4017

LOXL2

The transcription factor Snail controls epithelial-mesenchymal transitions (EMT) by repressing E-cadherin expression and other epithelial genes. However, the mechanisms involved in the regulation of Snail function are not fully understood. Here we show that lysyl-oxidase-like 2 and 3 (LOXL2 and LOXL3), two members of the lysyl-oxidase gene family, interact and cooperate with Snail to downregulate E-cadherin expression. Snail's lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2/3 and induction of EMT. Overexpression of LOXL2 or LOXL3 in epithelial cells induces an EMT process, supporting their implication in tumor progression. The biological importance of LOXL2 is further supported by RNA interference of LOXL2 in Snail-expressing metastatic carcinoma cells, which led to a strong decrease of tumor growth associated to increased apoptosis and reduced expression of mesenchymal and invasive/angiogenic markers. Taken together, these results establish a direct link between LOXL2 and Snail in carcinoma progression.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

LOXL2 is a copper- and lysine tyrosylquinone-dependent amine oxidase that has been proposed to function both extracellularly and intracellularly to activate oncogenic signaling pathways leading to EMT and invasion of breast cancer cells. In this study, we selected MCF-7 cells that stably express forms of recombinant LOXL2 differing in their subcellular localizations and catalytic competencies. This enabled us to dissect the molecular functions of intracellular and extracellular LOXL2s and examine their contributions to breast cancer metastasis/invasion. We discovered that secreted LOXL2 (~100-kDa) is N-glycosylated at Asn-455 and Asn-644, whereas intracellular LOXL2 (~75-kDa) is nonglycosylated and N-terminally processed, and is primarily associated with the nucleus. Both forms of LOXL2 can oxidize lysine in solution. However, we found that expression of intracellular LOXL2 is more strongly associated with EMT and invasiveness than secreted LOXL2 in vitro. The results indicate that nuclear associated LOXL2 contributes to the stabilization of Snail1 transcription factor at the protein level to induce EMT and promote invasion in vitro, through repression of E-cadherin, occludin, and estrogen receptor-alpha, and up-regulation of vimentin, fibronectin, and MT1-MMP.

Recurrence of breast cancer disease years after treatment appears to arise from disseminated dormant tumor cells (DTC). The mechanisms underlying the outgrowth of DTC remain largely unknown. Here we demonstrate that dormant MCF-7 cells expressing LOXL2 acquire a cancer stem cell (CSC)-like phenotype, mediating their outgrowth in the 3D BME system that models tumor dormancy and outgrowth. Similarly, MCF-7-LOXL2 cells colonizing the lung transitioned from dormancy to metastatic outgrowth whereas MCF-7 cells remained dormant. Notably, epithelial to mesenchymal transition (EMT) of MCF-7-LOXL2 cells was required for their CSC-like properties and their transition to metastatic outgrowth. These findings were further supported by clinical data demonstrating that increase in LOXL2 mRNA levels correlates with increase in the mRNA levels of EMT and stem cells markers, and is also associated with decrease in relapse free survival of breast cancer patients. Notably, conditional hypoxia induced expression of endogenous LOXL2 in MCF-7 cells promoted EMT and the acquisition of a CSC-like phenotype, while knockdown of LOXL2 inhibited this transition. Overall, our results demonstrate that expression of LOXL2 endowed DTC with CSC-like phenotype driving their transition to metastatic outgrowth and this stem-like phenotype is dependent on EMT that can be driven by the tumor microenvironment.

BACKGROUND: The incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1alpha functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1alpha promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1alpha in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism. METHODS: HIF-1alpha, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1alpha was used to determine whether LOXL2 been increased by HIF-1alpha. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1alpha induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip(R) Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl2. RESULTS: In clinical HCC tissues, it confirmed a positive relationship between HIF-1alpha and LOXL2 protein. Importantly, HIF-1alpha and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1alpha was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed. CONCLUSION: HIF-1alpha plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

In the present study, we investigated the role of lysyl oxidaselike 2 (LOXL2), the correlation between LOXL2 and epithelial to mesenchymal transition (EMT) and the effects of using betaaminopropionitrile (BAPN) to inhibit LOXL2 with the aim of reducing tumor progression in hepatocellular carcinoma (HCC). The expression level of LOXL2 was evaluated in HCC and adjacent noncancerous tissues using quantitative reverse transcription polymerase chain reaction and clinicopathological analyses. The effects of BAPN on cell proliferation, migration and invasion were investigated in vitro. Additionally, LOXL2 expression was assessed in the culture supernatants of HCC cell lines. Our results revealed that LOXL2 expression was higher in HCC cell lines and tissues. There was a significant correlation between EMT status and LOXL2 levels (P=0.004). BAPN reduced migration and invasion in HCC cells. HCC patients with high levels of LOXL2 expression had relatively shorter diseasefree survival (P=0.009) and overall survival (P=0.035). The expression level of LOXL2 was similar between cell supernatants and HCC cell lines. A multivariate analysis demonstrated that portal vein invasion (P=0.015), venous invasion (P=0.026), serum AFP (alphafetoprotein) levels (P=0.019) and LOXL2 expression (P=0.009) were independent prognostic factors. Our results indicated that a higher level of LOXL2 may contribute to tumor progression, indicating that LOXL2 has clinical value as a therapeutic target in HCC.