Ishan Roy

CXCL12 chemokine expression suppresses human pancreatic cancer growth and metastasis.

Pancreatic ductal adenocarcinoma is an unsolved health problem with nearly 75% of patients diagnosed with advanced disease and an overall 5-year survival rate near 5%. Despite the strong link between mortality and malignancy, the mechanisms behind pancreatic cancer dissemination and metastasis are poorly understood. Correlative pathological and cell culture analyses suggest the chemokine receptor CXCR4 plays a biological role in pancreatic cancer progression. In vivo roles for the CXCR4 ligand CXCL12 in pancreatic cancer malignancy were investigated. CXCR4 and CXCR7 were consistently expressed in normal and cancerous pancreatic ductal epithelium, established cell lines, and patient-derived primary cancer cells. Relative to healthy exocrine ducts, CXCL12 expression was pathologically repressed in pancreatic cancer tissue specimens and patient-derived cell lines. To test the functional consequences of CXCL12 silencing, pancreatic cancer cell lines stably expressingthe chemokine were engineered. Consistent with a role for CXCL12 as a tumor suppressor, cells producing the chemokine wereincreasingly adherent and migration deficient in vitro and poorly metastatic in vivo, compared to control cells. Further, CXCL12 reintroduction significantly reduced tumor growth in vitro, with significantly smaller tumors in vivo, leading to a pronounced survival advantage in a preclinical model. Together, these data demonstrate a functional tumor suppressive role for the normal expression of CXCL12 in pancreatic ducts, regulating both tumor growth andcellulardissemination to metastatic sites.

Chemokines and chemokine receptors: Update on utility and challenges for the clinician

Chemokines, or chemotactic cytokines, represent alarge family of secreted proteins that have a wide range of function in normal physiology. Chemokine functions include direction of immune cell trafficking, angiogenesis, and wound healing, all critically important to patients being considered for surgery or anticancer therapy. In recent years, perturbations in expression of chemokines or their cognate receptors have been associated with several inflammatory disorders and both solid-tumor and hematologic malignancies. As such, chemokines and chemokine receptors have made attractive targets for biomarker identification and drug discovery. To move chemokine laboratory science to the bedside application for biomarker and drug discovery, we will need a more detailed understanding of the mechanisms by which chemokines are expressed and function. Specific areas that require further study include the structure-function relationship of chemokines and their receptors, differential signaling of ligands, concentration gradient-dependent signaling, and the genetic or epigenetic mechanisms that regulate chemokine ligand or receptor gene expression. Chemokines provide a powerful opportunity for translational investigation. Chemokines are secreted proteins that travel in the circulation, move through the parenchyma and extracellular matrix of tissues, and bind to and activate the extracellular domain of their cognate receptors present on individual cell types. Consequently, chemokines can serve both as potential biomarkers and as promising targets for pharmaceutical intervention. Similar to other cytokines, chemokines altered in expression during specific disease states can serve as useful diagnostic or prognostic biomarkers. For example, chemokines such as CCL2, CCL5, and CCL20 are potential candidate biomarkers in atherosclerosis, diabetes, and inflammatory diseases of the skin and gut. Herein, we outline the numerous inflammatory diseases and cancers associated with aberrant chemokine expression and activity (Table). Our laboratory is currently investigating the role of several chemokines in pancreatic cancer biology, where inflammation is a key component of tumor initiation, growth, and possibly metastasis. We also discuss the existing opportunities for pharmaceutic targeting of aberrant chemokine activity to alleviate disease. In particular, the chemokine CXCL12 is a potential target for pharmaceutic intervention in several malignancies, including colorectal, breast, and lung cancer. Finally, we explore the emerging pharmaceutic strategies to target more specifically chemokine signaling, leading to a more favorable therapeutic profile. Table Changes in chemokine ligand and receptor expression in human disease

Structural basis for chemokine recognition by a G protein–coupled receptor and implications for receptor activation

Structural analysis of the interactions between a receptor and monomeric or dimeric forms of its ligand may aid in drug design. How receptors view monomers versus dimers Chemokines are proteins that stimulate cell migration in processes such as development, immune responses, and metastasis. Monomeric, dimeric, and oligomeric forms of chemokines can engage their cognate G protein–coupled receptors. Both the G protein–dependent and β-arrestin–dependent signaling pathways downstream of chemokine receptors must be activated to induce cell migration. Previous studies showed that a locked dimeric form of CXCL12 (LD CXCL12) fails to activate β-arrestin–dependent signaling after binding to its receptor CXCR4. Ziarek et al. solved the NMR structure of CXCR4 bound to a locked monomeric form of CXCL12 (LM CXCL12). LM CXCL12 physically interacted with the receptor differently than did the dimeric chemokine, and it stimulated both CXCR4-dependent signaling pathways to induce migration. Analysis of a hybrid NMR- and x-ray–based structure provided insights into the conformational changes required for chemokine receptor signaling, which may aid in designing drugs to target the chemokine family. Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein–dependent and β-arrestin–dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein–dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance–based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical “two-site model” for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor–mediated signal transduction.

Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells

Aggressive dissemination and metastasis of pancreatic ductal adenocarcinoma (PDAC) results in poor prognosis and marked lethality. Rho monomeric G protein levels are increased in pancreatic cancer tissue. As the mechanisms underlying PDAC malignancy are little understood, we investigated the role for cAMP in regulating monomeric G protein regulated invasion and migration of pancreatic cancer cells. Treatment of PDAC cells with cAMP elevating agents that activate adenylyl cyclases, forskolin, protein kinase A (PKA), 6‐Bnz‐cAMP, or the cyclic nucleotide phosphodiesterase inhibitor cilostamide significantly decreased migration and Matrigel invasion of PDAC cell lines. Inhibition was dose‐dependent and not significantly different between forskolin or cilostamide treatment. cAMP elevating drugs not only blocked basal migration, but similarly abrogated transforming‐growth factor‐β‐directed PDAC cell migration and invasion. The inhibitory effects of cAMP were prevented by the pharmacological blockade of PKA. Drugs that increase cellular cAMP levels decreased levels of active RhoA or RhoC, with a concomitant increase in phosphorylated RhoA. Diminished Rho signaling was correlated with the appearance of thickened cortical actin bands along the perimeter of non‐motile forskolin or cilostamide‐treated cells. Decreased migration did not reflect alterations in cell growth or programmed cell death. Collectively these data support the notion that increased levels of cAMP specifically hinder PDAC cell motility through F‐actin remodeling.

Pancreatic Cancer Cell Migration and Metastasis Is Regulated by Chemokine-Biased Agonism and Bioenergetic Signaling

Patients with pancreatic ductal adenocarcinoma (PDAC) invariably succumb to metastatic disease, but the underlying mechanisms that regulate PDAC cell movement and metastasis remain little understood. In this study, we investigated the effects of the chemokine gene CXCL12, which is silenced in PDAC tumors, yet is sufficient to suppress growth and metastasis when re-expressed. Chemokines like CXCL12 regulate cell movement in a biphasic pattern, with peak migration typically in the low nanomolar concentration range. Herein, we tested the hypothesis that the biphasic cell migration pattern induced by CXCL12 reflected a biased agonist bioenergetic signaling that might be exploited to interfere with PDAC metastasis. In human and murine PDAC cell models, we observed that nonmigratory doses of CXCL12 were sufficient to decrease oxidative phosphorylation and glycolytic capacity and to increase levels of phosphorylated forms of the master metabolic kinase AMPK. Those same doses of CXCL12 locked myosin light chain into a phosphorylated state, thereby decreasing F-actin polymerization and preventing cell migration in a manner dependent upon AMPK and the calcium-dependent kinase CAMKII. Notably, at elevated concentrations of CXCL12 that were insufficient to trigger chemotaxis of PDAC cells, AMPK blockade resulted in increased cell movement. In two preclinical mouse models of PDAC, administration of CXCL12 decreased tumor dissemination, supporting our hypothesis that chemokine-biased agonist signaling may offer a useful therapeutic strategy. Our results offer a mechanistic rationale for further investigation of CXCL12 as a potential therapy to prevent or treat PDAC metastasis.

