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.

Cutibacterium acnes-derived extracellular vesicles promote tumor growth in renal cell carcinoma.

Bacterial flora are present in various parts of the human body, including the intestine, and are thought to be involved in the etiology of various diseases such as multiple sclerosis, intestinal diseases, cancer, and uterine diseases. In recent years, the presence of bacterial 16S rRNA genes has been revealed in blood, which was previously thought to be a sterile environment, and characteristic blood microbiomes have been detected in various diseases. However, the mechanism and the origin of the bacterial information are unknown. In this study, we performed 16S rRNA metagenomic analysis of bacterial DNA in serum extracellular vesicles from five healthy donors and seven patients with renal cell carcinoma and detected Cutibacterium acnes DNA as a characteristic bacterial DNA in the serum extracellular vesicles of patients with renal cell carcinoma. In addition, C. acnes DNA was significantly reduced in postoperative serum extracellular vesicles from patients with renal cell carcinoma compared with that in preoperative serum extracellular vesicles from these patients and was also detected in tumor tissue and extracellular vesicles from tumor tissue-associated microbiota, suggesting an association between C. acnes extracellular vesicles and renal cell carcinoma. C. acnes extracellular vesicles were taken up by renal carcinoma cells to enhance their proliferative potential. C. acnes extracellular vesicles also exhibited tumor-promoting activity in a mouse model of renal cancer allografts with enhanced angiogenesis. These results suggest that extracellular vesicles released by C. acnes localized in renal cell carcinoma tissues act in a tumor-promoting manner.

Circulating extracellular vesicles carrying Firmicutes reflective of the local immune status may predict clinical response to pembrolizumab in urothelial carcinoma patients.

Bacterial flora has clinical significance for the host. The metabolic environment created by this flora influences immunotherapy in urothelial carcinoma. However, there are no reports on the clinical significance of bacterial flora in the host bloodstream. We aimed to clarify the correlation between extracellular vesicle (EV)-derived blood microflora information and tumor immunological status in urothelial carcinoma (UC) patients. Serum samples were collected from 20 healthy donors, 50 patients with localized UC, and 31 patients with metastatic UC (mUC) who had undergone pembrolizumab treatment. Bacterial DNA in EVs was extracted from each sample. Metagenomic sequencing was performed after amplification of the V1-V2 region of the bacterial 16S rRNA gene. Using the matched tumor tissue and serum samples, we revealed that the smaller amount of peripheral EVs carrying Firmicutes DNA was significantly correlated with the higher number of infiltrating T cells within tumor tissues (CD3; p = 0.015, CD4; p = 0.039, CD8; p = 0.0084) and the higher expression of activation markers on their surface (ICOS on both CD4; p = 0.0013 and CD8 T cells; p = 0.016 and 4-1BB on CD4 T cells; p = 0.016). In terms of circulating metabolic information, L-Ser and L-Pro levels, which play important roles in T cell expansion and proliferation, were significantly higher in the Firmicutes-low group (p = 0.010). All of the patients with higher Firmicutes abundance had disease progression without any clinical response (p = 0.026) and significantly inferior prognosis for pembrolizumab therapy (p = 0.035). This is the first study on the importance of peripheral bacterial EVs in cancer patients treated with cancer immunotherapy.

Proteomic analysis of urinary and tissue-exudative extracellular vesicles to discover novel bladder cancer biomarkers.

Proteomic analysis of urinary extracellular vesicles (EVs) is a powerful approach to discover potential bladder cancer (BCa) biomarkers, however urine contains numerous EVs derived from the kidney and normal urothelial epithelium, which can obfuscate information related to BCa cell-derived EVs. In this study, we combined proteomic analysis of urinary EVs and tissue-exudative EVs (Te-EVs), which were isolated from culture medium of freshly resected viable BCa tissues. Urinary EVs were isolated from urine samples of 11 individuals (7 BCa patients and 4 healthy individuals), and Te-EVs were isolated from 7 BCa tissues. We performed tandem mass tag (TMT)-labeling liquid chromatography (LC-MS/MS) analysis for both urinary EVs and Te-EVs and identified 1960 proteins in urinary EVs and 1538 proteins in Te-EVs. Most of the proteins identified in Te-EVs were also present in urinary EVs (82.4%), with 55 of these proteins showing upregulated levels in the urine of BCa patients (fold change > 2.0; P < .1). Among them, we selected 22 membrane proteins as BCa biomarker candidates for validation using selected reaction monitoring/multiple reaction monitoring (SRM/MRM) analysis on urine samples from 70 individuals (40 BCa patients and 30 healthy individuals). Six urinary EV proteins (heat-shock protein 90, syndecan-1, myristoylated alanine-rich C-kinase substrate (MARCKS), MARCKS-related protein, tight junction protein ZO-2, and complement decay-accelerating factor) were quantified using SRM/MRM analysis and validated as significantly upregulated in BCa patients (P < .05). In conclusion, the novel strategy that combined proteomic analysis of urinary EVs and Te-EVs enabled selective detection of urinary BCa biomarkers.

