Precursor Pancreatic Intraepithelial Neoplasia Research Articles

Abstract 2577: AGO2 interaction limits wild type RAS activation yet essential for disease progression in oncogenic KRAS driven cancers

Abstract KRAS mutations drive over 30% of all cancers and 90% of pancreatic cancer. To investigate additional potential modulators of RAS-mediated oncogenesis, we previously performed a screen for direct interactors of RAS in a panel of cancer cell lines and identified a direct interaction between KRAS and Argonaute 2 (AGO2), independent of KRAS mutation status. To define the effects of conditional loss of AGO2 in KRASG12D-driven cancer, we employed a genetically engineered mouse model of pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevented progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This resulted in increased of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RASWT-phospho ERK signaling including reduced baseline autophagy in mice with AGO2 loss. Interestingly, in the KPC model simultaneous expression of oncogenic KRAS and loss of a p53 allele abrogates this phenotype, suggesting that p53 deficiency overcomes the OIS check by AGO2. However, in both mouse and human PDAC, AGO2 expression increased with disease progression and, interestingly, was predominant at the plasma membrane, where it co-localized with RAS. These in vivo analyses support a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and WT RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the KRASG12D driven lung cancer mouse model, we see a similar requirement for AGO2 for diseases progression–but not for initiation–despite p53 loss indicating a context dependent requirement for AGO2. Given that AGO2 binds both the mutant and the WT forms of KRAS, we also investigated the role of RASWT-AGO2 interaction. Analysis using purified proteins showed that AGO2 competes with SOS1 to limit RASWT activation. AGO2 had no effect on the intrinsic GTPase activity of RAS or NF1-mediated GAP activity. In line with this data, AGO2 null mouse embryonic fibroblasts showed increased RAS activity and activation of MAPK/PI3K pathways. Further, using cell line models, we also showed that phosphorylation of AGO2Y393 by EGFR disrupted the interaction of RASWT with AGO2 at the membrane, but did not affect the interaction of mutant KRAS with AGO2. On the other hand, ARS-1620, a G12C-specific inhibitor, disrupted the KRASG12C-AGO2 interaction specifically in cells harboring this mutant, demonstrating that the oncogenic KRAS-AGO2 interaction can be pharmacologically targeted. Altogether, our findings reveal that the AGO2 interaction regulates RASWT and is essential for mutant KRAS driven oncogenesis. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Seema Chugh, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Xiao-Ming Wang, Sanjana Eyunni, Andrew Goodrum, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Xuhong Cao, Jiaqi Shi, Sethuramsundaram Pitchiaya, Chandan Kumar-Sinha, Howard C. Crawford, Arul M. Chinnaiyan. AGO2 interaction limits wild type RAS activation yet essential for disease progression in oncogenic KRAS driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2577.

Abstract A20: An essential role for Argonaute 2 in mouse models of KRAS driven cancers

Abstract In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing that is required for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed the genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in survival of KRASG12D mutant mice deficient in AGO2 expression. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis; however rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Since PanIN development requires EGFR and is not AGO2 dependent, we probed the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS (using co-IP and PLA analyses). In wild-type RAS expressing cells, grown in media containing serum, RAS-AGO2 co-localization was limited to the intracellular regions of the cells, which dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular basal levels. Using phosphorylation-deficient AGO2 mutants, we demonstrate that the disruption of wild type-RAS-AGO2 interaction is due to AGO2Y393 phosphorylation, a target of EGFR. Interestingly, the mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild-type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the lung cancer mouse model, we also observed a similar dependence of AGO2 in KRAS-driven lung adenocarcinoma. Along with related abstracts detailing the mechanisms of OIS mediated by AGO2 (Ronald Siebenaler) and evidence of direct interaction between oncogenic KRAS and AGO2 with an affinity of 200nM (Jessica Waninger), we present our latest studies related to the KRAS-AGO2 interaction. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Xiao-Ming Wang, Kristin M. Juckette, Alice Xu, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Lisha Wang, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS driven cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A20.

