Exosomes Produced by Mesenchymal Stem Cells Drive Differentiation of Myeloid Cells into Immunosuppressive M2-Polarized Macrophages in Breast Cancer.

Tumor-associated macrophages are major contributors to malignant progression and resistance to immunotherapy, but the mechanisms governing their differentiation from immature myeloid precursors remain incompletely understood. In this study, we demonstrate that exosomes secreted by human and mouse tumor-educated mesenchymal stem cells (MSCs) drive accelerated breast cancer progression by inducing differentiation of monocytic myeloid-derived suppressor cells into highly immunosuppressive M2-polarized macrophages at tumor beds. Mechanistically, MSC-derived exosomes but not exosomes from tumor cells contain TGF-ß, C1q, and semaphorins, which promote myeloid tolerogenic activity by driving PD-L1 overexpression in both immature myelomonocytic precursors and committed CD206+ macrophages and by inducing differentiation of MHC class II+ macrophages with enhanced l-Arginase activity and IL-10 secretion at tumor beds. Accordingly, administration of tumor-associated murine MSC-derived exosomes accelerates tumor growth by dampening antitumor immunity, and macrophage depletion eliminates exosome-dependent differences in malignant progression. Our results unveil a new role for MSC-derived exosomes in the differentiation of myeloid-derived suppressor cells into macrophages, which governs malignant growth.

RelA driven co-expression of CXCL13 and CXCR5 is governed by a multifaceted transcriptional program regulating breast cancer progression.

Lethal metastasis of primary breast tumors to lymph nodes has been found to be associated with the co-expression of chemokine CXCL13 and its receptor CXCR5. To date, however, the precise molecular events regulating the co-expression of CXCL13 and CXCR5 in the context of breast cancer progression have not been identified. Therefore, to extend our understanding of the drivers of breast cancer metastasis, we undertook a line of investigation in this study in which we demonstrate that the transcriptional regulation of CXCL13 is mediated by the reciprocal activity of RelA and Nrf2, while CXCR5 is transcriptionally silenced by CpG island methylation within its promoter. Critically, we show that intra-tumoral CXCL13 and CXCR5 mRNA expression is positively correlated with intra-tumoral RelA expression within the primary tumor of breast cancer (BCa) patients (n = 98). We demonstrate a role for Nrf2 in the negative transcriptional regulation of cxcl13. Furthermore, using a luciferase assay and deletion analysis of the cxcl13 gene promoter, we demonstrate that RelA and Nrf2 directly act upon the cxcl13 promoter to regulate transcription. Chromatin immunoprecipitation PCR, supported by in silico docking analyses, confirmed that RelA and Nrf2 both occupy multiple positions within the cxcl13 promoter. Collectively, in RelA high conditions, low Nrf2 and lack of cxcr5 promoter DNA-methylation govern CXCL13-CXCR5 co-expression within breast tumors, and thus drive disease progression and metastasis.

Heterodimer formation by Oct4 and Smad3 differentially regulates epithelial-to-mesenchymal transition-associated factors in breast cancer progression.

The multifunctional cytokine TGF-ß crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-ß and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-ß in late stages compared to early stages of the disease. In vitro studies showed that TGF-ß elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-ß-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-ß, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-ß signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients.

CCL2 conditionally determines CCL22-dependent Th2-accumulation during TGF-ß-induced breast cancer progression.

We investigated expressions of -CC chemokine ligand 2 (CCL2) and CCL5 in tumor samples from 147 breast cancer (BCa) patients and correlated with transforming growth factor-ß (TGF-ß) expression. We observed an inverse correlation of TGF-ß expression with CCL2, CCL5 expression in early stages of BCa. On contrary, in late stages, CCL2, not CCL5, expression was found to be directly proportional with TGF-ß expression. TGF-ß stimulated MDA-MB-231 cells to express CCL2, however, downregulated both CCL2 and CCL5 in MCF-7. Interestingly, a significant swing of Th1-Th2 ratio towards Th2 is seen within the primary tumors expressing moderate/high-CCL2-low/negative-CCL5. We observed that CCL2-CCR2 interaction induces monocytes/macrophages to secrete Th2-attracting chemokine CCL22 in vitro. Therefore, CCL2 secreted from the tumor microenvironment may attract and interact with monocytes/macrophages, and favor Th2 accumulation by inducing CCL22 secretion. Study in 4T1-BALB/c BCa mouse model demonstrated significant (p<0.05) decrease in CCL2, CCL5 and CCL22 levels and reduction in lung metastatic nodule numbers upon administering TGF-ß inhibitor. These findings collectively indicate that TGF-ß regulates CCL2 and CCL5 expression in a stage-dependent manner during BCa progression, which in turn, determines Th1-Th2 balance within the tumor microenvironment.

Bacterial fucose-rich polysaccharide stabilizes MAPK-mediated Nrf2/Keap1 signaling by directly scavenging reactive oxygen species during hydrogen peroxide-induced apoptosis of human lung fibroblast cells.

Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼ 42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and (1)H/(13)C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities.

CXCL13-CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis.

We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.