Overexpression of Slc22a18 facilitates fat accumulation in mice.

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Organic Anion Transporters (OAT) and Other SLC22 Transporters in Progression of Renal Cell Carcinoma.

(1) Background: Many transporters of the SLC22 family (e.g., OAT1, OAT3, OCT2, URAT1, and OCTN2) are highly expressed in the kidney. They transport drugs, metabolites, signaling molecules, antioxidants, nutrients, and gut microbiome products. According to the Remote Sensing and Signaling Theory, SLC22 transporters play a critical role in small molecule communication between organelles, cells and organs as well as between the body and the gut microbiome. This raises the question about the potential role of SLC22 transporters in cancer biology and treatment. (2) Results: In two renal cell carcinoma RNA-seq datasets found in TCGA, KIRC and KIRP, there were multiple differentially expressed (DE) SLC22 transporter genes compared to normal kidney. These included SLC22A6, SLC22A7, SLC22A8, SLC22A12, and SLC22A13. The patients with disease had an association between overall survival and expression for most of these DE genes. In KIRC, the stratification of patient data by pathological tumor characteristics revealed the importance of SLC22A2, SLC22A6, and SLC22A12 in disease progression. Interaction networks combining the SLC22 with ADME genes supported the centrality of SLC22 transporters and other transporters (ABCG2, SLC47A1) in disease progression. (3) Implications: The fact that many of these genes are uric acid transporters is interesting because altered uric acid levels have been associated with kidney cancer. Moreover, these genes play key roles in processing metabolites and chemotherapeutic compounds, thus making them potential therapeutic targets. Finally, our analyses raise the possibility that current approaches may undertreat certain kidney cancer patients with low SLC22 expression and only localized disease while possibly overtreating more advanced disease in patients with higher SLC22 expression. Clinical studies are needed to investigate these possibilities.

Expression of SLC22A18 regulates oxaliplatin resistance by modulating the ERK pathway in colorectal cancer.

Although oxaliplatin-based chemotherapy is the current standard adjuvant therapy for colorectal cancer (CRC), the molecular mechanisms underlying oxaliplatin resistance remain unclear. Here, we examined the molecular mechanisms underlying SLC22A18-associated oxaliplatin resistance and strategies for overcoming oxaliplatin resistance. We evaluated the association between SLC22A18 and prognosis in 337 patients with CRC and its functional significance and studied the mechanisms through which SLC22A18 affects oxaliplatin resistance development in CRC cells, using CRC cell lines and patient-derived cells (PDCs). SLC22A18 downregulation was positively correlated with worse survival in patients with CRC. Low SLC22A18-expressing cells showed relatively lower sensitivity to oxaliplatin than high SLC22A18-expressing cells. In addition, ERK activation was found to be involved in the mechanisms underlying SLC22A18-related oxaliplatin resistance. To confirm ERK pathway dependence, we used an ERK inhibitor and found that combined treatment with oxaliplatin and the ERK inhibitor overcame oxaliplatin resistance in the low SLC22A18-expressing cells. Ex vivo approaches using PDC confirmed the correlation between SLC22A18 expression and oxaliplatin resistance. Results of the in vivo study showed that SLC22A18 expression regulated oxaliplatin efficacy, and that combined treatment with an ERK inhibitor could be a useful therapeutic strategy when SLC22A18 is downregulated. Together, our findings indicate that SLC22A18 could serve as a biomarker for the prediction of oxaliplatin resistance. In cases of oxaliplatin resistance due to low SLC22A18 expression, resistance can be overcome by combined treatment with an ERK inhibitor.

A Syndrome of Variable Allergy, Short Stature, and Fatty Liver.

SLC22A18 (solute carrier family 22 member 18) is an imprinted gene, but its role in growth and development is not clear. In the present study, we recorded the clinical information of six male patients of six unrelated families. Real-time quantitative PCR, Sanger sequencing, and DNA methylation sequencing were performed in these patients. The results suggested that the patients with the clinical characteristics of allergic allergy, short stature, and fatty liver had a lower expression of SLC22A18. One novel variant (chr11: 2899732 delA) with clinical significance was found in the core promoter region of the patients. Overall, this study found a syndrome associated with SLC22A18.

The Roles of Imprinted SLC22A18 and SLC22A18AS Gene Overexpression Caused by Promoter CpG Island Hypomethylation as Diagnostic and Prognostic Biomarkers for Non-Small Cell Lung Cancer Patients.