Endoplasmic Reticulum stress-dependent expression of ERO1L promotes aerobic glycolysis in Pancreatic Cancer.

Rationale: Endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) is an endoplasmic reticulum (ER) luminal glycoprotein that has a role in the formation of disulfide bonds of secreted proteins and membrane proteins. Emerging data identify ERO1L as a tumor promoter in a wide spectrum of human malignancies. However, its molecular basis of oncogenic activities remains largely unknown.Methods: Pan-cancer analysis was performed to determine the expression profile and prognostic value of ERO1L in human cancers. The mechanism by which ERO1L promotes tumor growth and glycolysis in pancreatic ductal adenocarcinoma (PDAC) was investigated by cell biological, molecular, and biochemical approaches.Results: ERO1L was highly expressed in PDAC and its precursor pancreatic intraepithelial neoplasia and acts as an independent prognostic factor for patient survival. Hypoxia and ER stress contributed to the overexpression pattern of ERO1L in PDAC. ERO1L knockdown or pharmacological inhibition with EN460 suppressed PDAC cell proliferation in vitro and slowed tumor growth in vivo. Ectopic expression of wild type ERO1L but not its inactive mutant form EROL-C394A promoted tumor growth. Bioinformatics analyses and functional analyses confirmed a regulatory role of ERO1L on the Warburg effect. Notably, inhibition of tumor glycolysis partially abrogated the growth-promoting activity of ERO1L. Mechanistically, ERO1L-mediated ROS generation was essential for its oncogenic activities. In clinical samples, ERO1L expression was correlated with the maximum standard uptake value (SUVmax) in PDAC patients who received 18F-FDG PET/CT imaging preoperatively. Analysis of TCGA cohort revealed a specific glycolysis gene expression signature that is highly correlated with unfolded protein response-related gene signature.Conclusion: Our findings uncover a key function for ERO1L in Warburg metabolism and indicate that targeting this pathway may offer alternative therapeutic strategies for PDAC.

Abstract 957: An essential role for Argonaute 2 in mouse models of KRAS-driven cancers

Abstract In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing, that is essential for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in the survival of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RAS-phosphoERK signaling in the absence of AGO2. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. While AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, these lesions undergo OIS rather than progressing to PDAC. Further, we used co-IP and PLA analyses to probe the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS. In wild-type RAS expressing cells, RAS-AGO2 co-localization and interaction were limited to the intracellular regions of the cells, and dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular levels. Using phosphorylation-deficient AGO2 mutants, we further demonstrate that the disruption of wild-type RAS-AGO2 interaction is due to EGFR-mediated AGO2Y393 phosphorylation. Interestingly, mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. We will discuss our ongoing studies evaluating the effects of AGO2 ablation in the KRASG12Ddriven lung cancer mouse model and PDAC progression with p53 loss (KPC model). Our recent in vivo work supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Alice Xu, Xiao-Ming Wang, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 957.

PRDM14 is overexpressed in chronic pancreatitis prior to pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer that is typically diagnosed at a later stage with metastases and is difficult to treat. Therefore, investigating the mechanism of PDAC initiation is important to aid early‐stage cancer detection. PRDM14 is a transcription factor that maintains pluripotency in embryonic stem cells and is overexpressed in several cancers. We previously reported that PRDM14 is overexpressed and regulates cancer stem‐like phenotypes in PDAC, and herein, we assess whether PRDM14 expression increases prior to tumorigenesis. Through immunohistochemistry analyses of clinical tissues, we detected PRDM14‐positive cells in precursor pancreatic intraepithelial neoplasia and chronic pancreatitis, which is a risk factor for PDAC, lesions. PRDM14 staining in chronic pancreatitis was as high as that in PDAC and cancer adjacent tissues. We induced pancreatitis in mouse models by cerulein injection, and observed that PRDM14 expression increased in chronic pancreatitis models but not in control or acute pancreatitis mice. Moreover, cerulein treatment increased PRDM14 expression in PK‐1 and AsPC‐1 pancreatic cancer cell lines. Our results suggest that inflammation increases the expression of PRDM14, which regulates cancer stem‐like phenotypes, and this occurs prior to PDAC initiation and progression.

Integrated expression profiling of potassium channels identifys KCNN4 as a prognostic biomarker of pancreatic cancer

Dysregulated potassium (K+) channels have previously been shown to promote the development and progression of many types of cancers. Meanwhile, K+ channels are particularly important in regulating the endocrine and exocrine functions of pancreas. However, the expression pattern and prognostic significance of K+ channels in pancreatic ductal adenocarcinoma (PDAC) remain unknown. In this study, by screening a GEO dataset containing 36 microdissected PDAC and matching normal pancreatic tissue samples, four differentially expressed K+ channels (KCNJ5, KCNJ16, KCNN4 and KCNK1) were identified in PDAC. by immunohistochemical analysis of pancreatic tissue sections from Pdx1-Cre; LSL-KrasG12D/+ mice (KC), Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ mice (KPC) and human PDAC tissue microarrays, we found that Ca2+-activated K+ channel KCNN4 was significantly elevated in pancreatic intraepithelial neoplasia (PanIN) and PDAC epithelia compared with untransformed pancreas tissues. Higher epithelial KCNN4 expression was closely correlated with advanced TNM stages and predicted a poor prognosis in patients with PDAC. Elevated KCNN4 expression was significantly associated with shorter survival in univariable and multivariable analyses. Collectively, the identification of expression pattern of K+ channels in PDAC and its precursor PanIN demonstrates the importance of KCNN4 channel during the malignant transformation of PDAC. On the basis of the prognostic signals from two independent cohorts, KCNN4 should be considered as a promising therapeutic target.

Abstract IA6: Identification and characterization of factors required for KRAS-driven pancreatic tumorigenesis

Abstract Activating mutations in the KRAS oncogene occur in the vast majority of pancreatic ductal adenocarcinomas (PDAC). These mutations are present in early precursor pancreatic intra-epithelial neoplasia (PanIN) lesions and are considered initiating driver mutations in this disease. Moreover, expression of an activated Kras allele potently initiates PanIN formation and PDAC development in the mouse. Therefore, inhibiting mutant KRAS-mediated signaling is potentially an effective therapeutic strategy for PDAC. While efforts directly targeting mutant KRAS are underway, an alternative approach is to target key signaling molecules that are stimulated downstream by activated KRAS. We hypothesized that factors required for the development of KRAS-driven pancreatic tumors will be similarly required during late stage disease and therefore represent potential therapeutic targets. We therefore utilize in vivo and in vitro systems to identify and characterize these factors. Mutated KRAS promotes the proliferation of primary pancreatic ductal epithelial cells (PDECs) as well as their survival following challenge with apoptotic stimuli. KRAS-induced survival in PDECs is dependent on signaling through the RAF/MEK/ERK and PI3K/AKT signaling cascades. Interestingly, we found that the insulin-like growth factor receptor (IGF1R) is required for KRASG12D-induced stimulation of the PI3K/AKT signaling axis. We determined that KRASG12D induces the expression of the IGF1R ligand IGF2 downstream of the MEK/ERK cascade and IGF2 stimulates IGF1R, and subsequently PI3K/AKT signaling, in an autocrine manner. Consistent with this, ectopic IGF2 expression restored KRASG12D-induced survival in cells treated with a MEK inhibitor but not a PI3K inhibitor. Critically, IGF1R is required for KRASG12D-induced survival after exposure to apoptotic stimuli, and inhibition of IGF1R impairs pancreatic tumor formation in an orthotopic mouse model. In PDAC cell lines, shRNA-mediated knockdown of IGF1R impairs their anchorage-independent growth and migration activity. Thus, IGF1R-mediated signaling is required for KRASG12D-driven pancreatic tumorigenesis in vivo and impacts the transformation of pancreatic cancer cells in culture. The mTOR signaling complex TORC2 promotes the phosphorylation of AKT on Ser473, an event required for full activation of AKT. Given the importance of IGF1R and PI3K/AKT signaling in promoting KRASG12D-induced survival and pancreatic tumor formation, we explored the requirement for TORC2 activity in KRAS-driven PDAC development in vivo. We inactivated TORC2 through the pancreas-specific genetic ablation of the essential subunit Rictor. Rictor deficient pancreata are architecturally normal but smaller than wild-type pancreata. Interestingly, Rictor deletion significantly impaired KRASG12D-driven PanIN formation. Moreover, the absence of Rictor also impaired KRASG12D-driven PanIN formation following caerulein-induced pancreatic injury. Together, these data indicate an important role for TORC2 signaling during PDAC initiation. We additionally characterized the role of TORC2 in PDAC cell lines. ShRNA-mediated inhibition of Rictor impaired PDAC cell proliferation and transformation as assessed by anchorage-independent growth, suggesting an important role for the TORC2 signaling complex in maintenance of the transformed phenotype of PDAC cells. Analysis of TORC substrate proteins identified reduced phosphorylation and activation of AKT and SGK1, suggesting these downstream molecules as potential mediators of TORC2-dependent phenotypes in pancreatic tumorigenesis. Together, these data suggest that TORC2 activity plays a critical role in KRAS-driven PDAC initiation and progression. Collectively, our studies have identified critical factors required for KRAS-driven pancreatic tumorigenesis, suggesting additional therapeutic strategies for targeting PDAC. Citation Format: Brian C. Lewis. Identification and characterization of factors required for KRAS-driven 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 IA6.

Spatiotemporal Proteomic Analyses during Pancreas Cancer Progression Identifies Serine/Threonine Stress Kinase 4 (STK4) as a Novel Candidate Biomarker for Early Stage Disease

Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of <5%. Detection of resectable disease improves survival rates, but access to tissue and other biospecimens that could be used to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ∼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages.

MicroRNA-21 Is Induced Early in Pancreatic Ductal Adenocarcinoma Precursor Lesions

Pancreatic ductal adenocarcinoma (PDAC) has the poorest overall prognosis among gastrointestinal cancers; however, curative resection in early-stage PDAC greatly improves survival rates, indicating the importance of early detection. Because abnormal microRNA production is commonly detected in cancer, we investigated noninvasive precursor pancreatic intraepithelial neoplasia (PanIN) lesions for microRNA production as a potential early biomarker of PDAC. Pathologists identified and classified ductal lesions. We extracted total RNA from laser-capture microdissected PanIN tissue samples from a conditional KRAS(G12D) mouse model (n = 29) or of human origin (n = 38) (KRAS is v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). MicroRNA production was quantified by quantitative real-time PCR. Internal controls included 5S and U6 RNAs. Production of microRNAs miR-21, miR-205, and miR-200 paralleled PanIN progression in the KRAS(G12D) mouse model, compared with microRNA production in samples of nonpathologic ducts. miR-21 demonstrated the highest relative concentrations in the precursor lesions. Interestingly, miR-205 and miR-21 up-regulation preceded phenotypic changes in the ducts. The production of microRNAs miR-21, miR-221, miR-222, and let-7a increased with human PanIN grade, with peak production occurring in hyperplastic PanIN-2/3 lesions. In situ hybridization analysis indicated miR-21 production to be concentrated in pathologic ductal cells. miR-21 production was regulated by KRAS(G12D) and epidermal growth factor receptor in PDAC-derived cell lines. Aberrant microRNA production is an early event in the development of PanIN. Our findings indicate that miR-21 warrants further investigation as a marker for early detection of PDAC.

Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically.