Pancreatic Ductal Adenocarcinoma Initiation Research Articles

The Role of the Microbiome in Pancreatic Cancer.

Simple SummaryPancreatic cancer is deadly cancer characterized by dense stroma creating an immunosuppressive tumor microenvironment. Accumulating evidences indicate that the microbiome plays an important role in pancreatic cancer development and progression via the local and systemic inflammation and immune responses. The alteration of the microbiome modulates the tumor microenvironment and immune system in pancreatic cancer, which affects the efficacy of chemotherapies including immune-targeted therapies. Understanding the role of microbiome and underlying mechanisms may lead to novel biomarkers and therapeutic strategies for pancreatic cancer. This review summarizes the current evidence on the role of the microbiome in pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with little improvement in outcomes in recent decades, although the molecular and phenotypic characterization of PDAC has contributed to advances in tailored therapies. PDAC is characterized by dense stroma surrounding tumor cells, which limits the efficacy of treatment due to the creation of a physical barrier and immunosuppressive environment. Emerging evidence regarding the microbiome in PDAC implies its potential role in the initiation and progression of PDAC. However, the underlying mechanisms of how the microbiome affects the local tumor microenvironment (TME) as well as the systemic immune system have not been elucidated in PDAC. In addition, therapeutic strategies based on the microbiome have not been established. In this review, we summarize the current evidence regarding the role of the microbiome in the development of PDAC and discuss a possible role for the microbiome in the early detection of PDAC in relation to premalignant pancreatic diseases, such as chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN). In addition, we discuss the potential role of the microbiome in the treatment of PDAC, especially in immunotherapy, although the biomarkers used to predict the efficacy of immunotherapy in PDAC are still unknown. A comprehensive understanding of tumor-associated immune responses, including those involving the microbiome, holds promise for new treatments in PDAC.

Mechanistic and functional extrapolation of SET and MYND domain-containing protein 2 to pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal neoplasms worldwide and represents the vast majority of pancreatic cancer cases. Understanding the molecular pathogenesis and the underlying mechanisms involved in the initiation, maintenance, and progression of PDAC is an urgent need, which may lead to the development of novel therapeutic strategies against this deadly cancer. Here, we review the role of SET and MYND domain-containing protein 2 (SMYD2) in initiating and maintaining PDAC development through methylating multiple tumor suppressors and oncogenic proteins. Given the broad substrate specificity of SMYD2 and its involvement in diverse oncogenic signaling pathways in many other cancers, the mechanistic extrapolation of SMYD2 from these cancers to PDAC may allow for developing new hypotheses about the mechanisms driving PDAC tumor growth and metastasis, supporting a proposition that targeting SMYD2 could be a powerful strategy for the prevention and treatment of PDAC.

Differential Effects of Dietary Macronutrients on the Development of Oncogenic KRAS-Mediated Pancreatic Ductal Adenocarcinoma.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease and oncogenic KRAS mutations are prevalent in PDAC patients. Oncogenic KRAS is critical for the initiation and development of PDAC; however, it alone is insufficient to drive pancreatic cancer. Oncogenic KRAS is an important mediator in metabolic reprogramming, including glucose, glutamine, and fatty acid metabolisms, for cancer cell survival, proliferation, and invasion. Oncogenic KRAS and chronic high-fat diet synergize to promote PDAC, suggesting that dietary intake is critically involved in PDAC development. Here, by feeding mice expressing an endogenous level of oncogenic KRASG12D with a high-fat diet, high-carbohydrate diet, high-protein diet, or normal diet, we have demonstrated that different dietary macronutrients differentially impact pancreatic cancer susceptibility, and a high-protein diet may represent the desired, favorable dietary choice compared to high-fat diet and high-carbohydrate diet for pancreatic cancer patients. In addition, management of macronutrient intake aimed at thwarting inflammation is a promising preventive strategy for patients harboring oncogenic KRAS.KRAS mutations are prevalent in patients with pancreatic ductal adenocarcinoma (PDAC) and are critical to fostering tumor growth in part by aberrantly rewiring glucose, amino acid, and lipid metabolism. Obesity is a modifiable risk factor for pancreatic cancer. Corroborating this epidemiological observation, mice harboring mutant KRAS are highly vulnerable to obesogenic high-fat diet (HFD) challenges leading to the development of PDAC with high penetrance. However, the contributions of other macronutrient diets, such as diets rich in carbohydrates that are regarded as a more direct source to fuel glycolysis for cancer cell survival and proliferation than HFD, to pancreatic tumorigenesis remain unclear. In this study, we compared the differential effects of a high-carbohydrate diet (HCD), an HFD, and a high-protein diet (HPD) in PDAC development using a mouse model expressing an endogenous level of mutant KRASG12D specifically in pancreatic acinar cells. Our study showed that although with a lower tumorigenic capacity than chronic HFD, chronic HCD promoted acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions with increased inflammation, fibrosis, and cell proliferation compared to the normal diet (ND) in KrasG12D/+ mice. By contrast, chronic HPD showed no significant adverse effects compared to the ND. Furthermore, ablation of pancreatic acinar cell cyclooxygenase 2 (Cox-2) in KrasG12D/+ mice abrogated the adverse effects induced by HCD, suggesting that diet-induced pancreatic inflammation is critical for promoting oncogenic KRAS-mediated neoplasia. These results indicate that diets rich in different macronutrients have differential effects on pancreatic tumorigenesis in which the ensuing inflammation exacerbates the process. Management of macronutrient intake aimed at thwarting inflammation is thus an important preventive strategy for patients harboring oncogenic KRAS.

