ALKBH8 contributes to neurological function through oxidative stress regulation.

Transfer RNA (tRNA) modification is essential for proper protein translation, as these modifications play important roles in several biological functions and disease pathophysiologies. AlkB homolog 8 (ALKBH8) is one of the nine mammalian ALKBH family molecules known to regulate selenoprotein translation through the modification of the wobble uridine (U34) in tRNA; however, its specific biological roles remain unclear. In this study, we investigated the role of ALKBH8 using Alkbh8-knockout (Albkh8-/-) mice, which were observed to have reduced 5-methoxycarbonylmethyluridine (mcm5U) and (S)-5-methoxycarbonylhydroxymethyluridine levels; notably, the mcm5U level was partially compensated only in the brain. The results of the novel object recognition test showed reduction in time to explore a novel object in Albkh8-/- mice; increased latency to fall in the rotarod performance test and latency to the immobility period in the forced swim test were also observed. These abnormal behaviors indicate dysfunction of the central nervous system. Furthermore, we observed reduced brain weight and ischemic pathological changes in the cerebral cortex and hippocampus in the form of weak eosin staining in the fiber tracts adjacent to the hippocampal cornu ammonis 1 region and an increase in pyramidal cells in the temporal lobe. Concordantly, we identified the differential expression of oxidative stress-related proteins and metabolites in the cerebral cortex and hippocampus using omics analyses. Finally, neurons and glial cells derived from Albkh8-/- mice show reduced mitochondrial membrane potential. Collectively, these findings indicate that ALKBH8 maintains neural function through an oxidative stress-regulatory mechanism.

ALKBH4 is a novel enzyme that promotes translation through modified uridine regulation.

Epitranscriptomics studies the mechanisms of acquired RNA modifications. The epitranscriptome is dynamically regulated by specific enzymatic reactions, and the proper execution of these enzymatic RNA modifications regulates a variety of physiological RNA functions. However, the lack of experimental tools, such as antibodies for RNA modification, limits the development of epitranscriptomic research. Furthermore, the regulatory enzymes of many RNA modifications have not yet been identified. Herein, we aimed to identify new molecular mechanisms involved in RNA modification by focusing on the AlkB homolog (ALKBH) family molecules, a family of RNA demethylases. We demonstrated that ALKBH4 interacts with small RNA, regulating the formation and metabolism of the (R)-5-carboxyhydroxymethyl uridine methyl ester. We also found that the reaction of ALKBH4 with small RNA enhances protein translation efficiency in an in vitro assay system. These findings indicate that ALKBH4 is involved in the regulation of uridine modification and expand on the role of tRNA-mediated translation control through ALKBH4.

A novel application of hectorite nanoclay for preparation of colorectal cancer spheroids with malignant potential.

Three-dimensional (3D) cell culture, which provides an in vivo-like environment in vitro unlike the conventional two-dimensional (2D) cell culture, has attracted much attention from researchers. Although various 3D cell culture methods have been developed, information on a method using inorganic nanoclay is scant. Here, we report that hectorite, an inorganic layered silicate, can be used as an auxiliary material for 3D cell culture. Human colon cancer cell lines cultured in a medium containing 0.01% synthetic hectorite spontaneously formed 3D spheroids in an adherent plate. Morphologically, these spheroids were more dispersed in all directions than control spheroids generated in an ultralow adherent plate. Microarray analysis showed that FGF19, TGM2, and SERPINA3, whose expression is reportedly increased in colon cancer tissues and is related to tumorigenesis or metastasis, were upregulated in HT-29 spheroids formed using synthetic hectorite compared with those in control spheroids. Gene ontology analysis revealed upregulation of genes associated with morphogenesis, cytoskeleton, extracellular matrix, cellular uptake and secretion, signaling pathways, and gene expression regulation. Moreover, fluorescence-labeled hectorite particles were localized in the cytoplasm of individual cells in spheroids. These results suggest that the synthetic hectorite modified the physiological state of and gene expression within the cells, triggering spheroid formation with malignant characteristics. Our findings highlight a novel application of synthetic hectorite for 3D cell culture.

Discovery of two novel ALKBH5 selective inhibitors that exhibit uncompetitive or competitive type and suppress the growth activity of glioblastoma multiforme.

A group of RNA methylation enzymes is currently of interest as a new target for cancer therapy. Alpha-ketoglutarate-dependent dioxygenase B (AlkB) homolog 5 (ALKBH5) is an N6 -methyladenosine (m6 A) demethylation enzyme, and by high-throughput screening from pure small molecule compounds, we identified two novel inhibitors, Ena15 and Ena21, against it. Each compound showed either uncompetitive or competitive inhibition for 2-oxoglutarate (2OG). In addition, Ena21 had little inhibitory activity for fat mass and obesity-associated protein (FTO), which is another N6 -methyladenosine demethylation enzyme, while Ena15 enhanced the demethylase activity of FTO. The predicted binding poses of both compounds with the crystal structure of ALKBH5 (PDB ID: 4NRO) were comparable with these observations pertaining to the interaction of the 2OG catalytic site in this enzyme kinetics. Furthermore, either knockdown of ALKBH5 or inhibition with Ena15 or Ena21 inhibited cell proliferation of glioblastoma multiforme-derived cell lines, decreased cell population in the synthesis phase of the cell cycle, increased m6 A RNA level, and stabilized FOXM1 mRNA. Based on these results, Ena15 and Ena21 were found to be potential candidates that might help in further research into the biological function of ALKBH5.

Pharmacological Inhibition of miR-130 Family Suppresses Bladder Tumor Growth by Targeting Various Oncogenic Pathways via PTPN1.

Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrcTyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrcTyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrcTyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.

ALKBH4 promotes tumourigenesis with a poor prognosis in non-small-cell lung cancer.

The human AlkB homolog family (ALKBH) of proteins play a critical role in some types of cancer. However, the expression and function of the lysine demethylase ALKBH4 in cancer are poorly understood. Here, we examined the expression and function of ALKBH4 in non-small-cell lung cancer (NSCLC) and found that ALKBH4 was highly expressed in NSCLC, as compared to that in adjacent normal lung tissues. ALKBH4 knockdown significantly induced the downregulation of NSCLC cell proliferation via cell cycle arrest at the G1 phase of in vivo tumour growth. ALKBH4 knockdown downregulated E2F transcription factor 1 (E2F1) and its target gene expression in NSCLC cells. ALKBH4 and E2F1 expression was significantly correlated in NSCLC clinical specimens. Moreover, patients with high ALKBH4 expression showed a poor prognosis, suggesting that ALKBH4 plays a pivotal tumour-promoting role in NSCLC.

Development of a highly sensitive method for the quantitative analysis of modified nucleosides using UHPLC-UniSpray-MS/MS.

There are more than 150 types of naturally occurring modified nucleosides, which are believed to be involved in various biological processes. Recently, an ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) technique has been developed to measure low levels of modified nucleosides. A comprehensive analysis of modified nucleosides will lead to a better understanding of intracellular ribonucleic acid modification, but this analysis requires high-sensitivity measurements. In this perspective, we established a highly sensitive and quantitative method using the newly developed ion source, UniSpray. A mass spectrometer was used with a UniSpray source in positive ion mode. Our UHPLC-UniSpray-MS/MS methodology separated and detected the four major nucleosides, 42 modified nucleosides, and dG15N5 (internal standard) in 15 min. The UniSpray method provided good correlation coefficients (>0.99) for all analyzed nucleosides, and a wide range of linearity for 35 of the 46 nucleosides. Additionally, the accuracy and precision values satisfied the criteria of <15% for higher concentrations and <20% for the lowest concentrations of all nucleosides. We also investigated whether this method could measure nucleosides in biological samples using mouse tissues and non-small cell lung cancer clinical specimens. We were able to detect 43 and 31 different modified nucleosides from mouse and clinical tissues, respectively. We also found significant differences in the levels of N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), 1-methylinosine (m1I), 2'-O-methylcytidine (Cm), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5S2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) between cancerous and noncancerous tissues. In conclusion, we developed a highly sensitive methodology using UHPLC-UniSpray-MS/MS to simultaneously detect and quantify modified nucleosides, which can be used for analysis of biological samples.

Cancer type­SLCO1B3 promotes epithelial­mesenchymal transition resulting in the tumour progression of non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) is one of the most common histologically defined subtypes of lung cancer. To identify a promising molecular target for NSCLC therapy, we performed gene expression analysis at the exon level using postoperative specimens of NSCLC patients. Exon array and real­time PCR analyses revealed that an alternative splicing variant of solute carrier organic anion transporter family member 1B3 (SLCO1B3) called cancer type­SLCO1B3 (Ct­SLCO1B3) was significantly upregulated in the NSCLC samples. SLCO1B3 expressed in the liver [liver type (Lt)­SLCO1B3] was found to be localised in the cell membrane, whereas Ct­SLCO1B3 was detected in the cytoplasm of NSCLC cells. RNAi­mediated knockdown of Ct­SLCO1B3 inhibited in vitro anchorage­independent cell growth, cell migration, and in vivo tumour growth of A549 cells. Overexpression of Ct­SLCO1B3 but not Lt­SLCO1B3 upregulated anchorage­independent cell growth and cell migration of NCI­H23 cells. Mechanistically, Ct­SLCO1B3 was found to regulate the expression of epithelial­mesenchymal transition (EMT)­related genes. The upregulation of E­cadherin was discovered to be especially pivotal to phenotypes of Ct­SLCO1B3­suppressed A549 cells. These findings suggest that Ct­SLCO1B3 functions as a tumour­promoting factor via regulating EMT­related factors in NSCLC.

