Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against KRAS, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K in vitro, successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1a Cre/þ ;LSL-Kras G12D/þ ;Tgfbr2 flox/flox (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.
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/β-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/β-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.
Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDKs) that induce cell cycle progression. p16 INK4a (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 may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared to normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6 inhibition (CDK4/6i) results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared to 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 to treatment with vehicle or individual agents alone in Ptf1a cre/+ ;LSL-KRAS G12D/+ ;Tgfbr2 flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescenceassociated 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.
<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against <i>KRAS</i>, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K <i>in vitro,</i> successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1a<sup>Cre/+</sup>;LSL-Kras<sup>G12D/+</sup>;Tgfbr2<sup>flox/flox</sup> (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A–treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.</p></div>
<p>Supplementary Figure S5 shows densitometry analyses of pAKT and pP70S6K in HPNE and HPNE-KRAS cells.</p>
Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Chronic alcohol (>60 grams/day) consumption is strongly associated with the risk of PDAC development. The metabolites generated from alcohol have been shown to cause significant pancreatic injury. Although the association of alcohol with PDAC progression has been established, the details of the underlying molecular and cellular mechanisms governing this process are unknown. Our study revealed that alcohol-associated pancreatic carcinogenesis correlates with CXCL12-induced activation of cyclic AMP response element binding protein (CREB). In this study, we examined newly developed CREB knockout (CREBfl/fl) mouse in pancreas. Methods: Human pancreatic epithelial lines (HPNE), HPNE cells with KRAS (HPNE-KRAS), pancreatic stellate cells (PSCs), pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) and cancer-associated fibroblasts (CAFs) cells were exposed to chronic alcohol (50 mM) and analyzed for phospho-kinases in cell lysates and cytokines in conditioned media. Inducible Ptf1aCreERTM;KRASG12D/+ (iPK) mice and CREB knockout (CREBfl/fl) with KRAS activation [Ptf1aCreERTM;KRASG12D/+;CREBfl/fl (iPKCREBKO)] mice were used to investigate the effect of alcohol on CREB activation and the role of CREB in alcohol-associated PDAC pathogenesis. iPK and iPKCREBKO mice were exposed to Lieber-DeCarli alcohol diet for up to 14 weeks with or without caerulein injections. The number of acinar cells (amylase), ducts (cytokeratin 19), PanIN lesions (alcian blue positive), fibrosis (sirius red) and activation of CREB were measured by immunohistochemistry at 6 and 14 weeks of alcohol exposure in vivo. Serum obtained from alcohol-fed iPK and iPKCREBKO mice were analyzed for significant cytokine release upon alcohol exposure in vivo. We then determined the biologic effects of pharmacologic CREB inhibition in iPK mice exposed to alcohol diet. Results: We found increased pCREB levels in HPNE and HPNE-KRAS cells upon treatment with alcohol, which was further associated with the up-regulation of the CXCL12 protein in the conditioned media. Serum CXCL12 and tissue pCREB levels were high in alcohol fed iPK mice when compared to their corresponding control diet-fed mice. Blocking CXCL12 with Plerixafor, a CXCL12-CXCR4 axis inhibitor, decreased alcohol-induced pCREB levels. Finally, exposure of iPK mice to an alcohol diet coupled with cerulein administration significantly increased the number of PanIN lesions (alcian blue+ cells), and decreased acinar cells when compared to alcohol-fed iPKCREBKO mice. Conclusion: These findings implicate CREB as a critical oncogenic driver in alcohol addiction-induced pancreatic carcinogenesis. Additionally, CXCL12 represents an important mediator of CREB activation, which can be pharmacologically targeted. Citation Format: Tulasigeri Totiger, Supriya Srinivasan, Michael VanSaun, Chandrashekhar Joshi, Chanjuan Shi, Xizi Dai, Rajinder Dawra, Alexander Gaidarski, Eric Nestler, Nipun Merchant, Nagaraj Nagathihalli. CREB transgenic mice to study alcohol-associated pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5042.
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