Breast cancer: coordinated regulation of CCL2 secretion by intracellular glycosaminoglycans and chemokine motifs.

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 ((45)TIVA(48)). We have identified partial roles for R18+K19, H66, and the (45)TIVA(48) motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+(45)TIVA(48), the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, (45)TIVA(48) and R18+K19+(45)TIVA(48) domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

Mechanisms regulating the secretion of the promalignancy chemokine CCL5 by breast tumor cells: CCL5's 40s loop and intracellular glycosaminoglycans.

The chemokine CCL5 (RANTES) plays active promalignancy roles in breast malignancy. The secretion of CCL5 by breast tumor cells is an important step in its tumor-promoting activities; therefore, inhibition of CCL5 secretion may have antitumorigenic effects. We demonstrate that, in breast tumor cells, CCL5 secretion necessitated the trafficking of CCL5-containing vesicles on microtubules from the endoplasmic reticulum (ER) to the post-Golgi stage, and CCL5 release was regulated by the rigidity of the actin cytoskeleton. Focusing on the 40s loop of CCL5, we found that the (43)TRKN(46) sequence of CCL5 was indispensable for its inclusion in motile vesicles, and for its secretion. The TRKN-mutated chemokine reached the Golgi, but trafficked along the ER-to-post-Golgi route differently than the wild-type (WT) chemokine. Based on the studies showing that the 40s loop of CCL5 mediates its binding to glycosaminoglycans (GAG), we analyzed the roles of GAG in regulating CCL5 secretion. TRKN-mutated CCL5 had lower propensity for colocalization with GAG in the Golgi compared to the WT chemokine. Secretion of WT CCL5 was significantly reduced in CHO mutant cells deficient in GAG synthesis, and the WT chemokine acquired an ER-like distribution in these cells, similar to that of TRKN-mutated CCL5 in GAG-expressing cells. The release of WT CCL5 was also reduced after inhibition of GAG presence/synthesis by intracellular expression of heparanase, inhibition of GAG sulfation, and sulfate deprivation. The need for a (43)TRKN(46) motif and for a GAG-mediated process in CCL5 secretion may enable the future design of modalities that prevent CCL5 release by breast tumor cells.

Inflammatory mediators in breast cancer: coordinated expression of TNFα & IL-1ß with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition.

BACKGROUND: The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1ß were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course. METHODS: The expression of CCL2, CCL5, TNFα and IL-1ß was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments. RESULTS: CCL2, CCL5, TNFα and IL-1ß were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1ß in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1ß expression. These results suggest progression-related roles for TNFα and IL-1ß in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1ß compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1ß) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1ß acts jointly with other pro-malignancy factors to promote disease relapse. CONCLUSIONS: Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1ß may be important for disease course, and that TNFα & IL-1ß may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

Chemokines in human breast tumor cells: modifying their expression levels and determining their effects on the malignancy phenotype.

Chemokines have been recently recognized as important regulators of breast malignancy; however, much remains unknown regarding their roles in this disease. Improved understanding of chemokine contribution to breast cancer often requires studies in which the expression levels of chemokines by the tumor cells are modified (increased or decreased). In addition, it is essential to determine the roles of various chemokines in experimental in vivo model systems of breast cancer, using hormone-dependent or -independent human breast tumor cells (such as MCF-7, T47D and MDA-MB-231 cells). Since investigators often encounter difficulties in implementing these techniques in their studies of breast cancer, we hereby provide a detailed description of microporation approaches for modifying chemokine expression levels in human breast tumor cells, and of the measures required for establishment of xenograft models of primary tumors and of metastasis by such cells. In the breast malignancy context, the guidelines presented herein should enable researchers in the field to establish essential means for determination of chemokine roles in this disease.

Concomitant expression of the chemokines RANTES and MCP-1 in human breast cancer: a basis for tumor-promoting interactions.

The chemokines RANTES (CCL5) and MCP-1 (CCL2) were suggested to contribute, independently, to breast malignancy. In the present study, we asked if the two chemokines are jointly expressed in clinical samples of breast cancer patients, and do they interact in breast tumor cells. We found that RANTES and MCP-1 were expressed by breast tumor cells in primary tumors of Ductal Carcinoma In Situ and of Invasive Ductal Carcinoma, but minimally in normal breast epithelial duct cells. The chemokines were also detected in metastases and pleural effusions. Novel findings showed that co-expression of RANTES and MCP-1 in the same tumor was associated with more advanced stages of disease, suggesting that breast tumors "benefit" from interactions between the two chemokines. Accordingly, MCP-1 significantly promoted the release of RANTES from endogenous pre-made vesicles, in an active process that depended on calcium from intracellular and extracellular sources, and on intracellular transport of RANTES towards exocytosis. Our findings show a chemokine-triggered release of stored pro-malignancy chemokine from breast tumor cells. These observations support a major tumor-promoting role for co-expression of the chemokines in breast malignancy, and agree with the significant association of joint RANTES and MCP-1 expression with advanced stages of breast cancer.

The inflammatory chemokines CCL2 and CCL5 in breast cancer.

A causal role was recently attributed to inflammation in many malignant diseases, including breast cancer. The different inflammatory mediators that are involved in this disease include cells, cytokines and chemokines. Of these, many studies have addressed the involvement and roles of the inflammatory chemokines CCL2 (MCP-1) and CCL5 (RANTES) in breast malignancy. While minimally expressed by normal breast epithelial duct cells, both chemokines are highly expressed by breast tumor cells at primary tumor sites, indicating that CCL2 and CCL5 expression is acquired in the course of malignant transformation, and suggesting that the two chemokines play a role in breast cancer development and/or progression. Supporting this possibility are findings showing significant associations between CCL2 and CCL5 and more advanced disease course and progression. Furthermore, studies in animal model systems have shown active and causative roles for the two chemokines in this disease. In line with the tumor-promoting roles of CCL2 and CCL5 in breast cancer, the two chemokines were shown to mediate many types of tumor-promoting cross-talks between the tumor cells and cells of the tumor microenvironment: (1) they shift the balance at the tumor site between different leukocyte cell types by increasing the presence of deleterious tumor-associated macrophages (TAM) and inhibiting potential anti-tumor T cell activities; (2) of the two chemokines, mainly CCL2 promotes angiogenesis; (3) CCL2 and CCL5 which are expressed by cells of the tumor microenvironment osteoblasts and mesenchymal stem cells play a role in breast metastatic processes. In addition, both chemokines act directly on the tumor cells to promote their pro-malignancy phenotype, by increasing their migratory and invasion-related properties. Together, the overall current information suggests that CCL2 and CCL5 are inflammatory mediators with pro-malignancy activities in breast cancer, and that they should be considered as potential therapeutic targets for the limitation of this disease.

CXCL8-induced FAK phosphorylation via CXCR1 and CXCR2: cytoskeleton- and integrin-related mechanisms converge with FAK regulatory pathways in a receptor-specific manner.

CXCL8 is a potent chemokine, inducing focal adhesion kinase (FAK) phosphorylation, and migration via a FAK-mediated pathway. Since, unlike growth factors, chemokines directly control integrins and cytoskeleton rearrangements, we determined whether these elements regulate CXCL8-induced FAK phosphorylation. The analysis intentionally dissociated between the CXCL8 receptors CXCR1 and CXCR2. In both CXCR1- and CXCR2-expressing cells, actin and microtubules were required for CXCL8-induced FAK phosphorylation, and CXCL8-induced cell spreading was accompanied by concordant re-localization of FAK with actin and beta-tubulin. The phosphorylation of five FAK sites depended on intact actin filaments and microtubules. While in CXCR2-expressing cells FAK phosphorylation was adhesion-dependent and was stimulated by fibronectin, in CXCR1-expressing cells FAK phosphorylation was adhesion-independent. Of note, even in the absence of integrin stimulation, the CXCL8-induced phosphorylation of FAK in CXCR1-expressing cells required cytoskeletal elements. CXCL8-induced migration in both cell types was highly reliant on actin filaments, but only the migration of CXCR1-expressing cells was fully dependent on microtubules. Overall, several aspects of CXCL8-induced FAK phosphorylation and migration are regulated in a receptor-specific manner. These observations lay the basis for future investigation of the equilibrium between CXCR1 and CXCR2 in cells expressing both receptors together, such as neutrophils, endothelial cells and tumor cells.