Genomic imprinting is a process that involves one gene copy turned-off in a parent-of-origin-dependent manner. The regulation of imprinted genes is broadly dependent on promoter methylation marks, which are frequently associated with both oncogenes and tumor suppressors. The purpose of this study was to assess the DNA methylation patterns of the imprinted solute-carrier family 22 member 18 (SLC22A18) and SLC22A18 antisense (SLC22A18AS) genes in non-small cell lung cancer (NSCLC) patients to study their relevance to the disease. We found that both genes were hypomethylated in adenocarcinoma and squamous cell carcinoma patients. Due to this imprinting loss, SLC22A18 and SLC22A18AS were found to be overexpressed in NSCLC tissues, which is significantly more evident in lung adenocarcinoma patients. These results were validated through analyses of public databases of NSCLC patients. The reversed gene profile of both genes was achieved in vitro by treatment with ademetionine. We then showed that high SLC22A18 and SLC22A18AS expression levels were significantly associated with worsening disease progression. In addition, low levels of SLC22A18AS were also correlated with better overall survival for lung adenocarcinoma patients. We found that SLC22A18 and SLC22A18AS knockdown inhibits cell proliferation in vitro. All these results suggest that both genes may be useful as diagnostic and prognostic biomarkers in NSCLC, revealing novel therapeutic opportunities.

Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress.

The SLC22 family of transporters is widely expressed, evolutionarily conserved, and plays a major role in regulating homeostasis by transporting small organic molecules such as metabolites, signaling molecules, and antioxidants. Analysis of transporters in fruit flies provides a simple yet orthologous platform to study the endogenous function of drug transporters in vivo. Evolutionary analysis of Drosophila melanogaster putative SLC22 orthologs reveals that, while many of the 25 SLC22 fruit fly orthologs do not fall within previously established SLC22 subclades, at least four members appear orthologous to mammalian SLC22 members (SLC22A16:CG6356, SLC22A15:CG7458, CG7442 and SLC22A18:CG3168). We functionally evaluated the role of SLC22 transporters in Drosophila melanogaster by knocking down 14 of these genes. Three putative SLC22 ortholog knockdowns-CG3168, CG6356, and CG7442/SLC22A-did not undergo eclosion and were lethal at the pupa stage, indicating the developmental importance of these genes. Additionally, knocking down four SLC22 members increased resistance to oxidative stress via paraquat testing (CG4630: p < 0.05, CG6006: p < 0.05, CG6126: p < 0.01 and CG16727: p < 0.05). Consistent with recent evidence that SLC22 is central to a Remote Sensing and Signaling Network (RSSN) involved in signaling and metabolism, these phenotypes support a key role for SLC22 in handling reactive oxygen species.

Knockdown of Orphan Transporter SLC22A18 Impairs Lipid Metabolism and Increases Invasiveness of HepG2 Cells.

PURPOSE: The aim of this work is to investigate the roles of solute carrier family 22 member 18 (SLC22A18) in lipid metabolism and in establishing the tumor phenotype of HepG2 cells. METHODS: SLC22A18-knockdown HepG2 cells were established by stable transfection with shRNA. Protein expression levels were measured by quantitative proteomics and Western blot analysis. Cell growth was examined by cell counting kit. Accumulation of triglyceride-rich lipid droplets was measured by Oil-Red O staining. Cell migration and invasion were examined by Transwell assays. RESULTS: SLC22A18-knockdown HepG2 cells accumulated triglyceride-rich lipid droplets and showed decreased expression levels of lysosomal/autophagic proteins, suggesting that lipid degradation is suppressed. Growth of HepG2 cells was decreased by SLC22A18 knockdown, but was restored by free fatty acid supplementation. In addition, SLC22A18 knockdown decreased the expression of insulin-like growth factor-binding protein 1 (IGFBP-1) and increased the invasion ability of HepG2 cells. Exogenous IGFBP-1 blocked the increase of invasion activity induced by SLC22A18 knockdown. CONCLUSION: Our results suggest that suppression of SLC22A18 decreased the supply of intracellular free fatty acids from triglyceride-rich lipid droplets by impairing the lysosomal/autophagy degradation pathway and reduced the invasive activity of HepG2 cells by decreasing IGFBP-1 expression.

Involvement of an Orphan Transporter, SLC22A18, in Cell Growth and Drug Resistance of Human Breast Cancer MCF7 Cells.