CXM: A New Tool for Mapping Breast Cancer Risk in the Tumor Microenvironment

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3(IL2Rγ) consomic model compared with the SS(IL2Rγ) parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3(IL2Rγ) background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk.

Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

The mechanisms by which the extreme desmoplasia observed in pancreatic tumors develops remain unknown and its role in pancreatic cancer progression is unsettled. Chemokines have a key role in the recruitment of a wide variety of cell types in health and disease. Transcript and protein profile analyses of human and murine cell lines and human tissue specimens revealed a consistent elevation in the receptors CCR10 and CXCR6, as well as their respective ligands CCL28 and CXCL16. Elevated ligand expression was restricted to tumor cells, whereas receptors were in both epithelial and stromal cells. Consistent with its regulation by inflammatory cytokines, CCL28 and CCR10, but not CXCL16 or CXCR6, were upregulated in human pancreatitis tissues. Cytokine stimulation of pancreatic cancer cells increased CCL28 secretion in epithelial tumor cells but not an immortalized activated human pancreatic stellate cell line (HPSC). Stellate cells exhibited dose- and receptor-dependent chemotaxis in response to CCL28. This functional response was not linked to changes in activation status as CCL28 had little impact on alpha smooth muscle actin levels or extracellular matrix deposition or alignment. Co-culture assays revealed CCL28-dependent chemotaxis of HPSC toward cancer but not normal pancreatic epithelial cells, consistent with stromal cells being a functional target for the epithelial-derived chemokine. These data together implicate the chemokine CCL28 in the inflammation-mediated recruitment of cancer-associated stellate cells into the pancreatic cancer parenchyma.

Chemokines in colitis: microRNA control

Huang et al 1 identified and tested the role of a specific microRNA (miRNA) in the pathogenesis of IBD. The study of microRNAs is a burgeoning field within epigenetics. These small non-coding RNAs mediate translation-level repression of protein expression by binding to the 3′ -untranslated region of specific messenger RNA transcripts. In the innate and adaptive immune response, miRNAs play an important role in negative regulation of inflammatory conditions in the intestine. Inflammatory regulators such as IL-6, tumor necrosis factor (TNF) and toll-like receptors have been shown to induce miRNA expression in both acute and chronic inflammation. The roles for miRNAs in IBD are emerging from recent studies that compare miRNA expression in colonoscopic and peripheral blood draw biopsies from colitis patients with healthy individuals.2 Despite this, the vast majority of miRNAs identified in microarray analyses of colitis patients have yet to be investigated in experimental models of colitis or assigned specific mechanisms in the pathophysiology of human disease. In a novel approach, Huang et al 1 used array analyses to assay changes in microRNA expression from two different experimental models of colitis in order to map specific overlapping miRNA expression patterns, which were subsequently compared against analyses completed on human colitis patient specimens. These authors then continued their analyses to mechanistically determine that miR-141, a miRNA aberrantly expressed in both animal models of colitis and human patients, specifically inhibited expression of the β-isoform of CXCL12, a …

Exploiting agonist biased signaling of chemokines to target cancer

As knowledge of growth‐independent functions of cancer cells is expanding, exploration into the role of chemokines in modulating cancer pathogenesis, particularly metastasis, continues to develop. However, more study into the mechanisms whereby chemokines direct the migration of cancer cells is needed before specific therapies can be generated to target metastasis. Herein, we draw attention to the longstanding conundrum in the field of chemokine biology that chemokines stimulate migration in a biphasic manner; and explore this phenomenons impact on chemokine function in the context of cancer. Typically, low concentrations of chemokines lead to chemotactic migration and higher concentrations halt migration. The signaling mechanisms that govern this phenomenon remain unclear. Over the last decade, we have defined a novel signaling mechanism for regulation of chemokine migration through ligand oligomerization and biased agonist signaling. We provide insight into this new paradigm for chemokine signaling and discuss how it will impact future exploration into chemokine function and biology. In the pursuit of producing more novel cancer therapies, we suggest a framework for pharmaceutical application of the principles of chemokine oligomerization and biased agonist signaling in cancer.

Abstract A21: Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma

Multiple studies have shown that the extreme desmoplasia observed in pancreatic tumors modulates the effectiveness of chemotherapeutic agents. Despite this, the specific mechanisms by which stroma develops in pancreatic tumors are unknown and the role that it plays in pathogenesis of the disease is unsettled. Chemokines are a family of nearly 50 different secreted proteins playing a key role in the recruitment of a wide variety of cell types in health and disease. While we and others have extensively explored the role of the chemokine CXCL12 in pancreatic cancer, the expression and potential role of other chemokines, or their cognate receptors, in pancreatic cancer remains an area of active exploration. To this end, we profiled the expression of the chemokine family in pancreatic cancer. RT-PCR of a battery of established human pancreatic cancer cell lines revealed elevated transcript levels of the chemokine ligands CCL28 and CXCL16, as well as their cognate receptors CCR10 and CXCR6, respectively. These results were mirrored in cell lines derived from transgenic murine models of pancreatic cancer. Immunohistochemical analysis of both diseased and normal human pancreatic tissue showed that the expression of CCL28, CXCL16, CCR10, and CXCR6 proteins were upregulated in pancreatic adenocarcinoma compared with normal exocrine ductal epithelium. Interestingly, CCR10 and CXCR6 were also expressed in pancreatic tumor fibroblasts, while their cognate ligands, CCL28 and CXCL16 were absent in the stromal compartment. Based on those data we hypothesized that expression of chemokines in cancer cells with the concomitant expression of their cognate receptors by activated fibroblasts facilitates recruitment of those cancer-associated fibroblasts into the tumor microenvironment. Analysis of tissue from patients with pancreatitis revealed that CCL28/CCR10, but not CXCL16/CXCR6, proteins were upregulated. Consistent with its known regulation by inflammatory mediators, we found that interferon-gamma stimulated significant increases in CCL28 secretion by human pancreatic cancer cell lines. In contrast, interferon-gamma had little if any effect on CCL28 secretion by an immortalized activated human pancreatic stellate cell (HPSC) line. Stimulation with exogenous CCL28 elicited the prototypical dose dependent calcium signaling expected of a ligand activated chemokine receptor. CCL28 stimulated the dose dependent directional migration of HPSC without altering cell viability. These data are consistent with HPSC being a functional target for the chemokine. Finally, in a 2-D co-culture model, CCL28 secreted from human pancreatic cancer cells induced HPSC chemotactic migration. Pretreatment with neutralizing antibody to the chemokine blocked HPSC chemotaxis toward the cancer cells. Together, these data suggest that the CCL28-CCR10 chemokine axis plays a role in the inflammation-mediated recruitment of cancer-associated fibroblasts into the stromal compartment of pancreatic tumors. More broadly, we have demonstrated a new role for chemokines in the pancreatic tumor microenvironment, suggestive of their potential role in stromal remodeling in pancreatic cancer. This is the first study to establish the differential expression and functional role for the CCL28 chemokine in pancreatic disease. Citation Format: Ishan Roy, Kathleen A. Boyle, A Craig Mackinnon, Rosa F. Hwang, Susan Tsai, Douglas B. Evans, Michael B. Dwinell.{Authors}. Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A21.