SLIT2

Gene Summary

Gene:SLIT2; slit homolog 2 (Drosophila)
Aliases: SLIL3, Slit-2
Location:4p15.2
Summary:-
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:slit homolog 2 protein
HPRD
Source:NCBIAccessed: 28 February, 2015

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 28 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 28 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: SLIT2 (cancer-related)

Cai H, Liu W, Xue Y, et al.
Roundabout 4 regulates blood-tumor barrier permeability through the modulation of ZO-1, Occludin, and Claudin-5 expression.
J Neuropathol Exp Neurol. 2015; 74(1):25-37 [PubMed] Related Publications
The blood-tumor barrier (BTB) restricts the delivery of chemotherapeutic drug molecules to tumor tissues. We found that the endothelial cell (EC) receptor molecule Roundabout 4 (Robo4) is endogenously expressed in human brain microvascular ECs and that it is upregulated in a BTB model of glioma cocultured ECs. Knockdown of Robo4 in this BTB model increased permeability; short hairpin RNA targeting Robo4 (shRobo4) led to decreased transendothelial electric resistance values, increased BTB permeability, and downregulated expression of the EC tight junction proteins ZO-1, occludin, and claudin-5. Roundabout 4 influenced BTB permeability via binding with its ligand, Slit2. Short hairpin RNA targeting Robo4 also increased matrix metalloproteinase-9 (MMP-9) activity and expression in glioma cocultured ECs; pretreatment with the MMP inhibitor GM6001 partially blocked the effects of shRobo4 on the transendothelial electric resistance values and ZO-1 and occludin expression. Short hairpin RNA targeting Robo4 also upregulated the phosphorylation of Src and Erk1/2; the Src inhibitor PP2 and the Erk1/2 inhibitor PD98059 blocked shRobo4-mediated alteration in ZO-1 and occludin expression. Together, our results indicate that knockdown of Robo4 increased BTB permeability by reducing EC tight junction protein expression, and that the Src-Erk1/2-MMP-9 signal pathways are involved in this process. Thus, Robo4 may represent a useful future therapeutic target for enhancing BTB permeability.

Sher YP, Wang LJ, Chuang LL, et al.
ADAM9 up-regulates N-cadherin via miR-218 suppression in lung adenocarcinoma cells.
PLoS One. 2014; 9(4):e94065 [PubMed] Free Access to Full Article Related Publications
Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition) and ADAM9 (a type I transmembrane protein) are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e.g., cancer) by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma.

Vasiljević N, Scibior-Bentkowska D, Brentnall AR, et al.
Credentialing of DNA methylation assays for human genes as diagnostic biomarkers of cervical intraepithelial neoplasia in high-risk HPV positive women.
Gynecol Oncol. 2014; 132(3):709-14 [PubMed] Free Access to Full Article Related Publications
OBJECTIVE: Testing for high risk human papillomavirus (HR-HPV) is increasing; however due to limitations in specificity there remains a need for better triage tests. Research efforts have focused recently on methylation of human genes which show promise as diagnostic classifiers.
METHODS: Methylation of 26 genes: APC, CADM1, CCND2, CDH13, CDKN2A, CTNNB1, DAPK1, DPYS, EDNRB, EPB41L3, ESR1, GSTP1, HIN1, JAM3, LMX1, MAL, MDR1, PAX1, PTGS2, RARB, RASSF1, SLIT2, SOX1, SPARC, TERT and TWIST1 was measured by pyrosequencing in cytology specimens from a pilot set of women with normal or cervical intraepithelial neoplasia grade 3 (CIN3) histology. Six genes were selected for testing in Predictors 1, a colposcopy referral study comprising 799 women. The three genes EPB41L3, DPYS and MAL were further tested in a second colposcopy referral study, Predictors 2, comprising 884 women.
RESULTS: The six genes selected from the pilot: EPB41L3, EDNRB, LMX1, DPYS, MAL and CADM1 showed significantly elevated methylation in CIN2 and CIN3 (CIN2/3) versus ≤CIN1 in Predictors 1 (p<0.01). Highest methylation was observed in cancer tissues. EPB41L3 methylation was the best single classifier of CIN2/3 in both HR-HPV positive (p<0.0001) and negative samples (p=0.02). Logistic regression modeling showed that other genes did not add significantly to EPB41L3 and in Predictors 2, its classifier value was validated with AUC 0.69 (95% CI 0.65-0.73).
CONCLUSION: Several methylated genes show promise for detecting CIN2/3 of which EPB41L3 seems the best. Methylated human gene biomarkers used in combination may be clinically useful for triage of women with HR-HPV infections.

