The tumor microenvironment (TME) plays key roles in promoting disease progression in the aggressive triple-negative subtype of breast cancer (TNBC; Basal/Basal-like). Here, we took an integrative approach and determined the impact of tumor-stroma-inflammation networks on pro-metastatic phenotypes in TNBC. With the TCGA dataset we found that the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), as well as their target pro-metastatic chemokines CXCL8 (IL-8), CCL2 (MCP-1), and CCL5 (RANTES) were expressed at significantly higher levels in basal patients than luminal-A patients. Then, we found that TNFα- or IL-1β-stimulated co-cultures of TNBC cells (MDA-MB-231, MDA-MB-468, BT-549) with mesenchymal stem cells (MSCs) expressed significantly higher levels of CXCL8 compared to non-stimulated co-cultures or each cell type alone, with or without cytokine stimulation. CXCL8 was also up-regulated in TNBC co-cultures with breast cancer-associated fibroblasts (CAFs) derived from patients. CCL2 and CCL5 also reached the highest expression levels in TNFα/IL-1β-stimulated TNBC:MSC/CAF co-cultures. The elevations in CXCL8 and CCL2 expression partly depended on direct physical contacts between the tumor cells and the MSCs/CAFs, whereas CCL5 up-regulation was entirely dependent on cell-to-cell contacts. Supernatants of TNFα-stimulated TNBC:MSC “Contact” co-cultures induced robust endothelial cell migration and sprouting. TNBC cells co-cultured with MSCs and TNFα gained migration-related morphology and potent migratory properties; they also became more invasive when co-cultured with MSCs/CAFs in the presence of TNFα. Using siRNA to CXCL8, we found that CXCL8 was significantly involved in mediating the pro-metastatic activities gained by TNFα-stimulated TNBC:MSC “Contact” co-cultures: angiogenesis, migration-related morphology of the tumor cells, as well as cancer cell migration and invasion. Importantly, TNFα stimulation of TNBC:MSC “Contact” co-cultures in vitro has increased the aggressiveness of the tumor cells in vivo , leading to higher incidence of mice with lung metastases than non-stimulated TNBC:MSC co-cultures. Similar tumor-stromal-inflammation networks established in-culture with luminal-A cells demonstrated less effective or differently-active pro-metastatic functions than those of TNBC cells. Overall, our studies identify novel tumor-stroma-inflammation networks that may promote TNBC aggressiveness by increasing the pro-malignancy potential of the TME and of the tumor cells themselves, and reveal key roles for CXCL8 in mediating these metastasis-promoting activities.
IntroductionBreast 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 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β 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).MethodsChemokine 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-1β). 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.ResultsTNF-α (and IL-1β) 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-1β) 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0080-7) contains supplementary material, which is available to authorized users.
The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.
Stromal cells and pro-inflammatory cytokines play key roles in promoting the aggressiveness of triple-negative breast cancers (TNBC; Basal/Basal-like). In our previous study we demonstrated that stimulation of TNBC and mesenchymal stem cells (MSCs) co-cultures by the pro-inflammatory cytokine tumor necrosis factor α (TNFα) has led to increased metastasis-related properties in vitro and in vivo . In this context, elevated release of the pro-metastatic chemokines CXCL8 (IL-8) and CCL5 (RANTES) was noted in TNFα- and interleukin-1β (IL-1β)-stimulated TNBC:MSC co-cultures; the process was partly (CXCL8) and entirely (CCL5) dependent on physical contacts between the two cell types. Here, we demonstrate that DAPT, inhibitor of γ-secretase that participates in activation of Notch receptors, inhibited the migration and invasion of TNBC cells that were grown in “Contact” co-cultures with MSCs or with patient-derived cancer-associated fibroblasts (CAFs), in the presence of TNFα. DAPT also inhibited the contact-dependent induction of CXCL8, but not of CCL5, in TNFα- and IL-1β-stimulated TNBC:MSC/CAF co-cultures; some level of heterogeneity between the responses of different TNBC cell lines was noted, with MDA-MB-231:MSC/CAF co-cultures being the most sensitive to DAPT. Patient dataset studies comparing basal tumors to luminal-A tumors, and mRNA analyses of Notch receptors in TNBC and luminal-A cells pointed at Notch1 as possible mediator of CXCL8 increase in TNFα-stimulated TNBC:stroma “Contact” co-cultures. Accordingly, down-regulation of Notch1 in TNBC cells by siRNA has substantially reduced the contact-dependent elevation in CXCL8 in TNFα- and also in IL-1β-stimulated TNBC:MSC “Contact” co-cultures. Then, studies in which CXCL8 or p65 (NF-κB pathway) were down-regulated (siRNAs; CRISPR/Cas9) in TNBC cells and/or MSCs, indicated that upon TNFα stimulation of “Contact” co-cultures, p65 was activated and led to CXCL8 production mainly in TNBC cells. Moreover, our findings indicated that when tumor cells interacted with stromal cells in the presence of pro-inflammatory stimuli, TNFα-induced p65 activation has led to elevated Notch1 expression and activation, which then gave rise to elevated production of CXCL8. Overall, tumor:stroma interactions set the stage for Notch1 activation by pro-inflammatory signals, leading to CXCL8 induction and consequently to pro-metastatic activities. These observations may have important clinical implications in designing novel therapy combinations in TNBC.
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