The inflammatory chemokine CCL2 promotes pro-cancerous effects in breast cancer cells and in cells of the tumor microenvironment. The secretion of CCL2 by breast tumor cells is a key regulatory step in these processes, therefore understanding the mechanisms regulating its secretion may enable the design of improved therapeutic modalities.
In search for cellular components that regulate CCL2 secretion, we focused on intracellular glycosaminoglycans (GAG). Using mutant CHO cells deficient in GAG expression revealed that there was partial involvement of intracellular GAG in the secretion of WT-CCL2. Then, by producing three modified forms of the chemokine, we have shown that GAG-binding motifs in CCL2 are required for chemokine secretion by breast cancer cells. Each of the mutants retained only partial secretion ability compared to WT-CCL2. Combining intracellular GAG deficiency and mutated GAG-binding motifs in CCL2 has led to substantial inhibition of CCL2 secretion, accompanied by its reduced ability to exit from the ER towards the Golgi.
By triple-dye confocal analyses we have shown that WT-CCL2 was highly co-localized with heparane sulfate (HS) and with chondriotin sulfate (CS) in the Golgi in breast tumor cells. The possibility that heparane sulfate was involved in CCL2 secretion was supported by over-expression of heparanase, leading to inhibition of CCL2 secretion by breast tumor cells. Further analyses with shRNA to Ext1, the key enzyme involved in HS synthesis, revealed complex regulatory roles for HS and CS in CCL2 secretion by the tumor cells.
Overall, our findings indicate that HS/CS-mediated mechanisms regulate the secretion of CCL2 by breast tumor cells, and have identified three GAG-binding domains in CCL2 which are required for its release. Targeting the GAG-mediated mechanisms that lead CCL2 toward secretion may inhibit the release of the chemokine by breast tumor cells, and thus may reduce its tumor-promoting activities in this disease.
