Michael B. Dwinell

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and β-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

Silencing of epithelial CXCL12 expression by DNA hypermethylation promotes colonic carcinoma metastasis

Cellular metastasis is the most detrimental step in carcinoma disease progression, yet the mechanisms that regulate this process are poorly understood. CXCL12 and its receptor CXCR4 are co-expressed in several tissues and cell types throughout the body and play essential roles in development. Disruption of either gene causes embryonic lethality due to similar defects. Post-natally, CXCL12 signaling has a wide range of effects on CXCR4-expressing cells, including the directed migration of leukocytes, lymphocytes and hematopoietic stem cells. Recently, this signaling axis has also been described as an important regulator of directed carcinoma cell metastasis. We show herein that while CXCR4 expression remains consistent, constitutive colonic epithelial expression of CXCL12 is silenced by DNA hypermethylation in primary colorectal carcinomas as well as colorectal carcinoma-derived cell lines. Inhibition of DNA methyltransferase (Dnmt) enzymes with 5-aza-2′-deoxycytidine or genetic ablation of both Dnmt1 and Dnmt3b prevented promoter methylation and restored CXCL12 expression. Re-expression of functional, endogenous CXCL12 in colorectal carcinoma cells dramatically reduced metastatic tumor formation in mice, as well as foci formation in soft agar. Decreased metastasis was correlated with increased caspase activity in cells re-expressing CXCL12. These data constitute the unique observation that silencing CXCL12 within colonic carcinoma cells greatly enhances their metastatic potential.

Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells

Expression of the chemokine receptor CXCR4 has been linked with increased metastasis and decreased clinical prognosis in breast cancer. The current paradigm dictates that CXCR4 fosters carcinoma cell metastasis along a chemotactic gradient to organs expressing the ligand CXCL12. The present study asked if alterations in autocrine CXCR4 signaling via dysregulation of CXCL12 in mammary carcinoma cells modulated their metastatic potential. While CXCR4 was consistently detected, expression of CXCL12 characteristic of human mammary epithelium was silenced by promoter hypermethylation in breast cancer cell lines and primary mammary tumors. Stable re-expression of functional CXCL12 in ligand null cells increased orthotopic primary tumor growth in the mammary fat-pad model of tumorigenesis. Those data parallel increased carcinoma cell proliferation measured in vitro with little-to-no-impact on apoptosis. Moreover, re-expression of autocrine CXCL12 markedly reduced metastatic lung invasion assessed using in vivo bioluminescence imaging following tail vein injection. Consistent with those data, decreased metastasis reflected diminished intracellular calcium signaling and chemotactic migration in response to exogenous CXCL12 independent of changes in CXCR4 expression. Together these data suggest that an elevated migratory signaling response to ectopic CXCL12 contributes to the metastatic potential of CXCR4-expressing mammary carcinoma cells, subsequent to epigenetic silencing of autocrine CXCL12.

Chemokines and chemokine receptors in mucosal homeostasis at the intestinal epithelial barrier in inflammatory bowel disease

Chemokines, a large family of small chemoattractive cytokines, and their receptors play an integral role in the regulation of the immune response and homeostasis. The ability of chemokines to attract specific populations of immune cells sets them apart from other chemoattractants. Chemokines produced within the gastrointestinal mucosa are critical players in directing the balance between physiological and pathophysiological inflammation in health, inflammatory bowel disease (IBD), and the progression to colon cancer. In addition to the well-characterized role of chemokines in directed trafficking of immune cells to the gut mucosa, the expression of chemokine receptors on the cells of the epithelium makes them active participants in the chemokine signaling network. Recent findings demonstrate an important role for chemokines and chemokine receptors in epithelial barrier repair and maintenance as well as an intricate involvement in limiting metastasis of colonic carcinoma. Increased recognition of the association between barrier defects and inflammation and the subsequent progression to cancer in IBD thus implicates chemokines as key regulators of mucosal homeostasis and disease pathogenesis.

Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and restitution in model intestinal epithelia.

Chemokines are critical regulatory factors that direct migration, proliferation and maturation of receptor expressing target cells within gut mucosa. The aim of the present study was to define the cellular mechanisms whereby engagement of the essential chemokine CXCL12 to CXCR4 regulates restitutive epithelial cell migration. Non-transformed IEC-6 cells or polarized T84 epithelial monolayers were wounded and F-actin accumulation assessed using fluorescence microscopy and flow cytometry. Immunoblot analysis, pull-down assays, fluorescence microscopy and wound healing assays defined activation of Rho, Rho-kinase (ROCK), and myosin light chain (MLC) and the role for those Rho effectors in CXCL12-regulated epithelial restitution. CXCL12 increased RhoGTP and F-actin localization to the leading edge of wounded IEC-6 and T84 monolayers. CXCL12 congruently stimulated an increase in active MLC that was inhibited by blockade of ROCK and myosin light chain kinase and regulated epithelial migration. Our data in model intestinal epithelia suggest CXCR4 and CXCL12 may function as an autocrine and paracrine mucosal signaling network regulating the competency of the epithelial barrier to withstand injury and mediate repair following damage.

CCR6 Regulation of the Actin Cytoskeleton Orchestrates Human Beta Defensin-2- and CCL20-mediated Restitution of Colonic Epithelial Cells

Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

BackgroundRecent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells.MethodsIn this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed.ResultsBoth Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer.ConclusionsWe conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.

Flagellin-independent regulation of chemokine host defense in Campylobacter jejuni-infected intestinal epithelium.

ABSTRACT Campylobacter jejuni is a leading cause of bacterial food-borne diarrheal disease throughout the world and the most frequent antecedent of autoimmune neuropathy Guillain-Barré syndrome. While infection is associated with immune memory, little is known regarding the role of the epithelium in targeting dendritic cells (DC) for initiating the appropriate adaptive immune response to C. jejuni. The objective of this study was to define the role for the intestinal epithelium in the induction of the adaptive immune response in C. jejuni infection by assessing the production of DC and T-cell chemoattractants. Human T84 epithelial cells were used as model intestinal epithelia. Infection of T84 cells with C. jejuni dose- and time-dependently up-regulated DC and T-cell chemokine gene transcription and secretion. Induction required live bacteria and was in the physiologically relevant direction for attraction of mucosal immunocytes. C. jejuni-activated NF-κB signaling was shown to be essential for proinflammatory chemokine secretion. Notably, C. jejuni secretion occurred independently of flagellin identification by Toll-like receptor 5. Secretion of a DC chemoattractant by differing clinical C. jejuni isolates suggested adherence/invasion were key virulence determinants of epithelial chemokine secretion. The regulated epithelial expression of DC and T-cell chemoattractants suggests a mechanism for the directed trafficking of immune cells required for the initiation of adaptive immunity in campylobacteriosis. Chemokine secretion occurs despite Campylobacter evasion of the flagellin pattern recognition receptor, suggesting that alternate host defense strategies limit disease pathogenesis.

An Adenosine-Mediated Signaling Pathway Suppresses Prenylation of the GTPase Rap1B and Promotes Cell Scattering

Inhibition of adenosine receptors could reduce metastasis by enhancing prenylation-dependent signaling that promotes cell-cell adhesion. Signal to Scatter An early step in metastasis is the dissociation of cancer cells from the primary tumor mass. When localized to cell membranes, the small guanosine triphosphatase (GTPase) Rap1B promotes cell-cell adhesion, a function that was blocked by a signaling pathway identified by Ntantie et al. Activation of adenosine A2B receptors reduced the prenylation of Rap1B, a posttranslational modification that enables Rap1B to localize to cell membranes, and resulted in reduced cell-cell adhesion. Because tumors release adenosine, these findings suggest that inhibition of adenosine receptors could reduce cancer cell metastasis by enabling the prenylation and cell membrane localization of Rap1B, thereby promoting cell-cell adhesion. During metastasis, cancer cells acquire the ability to dissociate from each other and migrate, which is recapitulated in vitro as cell scattering. The small guanosine triphosphatase (GTPase) Rap1 opposes cell scattering by promoting cell-cell adhesion, a function that requires its prenylation, or posttranslational modification with a carboxyl-terminal isoprenoid moiety, to enable its localization at cell membranes. Thus, signaling cascades that regulate the prenylation of Rap1 offer a mechanism to control the membrane localization of Rap1. We identified a signaling cascade initiated by adenosine A2B receptors that suppressed the prenylation of Rap1B through phosphorylation of Rap1B, which decreased its interaction with the chaperone protein SmgGDS (small GTPase guanosine diphosphate dissociation stimulator). These events promoted the cytosolic and nuclear accumulation of nonprenylated Rap1B and diminished cell-cell adhesion, resulting in cell scattering. We found that nonprenylated Rap1 was more abundant in mammary tumors than in normal mammary tissue in rats and that activation of adenosine receptors delayed Rap1B prenylation in breast, lung, and pancreatic cancer cell lines. Our findings support a model in which high concentrations of extracellular adenosine, such as those that arise in the tumor microenvironment, can chronically activate A2B receptors to suppress Rap1B prenylation and signaling at the cell membrane, resulting in reduced cell-cell contact and promoting cell scattering. Inhibiting A2B receptors may be an effective method to prevent metastasis.

Constitutive CXCL12 Expression Induces Anoikis in Colorectal Carcinoma Cells

BACKGROUND & AIMS CXCL12 and CXCR4 signaling plays critical roles in development, homeostasis, and tumor metastasis. Previously, we have shown that epigenetic silencing of CXCL12 in colorectal and mammary carcinomas promotes metastasis. Anoikis is an essential process of colonic epithelial turnover and limits the metastatic progression of carcinoma. We sought to determine the role for anoikis in limiting tumor metastasis following reexpression of CXCL12 in human colorectal carcinoma cells. METHODS Tumor formation and metastasis of colonic carcinoma cells was monitored using in vivo bioluminescence imaging. Anoikis was defined by using caspase-3/7, focal adhesion kinase (FAK) and p130Cas cleavage, DNA fragmentation, and cell survival assays. Phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) was monitored by immunoblot and immunohistochemistry, and activity was inhibited by using U0126. RESULTS Constitutive expression of CXCL12 in human colorectal carcinoma cells reduced orthotopic tumor formation and inhibited metastasis in severe combined immunodeficient mice. Further, CXCL12 expression induced apoptosis specifically in nonadherent colorectal carcinoma cells. Apoptotic cell death was preceded by hypophosphorylation and cleavage of FAK and p130Cas, leading to increased cellular detachment in culture, and depended on alterations in the extracellular matrix. Similar to in vivo colonic epithelium, CXCL12-induced anoikis of carcinoma cells depended on basal ERK1/2 activation. CONCLUSIONS These data significantly expand the current paradigm of chemokine signaling in carcinogenesis by showing that endogenous CXCL12, in marked contrast to exogenous ligand, inhibits tumor metastasis through increased anoikis. Altered ERK1/2 signaling provides a mechanism for the dichotomy between the physiologic and pathophysiologic roles of CXCL12-CXCR4 signaling in the intestinal epithelium.