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.

MicroRNA-92b-3p is a prognostic oncomiR that targets TSC1 in clear cell renal cell carcinoma.

Although several studies have reported that microRNA (miR)-92b-3p is involved in various cellular processes related to carcinogenesis, its physiological role in clear cell renal cell carcinoma (ccRCC) remains unclear. To clarify the role of miR-92b-3p in ccRCC, we compared miR-92b-3p expression levels in ccRCC tissues and adjacent normal renal tissues. Significant upregulation of miR-92b-3p was observed in ccRCC tissues. Overexpression of miR-92b-3p using a miRNA mimic promoted proliferation, migration, and invasion activities of ACHN cells. Functional inhibition of miR-92b-3p by a hairpin miRNA inhibitor suppressed Caki-2 cell growth and invasion activities in vitro. Mechanistically, it was found that miR-92b-3p directly targeted the TSC1 gene, a known upstream regulator of mTOR. Overexpression of miR-92b-3p decreased the protein expression of TSC1 and enhanced the downstream phosphorylation of p70S6 kinase, suggesting that the mTOR signaling pathway was activated by miR-92b-3p in RCC cells. Importantly, a multivariate Cox proportion hazard model, based on TNM staging and high levels of miR-92b-3p, revealed that miR-92b-3p expression (high vs. low hazard ratio, 2.86; 95% confidence interval, 1.20-6.83; P = .018) was a significant prognostic factor for overall survival of ccRCC patients with surgical management. Taken together, miR-92b-3p was found to act as an oncomiR, promoting cell proliferation by downregulating TSC1 in ccRCC.

Diagnostic potential of TERT promoter and FGFR3 mutations in urinary cell-free DNA in upper tract urothelial carcinoma.

Most upper tract urothelial carcinomas (UTUC) are muscle invasive at the time of diagnosis. Current standard methods for the diagnosis of UTUC are invasive. Urine cytology is the only non-invasive test for detecting UTUC, but its sensitivity is low. A novel non-invasive assay for UTUC detection would improve patient outcome. This study aimed to investigate the mutation of cell-free DNA (cfDNA) in urine supernatant to develop a reliable diagnostic biomarker for UTUC patients. We studied urinary cfDNA from 153 individuals, including 56 patients with localized UTUC, and carried out droplet digital PCR assay for TERT promoter and FGFR3 hotspot mutations. We could detect mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR3 S249C in 9/56 (16.1%) patients. FGFR3 mutation was detected only in ≤pT1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%. Although these data need to be validated in a larger-scale cohort, mutation analysis of TERT promoter and FGFR3 in urinary cfDNA has the potential to be a non-invasive diagnostic marker and reliable factor for tumor staging.

Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma.

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be determined. Circulating tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. The present study aims to clarify the clinical utility of ctDNA for RCC. Fifty-three patients histologically diagnosed with clear cell RCC were enrolled. Targeted sequencing was carried out using plasma cell-free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR (ddPCR) to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics-based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50-166 bp) to large (167-250 bp) cfDNA fragments. Association of mutant allele frequency of ctDNA with clinical course was analyzed. Prognostic potential was evaluated using log-rank test. A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6) and VHL (n = 5), and median mutant allele frequency of ctDNA was 10%. We designed specific ddPCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. Positive ctDNA was significantly associated with a higher proportion of cfDNA fragments (P = .033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer-specific survival (P < .001, P = .011). In conclusion, our study shows the clinical utility of ctDNA status and cfDNA fragment size as biomarkers for prognosis and disease monitoring in RCC.

Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin.