MicroRNA-130b functions as an oncomiRNA in non-small cell lung cancer by targeting tissue inhibitor of metalloproteinase-2.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related death worldwide. Although many molecular-targeted drugs for NSCLC have been developed in recent years, the 5-year survival rate of patients with NSCLC remains low. Therefore, an improved understanding of the molecular mechanisms underlying the biology of NSCLC is essential for developing novel therapeutic strategies for the treatment of NSCLC. In this study, we examined the role of miR-130b in NSCLC. Our results showed that high expression of miR-130b in clinical specimens was significantly associated with poor overall survival in patients with NSCLC. Moreover, miR-130b expression was significantly increased in NSCLC clinical specimens from patients with vascular and lymphatic invasion. Consistent with this, overexpression of miR-130b promoted invasion and matrix metalloproteinase-2 (MMP-2) activity in A549 cells. Argonaute2 immunoprecipitation and gene array analysis identified tissue inhibitor of metalloproteinase-2 (TIMP-2) as a target of miR-130b. Invasion activity promoted by miR-130b was attenuated by TIMP-2 overexpression in A549 cells. Furthermore, TIMP-2 concentrations in serum were inversely correlated with relative miR-130b expression in tumor tissues from the same patients with NSCLC. Overall, miR-130b was found to act as an oncomiR, promoting metastasis by downregulating TIMP-2 and invasion activities in NSCLC cells.

Novel Metabolically Stable PCA-1/ALKBH3 Inhibitor Has Potent Antiproliferative Effects on DU145 Cells In Vivo.

Novel potent prostate cancer antigen-1 (PCA-1)/alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3) inhibitors both in vivo and in vivo were designed and evaluated by a stability assay in an S9 mixture, a mixture of rat liver homogenate and co-factors, and oral absorbability assay in rat, as well as enzyme and cell assays, and resulted in the synthesis of a novel potent PCA-1/ALKBH3 inhibitor in vivo. Among them, compound 7l exhibited potent inhibitory activities in a xenograft model bearing DU145 tumor at 10 mg/kg by subcutaneous administration without negative side-effects. This inhibitory activity in vivo was more potent than that of HUHS015 at 32 mg/kg, a known PCA-1/ALKBH3 inhibitor, or docetaxel at 2.5 mg/kg, the drug clinically used for androgen-independent prostate cancer.

Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma.

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03-1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.

TP53 gene status is a critical determinant of phenotypes induced by ALKBH3 knockdown in non-small cell lung cancers.

Human AlkB homolog 3 (ALKBH3) is overexpressed in non-small cell lung cancers (NSCLC) and its high expression is significantly correlated with poor prognosis. While ALKBH3 knockdown induces apoptosis in NSCLC cells, the underlying anti-apoptotic mechanisms of ALKBH3 in NSCLC cells remain unclear. Here we show that ALKBH3 knockdown induces cell cycle arrest or apoptosis depending on the TP53 gene status in NSCLC cells. In comparison to parental cells, TP53-knockout A549 cells showed DNA damage-responsive signal induced by ALKBH3 knockdown. TP53 knockout shifted the phenotypes of A549 cells induced by ALKBH3 knockdown from cell cycle arrest to apoptosis induction, suggesting that the TP53 gene status is a critical determinant of the phenotypes induced by ALKBH3 knockdown in NSCLC cells.

High miR-122 expression promotes malignant phenotypes in ccRCC by targeting occludin.

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. Although several studies have reported high expression of miR-122 in ccRCC, its physiological role remains unclear. To clarify the role of miR-122 in ccRCC, we compared miR-122 expression levels in non-cancerous tissue and ccRCC. Significant upregulation of miR-122 was observed in ccRCC specimens. Moreover, ccRCC patients with high miR-122 expression showed poor progression-free survival compared to those with low miR-122 expression. Overexpression of miR-122 using an miRNA mimic promoted proliferation, migration, and invasion activities of ccRCC cells. miR-122 directly targets occludin, a known component of tight junctions. Occludin knockdown promoted the cell migration activity but not proliferation or invasion activities of ccRCC cells. In human clinical specimens, miR-122 expression inversely correlated with occludin protein expression. These findings show that miR-122 is an oncomiR in ccRCC.

AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells.

The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion.

ALKBH8 promotes bladder cancer growth and progression through regulating the expression of survivin.

Human AlkB homolog 8 (ALKBH8) is highly expressed in high-grade, superficially and deeply invasive bladder cancer. Moreover, ALKBH8 knockdown induces apoptosis in bladder cancer cells. However, the underlying anti-apoptotic mechanism of ALKBH8 in bladder cancer cells has thus far remained unclear. Moreover, there is no direct evidence that highly expressed ALKBH8 is involved in tumor progression in vivo. We here show that ALKBH8 knockdown induced apoptosis via downregulating the protein expression of survivin, an anti-apoptotic factor also exhibiting increased levels in bladder cancer. We also clarify that ALKBH8 transgenic mice showed an accelerated rate of bladder tumor mass and invasiveness in an N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model. These findings suggest that the high expression of ALKBH8 is critical for the growth and progression of bladder cancer.

The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN.

Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer.

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3.

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.

Expression of miR-27a-3p is an independent predictive factor for recurrence in clear cell renal cell carcinoma.

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and function in tumor development and progression. We previously identified up-regulated miRNAs in clear cell renal cell carcinoma (ccRCC) compared to matched-pair normal kidney by microarray. Here, we identify miRNAs that are up-regulated in ccRCC and are also correlated with survival and/or recurrence. Twenty-four samples from ccRCC patients who underwent nephrectomies between 2011 and 2012 were divided into two groups: one of eleven patients who experienced recurrence (Group 1), and one of thirteen patients with no evidence of disease (Group 2) 2 years after surgery. Analyzing 22 miRNAs that were up-regulated in ccRCC in our previous study, we identify five miRNAs that were statistically up-regulated in Group 1 versus Group 2 by quantitative real-time PCR. We then evaluated these miRNAs in an independent cohort of 159 frozen ccRCC samples. High levels of miR-27a-3p (p < 0.01) correlated with a worse progression-free survival rate. Multivariate analysis revealed that miR-27a-3p was an independent predictive factor for recurrence. For functional analysis, miR-27a-3p controlled cell proliferation, migration and invasion in RCC cell lines. MiR-27a-3p could act as oncogenic miRNA and may be a candidate for targeted molecular therapy in ccRCC.

miR-629 Targets TRIM33 to Promote TGFß/Smad Signaling and Metastatic Phenotypes in ccRCC.

UNLABELLED: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFß/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFß-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFß on the expression of epithelial-mesenchymal transition-related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFß/Smad signaling pathway via TRIM33 in ccRCC. IMPLICATIONS: This study suggests that miR-629 has biomarker potential through its ability to regulate TGFß/Smad signaling and accelerate ccRCC cell motility and invasion.

LOXL2 status correlates with tumor stage and regulates integrin levels to promote tumor progression in ccRCC.

UNLABELLED: Clear cell renal cell carcinoma (ccRCC) is the most common histologically defined subtype of renal cell carcinoma (RCC). To define the molecular mechanism in the progression of ccRCC, we focused on LOX-like protein 2 (LOXL2), which is critical for the first step in collagen and elastin cross-linking. Using exon array analysis and quantitative validation, LOXL2 was shown to be significantly upregulated in clinical specimens of human ccRCC tumor tissues, compared with adjacent noncancerous renal tissues, and this elevated expression correlated with the pathologic stages of ccRCC. RNAi-mediated knockdown of LOXL2 resulted in marked suppression of stress-fiber and focal adhesion formation in ccRCC cells. Moreover, LOXL2 siRNA knockdown significantly inhibited cell growth, migration, and invasion. Mechanistically, LOXL2 regulated the degradation of both integrins α5 (ITGAV5) and ß1 (ITGB1) via protease- and proteasome-dependent systems. In clinical ccRCC specimens, the expression levels of LOXL2 and integrin α5 correlated with the pathologic tumor grades. In conclusion, LOXL2 is a potent regulator of integrin α5 and integrin ß1 protein levels and functions in a tumor-promoting capacity in ccRCC. IMPLICATIONS: This is the first report demonstrating that LOXL2 is highly expressed and involved in ccRCC progression by regulating the levels of integrins α5 and ß1.