Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation

Abstract

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...

Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis

“…We analyzed the effect of hypoxia (1% O 2 for 24 h) on the production of MVs by three human breast cancer cell lines: MCF-7 expresses the estrogen and progesterone receptors, but not HER2, and is classified as luminal subtype based on its gene expression pattern, whereas MDA-MB-231 and MDA-MB-435 cells (hereafter designated MDA-231 and MDA-435) do not express estrogen or progesterone receptors or HER2 (i.e., triple negative) and are classified as basallike/claudin-low subtype by gene expression criteria (43,44 (29)(30)(31).…”

“…In breast cancer, overexpression of HIF-1α or HIF-2α is associated with metastasis, treatment failure, and patient mortality (21)(22)(23)(24)(25)(26). HIFs are required for lymph node and lung metastasis in both autochthonous (27) and orthotopic (28)(29)(30) mouse models of breast cancer and activate the transcription of genes encoding proteins that are required for multiple steps in the invasion and metastasis of breast cancer cells (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”

HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells

“…This heterogeneous transcriptional response of breast cell lines to hypoxia is commonly observed. For example, analysis of mRNAs encoding lysyl oxidase (LOX) and LOX-like proteins (LOXL1-4) revealed that hypoxia induced increased expression of LOX and LOXL4 in MDA-MB-231, whereas LOXL2 expression was induced in MDA-MB-435 cells (12). We also observed heterogeneity with respect to requirements for HIF-α subunits, as CD47 induction required only HIF-1α in MCF7 cells, but both HIF-1α and HIF-2α in HCC1954 cells.…”

“…Specific HIF target genes have been identified that are induced by hypoxia in breast cancer cells and promote critical steps in the metastatic process, including stromal cell recruitment (8,9), cancer cell migration (10), invasion and intravasation (11)(12)(13)(14)(15), margination and extravasation (5), and premetastatic niche formation (12,16). Increased expression of HIF target genes in primary breast cancers is associated with increased patient mortality (17,18).…”

Expression and Significance of Hypoxia Inducible Factor-1α and Lysyl Oxidase in Non-small Cell Lung Cancer

“…In breast cancer, in addition to local effects on invasion, secreted LOX is critical for the formation of the premetastatic niche by accumulating at the distant sites, cross-linking collagen Ⅳ in the basement membrane and promoting the recruitment of bone marrow derived cells that stimulate angiogenesis (Wong et al, 2011;Wong et al, 2012). LOX also facilitates colorectal cancer cell migration and adhesion through the hydrogen peroxide regulation of FAK and SRC signal pathways (Baker et al, 2013).…”