Tumor-fibroblast interactions stimulate tumor vascularization by enhancing cytokine-driven production of MMP9 by tumor cells

Advance-stage breast carcinomas include significant amounts of fibroblasts and infiltrating immune cells which have been implicated in tumor growth, recurrence, and response to therapy. The present study investigated the contribution of fibroblasts to tumor growth using direct tumor-fibroblast co-cultures and tumor xenograft models. Our findings revealed that fibroblasts enhance breast carcinoma growth by promoting the tumor vasculature via the MMP9-dependent mechanism. In tumor-fibroblast cocultures, fibroblasts increased expression of TGF-beta, TNF, and IL-1 beta cytokines in tumor cells. These cytokines cooperatively induced expression of matrix metalloproteinase MMP9 in tumor cells. Knockdown of MMP9 by shRNA significantly reduced tumor vascularization induced by fibroblasts. Mechanistically, our findings argue that expression of MMP9 in tumor cells is regulated by crosstalk of TGF-beta with TNF and/or IL-1 beta cytokines. The mechanism of this cooperative response did not involve crossactivation of the canonical signaling pathways as TGF-beta did not activate RELA/p65 signaling, while TNF did not affect SMAD signaling. Instead, TGF-beta and TNF cytokines co-stimulated MAP kinases and expression of JUN and JUNB, AP1 transcription factor subunits, which together with RELA/p65 were essential for the regulation of MMP9. Depletion of JUN and JUNB or RELA in tumor cells blocked the cooperative induction of MMP9 by the cytokines. Thus, our studies uncovered a previously unappreciated role of tumor-fibroblast interactions in the stimulation of tumor angiogenesis, and an essential role of the MAPK-AP1 axis in the cooperative up-regulation of the angiogenic driver MMP9 by cytokine crosstalk.