Glycolysis addiction compensating for a defective pentose phosphate pathway confers gemcitabine sensitivity in SETD2-deficient pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy driven by genetic mutations and/or epigenetic dysregulation. Gemcitabine chemotherapy is the first-line regimen for pancreatic cancer but has limited efficacy. Our previous study revealed the role of SETD2-H3K36me3 loss in the initiation and metastasis of PDAC, but little is known about its role in tumor metabolism. Here, we found that SETD2-deficient PDAC enhanced glycolysis addiction via upregulation of glucose transporter 1 (GLUT1) to meet its large demand for glucose in progression. Moreover, SETD2 deficiency impaired nucleoside synthesis by directly downregulating the transcriptional level of transketolase (TKT) in the pentose phosphate pathway. The metabolic changes confer SETD2-deficient PDAC cells with increased sensitivity to gemcitabine under glycolysis restriction conditions. Collectively, our study provides mechanistic insights into how SETD2 deficiency reprograms glycolytic metabolism to compensate for insufficient nucleoside synthesis, suggesting that glycolysis restriction combined with gemcitabine might be a potential therapeutic strategy for PDAC patients with SETD2 deficiency.

Hypersialylation of Tumor Cells Promotes Pancreatic Cancer Progression

Tumor cells are well‐known to have elevated levels of sialylated surface glycoproteins. The addition of sialic acid (a negatively‐charged sugar) to select surface receptors modulates the structure and function of such receptors, leading to changes in intracellular signaling and gene expression. Increased tumor cell sialylation occurs, in part, through the upregulation of sialyltransferases such as ST6GAL1, an enzyme that adds an α2‐6 linked sialic acid to N‐glycosylated proteins. ST6GAL1 is overexpressed in numerous malignancies, including pancreatic ductal adenocarcinoma (PDAC), and high expression correlates with a poor prognosis. Prior work from our group revealed that ST6GAL1 confers a cancer stem cell phenotype, typified by invasiveness and apoptosis‐resistance, through the sialylation‐dependent activation of receptors such as EGFR. In the current investigation we used human Suit2 PDAC cells, as well as two metastatic Suit2 subclones, to show that ST6GAL1 activity promotes tumor progression and metastasis in tumor xenograft models. Moreover, high ST6GAL1 expression in this isogenic cell series induces EGFR activation and epithelial to mesenchymal transition. To complement tumor xenograft experiments, we developed a genetically‐engineered mouse (GEM) model with conditional ST6GAL1 expression in the pancreas (termed, “SC” mice) and crossed this line to the “KC” PDAC model, which expresses oncogenic Kras (KrasG12D) in the pancreas. Mice with dual expression of ST6GAL1 and KrasG12D (”KSC” mice) exhibit greatly accelerated PDAC initiation, progression, and mortality when compared with KC mice. In light of ST6GAL1's known role in conferring stem/progenitor properties, we hypothesized that ST6GAL1 activity contributes to PDAC initiation by fostering acinar to ductal metaplasia (ADM). During ADM, pancreatic acinar cells de‐differentiate into ductal‐like, progenitor cells and acquire greater proliferative potential as well as apoptosis resistance. Cells undergoing ADM are particularly vulnerable to oncogenic transformation. To interrogate whether ST6GAL1 promotes ADM, we compared SC mice with control, wild‐type littermates. Using a variety of analyses and model systems including RNA‐Seq of GEM pancreata, organoid lines derived from GEM pancreata, the canonical 266‐6 ADM cell model, and in vivo model of inflammation‐induced ADM, we determined that high expression of ST6GAL1 in acinar cells imparts an ADM‐like phenotype, as evidenced by the upregulation of stem and ductal genes and downregulation of genes associated with differentiated acinar cells. Furthermore, GEM tissues and organoids with ectopic ST6GAL1 expression display strikingly elevated levels of activated EGFR. Given that activation of EGFR is one the major drivers of ADM, these data suggest a potential mechanism by which ST6GAL1‐mediated sialylation facilitates ADM. Collectively these results point to a critical role for ST6GAL1 in both early and late stages of pancreatic malignancy.

