Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma

Pancreatic cancer is characterized by nearly universal activating mutations in KRAS. Among other somatic mutations, TP53 is mutated in more than 75% of human pancreatic tumors. Genetically engineered mice have proven instrumental in studies of the contribution of individual genes to carcinogenesis. Oncogenic Kras mutations occur early during pancreatic carcinogenesis and are considered an initiating event. In contrast, mutations in p53 occur later during tumor progression. In our model, we recapitulated the order of mutations of the human disease, with p53 mutation following expression of oncogenic Kras. Further, using an inducible and reversible expression allele for mutant p53, we inactivated its expression at different stages of carcinogenesis. Notably, the function of mutant p53 changes at different stages of carcinogenesis. Our work establishes a requirement for mutant p53 for the formation and maintenance of pancreatic cancer precursor lesions. In tumors, mutant p53 becomes dispensable for growth. However, it maintains the altered metabolism that characterizes pancreatic cancer and mediates its malignant potential. Further, mutant p53 promotes epithelial-mesenchymal transition (EMT) and cancer cell invasion. This work generates new mouse models that mimic human pancreatic cancer and expands our understanding of the role of p53 mutation, common in the majority of human malignancies

PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression

Pancreatic cancer: Advances and challenges

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Significant efforts have largely defined major genetic factors driving PDAC pathogenesis and progression. Pancreatic tumors are characterized by a complex microenvironment that orchestrates metabolic alterations and supports a milieu of interactions among various cell types within this niche. In this review, we highlight the foundational studies that have driven our understanding of these processes. We further discuss the recent technological advances that continue to expand our understanding of PDAC complexity. We posit that the clinical translation of these research endeavors will enhance the currently dismal survival rate of this recalcitrant disease

Mitogen-activated Protein Kinase Kinase Activity Maintains Acinar-to-Ductal Metaplasia and Is Required for Organ Regeneration in PancreatitisSummary

Background & Aims: Mitogen-activated protein kinase (MAPK) signaling in the exocrine pancreas has been extensively studied in the context of pancreatic cancer, where its potential as a therapeutic target is limited by acquired drug resistance. However, its role in pancreatitis is less understood. We investigated the role of mitogen-activated protein kinase kinase (MEK)-initiated MAPK signaling in pancreatitis to determine the potential for MEK inhibition in treating pancreatitis patients. Methods: To examine the role of MEK signaling in pancreatitis, we used both genetic and pharmacologic approaches to inhibit the MAPK signaling pathway in a murine model of cerulein-induced pancreatitis. We generated mice harboring inducible short hairpins targeting the MEK isoforms Map2k1 and/or Map2k2 specifically in the pancreatic epithelium. We also used the MEK inhibitor trametinib to determine the efficacy of systemic inhibition in mice with pancreatitis. Results: We demonstrated an essential role for MEK signaling in the initiation of pancreatitis. We showed that both systemic and parenchyma-specific MEK inhibition in established pancreatitis induces epithelial differentiation and stromal remodeling. However, systemic MEK inhibition also leads to a loss of the proliferative capacity of the pancreas, preventing the restoration of organ mass. Conclusions: MEK activity is required for the initiation and maintenance of pancreatitis. MEK inhibition may be useful in the treatment of chronic pancreatitis to interrupt the vicious cycle of destruction and repair but at the expense of organ regeneration. Keywords: Inflammation, Wound Healing, Tissue Regeneration, AD

Inhibiting the Hexosamine Biosynthetic Pathway Lowers OGlcNAcylation Levels and Sensitizes Cancer to Environmental Stress

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients

Pancreatic HIF2α Stabilization Leads to Chronic Pancreatitis and Predisposes to Mucinous Cystic Neoplasm

Tissue hypoxia controls cell differentiation in the embryonic pancreas, and promotes tumor growth in pancreatic cancer. The cellular response to hypoxia is controlled by the hypoxia-inducible factor (HIF) proteins, including HIF2α. Previous studies of HIF action in the pancreas have relied on loss-of-function mouse models, and the effects of HIF2α expression in the pancreas have remained undefined. Methods: We developed several transgenic mouse models based on the expression of an oxygen-stable form of HIF2α, or indirect stabilization of HIF proteins though deletion of von Hippel-Lindau, thus preventing HIF degradation. Furthermore, we crossed both sets of animals into mice expressing oncogenic KrasG12D in the pancreas. Results: We show that HIF2α is not expressed in the normal human pancreas, however, it is up-regulated in human chronic pancreatitis. Deletion of von Hippel-Lindau or stabilization of HIF2α in mouse pancreata led to the development of chronic pancreatitis. Importantly, pancreatic HIF1α stabilization did not disrupt the pancreatic parenchyma, indicating that the chronic pancreatitis phenotype is specific to HIF2α. In the presence of oncogenic Kras, HIF2α stabilization drove the formation of cysts resembling mucinous cystic neoplasm (MCN) in humans. Mechanistically, we show that the pancreatitis phenotype is linked to expression of multiple inflammatory cytokines and activation of the unfolded protein response. Conversely, MCN formation is linked to activation of Wnt signaling, a feature of human MCN. Conclusions: We show that pancreatic HIF2α stabilization disrupts pancreatic homeostasis, leading to chronic pancreatitis, and, in the context of oncogenic Kras, MCN formation. These findings provide new mouse models of both chronic pancreatitis and MCN, as well as illustrate the importance of hypoxia signaling in the pancreas

An Optimized Enzyme-Nucleobase Pair Enables <i>In Vivo</i> RNA Metabolic Labeling with Improved Cell-Specificity

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA

Multiomic characterization of pancreatic cancer-associated macrophage polarization reveals deregulated metabolic programs driven by the GM-CSF–PI3K pathway

The pancreatic ductal adenocarcinoma microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAMs) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment. However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEMs) in vitro and performed detailed, multiomic characterization (i.e., transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single-cell RNA sequencing dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K-AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization