CREB activation drives acinar to ductal reprogramming and promote pancreatic cancer progression in animal models of alcoholic chronic pancreatitis.

BACKGROUND AND AIMS: In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic Kras G12D/+ ( Kras* ) in promoting pancreatic cancer progression with ACP remains unexplored. METHODS: Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion ( Creb fl/fl ) in Ptf1a CreERTM/+ ;LSL-Kras G12D+/- (KC) genetic mouse models (KCC -/- ). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1a CreERTM+/- , KC and KCC -/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. RESULTS: ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. CONCLUSIONS: Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.

Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC.

Background: CXCR1/2 inhibitors are being implemented with immunotherapies in PDAC clinical trials. Cytokines responsible for stimulating these receptors include CXCL ligands, typically secreted by activated immune cells, fibroblasts, and even adipocytes. Obesity has been linked to poor patient outcome and altered anti-tumor immunity. Adipose-derived cytokines and chemokines have been implicated as potential drivers of tumor cell immune evasion, suggesting a possibility of susceptibility to targeting specifically in the context of obesity. Methods: RNA-sequencing of human PDAC cell lines was used to assess differential influences on the cancer cell transcriptome after treatment with conditioned media from peri-pancreatic adipose tissue of lean and obese PDAC patients. The adipose-induced secretome of PDAC cells was then assessed by cytokine arrays and ELISAs. Lentiviral transduction and CRISPR-Cas9 was used to knock out CXCL5 from a murine PDAC cell line for orthotopic tumor studies in diet-induced obese, syngeneic mice. Flow cytometry was used to define the immune profiles of tumors. Anti-PD-1 immune checkpoint blockade therapy was administered to alleviate T cell exhaustion and invoke an immune response, while the mice were monitored at endpoint for differences in tumor size. Results: The chemokine CXCL5 was secreted in response to stimulation of PDAC cells with human adipose conditioned media (hAT-CM). PDAC CXCL5 secretion was induced by either IL-1ß or TNF, but neutralization of both was required to limit secretion. Ablation of CXCL5 from tumors promoted an immune phenotype susceptible to PD-1 inhibitor therapy. While application of anti-PD-1 treatment to control tumors failed to alter tumor growth, knockout CXCL5 tumors were diminished. Conclusions: In summary, our findings show that known adipokines TNF and IL-1ß can stimulate CXCL5 release from PDAC cells in vitro. In vivo , CXCL5 depletion alone is sufficient to promote T cell infiltration into tumors in an obese setting, but requires checkpoint blockade inhibition to alleviate tumor burden. DATA AVAILABILITY STATEMENT: Raw and processed RNAseq data will be further described in the GEO accession database ( awaiting approval from GEO for PRJ number ). Additional raw data is included in the supplemental material and available upon reasonable request. WHAT IS ALREADY KNOWN ON THIS TOPIC: Obesity is linked to a worsened patient outcome and immunogenic tumor profile in PDAC. CXCR1/2 inhibitors have begun to be implemented in combination with immune checkpoint blockade therapies to promote T cell infiltration under the premise of targeting the myeloid rich TME. WHAT THIS STUDY ADDS: Using in vitro/ex vivo cell and tissue culture-based assays with in vivo mouse models we have identified that adipose derived IL-1ß and TNF can promote tumor secretion of CXCL5 which acts as a critical deterrent to CD8 T cell tumor infiltration, but loss of CXCL5 also leads to a more immune suppressive myeloid profile. HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: This study highlights a mechanism and emphasizes the efficacy of single CXCR1/2 ligand targeting that could be beneficial to overcoming tumor immune-evasion even in the obese PDAC patient population.

Combined PI3K and MAPK inhibition synergizes to suppress PDAC.

Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS as well as its target MAPK pathway effectors have shown limited success due to the difficulty to pharmacologically target KRAS, inherent drug resistance in PDAC cells, and acquired resistance through activation of alternative mitogenic pathways such JAK-STAT and PI3K-AKT. While KRAS canonically drives the MAPK signaling pathway via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Our therapeutic study targeted the PI3K-AKT pathway with the drug Omipalisib (p110α/ß/δ/γ and mTORC1/2 inhibitor) in combination with MAPK pathway targeting drug Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099), which is an inhibitor of the KRAS effector SHP2. Western blot analysis demonstrated that application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT) and in some instances increased pAKT levels. Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MEK and PI3K-AKT, to more effectively suppress pancreatic cancer. In vitro studies demonstrated that both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were generally more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. Additionally, we found that while combination Omipalisib/SHP099 treatment reduced implanted tumor growth in vivo , the Omipalisib/Trametinib treatment was significantly more effective. Therefore, we additionally tested the Omipalisib/Trametinib combination therapeutic in the highly aggressive PKT (Ptf1a cre , LSL-Kras G12D , TGFbR2 fl/fl ) spontaneous mouse model of PDAC. We subsequently found that PKT mice treated with the Omipalisib/Trametinib combination therapeutic survived significantly longer than mice treated with either drug alone, and more than doubled the mean survival time of vehicle control mice. Altogether, our data support the importance of a dual treatment strategy targeting both MAPK and PI3K-AKT pathways.

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-ß-Cyclodextrin against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin's clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-ß-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients.

SHP2 regulates adipose maintenance and adipocyte-pancreatic cancer cell crosstalk via PDHA1.

Adipocytes are the most abundant cell type in the adipose tissue, and their dysfunction is a significant driver of obesity-related pathologies, such as cancer. The mechanisms that (1) drive the maintenance and secretory activity of adipocytes and (2) mediate the cancer cellular response to the adipocyte-derived factors are not fully understood. To address that gap of knowledge, we investigated how alterations in Src homology region 2-containing protein (SHP2) activity affect adipocyte function and tumor crosstalk. We found that phospho-SHP2 levels are elevated in adipose tissue of obese mice, obese patients, and differentiating adipocytes. Immunofluorescence and immunoprecipitation analyses as well as in-silico protein-protein interaction modeling demonstrated that SHP2 associates with PDHA1, and that a positive association promotes a reactive oxygen species (ROS)-driven adipogenic program. Accordingly, this SHP2-PDHA1-ROS regulatory axis was crucial for adipocyte maintenance and secretion of interleukin-6 (IL-6), a key cancer-promoting cytokine. Mature adipocytes treated with an inhibitor for SHP2, PDHA1, or ROS exhibited an increased level of pro-lipolytic and thermogenic proteins, corresponding to an increased glycerol release, but a suppression of secreted IL-6. A functional analysis of adipocyte-cancer cell crosstalk demonstrated a decreased migration, invasion, and a slight suppression of cell cycling, corresponding to a reduced growth of pancreatic cancer cells exposed to conditioned media (CM) from mature adipocytes previously treated with inhibitors for SHP2/PDHA1/ROS. Importantly, PDAC cell growth stimulation in response to adipocyte CM correlated with PDHA1 induction but was suppressed by a PDHA1 inhibitor. The data point to a novel role for (1) SHP2-PDHA1-ROS in adipocyte maintenance and secretory activity and (2) PDHA1 as a regulator of the pancreatic cancer cells response to adipocyte-derived factors.

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.

Synthetic adiponectin-receptor agonist, AdipoRon, induces glycolytic dependence in pancreatic cancer cells.

Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.

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.

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.

Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer.

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 Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/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.

Tobacco Carcinogen-Induced Production of GM-CSF Activates CREB to Promote Pancreatic Cancer.

Although smoking is a significant risk factor for pancreatic ductal adenocarcinoma (PDAC), the molecular mechanisms underlying PDAC development and progression in smokers are still unclear. Here, we show the role of cyclic AMP response element-binding protein (CREB) in the pathogenesis of smoking-induced PDAC. Smokers had significantly higher levels of activated CREB when compared with nonsmokers. Cell lines derived from normal pancreas and pancreatic intraepithelial neoplasm (PanIN) exhibited low baseline pCREB levels compared with PDAC cell lines. Furthermore, elevated CREB expression correlated with reduced survival in patients with PDAC. Depletion of CREB significantly reduced tumor burden after tobacco-specific nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment, suggesting a CREB-dependent contribution to PDAC growth and progression in smokers. Conversely, NNK accelerated PanIN lesion and PDAC formation via GM-CSF-mediated activation of CREB in a PDAC mouse model. CREB inhibition (CREBi) in mice more effectively reduced primary tumor burden compared with control or GM-CSF blockade alone following NNK exposure. GM-CSF played a role in the recruitment of tumor-associated macrophages (TAM) and regulatory T cell (Treg) expansion and promotion, whereas CREBi significantly reduced TAM and Treg populations in NNK-exposed mice. Overall, these results suggest that NNK exposure leads to activation of CREB through GM-CSF, promoting inflammatory and Akt pathways. Direct inhibition of CREB, but not GM-CSF, effectively abrogates these effects and inhibits tumor progression, offering a viable therapeutic strategy for patients with PDAC.Significance: These findings identify GM-CSF-induced CREB as a driver of pancreatic cancer in smokers and demonstrate the therapeutic potential of targeting CREB to reduce PDAC tumor growth.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6146/F1.large.jpg Cancer Res; 78(21); 6146-58. ©2018 AACR.

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.

Adiponectin receptor agonists inhibit leptin induced pSTAT3 and in vivo pancreatic tumor growth.

Obesity is a significant risk factor for pancreatic cancer, harboring a chronic inflammatory condition characterized by dysregulation of the adipokines, leptin and adiponectin, that in turn alter oncogenic signaling pathways. We and others have shown that leptin promotes the proliferation and an invasive potential of pancreatic cancer cells through STAT3 mediated signaling. However, the role of adiponectin on the tumorigenicity of pancreatic cancer has not been elucidated. Adiponectin represents an important negative regulator of cytokines, which acts through two receptors, ADIPOR1 and ADIPOR2, to elicit pro-apoptotic, anti-inflammatory, and anti-angiogenic responses. We show that the level and expression of both adiponectin receptors are decreased in pancreatic tumors relative to normal pancreatic tissue. In vitro stimulation with adiponectin or a small molecule adiponectin receptor agonist, AdipoRon, increases apoptosis while inhibiting pancreatic cancer cell proliferation, colony formation, and anchorage independent growth. In addition, adiponectin receptor agonism inhibits leptin mediated STAT3 activation. In vivo, treatment of mice with AdipoRon inhibits orthotopic pancreatic tumor growth. These results demonstrate that adiponectin receptor activation is a key regulator of pancreatic cancer growth and AdipoRon provides a rational agent for the development of novel therapeutic strategies for pancreatic cancer.

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.

Modulation of the leptin receptor mediates tumor growth and migration of pancreatic cancer cells.

Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.

Host and tumor derived MMP13 regulate extravasation and establishment of colorectal metastases in the liver.

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the United States and worldwide. Our studies have previously shown an increase in metastatic burden in steatotic vs. normal livers using a mouse model of diet induced steatosis. In the present study we aim to identify and evaluate the molecular factors responsible for this increase in tumor burden. METHODS: We assessed changes in expression of a panel of matrix metalloproteinases (MMPs) using qRT-PCR between normal and steatotic livers and validated them with western blot analysis of protein levels. To evaluate the role of MMP13 on tumor development, we utilized a splenic injection model of liver metastasis in Wildtype and Mmp13 deficient mice, using either parental or stable Mmp13 knockdown cell lines. Further, to evaluate changes in the ability of tumor cells to extravasate we utilized whole organ confocal microscopy to identify individual tumor cells relative to the vasculature. MTT, migration and invasion assays were performed to evaluate the role of tumor derived MMP13 on hallmarks of cancer in vitro. RESULTS: We found that MMP13 was significantly upregulated in the steatotic liver both in mice as well as human patients with NAFLD. We showed a decrease in metastatic tumor burden in Mmp13-/- mice compared to wildtype mice, explained in part by a reduction in the number of tumor cells extravasating from the hepatic vasculature in the Mmp13-/- mice compared to wildtype mice. Additionally, loss of tumor derived MMP13 through stable knockdown in tumor cell lines lead to decreased migratory and invasive properties in vitro and metastatic burden in vivo. CONCLUSIONS: This study demonstrates that stromal as well as tumor derived MMP13 contribute to tumor cell extravasation and establishment of metastases in the liver microenvironment.

New tools for the quantitative assessment of prodrug delivery and neurotoxicity.

Systemic off-target toxicities, including neurotoxicity, are prevalent side effects in cancer patients treated with a number of otherwise highly efficacious anticancer drugs. In the current study, we have: (1) developed a new analytical metric for the in vivo preclinical assessment of systemic toxicities/neurotoxicity of new drugs and delivery systems; and (2) evaluated, in mice, the in vivo efficacy and toxicity of a versatile and modular NanoDendron (ND) drug delivery and imaging platform that we recently developed. Our paclitaxel-carrying ND prodrug, ND(PXL), is activated following proteolytic cleavage by MMP9, resulting in localized cytotoxic chemotherapy. Using click chemistry, we combined ND(PXL) with a traceable beacon, ND(PB), yielding ND(PXL)-ND(PB) that functions as a theranostic compound. In vivo fluorescence FRET imaging of this theranostic platform was used to confirm localized delivery to tumors and to assess the efficiency of drug delivery to tumors, achieving 25-30% activation in the tumors of an immunocompetent mouse model of breast cancer. In this model, ND-drug exhibited anti-tumor efficacy comparable to nab-paclitaxel, a clinical formulation. In addition, we combined neurobehavioral metrics of nociception and sensorimotor performance of individual mice to develop a novel composite toxicity score that reveals and quantifies peripheral neurotoxicity, a debilitating long-term systemic toxicity of paclitaxel therapy. Importantly, mice treated with nab-paclitaxel developed changes in behavioral metrics with significantly higher toxicity scores indicative of peripheral neuropathy, while mice treated with ND(PXL) showed no significant changes in behavioral responses or toxicity score. Our ND formulation was designed to be readily adaptable to incorporate different drugs, imaging modalities and/or targeting motifs. This formulation has significant potential for preclinical and clinical tools across multiple disease states. The studies presented here report a novel toxicity score for assessing peripheral neuropathy and demonstrate that our targeted, theranostic NDs are safe and effective, providing localized tumor delivery of a chemotherapeutic and with reduced common neurotoxic side-effects.

Hepatocellular proliferation correlates with inflammatory cell and cytokine changes in a murine model of nonalchoholic fatty liver disease.

Nonalchoholic fatty liver disease (NAFLD) is a problem of increasing prevalence and clinical significance worldwide and is associated with increased risk of development of end stage liver disease and cirrhosis, and can be complicated by hepatocellular carcinoma (HCC). NAFLD is characterized by physical and molecular changes in the liver microenvironment which include an influx of inflammatory cell populations, fibrosis, changes in gene expression, and cytokine production. To better understand changes to the liver in the setting of steatosis, we used a murine model of diet induced hepatic steatosis and corroborated our results with human patient samples of NAFLD. Among the cellular changes, we identified a significant increase in hepatocellular proliferation in the setting of steatosis as compared to controls. Analysis of inflammatory cell populations revealed increased infiltration of CD11b positive myeloid and CD3 positive lymphocytic cell populations in steatotic livers compared to normal livers. Resident Kupffer cells of the liver comprise the largest percentage of these myeloid cells and appear to be responsible for important cytokine alterations impacting proliferation of cells in the liver microenvironment. Significant alterations in cytokine profiles in the plasma and liver tissue lysates from normal and steatotic mice were detected including leptin, CXCL1, CXCL2, and CXCL16 that were further shown to directly increase hepatocyte proliferation in vitro. This increased hepatocellular proliferation and turnover in the setting of steatosis may play important roles in the progression and complications of NAFLD.

Targeting the Wnt pathway in synovial sarcoma models.

UNLABELLED: Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/ß-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of ß-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/ß-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/ß-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents. SIGNIFICANCE: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT­SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans.