Novel Machine Learning Approaches Revolutionize Pancreatic Malignancy Prognosis: Exploring Programed Cell Death

Carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) has been well studied using genetically engineered mouse (GEM) models with abnormalities in activation of both RAS and E2 factor (E2F)-which is associated with the Rb-E2F pathway, and dysfunction of p53-which is associated with the transformed 3T3 cell double minute 2 (MDM2)-p53 pathway. Catastrophic progression of the malignancy is one of unique manifestation, which has not been recapitulated in the GEMs to study underlined mechanisms, while we have recently reported a new GEM developing PDAC to die in 3 weeks after birth due to the catastrophic progression [Yamaguchi T. et al, J Pathol. 2014; 234(2):228-138]. The GEM had the specific expression of SV40 tsA58 large T antigen (tsTAg) to inhibit p53 and Rb family molecules (Rb1, Rbl1 and Rbl2) on pancreas with the expression of oncogenic Kras (KrasG12D) by Pdx1 promoter-mediated Cre-loxP recombination. In this study, we sought to determine if the induced tsTAg on pancreatic epithelial cells with constitutive KrasG12D expression are enough to develop PDAC and catastrophic manifestation, by the generation of a new GEM based on Cre-loxP recombination and Tet-On system. In the GEM, KrasG12D constitutively expresses on pancreatic epithelial cells after the Cre-loxP recombination under the control Pdx1 promoter, while tsTAg expression can be regulated by Tet-On system after the recombination by Pdx1 promoter. Any of the mice did not developed PDAC within 6 month, but all of the mice developed PDAC by Dox administration in within 1 week and died by the progression of PDAC within 3 weeks after the start.The rapid progression and development of PDAC appeared to be associated with the stronger tsTAg expression depending on reverse tetracycline trans-activator. These results demonstrated that KrasG12D driven PDAC carcinogenesis was promoted by the presence of tsTAg expression, resulting in production of PDAC cells with aggressive growth. In conclusion, our established new mouse model can contribute to increase knowledge about catastrophic PDAC. Citation Format: Takashi Yamaguchi, Sanae K. Ikehara, Hayao Nakanishi, Yuzuru Ikehara. Genetically engineered mouse models of catastrophic pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 822. doi:10.1158/1538-7445.AM2015-822

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer. Despite major advances in defining the molecular mutations driving PDAC, this disease remains universally lethal with an overall 5-year survival rate of only about 7–8%. Genetic alterations in PDAC are exemplified by four critical genes (KRAS, TP53, CDKN2A, and SMAD4) that are frequently mutated. Among these, KRAS mutation ranges from 88% to 100% in several studies. Hippo signaling is an evolutionarily conserved network that plays a key role in normal organ development and tissue regeneration. Its core consists of the serine/threonine kinases mammalian sterile 20-like kinase 1 and 2 (MST1/2) and large tumor suppressor 1 and 2. Interestingly, pancreas-specific MST1/2 double knockout mice have been reported to display a decreased pancreas mass. Many of the genes involved in the Hippo signaling pathway are recognized as tumor suppressors, while the Hippo transducers Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are identified as oncogenes. By dephosphorylation, YAP and TAZ accumulate in the nucleus and interact with transcription factors such as TEA domain transcription factor-1, 2, 3, and 4. Dysregulation of Hippo signaling and activation of YAP/TAZ have been recognized in a variety of human solid cancers, including PDAC. Recent studies have elucidated that YAP/TAZ play a crucial role in the induction of acinar-to-ductal metaplasia, an initial step in the progression to PDAC, in genetically engineered mouse models. YAP and TAZ also play a key role in the development of PDAC by both KRAS-dependent and KRAS-independent bypass mechanisms. YAP/TAZ have become extensively studied in PDAC and their biological importance during the development and progression of PDAC has been uncovered. In this review, we summarize the biological significance of a dysregulated Hippo signaling pathway or activated YAP/TAZ in PDAC and propose a role for YAP/TAZ as a therapeutic target.