Cancer-associated extracellular vesicles (EVs) are intimately involved in establishment of tumor microenvironment and occurrence of metastasis. However, previous studies have mainly relied on experiments with cultured cell lines or mouse models, making it difficult to gain a full understanding of EV functions in human body. Hence, we extracted EVs directly from surgically resected viable clear cell renal cell carcinoma (ccRCC) tissues and adjacent normal renal tissues (n = 20). Quantitative LC/MS analysis identified 3,871 tissue-exudative EV (Te-EV) proteins, among which azurocidin (AZU1) was highly enriched in tumor Te-EVs (p = 2.85 × 10-3 , fold-change = 31.59). Importantly, AZU1 content was also significantly higher in serum EVs from ccRCC patients compared to those from healthy donors. We further found that ccRCC-derived EVs had AZU1-dependent membrane permeabilizing activity for the vascular endothelial cell layer. Thus Te-EVs should be ideal resource for investigation of physiological EV functions.

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.

Peripheral blood monocyte count reflecting tumor-infiltrating macrophages is a predictive factor of adverse pathology in radical prostatectomy specimens.

BACKGROUND: Tumor-infiltrating macrophages, which are thought to be derived from blood monocytes, interact with tumor cells to promote cancer progression. The aim of this study was to assess the association of peripheral blood monocyte count with pathological findings and local tumor-infiltrating macrophages in prostatectomy specimens. METHODS: Preoperative peripheral blood monocyte counts were retrospectively assessed for their associations with pathological findings (pathological T stage, Gleason Score, extraprostatic extension, seminal vesicle invasion, and surgical margin) and biochemical recurrence of 248 patients who underwent radical prostatectomy. Local tumor-infiltrating macrophages were also evaluated immunohistochemically for their association with peripheral monocyte counts. RESULTS: The peripheral monocyte counts of the patients with extraprostatic extension, seminal vesicle invasion, or primary Gleason ≥4 were significantly higher than those of the patients without each of these pathological findings (P < 0.001, P = 0.034, and P = 0.004, respectively). Peripheral monocyte count was a significant predictor of adverse pathology and postoperative biochemical recurrence in localized prostate cancer by multivariate analysis (P = 0.001 and P = 0.041, respectively). Both the density and the count of tumor-infiltrating macrophages correlated significantly with the peripheral blood monocyte count (Spearman rank correlation coefficients were 0.463 and 0.649, respectively, P < 0.001). CONCLUSIONS: Peripheral blood monocyte count reflecting local tumor-infiltrating macrophages was a predictive factor for tumor progression and prognosis in patients with localized prostate cancer. Elucidating the mechanism of the interaction of peripheral monocytes with tumor-infiltrating macrophages is necessary.

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.

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.

Differentiation-inducing factor-1 suppresses the expression of c-Myc in the human cancer cell lines.

Differentiation-inducing factor-1 (DIF-1), a morphogen for Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by suppressing the Wnt/ß-catenin signaling pathway. In this study, we examined the effect of DIF-1 on c-Myc, a target gene product of the Wnt/ß-catenin signaling pathway, mainly using HCT-116 colon cancer cells. DIF-1 strongly reduced the amount of c-Myc protein in time- and concentration-dependent manners and reduced c-Myc mRNA expression by inhibiting promoter activity through the TCF binding sites. The effect of DIF-1 on c-Myc was also confirmed using the human cervical cell line HeLa. Pretreatment with the proteasome inhibitor MG132 or glycogen synthase kinase-3ß (GSK-3ß) inhibitors (LiCl and SB216763) attenuated the effect of DIF-1, suggesting that DIF-1 induced c-Myc protein degradation through GSK-3ß activation. Furthermore, we examined whether c-Myc was involved in the anti-proliferative effect of DIF-1 using c-Myc-overexpressing cells and found that c-Myc was associated with the anti-proliferative effect of this compound. These results suggest that DIF-1 inhibits c-Myc expression by inhibiting promoter activity and inducing protein degradation via GSK-3ß activation, resulting in the inhibition of cell proliferation. Since c-Myc seems to be profoundly involved in accelerated proliferation of various malignant tumors, DIF-1 may have a potential to develop into a novel anti-cancer agent.

Hypoxia regulates tumour characteristic RNA modifications in ovarian cancers.

