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.

Dosch AR ，Dai X ，Reyzer ML ，Mehra S ，Srinivasan S ，Willobee BA ，Kwon D ，Kashikar N ，Caprioli R ，Merchant NB ，Nagathihalli NS ... -  《-》

Signal Transducer and Activator of Transcription 3, Mediated Remodeling of the Tumor Microenvironment Results in Enhanced Tumor Drug Delivery in a Mouse Model of Pancreatic Cancer.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense desmoplastic reaction (stroma) that impedes drug delivery to the tumor. Attempts to deplete the tumor stroma have resulted in formation of more aggressive tumors. We have identified signal transducer and activator of transcription (STAT) 3 as a biomarker of resistance to cytotoxic and molecularly targeted therapy in PDAC. The purpose of this study is to investigate the effects of targeting STAT3 on the PDAC stroma and on therapeutic resistance. Activated STAT3 protein expression was determined in human pancreatic tissues and tumor cell lines. In vivo effects of AZD1480, a JAK/STAT3 inhibitor, gemcitabine or the combination were determined in Ptf1a(cre/+);LSL-Kras(G12D/+);Tgfbr2(flox/flox) (PKT) mice and in orthotopic tumor xenografts. Drug delivery was analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry. Collagen second harmonic generation imaging quantified tumor collagen alignment and density. STAT3 activation correlates with decreased survival and advanced tumor stage in patients with PDAC. STAT3 inhibition combined with gemcitabine significantly inhibits tumor growth in both an orthotopic and the PKT mouse model of PDAC. This combined therapy attenuates in vivo expression of SPARC, increases microvessel density, and enhances drug delivery to the tumor without depletion of stromal collagen or hyaluronan. Instead, the PDAC tumors demonstrate vascular normalization, remodeling of the tumor stroma, and down-regulation of cytidine deaminase. Targeted inhibition of STAT3 combined with gemcitabine enhances in vivo drug delivery and therapeutic response in PDAC. These effects occur through tumor stromal remodeling and down-regulation of cytidine deaminase without depletion of tumor stromal content.

Nagathihalli NS ，Castellanos JA ，Shi C ，Beesetty Y ，Reyzer ML ，Caprioli R ，Chen X ，Walsh AJ ，Skala MC ，Moses HL ，Merchant NB ... -  《-》

Combined blockade of Src kinase and epidermal growth factor receptor with gemcitabine overcomes STAT3-mediated resistance of inhibition of pancreatic tumor growth.

We previously established a mechanistic rationale for Src inhibition as a novel therapeutic target in pancreatic cancer and have identified activated STAT3 as a potential biomarker of resistance to Src inhibition. The purpose of this study was to translate the current understanding of complementary activated tyrosine kinase signaling pathways by targeting Src kinase and epidermal growth factor receptor (EGFR). IC(50) values for dasatinib, a Src kinase inhibitor, erlotinib, an EGFR tyrosine kinase inhibitor and gemcitabine were determined and sensitive and resistant pancreatic cancer cell lines were identified. The in vitro and in vivo effects of these agents on multiple signaling pathways and tumorigenicity in pancreatic cancer were investigated. The combination of dasatinib, erlotinib, and gemcitabine resulted in cooperative inhibition of cell migration and invasion of both sensitive and resistant pancreatic cancer cells as well as cooperative inhibition of multiple signaling pathways including FAK, AKT, ERK, JNK, MAPK, and STAT3 at concentrations that were ineffective as individual agents or as double combinations of agents. The triple combination of agents was also most effective at inhibiting the growth of xenografts of both sensitive and resistant pancreatic cancer cells in vivo without increasing toxicity. Furthermore, combined inhibition of Src and EGFR with gemcitabine inhibited constitutively activated STAT3 in vitro and in vivo. These results provide evidence that combined targeted biological therapy in addition to cytotoxic chemotherapy can overcome treatment resistance. Such treatment strategies may be used to tailor therapy based on identified biomarkers of resistance to targeted monotherapy.

Nagaraj NS ，Washington MK ，Merchant NB 《-》

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.

Nagathihalli NS ，Castellanos JA ，Lamichhane P ，Messaggio F ，Shi C ，Dai X ，Rai P ，Chen X ，VanSaun MN ，Merchant NB ... -  《-》

Inhibiting signal transducer and activator of transcription-3 increases response to gemcitabine and delays progression of pancreatic cancer.

Venkatasubbarao K ，Peterson L ，Zhao S ，Hill P ，Cao L ，Zhou Q ，Nawrocki ST ，Freeman JW ... -  《Molecular Cancer》