford.{Authors}. Initiation of pancreatic metaplasia requires ADAM17-mediated autocrine signaling, not paracrine signaling derived from macrophages. [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 A65.

Abstract A62: ATDC is required for KRAS-induced pancreatic tumorigenesis

Abstract We have recently demonstrated that ATDC, a novel oncogenic protein, serves as an invasive switch in pancreatic cancer (PDA) by activation of beta–catenin signaling and upregulation of CD44, resulting in EMT and an invasive phenotype during PanIN progression. To further explore the tumorigenic function of ATDC, we generated a floxed ATDC mouse (A F/F) to evaluate the impact of conditional knockout of ATDC on oncogenic Kras-induced PDA initiation and progression. Pancreas-specific ATDC knockout did not cause any histologic abnormalities in pancreas, up to 1 year of age (n=8). Through a series of crosses of LSL-KrasG12D (K), p53F/+ (P), RosaYFP (Y), Pdx1-Cre (C) and AF/F mice, KrasG12D; CY (KCY); KrasG12D; p53+/-; CY (KPCY), KCYA-/- KPCYA-/- mice were generated. Knockout of ATDC in KPCY mice completely prevented the development of ADM and PanIN lesions in 3 month old mice (n= 8), and resulted in the formation of very rare ADM and PanIN1 lesions (2 out of 8) in KPCYA-/- mice at 12 months of age (n=8). In contrast, all KPCY mice developed extensive PanIN (low and high grade) at 3 months of age (n= 8), with the subsequent development of invasive and metastatic cancer at frequencies similar to that reported in the literature. To determine the possible mechanisms by which ATDC inhibited KrasG12D-induced acinar-ductal metaplasia (ADM), we isolated acini from 1.5 month old KCY and KCYA-/- pancreata and performed in vitro 3D cultures and ADM assays. ADM lesions readily formed in 3-D cultures of acini from KCY mice at 5 days, and this was significantly inhibited in acini isolated from KCYA-/- mice (duct-like structures: 95.1±3.5% to 28.0±2.2%*, KCY vs KCYA-/-, n=3, *p<0.05). Expression of ATDC specific shRNA in acini from KCY mice also effectively decreased ADM formation in 3D culture, an effect that was completely reversed by ATDC overexpression using an ATDC-shRNA-resistant expression vector. To further evaluate the role of ATDC in ADM and PanIN formation, we induced caerulein-mediated acute pancreatitis in 1.5 month old WT, CYA-/-, KCY, KCYA-/- mice and analyzed pancreatic tissue 1 and 7 days following cerulein treatment. 1 day post-caerulein treatment, KCY, KCYA-/-, CYA-/- and WT mice exhibited widespread ADM, which was replaced by normal acini by 7 days in WT, CYA-/- and KCYA-/- mice. However, in KCY mice 7 days post-cerulein treatment, extensive ADM and PanIN lesions were present, suggesting that ATDC is required for oncogenic KRAS to promote ADM and PanIN formation. Conclusions: Knockout of ATDC markedly reduces KrasG12D-induced ADM and PanIN formation, highlighting a key biologic function for ATDC in this process and its role in driving progression of KRAS-induced tumorigenesis in the pancreas. Citation Format: Lidong Wang, Huibin Yang, Ethan V. Abel, Phillip L. Palmbos, Christophe Halbrook, Kenneth Takeuchi, Jiaqi Shi, Yaqing Zhang, Sumithra Urs, Meghna Waghray, Marina Pasca di Magliano, Andrew D. Rhim, Howard C. Crawford, Diane M. Simeone.{Authors}. ATDC is required for KRAS-induced pancreatic tumorigenesis. [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 A62.

