CXCL12 Chemokine Expression and Secretion Regulates Colorectal Carcinoma Cell Anoikis through Bim-Mediated Intrinsic Apoptosis

BACKGROUND: Resistance to anoikis, apoptosis triggered by a loss of cellular adhesion to the underlying extracellular matrix, is a hallmark of metastatic cancer. Previously we have shown re-establishment of CXCL12 expression in colorectal carcinoma cells inhibits metastasis by enhancing anoikis sensitivity. The objective of these studies was to define the signaling mechanisms regulating CXCL12-mediated anoikis. METHODOLOGY/PRINCIPAL FINDINGS: Adhesion, examined by crystal violet staining, immunofluorescence microscopy, and immunoblot analysis indicated decreased focal adhesion signaling corresponding with loss of adhesion in cells constitutively simulated by CXCL12. Loss of adhesion was inhibited by pertussis toxin treatment, indicating CXCL12 regulating anoikis through G(αi)-protein coupled receptors. Non-adherent HCT116 and HT29 colorectal carcinoma cells expressing CXCL12 exhibited enhanced anoikis sensitivity by propidium iodide staining, caspase activity assays, and immunoblot compared to GFP control cells. CXCL12 producing carcinomas cultured on poly-HEMA displayed heightened Bim and loss of Mcl-1 and Bcl-2 preceding cytochrome c release, and caspase-9 activation. RNAi knockdown of Bim reversed anoikis sensitivity of CXCL12-expressing cells and fostered increased soft-agar foci formation and hepatic tumors in an orthotopic mouse model of metastasis. CONCLUSIONS/SIGNIFICANCE: These data indicate CXCL12 provides a barrier to metastasis by increasing anoikis via activation of a Bim-mediated intrinsic apoptotic pathway. These results underscore the importance of retaining CXCL12 expression to sensitize colorectal carcinomas to anoikis and minimize tumor progression

CXCR4 Negatively Regulates Keratinocyte Proliferation in IL-23-Mediated Psoriasiform Dermatitis

CXCR4 is expressed by basal keratinocytes (KCs), but little is known about its function in inflamed skin. We crossed K14-Cre and CXCR4flox/flox (f/f) transgenic mice, resulting in mice with specific loss of the CXCR4 gene in K14-expressing cells (K14-CXCR4KO), including basal KCs. K14-CXCR4KO pups had no obvious skin defects. We compared K14-CXCR4KO and CXCR4f/f control mice in an IL-23-mediated psoriasiform dermatitis model and measured skin edema, and histologic and immunohistological changes. IL-23-treated K14-CXCR4KO mice showed a 1.3-fold increase in mean ear swelling, a 2-fold increase in epidermal thickness, and greater parakeratosis. IL-23-treated wild-type (WT) mice showed weak CXCR4 expression in areas of severe epidermal hyperplasia, but strong CXCR4 expression in nonhyperplastic regions, suggesting that CXCR4 may regulate KC proliferation. To test this hypothesis, we overexpressed CXCR4 in HaCaT KC cells and treated them with IL-22 and/or CXCL12 (chemokine (C-X-C motif) ligand 12). CXCL12 blocked IL-22-mediated HaCaT cell proliferation in vitro and synergized with IL-22 in upregulating SOCS3 (suppressor of cytokine signaling 3), a key regulator of STAT3 (signal transducer and activator of transcription 3). SOCS3 was required for CXCR4-mediated growth inhibition. In human psoriatic skin, both CXCR4 and SOCS3 were upregulated in the junctional region at the border of psoriatic plaques. Thus, CXCR4 has an unexpected role in inhibiting KC proliferation and mitigating the effects of proliferative T helper type 17 cytokines

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC

Flagellin-Independent Regulation of Chemokine Host Defense in Campylobacter jejuni-Infected Intestinal Epithelium

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