Summary Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.

Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER + /PR + breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.digoxin | mammary fat pad implantation | tumor relapse

Primary tumors facilitate metastasis by directing bone marrowderived cells (BMDCs) to colonize the lungs before the arrival of cancer cells. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of breast cancer metastatic niche formation through induction of multiple members of the lysyl oxidase (LOX) family, including LOX, LOX-like 2, and LOX-like 4, which catalyze collagen cross-linking in the lungs before BMDC recruitment. Only a subset of LOX family members was expressed in any individual breast cancer, but HIF-1 was required for expression in each case. Knockdown of HIF-1 or hypoxia-induced LOX family members reduced collagen cross-linking, CD11b+ BMDC recruitment, and metastasis formation in the lungs of mice after orthotopic transplantation of human breast cancer cells. Metastatic niche formation is an HIF-1-dependent event during breast cancer progression. extracellular matrix | lung metastasis I ntratumoral hypoxia is a common finding that is attributable to inadequate O 2 delivery to regions of rapidly growing cancers that are distant from functional blood vessels (1). Reduced O 2 availability leads to increased activity of hypoxia-inducible factors (HIFs), which consist of an O 2 -regulated HIF-1α or HIF-2α subunit and the constitutively expressed HIF-1β subunit (2, 3). HIF inhibition blocks tumor xenograft growth (2, 4).Metastasis is responsible for 90% of deaths among patients who have breast cancer and involves multiple steps, including cancer cell invasion through ECM, intravasation, extravasation, and colonization of distant organs (5). Recent studies have reported that prior recruitment of bone marrow-derived cells (BMDCs) to the metastatic site promotes subsequent colonization by cancer cells (6). The primary tumor is responsible for BMDC recruitment to the metastatic site. Breast tumors secrete lysyl oxidase (LOX), which localizes at metastatic sites in the lungs and remodels collagen, thereby facilitating BMDC recruitment (7,8). LOX oxidatively deaminates the ε-amino groups of lysine residues, resulting in intramolecular and intermolecular cross-linking of collagen molecules (9). Crosslinking stabilizes collagen by assembly into fibrils and fibers, which enhance ECM tensile strength, leading to focal adhesion formation and PI3K signaling (10). The LOX family is composed of LOX and LOX-like (LOXL) proteins LOXL1-4. So far, only LOX has been implicated in metastatic niche formation (7). In this study, we demonstrate that HIF-1 regulates metastatic niche formation by activating expression of LOX and LOXL proteins. HIF-1 silencing suppresses metastatic niche formation and metastasis regardless of which LOX family member is involved. ResultsHypoxia-Induced LOX/LOXL Expression in Breast Cancer Cell Lines.Two metastatic breast cancer cell lines, MDA-MB-231 (MDA-231) and MDA-MB-435 (MDA-435), as well as a nonmetastatic line, MCF-7, were cultured under standard, nonhypoxic tissue culture conditions of 95% air/5% CO 2 (vol/vol; 20% O 2 ) and under hypoxic culture conditi...

Most cases of breast cancer mortality are due to vascular metastasis. Breast cancer cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor activity (HIF) in breast cancer cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of breast cancer cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of breast cancer cells to ECs. Interference with HIF, ANGPTL4, or L1CAM expression inhibits vascular metastasis of breast cancer cells to the lungs.