ST6Gal1 Contributes to Pancreatic Cancer Initiation by Promoting Pancreatitis‐Induced Acinar to Ductal Metaplasia

Aberrant glycosylation is one of the earliest known hallmarks of cancer. Amongst the predominate changes in tumor cell glycosylation is an increase in α2‐6 sialylation on N‐glycans, a modification elaborated by the ST6Gal1 sialyltransferase. ST6Gal1 activity dramatically alters cellular function via the sialylation of various surface receptors such as EGFR and TNFR1, resulting in altered signal transduction and gene expression. ST6Gal1 expression is upregulated in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC). Recent studies from our group suggest that the upregulation of ST6Gal1 in pancreatic acinar cells may promote PDAC initiation by facilitating acinar to ductal metaplasia (ADM), a process in which acinar cells de‐differentiate into ductal‐like, progenitor cells. In the nonmalignant pancreas, ADM is induced primarily by pancreatitis, a known risk factor for PDAC. Pancreatitis causes acinar cell apoptosis, however some cells undergo ADM and acquire regenerative capabilities essential for tissue healing. Notably, ADM‐like cells are particularly susceptible to oncogenic transformation. In the current study we determined that ST6Gal1 was strikingly upregulated in pancreatic tissues from chronic pancreatitis patients and mice with experimental pancreatitis. Furthermore, ST6Gal1 was selectively expressed in the ADM‐like cells, as identified both morphologically and by staining for ADM‐specific markers. ST6Gal1 activity was associated with a proliferative phenotype. More than 50% of cells with upregulated ST6Gal1 co‐expressed the proliferative marker, Ki67, whereas activated caspase 8 was undetectable in ST6Gal1‐positive cells. To interrogate a functional role for ST6Gal1 in ADM, we developed genetically‐engineered mice with ST6Gal1 expression in the pancreas (“SC” mice). Importantly, acinar cells from SC mice exhibited ADM‐like characteristics, indicated by increased expression of classic stem and ductal genes. RNASeq analyses comparing SC and WT pancreata indicated that SC cells had an enrichment in: stem cell pathways; a pancreatic ductal cell program; and gene networks associated with pancreatic cancer. Additionally, organoids derived from SC pancreata displayed increased expression of stem and ductal genes relative to WT organoids, as well as greater organoid‐forming potential and growth. To evaluate inflammation‐induced ADM, SC and WT mice were injected with cerulein to induce pancreatitis, and the ADM‐like cells were quantified by examining surface ADM markers by flow cytometry. Following induction of pancreatitis, more ADM‐like cells were present in SC versus WT pancreata. Finally, we crossbred SC mice to the “KC” PDAC model, which expresses oncogenic KRas (KrasG12D) in the pancreas. Mice with dual expression of ST6Gal1 and KrasG12D had greatly accelerated PDAC initiation, progression and mortality as compared with KC mice. Together these data suggest that the upregulation of ST6Gal1 during chronic pancreatitis may prime acinar cells for neoplastic transformation by facilitating ADM.

