Human lysyl oxidase-like 2

Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu²⁺- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.     

类赖氨酰氧化酶2与肿瘤转移和发生

类赖氨酰氧化酶2（lysyl oxidase—like 2,LOXL2）是赖氨酰氧化酶（1ysyl oxidase,LOX）基因家族的成员之一,其表达产物能促进胶原沉积。LOXL2的过表达能促进纤维化,并与肿瘤侵袭、转移及不良预后有关。目前大部分学者认为LOXL2是一种转移促进基因,也有实验支持其是一种肿瘤抑制基因。研究发现LOXL2可以通过激活Snail／Ecadherin通路或Src／FAK通路促进转移。LOXL2有望作为肿瘤生物标志物,用于预后判断,成为一个新的治疗靶点。     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

The Lysyl Oxidase Pro-peptide Attenuates Fibronectin-mediated Activation of Focal Adhesion Kinase and p130Cas in Breast Cancer Cells

The lysyl oxidase (LOX) gene encodes an enzyme (LOX) critical for extracellular matrix maturation. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. In particular, the pro-peptide domain (LOX-PP) released from the secreted precursor protein (Pro-LOX) was found to inhibit the transformed phenotype of breast, lung, and pancreatic cancer cells. However, the mechanisms of action of LOX-PP remained to be determined. Here, the ability of LOX-PP to attenuate the integrin signaling pathway, which leads to phosphorylation of focal adhesion kinase (FAK), and the activation of its downstream target p130^Cas, was determined. In NF639 breast cancer cells driven by Her-2/neu, which signals via Ras, ectopic Pro-LOX and LOX-PP expression inhibited fibronectin-stimulated protein tyrosine phosphorylation. Importantly, phosphorylation of FAK on Tyr-397 and Tyr-576, and p130^Cas were substantially reduced. The amount of endogenous p130^Cas in the Triton X-100-insoluble protein fraction, and fibronectin-activated haptotaxis were decreased. Interestingly, expression of mature LOX enzyme enhanced fibronectin-stimulated integrin signaling. Of note, treatment with recombinant LOX-PP selectively reduced fibronectin-mediated haptotaxis of NF639, MDA-MB-231, and Hs578T breast cancer cells. Thus, evidence is provided that one mechanism of action of LOX-PP tumor suppression is to block fibronectin-stimulated signaling and cell migration.The lysyl oxidase (LOX)² gene family is comprised of five members LOX, LOXL1, LOXL2, LOXL3, and LOXL4, which encode enzymes that modify extracellular matrix (ECM) proteins to promote their cross-linking and deposition (1). The LOX gene is the best characterized and codes for the synthesis of a secreted 50-kDa glycosylated pro-enzyme (Pro-LOX). Pro-LOX is extracellularly processed by proteolytic cleavage to a mature active 32-kDa enzyme (LOX) and an 18-kDa pro-peptide (LOX-PP) by the procollagen C proteinases bone morphogenic protein-1 (BMP-1), and the related tolloid-like proteins TLL1 and TLL2 (2–4). In murine Pro-LOX, proteolytic processing occurs between amino acids Gly-162 and Asp-163, generating LOX-PP containing 141 amino acids (5). LOX-PP contains two consensus N-glycosylation sites, Asn-91 and Asn-138 (murine sequence) (2) and several O-glycosylation sites.³ LOX-PP does not contain any known protein domains, and structural prediction analysis indicates that LOX-PP assembles as an intrinsically disordered protein (6). Among the LOX family members, the C-terminal ends encode the enzyme domain and are highly conserved, whereas the N-terminal ends that encode the pro-peptide region have variable sequences. Based on structural and sequence similarities of the pro-peptide regions, the LOX family members can be divided into two subgroups: LOXL2, LOXL3, and LOXL4 as one group whose propeptide regions contain four scavenger receptor cysteine-rich domains, and LOX and LOXL1 as a separate group with much simpler and smaller pro-peptide region containing no cysteine residues (reviewed in Ref. 1). In contrast to Pro-LOX, the exact maturation site of Pro-LOXL1 is still unidentified.LOX is essential in the formation of blood vessels and in maintaining their normal characteristics (7–9). Up-regulation of LOX expression has been described in stromal cells that surround ductal breast and broncho-pulmonary carcinomas (10).Expression of the LOX gene was found to inhibit the transforming activity of the Ras oncogene in NIH 3T3 fibroblasts and hence was named the “ras recision” gene (rrg) (11, 12). The LOX gene was shown to inhibit growth in soft agar of NIH 3T3 fibroblasts and to attenuate Ras-mediated activation of phosphatidylinositol 3-kinase (PI3K), Akt, and Erk1/2 kinases and NF-κB activation (13). More recently, the rrg activity was mapped to the 18-kDa LOX-PP. Specifically, LOX-PP was shown to inhibit Ras-mediated transformation of fibroblasts as determined by reduced growth in soft agar, localization of PDK1 to the membrane, and activation of NF-κB (14). Furthermore, the inhibitory effects of LOX-PP on Ras signaling were extended to breast, pancreatic, and lung cancer cells (6, 14, 15). LOX-PP expression in these carcinoma cells reverted Her-2/neu- and Ras-mediated epithelial to mesenchymal transition (EMT), leading to increased expression of E-cadherin and γ-catenin, and reduced levels of Snail, vimentin, and/or BCL-2 (7, 15). Furthermore, LOX-PP expression reduced tumor formation in a xenograft model by Her-2/neu-overexpressing NF639 cells (6).Acquisition of the ability to invade the ECM is essential to EMT. The ECM has multiple mechanical and signaling functions. The ECM defines interfaces between tissues, provides a scaffold for cell traction, and a substrate for cell migration and adhesion. It is composed of a complex of proteins such as collagens, fibronectin, and laminin, which can interact and bind various growth factors (16). Fibronectin is of particular interest because it was recently shown to interact with the C terminus of Pro-LOX (17). Binding of fibronectin to its receptors (e.g. integrins α₅β₁ or α_vβ₁) stimulates the tyrosine phosphorylation of cellular proteins, in particular that of focal adhesion kinase (FAK) (18). Little is known about the mechanism of action of LOX-PP. Here, we have asked whether the tumor suppressor activity of LOX-PP attenuates the activation of the integrin signaling pathway in breast cancer cells. We report that LOX-PP attenuates FAK signaling and activation of its downstream target p130^Cas and is a robust inhibitor of fibronectin-stimulated cell migration.     