Abstract B04: IKBKE integrates Gli and mTOR-AKT activation during Kras-induced pancreatic tumorigenesis

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest malignancies lacking effective therapeutic strategies. Here we show that the non-canonical IκB-related kinase, IKBKE, acts downstream of the Gli transcription factors and engages in reciprocal regulation of Gli signaling in pancreatic cancer cells. We find that loss of IKBKE inhibits the initiation and progression of pancreatic tumors in mice carrying pancreatic specific KRAS activation. Mechanistically, IKBKE does not play a major role in NF-κB activation in pancreatic cancer; but is critical for AKT activation in vivo. Moreover, we find that IKBKE promotes reactivation of AKT post-inhibition of mTOR in PDAC cells, and that combined inhibition of IKBKE and mTOR synergistically blocks pancreatic tumor growth. Together, our findings highlight the functional interaction of IKBKE with Hh/Gli and mTOR/AKT signaling in pancreatic cancer, and suggest IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic. Citation Format: Mihir Rajurkar, Martin Fernandez-Zapico, Brian Lewis, Junhao Mao. IKBKE integrates Gli and mTOR-AKT activation during Kras-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B04.

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

Abstract 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.

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

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

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. 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. 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. KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

EGF Receptor Is Required for KRAS-Induced Pancreatic Tumorigenesis

Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis invivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation.

The activity of Gli transcription factors is essential for Kras-induced pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here we find that transcriptional activation mediated by the Gli family of transcription factors, although dispensable for pancreatic development, is required for Kras-induced proliferation and survival in primary pancreatic epithelial cells in culture and for Kras-driven pancreatic intraepithelial neoplasia and PDAC formation in vivo. Further, ectopic Gli1 activation in the mouse pancreas accelerates Kras-driven tumor formation, underscoring the importance of Gli transcription factors in pancreatic tumorigenesis. Interestingly, we demonstrate Gli-regulated I-kappa-B kinase epsilon (IKBKE) and NF-κB activity in pancreatic cancer cells and show that this activity is a critical downstream mediator for Gli-dependent PDAC cell transformation and survival. Together, these studies demonstrate the requirement for Gli in Kras-dependent pancreatic epithelial transformation, suggest a mechanism of Gli-NF-κB oncogenic activation, and provide genetic evidence supporting the therapeutic targeting of Gli activity in pancreatic cancer.

The Requirement of GLI Transcriptional Activity in KRAS-Induced Pancreatic Neoplasia

Background and Aim: Kras mutations are detected in 90% of human pancreatic ductal adenocarcinoma (PDAC) and are believed to be the critical oncogenic event during pancreatic tumorigenesis. However, the downstream transcriptional mechanisms by Kras in pancreatic cancer still remain poorly understood. Recent In Vitro studies suggested that Gli transcriptional factors, the intracellular mediator of Hedgehog signaling, are important for Kras activity in human PDAC cell lines. However, a requirement for Gli activation in pancreatic malignancy has not been demonstrated In Vivo. Here we carried out both loss-of-function and gain-offunction genetic analyses to establish the functional significance of Gli transcriptional activation in Kras-Induced pancreatic tumorigenesis using genetically modified mouse models. Methods: We utilized a conditional Rosa26 allele of Gli3T, R26-Gli3T. Gli3T is a dominant repressor of Gli3 that blocks Gli transcriptional activation. The R26-Gli3T mice were crossed to the Ptf1a-Cre;LDL-KrasG12D mice, a mouse model that allows pancreatic-specific Kras activation and tumor formation. A cohort of Ptf1a-Cre;LDL-KrasG12D;R26-Gli3T mice was generated and monitored for pancreatic tumor development. For Gli gain of function studies, we ectopically expressed Gli1 along with oncogenic Kras in mouse pancreas to determine whether Gli1 cooperates with Kras to promote tumorigenesis. Results: Our results showed that Gli3T expression caused a severe growth defect and loss of tumorigenicity in human PDAC cell lines. More importantly, the mice carrying Gli3T along with oncogenic Kras failed to develop PanIN lesions, in contrast to the mice with Kras activation alone. Mice expressing Gli1 along with activated Kras in pancreas also showed marked acceleration of tumor formation. Furthermore, our gene expression profiling identified a distinct transcriptional program mediated by Gli proteins in pancreatic tumor cells. Conclusions: Gli transcriptional activation not only accelerates pancreatic tumorigenesis in cooperation with Kras, but is also required for Kras-induced tumorigenesis In Vivo.