Candidate diagnostic and prognostic markers for pancreatitis and pancreatic ductal adenocarcinoma

Diagnostic markers are desperately needed for early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity of PDA marker expression was analyzed by interrogating quantitative dot plots of single cell (sc) RNAseq for 3021 candidate genes in tumor samples from a mouse model for PDA (KIC) at early and late stages of PDA progression compared to normal pancreas. Protein markers were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines. Additional candidate genes were identified from signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes. Developmental progression of cancer was revealed in seven classes of PDA markers expressed in cancer cells and stroma. The earliest diagnostic markers (47 genes) were specifically expressed in epithelial cancer cells in early, but not late, PDA tumors. Other markers were first elevated in epithelial cancer cells of late stage tumors (288 genes), or in both epithelial and mesenchymal cells (740 genes), or only in mesenchymal cells (216 genes). Stromal markers were differentially expressed in early‐ and/or late‐PDA neoplasia (594 genes). Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and acinar‐specific homozygous deletion of differentiation transcription factors (dTFs), were screened for early expression of all PDA markers identified in KIC neoplasia. Select diagnostic markers were validated by immunohistochemistry in mouse and human samples of normal pancreas, early‑ and late‐stage PDA. Prognostic markers from TCGA were identified that showed differential and cell type‐specific expression in PDA. Here we present diagnostic and prognostic markers for disease progression from pancreatitis to late‐stage PDA.

SULF2 enhances GDF15-SMAD axis to facilitate the initiation and progression of pancreatic cancer

Owing to the lack of early diagnosis, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal tumours. Because acinar-to-ductal metaplasia (ADM) is a critical process to pancreatic regeneration and PDAC initiation, we applied GSE65146, a dataset composed of transcripts at different time points in wild-type and KrasG12D mutant mice upon pancreatitis induction, to obtain regeneration- and tumour initiation-related genes. By overlapping with genes differentially expressed in human PDAC, we defined the initiation- and progression-related genes, and the most prognostic gene, SULF2, was selected for further verification. By using multiple PDAC genetically engineered murine models (GEMMs), we further verified that the expression of SULF2 was increased at the ADM and PDAC stages. Functionally, SULF2 was able to promote the dedifferentiation of acinar cells as well as the metastatic ability of PDAC. Additionally, our study revealed that SULF2 could enhance TGFβ-SMAD signalling via GDF15. More importantly, serum SULF2 was elevated in patients with PDAC, and in combination with CA19-9, it provided a better method for PDAC diagnosis. Herein, our study screened out key genes for the initiation and progression of PDAC, providing potential indicators for the diagnosis of the disease.

Molecular signaling in pancreatic ductal metaplasia: emerging biomarkers for detection and intervention of early pancreatic cancer.

Pancreatic ductal metaplasia (PDM) is the transformation of potentially various types of cells in the pancreas into ductal or ductal-like cells, which eventually replace the existing differentiated somatic cell type(s). PDM is usually triggered by and manifests its ability to adapt to environmental stimuli and genetic insults. The development of PDM to atypical hyperplasia or dysplasia is an important risk factor for pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDA). Recent studies using genetically engineered mouse models, cell lineage tracing, single-cell sequencing and others have unraveled novel cellular and molecular insights in PDM formation and evolution. Those novel findings help better understand the cellular origins and functional significance of PDM and its regulation at cellular and molecular levels. Given that PDM represents the earliest pathological changes in PDA initiation and development, translational studies are beginning to define PDM-associated cell and molecular biomarkers that can be used to screen and detect early PDA and to enable its effective intervention, thereby truly and significantly reducing the dreadful mortality rate of PDA. This review will describe recent advances in the understanding of PDM biology with a focus on its underlying cellular and molecular mechanisms, and in biomarker discovery with clinical implications for the management of pancreatic regeneration and tumorigenesis.

The Intricate Crosstalk Between Insulin and Pancreatic Ductal Adenocarcinoma: A Review From Clinical to Molecular.

Increased insulin level (or “hyperinsulinemia”) is a common phenomenon in pancreatic ductal adenocarcinoma (PDA) patients and signals poor clinical outcomes. Insulin is safe in low PDA risk population, while insulin significantly promotes PDA risk in high PDA risk population. The correlation between insulin and PDA is a reciprocal self-reinforcing relationship. On the one hand, pancreatic cancer cells synthesize multiple molecules to cause elevated peripheral insulin resistance, thus enhancing hyperinsulinemia. On the other hand, insulin promotes pancreatic cancer initiation and sustains PDA development by eliciting tumorigenic inflammation, regulating lipid and glucose metabolic reprogram, overcoming apoptosis through the crosstalk with IGF-1, stimulating cancer metastasis, and activating tumor microenvironment formation (inflammation, fibrosis, and angiogenesis). Currently, taking glucose sensitizing agents, including metformin, SGLT-2 inhibitor, and GLP-1 agonist, is an effective way of lowering insulin levels and controlling PDA development at the same time. In the future, new drugs targeting insulin-related signal pathways may pave a novel way for suppressing PDA initiation and progression.

Inhibition of DCLK1 kinase reverses epithelial-mesenchymal transition and restores T-cell activity in pancreatic ductal adenocarcinoma

Immunotherapy has recently become a promising cancer therapy with extensive applications of immune checkpoint inhibitors (ICIs). However, pancreatic ductal adenocarcinoma (PDAC) appears to be unresponsive to immunotherapy due to the immunosuppressive microenvironment. Recent studies showed that cancer stem cell marker DCLK1 promoted the initiation and development of PDAC. Nevertheless, the mechanism driving this process remains unclear. Here, by performing gain-of-function investigations in PDAC cell lines, we demonstrate that both DCLK1 long (DCLK1-iso1, DCLK1-AS) and short (DCLK1-iso4, DCLK1-BL) isoforms can efficiently activate EMT leading to tumor migration and invasion. Consistent with experiments in vitro, bioinformatic analysis demonstrates that DCLK1 may act as a driver of EMT activation in PDAC. Further analysis showed that EMT was associated with an immunosuppressive microenvironment, which includes more immunosuppressive cells and chemokines, and patients with a higher EMT score were less sensitive to immune checkpoint inhibitors according to the TIDE (Tumor Immune Dysfunction and Exclusion) algorithm. Multiplexed immunofluorescence results demonstrated the close correlation between DCLK1, EMT and immunosuppression in PDAC patients. The findings were further confirmed in vivo reflected by decreased CD4+, CD8+ T cells and increased M2 macrophages as well as E-cad loss in DCLK1-overexpressing subcutaneous tumors. Importantly, the highly-specific DCLK1 inhibitor (DCLK1-IN-1) was able to effectively block EMT process and restore T-cell activity. Altogether, our data demonstrate that DCLK1 is strongly associated with tumor immune escape in PDAC and inhibiting DCLK1 kinase activity may be a promising therapeutic modality.

A shifting adversary: control of pancreatic cancer transcriptomic subtypes

Cellular plasticity, the dynamic ability of cells to adopt distinct transcriptional states, plays a well-known role in the pancreas during the initiation of pancreatic ductal adenocarcinoma (PDA), the most common form of pancreatic cancer. It is now becoming clear that plasticity also plays an important role after the emergence of PDA. PDA is composed of two major transcriptional subtypes, classical and basal-like, with important biological differences. Recent work has indicated that individual tumors can be comprised of cells of each subtype, and that tumor subtype can change during the evolution of a tumor. This suggests that PDA cells can transit between transcriptional states, with important implications for disease progression. This review discusses what is currently known about inter-subtype plasticity and how this process is controlled.

Abstract PR-011: Loss of compensatory feedback mechanism involving splicing factor SRSF1 accelerates KrasG12D-mediated pancreatic cancer initiation

