The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis,independent of RANK ligand, which disrupts normal bone homeostasisleading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications

Supplementary Figure 2 from Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

PDF file - 911K, S2: Inhibition of LOXL2 in 4T1 CM reduces fibroblast branching on collagen and collagen contraction</p

Supplementary Figure 3 from Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

PDF file - 205K, S3: Integrin Beta3 mediates LOXL2 driven fibroblast activation</p

Supplementary Figure Legend from Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

PDF file - 64K</p

Supplementary Figure 1 from Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

PDF file - 82K, S1: 4T1 shLOXL2 CM has lower levels of LOXL2 protein and activity</p

Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of alpha-smooth muscle actin (alpha-SMA). Using a marker for reticular fibroblasts, it was determined that expression of alpha-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared with control tumors which exhibited matrices with dense, parallel alignments. Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of alpha-SMA in fibroblasts grown on collagen matrices. Mechanistically, it was determined that LOXL2 activated fibroblasts through integrin mediated focal adhesion kinase activation. These results indicate that inhibition of LOXL2 in tumors not only reduces tumor cell invasion but also attenuates the activation of host cells in the tumor microenvironment. (C) 2013 AACR

ERK-MAPK signaling opposes Rho-kinase to promote endothelial cell survival and sprouting during angiogenesis

SummaryInhibition of ERK-MAPK signaling by expression of dominant-negative MEK1 in the tumor vasculature suppresses angiogenesis and tumor growth. In an organotypic tissue culture angiogenesis assay, ERK-MAPK inhibition during the migratory phase results in loss of bipolarity, detachment, and cell death of isolated endothelial cells and retraction of sprouting tubules. These effects are the consequence of upregulated Rho-kinase signaling. Transient inhibition of Rho-kinase rescues the effects of ERK-MAPK inhibition in vitro and in vivo, promotes sprouting, and increases vessel length in tumors. We propose a regulatory role of Rho-kinase by ERK-MAPK during angiogenesis that acts through the control of actomyosin contractility. Our data delineate a mechanism by which ERK-MAPK promotes endothelial cell survival and sprouting by downregulating Rho-kinase signaling

Supplementary Figure S2 from LOX-Mediated Collagen Crosslinking Is Responsible for Fibrosis-Enhanced Metastasis

&lt;p&gt;Supplementary Figure S2 PDF file - 171K, aSMA co-localisation during pulmonary fibrosis and LOX family member expression levels induced by liver fibrosis&lt;/p&gt;</jats:p

Hypoxia-Induced Lysyl Oxidase Is a Critical Mediator of Bone Marrow Cell Recruitment to Form the Premetastatic Niche

SummaryTumor cell metastasis is facilitated by “premetastatic niches” formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment and metastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease