Stromal-MDM2 Promotes Lung Cancer Cell Invasion through Tumor-Host Feedback Signaling.

Tumor-host interactions play a major role in malignancies' initiation and progression. We have reported in the past that tumor cells attenuate genotoxic stress-induced p53 activation in neighboring stromal cells. Herein, we aim to further elucidate cancer cells' impact on signaling within lung cancer stroma. Primary cancer-associated fibroblasts were grown from resected human lung tumors. Lung cancer lines as well as fresh cultures of resected human lung cancers were used to produce conditioned medium (CM) or cocultured with stromal cells. Invasiveness of cancer cells was evaluated by transwell assays, and in vivo tumor growth was tested in Athymic nude mice. We found CM of a large variety of cancer cell lines as well as ex vivo-cultured lung cancers to rapidly induce protein levels of stromal-MDM2. CM of nontransformed cells had no such effect. Mdm2 induction occurred through enhanced translation, was mTORC1-dependent, and correlated with activation of AKT and p70 S6 Kinase. AKT or MDM2 knockdown in fibroblasts reduced the invasion of neighboring cancer cells, independently of stromal-p53. MDM2 overexpression in fibroblasts enhanced cancer cells' invasion and growth of inoculated tumors in mice. Our results indicate that stromal-MDM2 participates in a p53-independent cancer-host feedback mechanism. Soluble cancer-originated signals induce enhanced translation of stromal-MDM2 through AKT/mTORC1 signaling, which in turn enhances the neighboring cancer cells' invasion ability. The role of these tumor-host interactions needs to be further explored. IMPLICATIONS: We uncovered a novel tumor-stroma signaling loop, which is a potentially new therapeutic target in lung cancer and possibly in additional types of cancer.

Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

INTRODUCTION: Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Here, we identified the impact of TNF-α and IL-1ß on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES). METHODS: Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1ß). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers. RESULTS: TNF-α (and IL-1ß) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1ß) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors. CONCLUSIONS: Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

Role of CXCR3 carboxyl terminus and third intracellular loop in receptor-mediated migration, adhesion and internalization in response to CXCL11.

The chemokine receptor CXCR3 is predominantly expressed on activated T and natural killer (NK) cells. CXCR3 and its ligands, CXCL11, CXCL10, and CXCL9, play a major role in T-helper 1 (Th1)-dependent inflammatory responses. CXCL11 is the most dominant physiological inducer of adhesion, migration, and internalization of CXCR3. To study the role of CXCR3 carboxyl-terminus and the third intracellular (3i) loop in chemokine-mediated migration, adhesion, and CXCR3 internalization, we generated CXCR3 receptors mutated in their distal (Ser-Thr domain) or proximal (trileucine domain) membrane carboxyl terminus, and/or the third intracellular loop. We found that migration of CXCR3-expressing HEK 293 cells toward CXCL11 was pertussis toxin-dependent and required the membrane proximal carboxyl terminus of CXCR3. Internalization induced by CXCL11 and protein kinase C (PKC) activation was also regulated by the membrane proximal carboxyl terminus; however, only CXCL11-induced internalization required the LLL motif of this region. Internalization and Ca(2+) flux induced by CXCL11 were independent of the 3i loop S245, whereas migration at high CXCL11 concentrations, integrin-dependent adhesion, and actin polymerization were S245 dependent. Our findings indicate that CXCL11-dependent CXCR3 internalization and cell migration are regulated by the CXCR3 membrane proximal carboxyl terminus, whereas adhesion is regulated by the 3i loop S245. Thus, distinct conformational changes induced by a given CXCR3 ligand trigger different downstream effectors of adhesion, motility, and CXCR3 desensitization.

IL-8-induced migratory responses through CXCR1 and CXCR2: association with phosphorylation and cellular redistribution of focal adhesion kinase.

CXCR1 and CXCR2 mediate migratory activities in response to IL-8 and other ELR+-CXC chemokines (e.g., GCP-2 and NAP-2). In vitro, activation of migration is induced by low IL-8 concentrations (10-50 ng/mL), whereas migratory shut-off is induced by high IL-8 concentrations (1000 ng/mL). The stimulation of CXCR1 and CXCR2 by IL-8 concentrations that result in migratory activation induced focal adhesion kinase (FAK) phosphorylation in a G(alpha)i-dependent manner. The expression of FRNK, a dominant negative mutant of FAK, perturbed migratory responses to the activating dose of 50 ng/mL IL-8. The migration-activating concentrations of 50 ng/mL GCP-2 and NAP-2 induced less potent migratory responses and FAK phosphorylation in CXCR2-expressing cells as compared with IL-8. These results indicate that FAK is phosphorylated, and required, for the chemotactic response under conditions of migratory activation by ELR+-CXC chemokines. In addition, FAK phosphorylation was determined following exposure to migration-attenuating concentrations of IL-8. In CXCR1-RBL cells this treatment resulted in FAK phosphorylation, in similar levels to those induced by activating concentrations of IL-8. In contrast, in CXCR2-RBL cells the migration-attenuating concentrations of IL-8 induced promoted levels of FAK phosphorylation and different patterns of FAK phosphorylation on its six potential tyrosine phosphorylation sites, as compared to activating concentrations of the chemokine. Exposure to IL-8 resulted not only in FAK phosphorylation but also in its cellular redistribution, indicated by the formation of defined contact regions with the substratum, enriched in phosphorylated FAK and vinculin. Overall, FAK phosphorylation was associated with, and found to be differently regulated upon, ELR+-CXC chemokine-induced migration.

Intracellular trafficking of human CXCR1 and CXCR2: regulation by receptor domains and actin-related kinases.

In this study we investigated the regulation of CXCR1 and CXCR2 intracellular trafficking. First, we produced a chimeric CXCR2 receptor that contained the internalization motifs of both CXCR2 and CXCR1 (CXCR2: LLKIL sequence; CXCR1: C-terminal phosphorylation sites). Elevated levels of internalization were induced by different ELR-expressing CXC chemokines on the chimeric receptor, as compared to wild-type CXCR2. Analysis of inter-relationships between CXCR1 and CXCR2 during internalization indicated that the exposure of cells that expressed both CXCR1 and CXCR2 to CXCL8 or CXCL6 resulted in decreased levels of CXCR1 internalization as compared to those in cells that expressed only CXCR1. To characterize the role of actin-related components in CXCR1 and CXCR2 trafficking, wortmannin, a potent inhibitor of phosphatidylinositol kinases, was used. The presence of wortmannin during receptor recycling inhibited CXCR1 and CXCR2 re-expression following CXCL8-induced internalization, and resulted in a marked disruption of the proper organization of actin filaments. The kinase-dependent recycling process required CXCR2 C-terminal phosphorylation sites. Our results suggest that actin-related kinases are required for the proper functionality of actin filaments, which are the instrumental factors needed for receptor recycling. In all, CXCR1 and CXCR2 internalization and recycling are tightly regulated by receptor domains and by actin-related kinases.