<div>Abstract<p>Perturbation of cell polarity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) progression. Scribble (SCRIB) is a well-characterized polarity regulator that has diverse roles in the pathogenesis of human neoplasms. To investigate the impact of SCRIB deficiency in PDAC development and progression, <i>Scrib</i> expression was genetically ablated in well-established mouse models of PDAC. <i>Scrib</i> loss in combination with <i>Kras</i><sup><i>G12D</i></sup> did not influence development of pancreatic intraepithelial neoplasms in mice. However, <i>Scrib</i> deletion cooperated with <i>Kras</i><sup><i>G12D</i></sup> and concomitant <i>Trp53</i> heterozygous deletion to promote invasive PDAC and metastatic dissemination, leading to reduced overall survival. Immunohistochemical and transcriptome analyses revealed that <i>Scrib</i>-null tumors display a pronounced reduction of collagen content and an abundance of cancer-associated fibroblasts (CAF). Mechanistically, IL1α levels were reduced in <i>Scrib</i>-deficient tumors, and <i>Scrib</i> knockdown downregulated IL1α in mouse PDAC organoids (mPDO), which impaired CAF activation. Furthermore, <i>Scrib</i> loss increased YAP activation in mPDOs and established PDAC cell lines, enhancing cell survival. Clinically, SCRIB expression was decreased in human PDAC, and SCRIB mislocalization was associated with poorer patient outcome. These results indicate that SCRIB deficiency enhances cancer cell survival and remodels the tumor microenvironment to accelerate PDAC development and progression, establishing the tumor suppressor function of SCRIB in advanced pancreatic cancer.</p><p> <b>Significance:</b> SCRIB loss promotes invasive pancreatic cancer development via both cell-autonomous and non–cell-autonomous processes and is associated with poorer outcomes, denoting SCRIB as a tumor suppressor and potential biomarker for the prediction of recurrence.</p></div>

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

<h3>Background</h3> Nuclear protein 1 (Nupr1) is a major factor in the cell stress response required for <i>Kras^G12D</i>-driven formation of pancreatic intraepithelial neoplastic lesions (PanINs). We evaluated the relevance of Nupr1...

Fascin Is Regulated by Slug, Promotes Progression of Pancreatic Cancer in Mice, and Is Associated With Patient Outcomes

Musashi 2 (MSI2), a member of the Musashi RNA-binding family, is reported to be an oncoprotein in pancreatic ductal adenocarcinoma (PDAC), but the mechanisms of MSI2 in the development and progression of PDAC have not been fully demonstrated. In this research, we studied the clinical significance, biologic effects and the underlying mechanism of MSI2 in the progression of PDAC. The expression of MSI2, Mps-binding protein 1 (MOB1) and Salvador family WW domain-containing protein 1 (SAV1) in PDAC tissues were analyzed immunohistochemically. The biologic effects of MSI2 regarding PDAC cell proliferation, migration and invasion were studied using gain- and loss-of-function assays. MSI2 regulated Hippo signaling pathway via SAV1 and MOB1 was tested in several PDAC cell lines, and the mechanisms were studied using molecular biologic methods. The expression of MSI2 was significantly increased in PDAC cell lines and tissues, and positively associated with tumor poorer differentiation, lymph nodes metastasis and TNM stages. Overexpression of MSI2 promoted PDAC cells proliferation, migration and invasion. Further studies demonstrated that MSI2 regulated the Hippo signaling pathway via directly binding to the mRNAs of SAV1 and MOB1, and controlled the translation and stability of SAV1 and the translation of MOB1. This study demonstrated that MSI2 regulated the Hippo signaling pathway via suppressing SAV1 and MOB1 at post-transcriptional level and promoted PDAC progression.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer deaths and is unresponsive to existing therapy. Smoking and alcohol-induced pancreatitis are among the risk factors for PDAC. We have previously reported that beta-adrenergic receptors (beta-ARs) stimulate the proliferation and migration of human PDAC cells in vitro by cAMP-dependent signaling and that the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates this pathway directly in vitro while additionally stimulating the release of noradrenaline/adrenaline by binding to alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) in hamsters. In this study, we have tested the hypothesis that the beta-AR antagonist propranolol prevents the development of PDAC induced in hamsters with ethanol-induced pancreatitis by NNK. We found that propranolol had strong cancer preventive effects in this animal model. Western blots of pancreatic duct cells and PDAC cells harvested by laser capture microscopy showed significant upregulation of the alpha7 nAChR associated with significant inductions of p-CREB, p-ERK1/2, and increases in epidermal growth factor and vascular endothelial growth factor in PDAC cells of hamsters not treated with propranolol. These effects were reversed by treatment with propranolol. Our data suggest that propranolol may prevent the development of PDAC by blocking cAMP-dependent intracellular signaling, cAMP-dependent release of epidermal growth factor, and PKA-dependent release of vascular endothelial growth factor while additionally downregulating the alpha7 nAChR by inhibiting cAMP-mediated subunit assembly. We conclude that increased cAMP signaling is an important factor that drives the development and progression of PDAC and that the inhibition of cAMP formation is a promising new target for the prevention and adjuvant therapy of PDAC.

Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains the 4th most deadly cancer in the US. Current chemotherapeutic regimens poorly control PDAC progression and no approved immunotherapeutic options currently exist. PDAC incidence and progression are increased by obesity, which promotes a potently protumor PDAC microenvironment characterized by inflammation, fibrosis, and immunosuppression. β Hydroxy β Methylbutyrate, found to be safe and well-tolerated in clinical trials over many years, blunts cancer cachexia; however it has not been tested in PDAC. Further, the role of HMB in modifying existing therapies has been understudied. We sought to determine if HMB treatment would alter therapeutic response of lean and obese mice to gemcitabine or anti-PD1 immunotherapy. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with gemcitabine or anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. In addition, bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without LPS. Tumor/macrophage transcriptomic analysis, followed by gene set enrichment analysis and enrichment mapping identified transcriptional responses. CIBERSORTx determined microenvironment immune cell composition. Results: a) HMB reduces obesity-associated PDAC progression; b) HMB treatment and gemcitabine together suppress PDAC growth in obese mice; c) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance; d) HMB enhances the efficacy of anti-PD1 immunotherapy; e) HMB promotes M1 polarization of BMDM. Conclusion: Overall, HMB evokes antitumor immune responses in the context of pro-inflammatory stimuli such as obesity or immunotherapy. Moreover, HMB promotes immune surveillance in PDAC, synergizing with both cytotoxic chemotherapy and immunotherapy. HMB has been found to be safe and well tolerated in clinical trials for many years. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. This research was supported by R35CA197627 to S. Hursting. Citation Format: Michael F. Coleman, Kristyn A. Liu, Suhas K. Etigunta, Alex J. Pfeil, Xiaohu Tang, Salvador Fabela, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β Hydroxy β Methylbutyrate synergizes with cytotoxic chemotherapy and immunotherapy in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3151.

&lt;div&gt;Abstract&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, and its incidence is increasing annually. It is critical to reveal and delineate the molecular mechanism promoting PDAC development and progression. Mammalian STE20-like kinase 1 (MST1) is a proapoptotic cytoplasmic kinase and also one of the core components of the Hippo pathway. Here, we showed that MST1 expression was decreased in PDAC, and restored expression of MST1 promoted PDAC cell death and suppressed the proliferation, migration, invasion, and cell spheroid formation of PDAC via caspase-1–induced pyroptosis. Further studies demonstrated that pyroptosis induced by MST1 was independent of the Hippo pathway, but mediated by reactive oxygen species (ROS). And ROS scavenger N-acetyl-cysteine attenuated the activation of caspase-1 induced by MST1 and the effect of MST1 in PDAC cell death, proliferation, migration, and invasion. Collectively, our study demonstrated that MST1 suppressed the progression of PDAC cells at least partly through ROS-induced pyroptosis.&lt;/p&gt;Implications:&lt;p&gt;In this study, we identified a new mechanism of MST1 in inhibiting PDAC development and progression and revealed that MST1 would be a potential prognostic and therapeutic target for PDAC.&lt;/p&gt;&lt;/div&gt;

Bioinformatics analyses of infiltrating immune cell participation on pancreatic ductal adenocarcinoma progression and in vivo experiment of the therapeutic effect of Shuangshen granules