The SLC22A18 gene, which encodes an orphan transporter, is located at the 11p15.5 imprinted region, an important tumor suppressor gene region. However, the role of SLC22A18 in tumor suppression remains unclear. Here, we investigated the involvement of SLC22A18 in cell growth, invasion, and drug resistance of MCF7 human breast cancer cell line. Western blot analysis indicated that SLC22A18 is predominantly expressed at intracellular organelle membranes. Quantitative proteomics showed that knockdown of SLC22A18 significantly altered the expression of 578 (31.0%) of 1867 proteins identified, including proteins related to malignancy and poor prognosis of breast cancer. SLC22A18 knockdown (1) increased MCF7 cell growth concomitantly with a >7-fold increase of annexin A8 (involved in cell growth and migration; a predictor of poor prognosis), (2) induced spherical morphology of MCF7 cells concomitantly with a nearly 3-fold increase of CD44 (involved in regulation of malignant phenotypes), and (3) increased chemosensitivity to vinca alkaloids concomitantly with a >80% reduction of doublecortin-like kinase 1 (involved in regulation of microtubule polymerization). Our results suggest that SLC22A18 may act as a tumor suppressor by regulating the expression levels of cell growth-related proteins, and vinca alkaloids might show therapeutic efficacy against low-SLC22A18-expressing breast cancer.

Characterization of SLC22A18 as a tumor suppressor and novel biomarker in colorectal cancer.

SLC22A18, solute carrier family 22, member 18, has been proposed to function as a tumor suppressor based on its chromosomal location at 11p15.5, mutations and aberrant splicing in several types of cancer and down-regulation in glioblastoma. In this study, we sought to demonstrate the significance of SLC22A18 as a tumor suppressor in colorectal cancer (CRC) and provide mechanistic bases for its function. We first showed that the expression of SLC22A18 is significantly down-regulated in tumor tissues using matched normal-tumor samples from CRC patients. This finding was also supported by publically accessible data from The Cancer Genome Atlas (TCGA). Functionally, SLC22A18 inhibits colony formation and induces of G2/M arrest consistent with being a tumor suppressor. Interestingly, suppression of KRAS by RNA interference promotes SLC22A18 expression, and expression of SLC22A18 in turn inhibits KRAS(G12D)-mediated anchorage independent growth of NIH3T3 cells indicating a mutual negative interaction. Finally, we evaluated diagnostic and prognostic values of SLC22A18 using clinical and gene expression data from TCGA which revealed a significantly worse long-term prognosis for patients with low level SLC22A18 expression. In sum, we established SLC22A18 as a tumor suppressor in colon epithelial cells and propose that SLC22A18 is potentially a marker of diagnostic and prognostic values.

Expression of special AT-rich sequence-binding protein 1 gene and its relationship with SLC22A18 protein expression in astrocytoma / 中华神经医学杂志

Objective To study the expression of special AT-rich sequence-binding protein 1(SATB1) gene and its relationship with SLC22A18 protein expression in astrocytoma.Methods Fifty-six patients with astrocytomas (12 with grade Ⅰ,13 with grade Ⅱ,15 with grade Ⅲ,and 16 with grade Ⅳ),performed surgical excision in our hospitals from September 2006 to June 2010 and from September 2003 to June 2006,were chosen in our study; another 10 brain tissues from patients performed decompression operation resulting from cerebral hernia were selected as the controls.RT-PCR and Western blotting were used to detect the mRNA and protein expressions of SATB1.The SLC22A18 protein expression was detected by immunohistochemical assay.The relations between SLC22A18expressions and SA TB1 levels,and these two and the degree of malignancy were analyzed.Results RT-PCR and Western blotting revealed that positive mRNA and protein expressions were noted in 35patients with astrocytomas; the mRNA and protein expression rate and value of SATB1 in the astrocytoma tissues were significantly different among different grades of tumors (P＜0.05); the higher the malignancy grade,the higher mRNA and protein expression rate and value ofSA TB1; the protein expression value of SA TB1 had a positive correlation with the malignancy grade of tumors (r=0.987,P=0.000).And a few expressions of SA TB1 mRNA and protein were found in the tissues of controls.Immunohistochemical assay indicated that positive protein expression of SLC22A18 was noted in 19 astrocytoma tissues,and the protein expression rate of SLC22A18 in the astrocytoma tissues was significantly different among different grades of tumors (P＜0.05); the higher the malignancy grade,the lower expression of SLC22A18.And the protein expression of SLC22A18 was found in all the tissues of controls.The SATB1 expression rate in the tissues with negative SLC22A18 expression (81.1％) was significantly higher than that in the tissues with positive SLC22A18 expression (26.3％,P＜0.05).Conclusion SATB1 expresses in the astrocytoma tissues,indicating that it may play an important role in the pathogenesis of astrocytoma;up-regulation of SATB1 expression and dysfunction of SLC22A18 may play synergetic roles in the process of carcinogenesis of astrocytoma.

Relationship between aberrant methylation of SLC22A18 gene promoter and SLC22A18 expression in glioma / 中华神经医学杂志

Objective To investigate the relationship between aberrant methylation of SLC22A18 gene promoter and SLC22A18 expression in human glioma. Methods Thirty patients with glioma and 10 patients with craniocerebral injury performed decompression were chosen in our study;their tissue samples were prepared. Methylation-specific PCR (MSP) was used to detect the methylation status of SLC22A18 gene promoter;and Western blotting and RT-PCR were employed to measure the protein and mRAN expressions of SLC22A18 in these tissue samples. U251 cells were cultured in vitro with demethylating agent 5-aza-2-deoxycytidine (experimental group, 2μmol/L) and common medium (control group), resepectively;the re-expression of SLC22A18 in U251 cells was measured by Western blotting and cell growth suppression induced by 5-aza-2-deoxycytidine was also observed 3, 5 and 7 d after the culture. Results The methlylation of SLC22A18 gene promoter existed in glioma tissues of 15 patients (50%) but that did not exist in the tissues of patients with craniocerebral injury. The protein and mRAN expressions of SLC22A18 in the tissue samples of these 15 patients were significantly decreased as compared with those in patients with craniocerebral injury (P<0.05);cell counting of U251 cells in the experimental group on the 5th and 7th d of culture was significantly decreased as compared with that of those in the control group (P<0.05). On the 7ht d of culture, Western blotting indicated that the protein b expression level of SLC22A18 in the experimental group was obviously higher than that in the control group. Conclusion The aberrant methylation of SLC22A18 gene promoter plays a key role in down-regulating SLC22A18 expression, and demethylation agents can restore the SLC22A18 expression and suppress the growth of U251 cells.

Expression of imprinted tumor suppressor gene SLC22A18 in breast eancer / 中华普通外科杂志

Objective To investigate the expression of sLc22A1 8,an impfinted tumor suppressor gene,in breast cancer and explore the relationship between expression of SLC22A18 and the pathogenesis of breast cancer. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction was applied on 46 cases of infiltrating duetal breast carcinoma(IDC),46 csges of corresponding adjacent noncancerous tissues and 20 benign breast tissues in order to detect mRNA expression of SLC22A18 gene.Protein expression was detected by immunohistochemistry.Statistical analysis was carried out to analyse the correlation between SLC22A18 gene expression and various elinical parameters in these breast cancer patients. Results SLC22A18 mRNA expression in 46 IDC tissues Was lower than that in all corresponding adjacent non-cancerous tissues(Z=-4.900,P＜0.01).SLC22A18 mRNA expression was lower in breast cancer eases,when compared with that in benign cases(Z=-3.182,P＜0.01).SLIC22A18 mRNA expression in 40 IDCs Was lower than that in 6 dutal carcinoma in situ(part of IDC)(Z=-2.022,P＜0.05).There was a decreased or completely diminished SLC22A18 protein expression in breast cancer.A significant difference of SLC22A18 protein expression was also observed in IDC and benign groups(P＜0.05).Neither mRNA nor protein expression of SLC22A18 gene correlated with clinieopathologic parameters such as age of patients,size of tumor,ehnical stage,pathologic subtype,histologlc grade or lymph node metastasis(P＞0.05).Condusion Decreased expression of SLC22A18 gene may play an important role in the carcinogenesis of IDC.

Methylation and mRNA expression of imprinted gene SLC22A18/TSSC5/BWR1A in breast cancer / 中国病理生理杂志

AIM:To study the mRNA expression and methylation status of imprinted gene SLC22A18 in infiltrating ductal carcinomas(IDCs),and the correlation between methylation status and clinical characteristics in IDCs.METHODS:The methylation status at the promoter regions of SLC22A18 gene was examined by methylation-specific polymerase chain reaction(MSP)in the specimens of IDC from 40 patients.The mRNA expression of SLC22A18 gene was detected by real-time reverse quantitative transcriptase polymerase chain reaction(real-time RT-PCR)in 40 specimens of IDC and the cell line MDA-MB-231.The cell line MDA-MB-231 was treated with 5-aza-2'-deoxycytidine(5'-aza-dc)and trichostatin A(TSA),then MSP and rea1-time RT-PCR were used to detect the methylation status and mRNA expression levels of SLC22A18 gene.RESULTS:SLC22A18 mRNA expression in 40 IDC tissues was lower than that in al1 corresponding adjacent non-cancerous tissues(P