Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxiainducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.orthotopic transplantation | triple negative breast cancer | oxygen | tumor microenvironment | mammary fat pad implantation

Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine β-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that IntroductionAn important advance in cancer biology has been the appreciation that, in addition to somatic mutations in oncogenes and tumor suppressor genes within cancer cells, a major mechanism driving disease progression is the interaction of cancer cells with the tumor microenvironment. The tumor stroma consists of extracellular matrix and various mesenchymal cell types, including vascular ECs and pericytes, fibroblasts, myofibroblasts, and various cells of bone marrow origin, including tumor-associated macrophages, bone marrow-derived angiogenic cells, neutrophils, mast cells, myeloid-derived suppressor cells, and mesenchymal stem cells (MSCs), which are recruited to the tumor and enhance primary tumor growth and/or promote metastasis (1). The molecular mechanisms by which stromal cells are attracted to, and communicate with, cancer cells are only understood in a limited number of contexts. For example, breast cancer cell (BCC) production of colony-stimulating factor 1 (CSF1) induces homing of CSF1 receptor-expressing tumor-associated macrophages that secrete epidermal growth factor, which binds to its receptor on cancer cells and stimulates their invasive properties (2, 3).The combination of cancer cell proliferation and stromal cell recruitment results in an imbalance between O 2 consumption and delivery. Tumor vasculature is structurally and functionally abnormal, leading to spatial and temporal heterogeneity in perfusion,

Intratumoral hypoxia induces the recruitment of stromal cells, such as macrophages and mesenchymal stem cells (MSCs), which stimulate invasion and metastasis by breast cancer cells (BCCs). Production of macrophage colony-stimulating factor 1 (CSF1) by BCCs is required for macrophage recruitment, but the mechanisms underlying CSF1 expression have not been delineated. Triplenegative breast cancers have increased expression of genes regulated by hypoxia-inducible factors (HIFs). In this study, we delineate two feed-forward signaling loops between human MDA-MB-231 triple-negative BCCs and human MSCs that drive stromal cell recruitment to primary breast tumors. The first loop, in which BCCs secrete chemokine (C-X-C motif) ligand 16 (CXCL16) that binds to C-X-C chemokine receptor type 6 (CXCR6) on MSCs and MSCs secrete chemokine CXCL10 that binds to receptor CXCR3 on BCCs, drives recruitment of MSCs. The second loop, in which MSCs secrete chemokine (C-C motif) ligand 5 that binds to C-C chemokine receptor type 5 on BCCs and BCCs secrete cytokine CSF1 that binds to the CSF1 receptor on MSCs, drives recruitment of tumor-associated macrophages and myeloid-derived suppressor cells. These two signaling loops operate independent of each other, but both are dependent on the transcriptional activity of HIFs, with hypoxia serving as a pathophysiological signal that synergizes with chemokine signals from MSCs to trigger CSF1 gene transcription in triple-negative BCCs.HIF-1 | mammary fat pad | orthotopic implantation | lymph node metastasis | lung metastasis B reast cancer metastasis transforms a local disease that is cured by surgical excision into a systemic disease that responds poorly to available therapies and is the major cause of patient mortality (1). Although somatic mutations have been cataloged in hundreds of human breast cancers and many genes that promote or suppress metastasis have been identified, the analysis of genetic alterations cannot reliably distinguish metastatic from nonmetastatic cancers (1-3). Multiple stromal cell types, including mesenchymal stem cells (MSCs) and tumorassociated macrophages (TAMs), are recruited to the tumor microenvironment and promote metastasis (4, 5). In mouse models, MSCs produce chemokines, including chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C-X-C motif) ligand 10 (CXCL10), which bind to their cognate receptors, chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 3 (CXCR3), respectively, on breast cancer cells (BCCs) to stimulate invasion and metastasis (6-9). TAMs are abundant in breast cancer and outnumber the BCCs in some cases (10). The density of TAMs in primary breast cancer biopsies is correlated with metastasis and patient mortality (11-13). In mouse models, macrophage colony-stimulating factor 1 (CSF1) and the chemokine CCL2 are secreted by BCCs and bind to their cognate receptors, CSF1 receptor (CSF1R) and CCR2, on TAMs, leading to their recruitment to the tumor microenvironment, where they produce EGF and other secreted proteins that promote ...