Prior to pancreatic ductal adenocarcinoma (PDAC), pancreatic acini cells change their morphology through pancreatic intraepithelial neoplasia (PanIN) stages, becoming increasingly dysplastic as stroma and tissue hypoxia increase. This increasing hypoxia forms a cancer promoting microenvironment, wherein we propose metabolic changes trigger aberrant mitochondrial networks form healthy tissue. We developed tissue superresolution imaging to directly quantify structural mitochondrial response through patient histology, whereby DNA-PAINT can provide super-resolution detail decoupled from photo bleaching to visualize mitochondria through tissue layers. Expanding from preliminary data, target mitochondrial dynamics proteins’ organization will also be correlated to PanIN stages. Introduction: Early oncogene involvement in PDAC progression links to metabolic and mitochondrial regulatory changes. Typically nutrient deprivation and hypoxia trigger cell death from increased reactive oxygen species production and organelle damage which trigger apoptosis, yet these prolonged effects can be cancer promoting when less severe. Mitochondrial dynamics and proteins related to mitochondrial fission and fusion can reduce apoptotic signaling, enhance aerobic glycolysis, and increase ROS to allow cancer progression. Materials and Methods: Adapting from previous proof of concept, mitochondria (TOM20) within formalin-fixed paraffin embedded (FFPE) tissue sections were imaged with stochastic optical reconstruction microscopy (STORM). In brief: cadaver healthy pancreas FFPE tissue samples underwent deparafinization, antigen retrieval, indirect immuno-labeling with AlexaFluor647, TIRF illumination with a 60x objective was used for data collection, whereby data processing was conducted using the open-source FIJI and custom MATLAB software. Results and Discussion: Preliminary data shows proof of principle that both STORM and DNA-PAINT can be correlated to histological staining of human pancreas. Conclusions: Superresolution imaging reveals ultrastructural details of mitochondria in FFPE patient samples not resolved via conventional fluorescence imaging. Through quantitative feature analysis, we would be able to correlate aberrant mitochondria structure and abundance to PDAC progression. Acknowledgements: Funding provided by the Cancer Early Detection Advanced Research (CEDAR) Center of OHSU Knight Cancer Institute. Citation Format: Matthew J. Rames, Fehmi Civitci, Ting Zheng, Josiah Wagner, Jason Link, Xiaolin Nan. Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology [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 799.

We have identified regulators of G protein signaling (Rgs8 and Rgs16) as a new class of tumor suppressor genes in a mouse model of pancreatic ductal adenocarcinoma (PDA). PDA is the 3rd leading cause of cancer related deaths in the United States. Kras mutations (e.g. KrasG12D) are associated with over 90% of human PDA and are an early event in the multistep process leading to PDA. Kras can be activated by protein kinase and G-Protein Coupled Receptor (GPCR) signaling. Rgs proteins regulate GPCR signaling by accelerating the GTPase activity of Gq- and Gi class alpha subunits. Activating alleles of Gq that are resistant to Rgs inhibition are associated with PDA in humans. We found Rgs8 and Rgs16 are in vivo reporters of Kras activity in pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and PDA progression (DMM 8, 2015). Rgs8 and Rgs16 are expressed in PanIN and IPMN, precursors of PDA, in KC mice (LSL-KrasG12D; p48::Cre). To test if Rgs8-16 function as tumor suppressor genes, we crossed the Rgs8-16 double knockout into KC (termed KCR8-16) mice. Compared to KC, PDA initiates earlier, is more aggressive, and KCR8-16 mice die earlier. Our study suggests that Rgs8 and Rgs16 control Kras-dependent PDA initiation and progression. Note: This abstract was not presented at the meeting. Citation Format: Shreoshi Pal Choudhuri, Yalda Zolghadri, Luke Mascarenhas, Ozhan Ocal, Thomas Wilkie. Rgs8 and Rgs16 are tumor suppressor genes in mouse pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5518. doi:10.1158/1538-7445.AM2017-5518

Expression and prognostic value of NSD1 and SETD2 in pancreatic ductal adenocarcinoma and its precursor lesions