Abstract KRAS is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC), triggering the formation of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). However, the majority of pancreatic cells from KC (LSL-KrasG12D/+; Pdx-1-Cre) mice carrying the KrasG12D mutation remain morphologically normal for a long time, suggesting the existence of compensatory feedback mechanisms that buffer KrasG12D signaling, and that additional steps are required for disrupting cell homeostasis and promoting transformation. Here we found that splicing factor SRSF1, which is associated with cell transformation, is downregulated in the majority of morphologically normal pancreas cells with the KrasG12D mutation as a compensatory feedback mechanism. To assess the role of SRSF1 in PDAC, we generated a transgenic mouse strain (SC) with Dox-inducible pancreatic-specific expression of SRSF1 protein. Elevated SRSF1 alone is sufficient to induce pancreatitis and ADM transformation. By further crossing the SC strain with KrasG12D alone (KSC) or together with Trp53R172H (KPSC), we found that increasing SRSF1 accelerated KrasG12D-mediated tumor initiation and resulted in more aggressive PDAC. To address the underlying mechanisms of SRSF1’s involvement in the homeostatic response to KrasG12D mutation and in PDAC initiation, we generated organoid lines from the above mouse strains. Following Dox induction, elevated SRSF1 with Kras WT or KrasG12D consistently activated MAPK signaling, which is one of the top negatively enriched pathways in response to KrasG12D. Furthermore, SRSF1 promoted the expression of Il1r1—an IL1 receptor associated with the activation of MAPK signaling—by regulating an alternative-splicing event in the 5’UTR to generate a more stable mRNA isoform. We conclude that decreased SRSF1 is a compensatory feedback mechanism in pancreatic cells against the KrasG12D mutation, whose disruption facilitates PDAC initiation. Citation Format: Ledong Wan, Kuan-Ting Lin, Mohammad A. Rahman, Zhikai Wang, Youngkyu Park, David A. Tuveson, Adrian R. Krainer. Loss of compensatory feedback mechanism involving splicing factor SRSF1 accelerates KrasG12D-mediated pancreatic cancer initiation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-011.

Abstract PO-104: Activation of WNT signaling in CD4+ T cells promotes immune suppression in pancreatic cancer

Abstract WNT ligand expression and activation of the WNT signaling have been associated with pancreatic ductal adenocarcinoma (PDA). Using a mouse model that recapitulates the stepwise progression of PDA, we previously showed that epithelial WNT signaling is required for PDA initiation and progression. Through single cell RNA sequencing, we discovered that, in addition to epithelial cells, infiltrating immune cells and in particular T cells express the WNT signaling machinery, including receptors, downstream effectors and target genes. In particular, T cells express high levels of Tcf7, encoding the transcription factor TCF1. TCF1 is important for T cell development, but its role in adult T cells and specifically in the setting of pancreatic cancer remained unknown. To address this, we generated mice that allow deletion of Tcf7 specifically in CD4+ T cells in an inducible manner. We then implanted orthotopic syngeneic tumors in control mice or in mice lacking Tcf7 in CD4+ T cells, and observed reduced tumor growth in the latter. Analysis of the tumors revealed an increase in apoptotic cells; while the prevalence of infiltrating T cells did not change, we observed an increase in cytotoxic marker expression, such as Granzyme B, consistent with increased T cell activation. We observed decreased regulatory T cells, possibly indicating less immune suppression. However, we also observed a compensatory infiltration of increased granulocytic myeloid derived suppressor cells. Deletion of Tcf7 in CD4+ T cells also re-polarized the immunosuppressive macrophages into pro-inflammatory macrophages, and alterations of inflammatory cytokines secreted by cancer associated fibroblasts. Taken together, this study reveals an important role of WNT signaling in CD4+ T cells in the PDA microenvironment. We are currently evaluating ways to exploit changes in the immune microenvironment upon inactivation of Tcf7 in CD4+ T cells in combination therapy approaches. Citation Format: Wenting Du, Rosa E. Menjivar, Katelyn Donahue, Ashley Velez-Delgado, Marina Pasca di Magliano. Activation of WNT signaling in CD4+ T cells promotes immune suppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-104.

MiR-802 Suppresses Acinar-to-Ductal Reprogramming During Early Pancreatitis and Pancreatic Carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood. The Ptf1aCre/+LSL-KrasG12D/+ and Ptf1aCre/+LSL-KrasG12D/+LSL-p53R172H/+ and caerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM. We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic KrasG12D-induced transformation. Genetic ablation of mir-802 cooperates with KrasG12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels ofductal and attenuated expression of acinar identity genes.Consistently with these findings, we show that this miR-802-RhoA-F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival. We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802-RhoA-F-actin network may be a new strategy to interfere with pancreatic carcinogenesis.

Acinar cell clonal expansion in pancreas homeostasis and carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide1. Studies in human tissues and in mouse models have suggested that for many cancers, stem cells sustain early mutations driving tumour development2,3. For the pancreas, however, mechanisms underlying cellular renewal and initiation of PDAC remain unresolved. Here, using lineage tracing from the endogenous telomerase reverse transcriptase (Tert) locus, we identify a rare TERT-positive subpopulation of pancreatic acinar cells dispersed throughout the exocrine compartment. During homeostasis, these TERThigh acinar cells renew the pancreas by forming expanding clones of acinar cells, whereas randomly marked acinar cells do not form these clones. Specific expression of mutant Kras in TERThigh acinar cells accelerates acinar clone formation and causes transdifferentiation to ductal pre-invasive pancreatic intraepithelial neoplasms by upregulating Ras-MAPK signalling and activating the downstream kinase ERK (phospho-ERK). In resected human pancreatic neoplasms, we find that foci of phospho-ERK-positive acinar cells are common and frequently contain activating KRAS mutations, suggesting that these acinar regions represent an early cancer precursor lesion. These data support a model in which rare TERThigh acinar cells may sustain KRAS mutations, driving acinar cell expansion and creating a field of aberrant cells initiating pancreatic tumorigenesis.

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53

Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation invivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents invivo. Of note, this therapeutic effect is efficient across various cancer types. Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Epigenetic Alterations in Pancreatic Cancer Metastasis.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States. Pancreatic ductal adenocarcinoma (PDA) is the most common (90%) and aggressive type of pancreatic cancer. Genomic analyses of PDA specimens have identified the recurrent genetic mutations that drive PDA initiation and progression. However, the underlying mechanisms that further drive PDA metastasis remain elusive. Despite many attempts, no recurrent genetic mutation driving PDA metastasis has been found, suggesting that PDA metastasis is driven by epigenetic fluctuations rather than genetic factors. Therefore, establishing epigenetic mechanisms of PDA metastasis would facilitate the development of successful therapeutic interventions. In this review, we provide a comprehensive overview on the role of epigenetic mechanisms in PDA as a critical contributor on PDA progression and metastasis. In particular, we explore the recent advancements elucidating the role of nucleosome remodeling, histone modification, and DNA methylation in the process of cancer metastasis.

Upregulated CD58 is associated with clinicopathological characteristics and poor prognosis of patients with pancreatic ductal adenocarcinoma

BackgroundCD58 has been demonstrated to be abnormally expressed in multiple hematopoietic malignancies and solid tumors and plays an essential role in tumorigenesis and progression; however, its clinical significance and prognostic value in pancreatic ductal adenocarcinoma (PDAC) remain unknown.MethodsBased on diverse online public databases and 81 PDAC samples of tissue microarray-based immunohistochemistry (IHC), we evaluated CD58 expression in PDAC patients and analyzed its association with clinicopathological characteristics, clinical outcomes, and infiltration of immune cells in PDAC. Furthermore, the correlation between CD58 and the cancer stem cell (CSC)-related, epithelial–mesenchymal transition (EMT)-related, and immune-related markers were detected. Besides, the functional enrichment analysis and related pathways were analyzed and visualized.ResultsCD58 expression was elevated in pancreatitis and PDAC tissues than normal pancreas or adjacent nontumor tissues. The positive cases of CD58 (e.g. more than 50% positive cells) in PDAC account for 95.06% (77/81). Upregulated CD58 in cancer tissues was associated with worse histological grade, larger tumor size, and poorer overall survival and disease-free survival in PDAC patients. Furthermore, Cox multivariate regression analysis revealed that CD58 was an independent prognostic factor in PDAC. CD58 expression was correlated with infiltrations of neutrophils, CD8+ T cells, and dendritic cells (DCs). In addition, correlation gene analysis indicated that CD58 expression was strongly correlated with immune-related, EMT-related, and CSC-related markers. Functional enrichment analysis and KEGG pathway manifested that CD58 might be involved in PDAC initiation and progression.ConclusionsCD58 expression is upregulated in PDAC tissues and its high expression is notably related to poor survival of PDAC. Therefore, CD58 may serve as a novel and effective marker for predicting the prognosis of PDAC patients.

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+ KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.