Analysis of ovarian cancer tissue and normal ovarian tissue revealed 31 RNA modifications with significant differences observed in cancer tissue compared with normal tissue. Moreover, we found Im and chm5U as characteristic RNA modifications in advanced and platinum-resistant ovarian cancers, respectively. Considering that these differences in RNA modifications may be due to the intra-tumour microenvironment, we xenografted the ovarian cancer cell line RMG-1 to create RMG-1 tumours and compared them with original RMG-1 cells. As a result, 14 of the 31 RNA modifications showed marked variations during tumorigenesis. Eight RNA modifications (m2,2G, t6A, m7G, m5U, m1G, i6A, m6t6A and m1A), which were upregulated in ovarian cancer tissues and in RMG-1-xenografted tumour, were also upregulated under hypoxic conditions. RNAseq analysis, using the matched RNA samples analysed for RNA modifications, showed that 2137 genes were highly expressed in ovarian cancer tissues compared with those in normal ovarian tissues. Of these, 134 genes, which were enriched in a gene set belonging to the hypoxia signalling pathway, were positively correlated with the above eight RNA modifications. These results suggest that the tumour microenvironment, including hypoxia, is important for cancer characteristic RNA modifications.

Bacteria-derived DNA in serum extracellular vesicles are biomarkers for renal cell carcinoma.

This is the first study to determine the clinical importance of circulating bacterial DNA in patients with renal cell carcinoma (RCC). We performed 16S rRNA metagenomic analysis of serum extracellular vesicles (EVs) from 88 patients with RCC and 10 healthy donors and identified three abundant bacterial DNA: Bacteroidia, TM7-1, and Sphingomonadales. Combining characteristic bacterial DNA information (three bacteria-derived DNA), a BTS index was created to diagnose patients with RCC. The BTS index showed high sensitivity not only in the discovery cohort, but also in the validation cohort, suggesting that it was useful as a screening test. Furthermore, in nivolumab treatment of RCC, patients with higher levels of Bacteroidia DNA in serum EVs had significantly poorer progression-free and overall survival than did those with lower levels. This study showed that circulating Bacteria-derived DNA could be used as a biomarker for RCC.

Azurocidin is loaded into small extracellular vesicles via its N-linked glycosylation and promotes intravasation of renal cell carcinoma cells.

Azurocidin (AZU1) is an antimicrobial protein secreted by neutrophils that acts as a chemoattractant for monocytes and macrophages and a permeabilizer of vascular endothelial cells. We previously identified AZU1 to be specifically present in extracellular vesicles (EVs) obtained from renal cell carcinoma (RCC) tissues. Here, we examined the relationship between N-linked glycosylation and AZU1 loading into small EVs (SEVs). Inhibition of N-linked glycosylation by introducing mutations in three glycosylation sites inhibited AZU1 loading into SEVs. Furthermore, SEVs released from AZU1-wild-type cells increased the Ca2+ concentration in endothelial cells and the endothelial permeability, whereas SEVs released from AZU1-mutant cells had no significant effect. Anti-AZU1 antibodies diminished the effect of SEVs on endothelial cell sheets. Collectively, we found that N-linked glycosylation of AZU1 directs its loading into SEVs, thereby enabling AZU1-positive SEVs to function as potent permeabilizers of endothelial cells and leading to enhanced transendothelial migration of RCC cells.

MiR-30b-3p and miR-126-3p of urinary extracellular vesicles could be new biomarkers for prostate cancer.

BACKGROUND: Extracellular vesicles (EVs) including exosomes are present in blood, urine, and saliva and contain proteins, microRNAs, and messenger RNAs. We investigated microRNAs in urinary EVs to discover new biomarkers of prostate cancer (PCa). METHODS: We isolated EVs from urine obtained following digital rectal examination (DRE) of 14 men with elevated levels of serum prostate-specific antigen (PSA) [negative biopsy (n=4) and PCa with Gleason scores of 6 (n=3), 7 (n=3), and 8-9 (n=4)]. MicroRNAs extracted from EVs were analyzed by microRNA microarray. RESULTS: MicroRNAs miR-30b-3p and miR-126-3p were identified as being overexpressed in urinary EVs of the PCa patients versus the biopsy-negative men, but no microRNAs were associated with the Gleason score. In the independent cohort as well, these two microRNAs were overexpressed in urinary EVs from the PCa patients versus the negative-biopsy men. Logistic regression analysis adjusted by age and PSA showed that these two microRNAs were significantly associated with the prediction of PCa in biopsy specimens. Sensitivity and specificity of miR-30b-3p and miR-126-3p for the prediction of PCa were 46.4% and 88.0% and 60.7% and 80.0%, respectively, which were better than those of serum PSA (53.5% and 64.0%, respectively). CONCLUSIONS: MiR-30b-3p and miR-126-3p in urinary EVs could be potential biomarkers of PCa.

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.

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.