Summary Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening and increased focal adhesions. Inducing collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 Kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibiting integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM, and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling and induced the invasion of a premalignant epithelium. Consistently, reducing lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/ migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with B-aminopropionitrile (BAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of BAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with BAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on BAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer. (Cancer Res 2005; 65(24): 11429-36)

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Lysyl oxidase-like 2 (LOXL2) belongs to an amine oxidase family whose members have been implicated in crosslink formation in stromal collagens and elastin, cell motility, and tumor development and progression. We previously demonstrated the association between increased LOXL2 expression and invasive/metastatic behavior in human breast cancer cells and mouse squamous and spindle cell carcinomas, interaction between LOXL2 and SNAIL in epithelial-mesenchymal transition, and localization of the LOXL2 gene to 8p21.2-21.3, within a minimally deleted region in several cancers, including colon and esophagus. In the present study, we analyzed LOXL2 expression in colon and esophageal tumors, and explored methylation as a regulator of LOXL2 expression. Immunohistochemistry using normal tissues demonstrated intracellular localization of LOXL2 in colonic enteroendocrine cells and esophageal squamous cells at the luminal surface, but not in mitotically active cells. Tissue array analysis of 52 colon adenocarcinomas and 50 esophageal squamous cell carcinomas revealed presence of LOXL2 expression in 83 and 92% of the samples, respectively, and a significant association between increased number of LOXL2-expressing cells and less-differentiated colon carcinomas. We determined that the methylation status of the 1150 bp 5' CpG island may contribute to the regulation of the gene. Loss of heterozygosity studies, using a microsatellite within intron 4 of the LOXL2 gene, revealed that loss of LOXL2 was unlikely to play a major role in either colon or esophageal tumors. These results suggest that increased LOXL2 expression in colon and esophageal cancer may contribute to tumor progression.

A growing number of studies indicate the importance of the lysyl oxidase family in the promotion of epithelial neoplasms towards their more aggressive forms. However, the role of individual family members in carcinoma progression has yet to be ascertained. In this study, we analyzed LOXL2 expression in malignantly transformed MCF-7 and normal MCF-10A mammary epithelial cell line clones stably transduced with LOXL2 in vitro, and in normal and cancerous breast tissue samples in vivo. We found LOXL2 to be catalytically active in both MCF-7 and MCF-10 clones. LOXL2 overexpression promoted a more mesenchymal morphology in both cell types, but LOXL2-induced increase in migratory ability could only be established in MCF-7 clones. We demonstrated altered localization of the LOXL2 protein in breast cancer tissue compared to normal mammary tissue, and altered localization and processing of LOXL2 protein in breast cancer cell lines compared to normal cell lines, which may allow LOXL2 to interact with different intra and extracellular components during tumor progression. Results support the role of LOXL2 in selectively promoting a metastatic phenotype in breast tumor cells. Additional data suggest epigenetic molecular mechanisms in tumor specific regulation of LOXL2 expression that could be explored as a molecular target in the prevention of breast cancer progression. ' UICCKey words: LOXL2; epigenetic regulation; breast cancer Lysyl oxidase-like 2 (LOXL2) belongs to a family of 5 proteins characterized by a conserved carboxy-terminal copper-binding domain and a lysyl-tyrosyl quinone cofactor, which are necessary for the amine oxidase activity of these extracellular matrix (ECM) enzymes.1,2 Indeed, catalytic activity for LOXL2 has been confirmed in Chinese hamster ovary cells.3 This study also reported that LOXL2 activity was not inhibited by b-aminoproprionitrile (BAPN), making it different from the BAPN-sensitive catalytic activity reported for LOX, 4 LOXL, 5,6 and LOXL4, 7 despite the high homology within their catalytic region. The relationship between the catalytic activity and physiologic or pathologic function of LOXL2 has yet to be characterized.In our previous studies, we reported high levels of LOXL2 mRNA expression in highly invasive, metastatic breast cancer cell lines and highly tumorigenic, metastatic mouse squamous and spindle cell carcinomas, compared to absent expression in noninvasive, nonmetastatic breast cancer cell lines and a nontumorigenic keratinocyte cell line. 8,9 At the tissue level, we reported the association of increased LOXL2 protein expression with poorly differentiated colon adenocarcinoma and mucinous carcinoma, both of which have aggressive behavior and high incidence of metastasis.10,11 This observation is supported by a recent report of increased LOXL2 mRNA expression in colon tumors and liver metastases compared to normal colon. 12 In breast cancer, increased LOXL2 protein expression was associated with higher tumor grade, decreased overall and disease-free survival in lymphnode negat...