E-cadherin's dark side: Possible role in tumor progression

In the context of cancer, E-cadherin has traditionally been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial–mesenchymal transition (EMT) in epithelial tumor progression. Germline or somatic mutations in the E-cadherin gene (CDH1) or downregulation by epigenetic mechanisms have been described in a small subset of epithelial cancers. However, recent evidence also points toward a promoting role of E-cadherin in several aspects of tumor progression. This includes preserved (or increased) E-cadherin expression in microemboli of inflammatory breast carcinoma, a possible “mesenchymal to epithelial transition” (MET) in ovarian carcinoma, collective cell invasion in some epithelial cancers, a recent association of E-cadherin expression with a more aggressive brain tumor subset, as well as the intriguing possibility of E-cadherin involvement in specific signaling networks in the cytoplasm and/or nucleus. In this review we address a lesser-known, positive role for E-cadherin in cancer.     

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC^low) and highly aggressive CSC-like cells (CSC^high) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC^high cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC^low cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC^high cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC^high cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.     

Salidroside ameliorated hypoxia-induced tumorigenesis of BxPC-3 cells via downregulating hypoxia-inducible factor (HIF)-1α and LOXL2

Herein, we found that salidroside suppressed hypoxia-inducible factor 1 alpha (HIF-1α) and lysyl oxidase-like protein 2 (LOXL2) within human pancreatic cancer BxPC-3 cells cultured both under normoxia and hypoxia condition. To investigate the effect of salidroside on tumorigenesis of BxPC-3 cells and whether HIF-1α and LXCL2 were involved in this process, cells transfected with or without LOXL2 overexpression vector, were treated with 50 μg/mL of salidroside or 50 μM of KC7F2 (a HIF-1α inhibitor) under hypoxia. Cell viability and invasion were assessed using CCK-8 and Transwell chamber assay, respectively. Expression of E-cadherin and matrix metalloproteinase 2/9 (MMP 2/9) was determined, by Western blot analysis, to assess cell mobility at molecular levels. We confirmed that hypoxia increased LOXL2 and induced tumorigenesis of BxPC-3 cells, as evidenced by promoted cell proliferation and invasion, enhanced MMP2/9 while reduced E-cadherin. Interestingly, hypoxia-induced carcinogenesis was significantly retarded by both salidroside and KC7F2, however, enhanced with LOXL2 overexpression. Besides, salidroside and KC7F2 reduced LOXL2, and reversed the tumorigenesis of BxPC-3 cells induced by LOXL2 overexpression. Given the inhibitory effect of salidroside on HIF-1α expression, our data suggested that: (1) LOXL2 was the mechanism, whereby salidroside and KC7F2 showed inhibitory effect on cancer progression of BxPC-3 cells; (2) salidroside exerted its anticancer effect, most likely, by a HIF-1α/LOXL2 pathway. In conclusion, salidroside was a novel therapeutic drug in pancreatic cancer, and downregulation of HIF-1α and LXCL2 was the underlying mechanism.