Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer.

BACKGROUND & AIMS: We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. METHODS: CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1. RESULTS: MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8+ T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit. CONCLUSIONS: Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.

Distinct mechanisms of innate and adaptive immune regulation underlie poor oncologic outcomes associated with KRAS-TP53 co-alteration in pancreatic cancer.

Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

Targeting Tumor-Stromal IL6/STAT3 Signaling through IL1 Receptor Inhibition in Pancreatic Cancer.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense, desmoplastic stroma and the consequent altered interactions between cancer cells and their surrounding tumor microenvironment (TME) that promote disease progression, metastasis, and chemoresistance. We have previously shown that IL6 secreted from pancreatic stellate cells (PSC) stimulates the activation of STAT3 signaling in tumor cells, an established mechanism of therapeutic resistance in PDAC. We have now identified the tumor cell-derived cytokine IL1α as an upstream mediator of IL6 release from PSCs that is involved in STAT3 activation within the TME. Herein, we show that IL1α is overexpressed in both murine and human PDAC tumors and engages with its cognate receptor IL1R1, which is strongly expressed on stromal cells. Further, we show that IL1R1 inhibition using anakinra (recombinant IL1 receptor antagonist) significantly reduces stromal-derived IL6, thereby suppressing IL6-dependent STAT3 activation in human PDAC cell lines. Anakinra treatment results in significant reduction in IL6 and activated STAT3 levels in pancreatic tumors from Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice. Additionally, the combination of anakinra with cytotoxic chemotherapy significantly extends overall survival compared with vehicle treatment or anakinra monotherapy in this aggressive genetic mouse model of PDAC. These data highlight the importance of IL1 in mediating tumor-stromal IL6/STAT3 cross-talk in the TME and provide a preclinical rationale for targeting IL1 signaling as a therapeutic strategy in PDAC.

Combined Blockade of MEK and CDK4/6 Pathways Induces Senescence to Improve Survival in Pancreatic Ductal Adenocarcinoma.

Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDK) that induce cell-cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition (CDK4/6i) may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared with normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6i results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared with monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared with treatment with vehicle or individual agents alone in Ptf1acre/+ ;LSL-KRASG12D/+ ;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.

Combined Src/EGFR Inhibition Targets STAT3 Signaling and Induces Stromal Remodeling to Improve Survival in Pancreatic Cancer.

Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics which hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src and EGFR pathways. Although it is well-established that concurrent EGFR and Src inhibition exert these antineoplastic properties through direct inhibition of mitogenic pathways in tumor cells, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In this study, we demonstrate in both orthotopic tumor xenograft and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, increases microvessel density, and prevents tissue fibrosis in vivo. Furthermore, the stromal changes induced by parallel EGFR and Src pathway inhibition resulted in improved overall survival in PKT mice when combined with gemcitabine. As a phase I clinical trial utilizing concurrent EGFR and Src inhibition with gemcitabine has recently concluded, these data provide timely translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. IMPLICATIONS: These findings demonstrate that Src/EGFR inhibition targets STAT3, remodels the tumor stroma, and results in enhanced delivery of gemcitabine to improve overall survival in a mouse model of PDAC.

Src kinase inhibition restores E-cadherin expression in dasatinib-sensitive pancreatic cancer cells.

The Src family of non-receptor tyrosine kinases are frequently activated in pancreatic ductal adenocarcinoma (PDAC), contributing to disease progression through downregulation of E-cadherin and induction of epithelial-to-mesenchymal transition (EMT). The purpose of this study was to examine the efficacy of Src kinase inhibition in restoring E-cadherin levels in PDAC. Immunohistochemical analysis of human PDAC samples showed Src activation is inversely correlated with E-cadherin levels. Protein and mRNA levels of E-cadherin, the gene expression of its various transcriptional repressors (Zeb1, Snail, Slug, LEF-1, TWIST), and changes in sub-cellular localization of E-cadherin/ß-catenin in PDAC cells were characterized in response to treatment with the Src inhibitor, dasatinib (DST). DST repressed Slug mRNA expression, promoted E-cadherin transcription, and increased total and membranous E-cadherin/ß-catenin levels in drug-sensitive PDAC cells (BxPC3 and SW1990), however no change was observed in drug-resistant PANC1 cells. BxPC3, PANC1, and MiaPaCa-2 flank tumor xenografts were treated with DST to examine changes in E-cadherin levels in vivo. Although DST inhibited Src phosphorylation in all xenograft models, E-cadherin levels were only restored in BxPC3 xenograft tumors. These results suggest that Src kinase inhibition reverses EMT in drug-sensitive PDAC cells through Slug-mediated repression of E-cadherin and identifies E-cadherin as potential biomarker for determining response to DST treatment.

Inverse Correlation of STAT3 and MEK Signaling Mediates Resistance to RAS Pathway Inhibition in Pancreatic Cancer.

Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) include Kras mutations, a dense desmoplastic stroma that prevents drug delivery to the tumor, and activation of redundant signaling pathways. We have previously identified a mechanistic rationale for targeting STAT3 signaling to overcome therapeutic resistance in PDAC. In this study, we investigate the molecular mechanisms underlying the heterogeneous response to STAT3 and RAS pathway inhibition in PDAC. Effects of JAK/STAT3 inhibition (STAT3i) or MEK inhibition (MEKi) were established in Ptf1acre/+; LSL-KrasG12D/+ ; and Tgfbr2flox/flox (PKT) mice and patient-derived xenografts (PDX). Amphiregulin (AREG) levels were determined in serum from human patients with PDAC, LSL-KrasG12D/+;Trp53R172H/+;Pdx1Cre/+ (KPC), and PKT mice. MEKi/STAT3i-treated tumors were analyzed for integrity of the pancreas and the presence of cancer stem cells (CSC). We observed an inverse correlation between ERK and STAT3 phosphorylation. MEKi resulted in an immediate activation of STAT3, whereas STAT3i resulted in TACE-induced, AREG-dependent activation of EGFR and ERK. Combined MEKi/STAT3i sustained blockade of ERK, EGFR, and STAT3 signaling, overcoming resistance to individual MEKi or STAT3i. This combined inhibition attenuated tumor growth in PDX and increased survival of PKT mice while reducing serum AREG levels. Furthermore, MEKi/STAT3i altered the PDAC tumor microenvironment by depleting tumor fibrosis, maintaining pancreatic integrity, and downregulating CD44+ and CD133+ CSCs. These results demonstrate that resistance to MEKi is mediated through activation of STAT3, whereas TACE-AREG-EGFR-dependent activation of RAS pathway signaling confers resistance to STAT3 inhibition. Combined MEKi/STAT3i overcomes these resistances and provides a novel therapeutic strategy to target the RAS and STAT3 pathway in PDAC.Significance: This report describes an inverse correlation between MEK and STAT3 signaling as key mechanisms of resistance in PDAC and shows that combined inhibition of MEK and STAT3 overcomes this resistance and provides an improved therapeutic strategy to target the RAS pathway in PDAC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6235/F1.large.jpg Cancer Res; 78(21); 6235-46. ©2018 AACR.

Pancreatic stellate cell secreted IL-6 stimulates STAT3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a dynamic tumor supported by several stromal elements such as pancreatic stellate cells (PSC). Significant crosstalk exists between PSCs and tumor cells to stimulate oncogenic signaling and malignant progression of PDAC. However, how PSCs activate intercellular signaling in PDAC cells remains to be elucidated. We have previously shown that activated signal transducer and activator of transcription 3 (STAT3) signaling is a key component in the progression of pancreatic neoplasia. We hypothesize that PSC secreted IL-6 activates STAT3 signaling to promote PanIN progression to PDAC. Human PDAC and mouse PanIN cells were treated with PSC-conditioned media (PSC-CM), and phospho- and total-STAT3 levels by immunoblot analysis were determined. IL-6 was quantified in PSC-CM and cell invasion and colony formation assays were performed in the presence or absence of a neutralizing IL-6 antibody and the JAK/STAT3 inhibitor AZD1480. Serum from Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) and LSL-KrasG12D/+; Trp53R172H/+; Pdx1Cre/+ (KPC) mice demonstrated increased levels of IL-6 compared to serum from non-PDAC bearing KC and PK mice. PSC secreted IL-6 activated STAT3 signaling in noninvasive, precursor PanIN cells as well as PDAC cells, resulting in enhanced cell invasion and colony formation in both cell types. There was a significant positive linear correlation between IL-6 concentration and the ratio of phosphorylated STAT3/total STAT3. IL-6 neutralization or STAT3 inhibition attenuated PSC-CM induced activation of STAT3 signaling and tumorigenicity. These data provide evidence that PSCs are directly involved in promoting the progression of PanINs towards invasive carcinoma. This study demonstrates a novel role of PSC secreted IL-6 in transitioning noninvasive pancreatic precursor cells into invasive PDAC through the activation of STAT3 signaling.

Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation.

Neural tissue engineering is one of the most promising approaches for healing nerve damage, which bypasses the limits of contemporary conventional treatments. In a previous study, we developed a fibrous scaffold via electrospinning poly (glycerol dodecanedioate) (PGD) and gelatin that mimics the structure of a native extracellular matrix (ECM) for soft tissue engineering application. In this study, fumaric acid (FA) was incorporated into the PGD synthesis process, which produced a PGD derivative referred to as poly (glycerol dodecanedioate co-fumarate) (PGDF). This introduced a new functional group, a double bond, into the polymer thus providing new modification possibilities. Arg-Gly-Asp-Cys (RGDC) and laminin peptides were chosen as biomolecules to modify the fiber and facilitate cell attachment and differentiation efficiency. The release of FA into the medium was quantified to investigate the bioreactivity of the derived scaffolds. In combination with UV crosslinking, the developed PGDF fiber mats were able to withstand degradation processes for up to 2 months, which ensures that neural tissue engineering applications are viable. Cell viability and motor neuron differentiation efficiency were demonstrated to be significantly improved with the addition of FA, RGDC and laminin peptides.

Electrospun fibrous scaffolds of Poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task.  Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named poly(glycerol-dodecanoate) (PGD)(1) by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun fiber scaffolds of poly (glycerol-dodecanedioate) and its gelatin blended polymers for soft tissue engineering.

For tissue engineering applications, biodegradable scaffolds play a vital role in supporting and guiding the seeded cells to form functional tissues by mimicking the structure and function of native extracellular matrices. Previously, we have developed a biodegradable elastomer poly (glycerol-dodecanedioate) (PGD) with mechanical properties suitable for soft tissue engineering. In the study, we found that the PGD and PGD blended with gelatin (PGD/gelatin) were able to be electrospun into fibrous scaffolds, and the diameters of the fibers could be adjusted by controlling the PGD concentration. When using our newly designed electrospinning collector, fibers could be easily harvested and the size of the fiber mat could be flexibly adjusted. The data of Raman spectra also confirmed the esterfication reaction in PGD polymerization and showed no significant structure change after electrospinning. Biocompatibility testing of the PGD and PGD/gelatin, by using human foreskin fibroblasts, indicated that gelatin could enhance cell adhesion and proliferation. Overall, electrospun fibers made from PGD and PGD/gelatin exhibited several advantages including easy synthesis from renewable raw materials, flexible fabrication by using less toxic solvents like ethanol, and good biocompatibility.