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.

Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of the hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through induction of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the expression of LOX and LOXL proteins, collagen cross-linking, CD11b+ BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1α-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor.