Continuous Inflammatory Stimulation Leads via Metabolic Plasticity to a Prometastatic Phenotype in Triple-Negative Breast Cancer Cells.

Chronic inflammation promotes cancer progression by affecting the tumor cells and their microenvironment. Here, we demonstrate that a continuous stimulation (~6 weeks) of triple-negative breast tumor cells (TNBC) by the proinflammatory cytokines tumor necrosis factor α (TNFα) + interleukin 1ß (IL-1ß) changed the expression of hundreds of genes, skewing the cells towards a proinflammatory phenotype. While not affecting stemness, the continuous TNFα + IL-1ß stimulation has increased tumor cell dispersion and has induced a hybrid metabolic phenotype in TNBC cells; this phenotype was indicated by a transcription-independent elevation in glycolytic activity and by increased mitochondrial respiratory potential (OXPHOS) of TNBC cells, accompanied by elevated transcription of mitochondria-encoded OXPHOS genes and of active mitochondria area. The continuous TNFα + IL-1ß stimulation has promoted in a glycolysis-dependent manner the activation of p65 (NF-kB), and the transcription and protein expression of the prometastatic and proinflammatory mediators sICAM-1, CCL2, CXCL8 and CXCL1. Moreover, when TNBC cells were stimulated continuously by TNFα + IL-1ß in the presence of a glycolysis inhibitor, their conditioned media had reduced ability to recruit monocytes and neutrophils in vivo. Such inflammation-induced metabolic plasticity, which promotes prometastatic cascades in TNBC, may have important clinical implications in treatment of TNBC patients.

Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells.

The pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1ß (IL-1ß) are expressed simultaneously and have tumor-promoting roles in breast cancer. In parallel, mesenchymal stem cells (MSCs) undergo conversion at the tumor site to cancer-associated fibroblasts (CAFs), which are generally connected to enhanced tumor progression. Here, we determined the impact of consistent inflammatory stimulation on stromal cell plasticity. MSCs that were persistently stimulated by TNFα + IL-1ß (generally 14-18 days) gained a CAF-like morphology, accompanied by prominent changes in gene expression, including in stroma/fibroblast-related genes. These CAF-like cells expressed elevated levels of vimentin and fibroblast activation protein (FAP) and demonstrated significantly increased abilities to contract collagen gels. Moreover, they gained the phenotype of inflammatory CAFs, as indicated by the reduced expression of α smooth muscle actin (αSMA), increased proliferation, and elevated expression of inflammatory genes and proteins, primarily inflammatory chemokines. These inflammatory CAFs released factors that enhanced tumor cell dispersion, scattering, and migration; the inflammatory CAF-derived factors elevated cancer cell migration by stimulating the chemokine receptors CCR2, CCR5, and CXCR1/2 and Ras-activating receptors, expressed by the cancer cells. Together, these novel findings demonstrate that chronic inflammation can induce MSC-to-CAF conversion, leading to the generation of tumor-promoting inflammatory CAFs.

Inflammation-Driven Breast Tumor Cell Plasticity: Stemness/EMT, Therapy Resistance and Dormancy.

Cellular heterogeneity poses an immense therapeutic challenge in cancer due to a constant change in tumor cell characteristics, endowing cancer cells with the ability to dynamically shift between states. Intra-tumor heterogeneity is largely driven by cancer cell plasticity, demonstrated by the ability of malignant cells to acquire stemness and epithelial-to-mesenchymal transition (EMT) properties, to develop therapy resistance and to escape dormancy. These different aspects of cancer cell remodeling are driven by intrinsic as well as by extrinsic signals, the latter being dominated by factors of the tumor microenvironment. As part of the tumor milieu, chronic inflammation is generally regarded as a most influential player that supports tumor development and progression. In this review article, we put together recent findings on the roles of inflammatory elements in driving forward key processes of tumor cell plasticity. Using breast cancer as a representative research system, we demonstrate the critical roles played by inflammation-associated myeloid cells (mainly macrophages), pro-inflammatory cytokines [such as tumor necrosis factor α (TNFα) and interleukin 6 (IL-6)] and inflammatory chemokines [primarily CXCL8 (interleukin 8, IL-8) and CXCL1 (GROα)] in promoting tumor cell remodeling. These inflammatory components form a common thread that is involved in regulation of the three plasticity levels: stemness/EMT, therapy resistance, and dormancy. In view of the fact that inflammatory elements are a common denominator shared by different aspects of tumor cell plasticity, it is possible that their targeting may have a critical clinical benefit for cancer patients.

Notch-Mediated Tumor-Stroma-Inflammation Networks Promote Invasive Properties and CXCL8 Expression in Triple-Negative Breast Cancer.

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-1ß (IL-1ß)-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-1ß-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-1ß-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.

Tumor-Stroma-Inflammation Networks Promote Pro-metastatic Chemokines and Aggressiveness Characteristics in Triple-Negative Breast Cancer.

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 1ß (IL-1ß), 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-1ß-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-1ß-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.