Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment.

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

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Laminin peptides influence cancer biology. We investigated the role of a lamininderived peptide C16 regulating invadopodia molecules in human prostate cancer cells (DU145). C16 augmented invadopodia activity of DU145 cells, and stimulated expression Tks4, Tks5, cortactin, and membrane-type matrix metalloproteinase 1.Reactive oxygen species generation is also related to invadopodia formation. This prompted us to address whether C16 would induce reactive oxygen species generation in DU145 cells. Quantitative fluorescence and flow cytometry showed that the peptide C16 increased reactive oxygen species in DU145 cells. Furthermore, significant colocalization between Tks5 and reactive oxygen species was observed in C16-treated cells. Results suggested that the peptide C16 increased Tks5 and reactive oxygen species in prostate cancer cells. The role of C16 increasing Tks and reactive oxygen species are novel findings on invadopodia activity. K E Y W O R D S extracellular matrix, invadopodia, laminin, prostate cancer, reactive oxygen species, Tks4, Tks5