Keith S. K. Fong

A molecular role for lysyl oxidase‐like 2 enzyme in Snail regulation and tumor progression

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. Snails 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.

Structural and functional diversity of lysyl oxidase and the LOX-like proteins.

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

Lysyl Oxidase Binds Transforming Growth Factor-β and Regulates Its Signaling via Amine Oxidase Activity

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-β1 (TGF-β1), a potent growth factor abundant in bone, the effect of LOX on TGF-β1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-β1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-β1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-β1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Lysyl oxidase-like 2 expression is increased in colon and esophageal tumors and associated with less differentiated colon tumors.

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.

Lysyl oxidase-like 2 promotes migration in noninvasive breast cancer cells but not in normal breast epithelial cells.

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.

Active lysyl oxidase (LOX) correlates with focal adhesion kinase (FAK)/paxillin activation and migration in invasive astrocytes

The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up‐regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross‐linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide‐mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I–IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher‐grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta‐aminopropionitrile inhibition of LOX activity and depletion of H2O2 via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein‐1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX‐mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure

ABSTRACT Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse. Summary: We propose an epigenetic mechanism establishing the regulation of genes that are crucial for neural tube closure. This mechanism could be a novel target for resolving such birth defects and associated disorders.

Transcriptome Analysis of PPARγ Target Genes Reveals the Involvement of Lysyl Oxidase in Human Placental Cytotrophoblast Invasion

Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by β-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion.

LOX (lysyl oxidase)

Review on LOX (lysyl oxidase), with data on DNA, on the protein encoded, and where the gene is implicated.

Midline craniofacial malformations with a lipomatous cephalocele are associated with insufficient closure of the neural tube in the tuft mouse

BACKGROUND Genetic variations affecting neural tube closure along the head result in malformations to the face and brain, posing a significant impact on health care costs and the quality of life. METHODS We have established a mouse line from a mutation that arose spontaneously in our wild-type colony that we called tuft. Tuft mice have heritable midline craniofacial defects featuring an anterior lipomatous cephalocele. RESULTS Whole-mount skeletal stains indicated that affected newborns had a broader interfrontal suture where the cephalocele emerged between the frontal bones. Mice with a cephalocele positioned near the rostrum also presented craniofacial malformations such as ocular hypertelorism and midfacial cleft of the nose. Gross and histological examination revealed that the lipomatous cephalocele originated as a fluid filled cyst no earlier than E14.5 while embryos with a midfacial cleft was evident during craniofacial development at E11.5. Histological sections of embryos with a midfacial cleft revealed the cephalic neuroectoderm remained proximal or fused to the frontonasal ectoderm about the closure site of the anterior neuropore, indicating a defect to neural tube closure. We found the neural folds along the rostrum of E9 to E10.5 embryos curled inward and failed to close as well as embryos with exencephaly and anencephaly at later stages. Whole-mount in situ hybridization of anterior markers Fgf8 and Sonic hedgehog indicated closure of the rostral site was compromised in severe cases. CONCLUSION We present a model demonstrating how anterior cranial cephaloceles are generated following a defect to neural tube closure and relevance to subsequent craniofacial morphogenesis in the tuft mouse.