Nones K, Waddell N, Song S, et al.
Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.
Int J Cancer. 2014; 135(5):1110-8 [PubMed] Related Publications
The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Yu H, Gao G, Jiang L, et al.
Decreased expression of miR-218 is associated with poor prognosis in patients with colorectal cancer.
Int J Clin Exp Pathol. 2013; 6(12):2904-11 [PubMed] Free Access to Full Article Related Publications
The identification of biomarkers in colorectal cancer (CRC) diagnosis and therapy is important in achieving early cancer diagnosis and improving patient outcomes. The aim of this study was to examine clinical significance of miR-218 expression in sera and tissues from CRC patients. A total of 189 cases and 30 healthy subjects were included. The expression levels of miR-218, SLIT2 and SLIT3 were measured by quantitative reverse transcription PCR (qRT-PCR). The relationship between miR-218 expression and clinicopathological characteristics was investigated. The expression levels of miR-218, SLIT2 and SLIT3 in CRC tissues were decreased than those in adjacent normal tissues (all P < 0.05). miR-218 expression was significantly associated with TNM stage, lymph node metastasis (LNM) and differentiation (all P < 0.05). Patients with low miR-218 expression had shorter survival time than those with high miR-218 expression (P = 0.036). Furthermore, the expression levels of serum miR-218 in CRC patients were lower than those in controls (P = 0.005). An increased level of serum miR-218 was found 1 month after surgery (P = 0.026). In conclusion, the miR-218 may has important roles in the development and progression of CRC and be a potential diagnostic and prognostic biomarker of CRC.

Ma WJ, Zhou Y, Lu D, et al.
Reduced expression of Slit2 in renal cell carcinoma.
Med Oncol. 2014; 31(1):768 [PubMed] Related Publications
Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is down-regulated in RCC partially due to promoter methylation.

Mano Y, Aishima S, Fukuhara T, et al.
Decreased roundabout 1 expression promotes development of intrahepatic cholangiocarcinoma.
Hum Pathol. 2013; 44(11):2419-26 [PubMed] Related Publications
Roundabout 1 (Robo1) is a transmembrane receptor of the immunoglobulin family. Slit2 is one of its ligands. The function of Slit2/Robo1 signaling in the development of intrahepatic cholangiocarcinoma (ICC) remains to be elucidated. We examined the immunohistochemical expression of Robo1 and Slit2 and their clinicopathologic implications in 132 cases of ICC. Also, small interfering RNA of Robo1 was transfected into a high-expression ICC cell line, and a Robo1 vector was transfected into a low-Robo1 expression ICC cell line. The effect of Robo1 suppression and overexpression in cell proliferation and migration of cultured ICC cells with Slit2 stimulation was investigated. Immunohistochemical study of ICC in the low-Robo1 expression group showed larger tumors (P = .015), a higher Ki-67 labeling index (P = .021), and low expression of Slit2 (P = .0005). The low-Slit2 expression group frequently showed perineural invasion (P = .036) and lymph node metastases (P = .013). Low Robo1 expression was associated with a poor prognosis (P = .0207). Robo1 suppression in Huh28 cells tended to promote cell proliferation and migration, whereas Robo1 overexpression in RBE cells significantly suppressed cell proliferation and migration. Low Robo1 expression was associated with cell proliferation and migration in ICC and was one of the adverse prognostic factors in patients with these tumors.

Shi R, Liu W, Liu B, et al.
Slit2 expression and its correlation with subcellular localization of β-catenin in gastric cancer.
Oncol Rep. 2013; 30(4):1883-9 [PubMed] Related Publications
Gastric cancer is the fourth most common cancer worldwide. Several signaling pathways are involved in gastric cancer development and progression. Slit2 was recently found to be involved in cancer; however, its expression pattern in gastric cancer has not been discovered yet. In the present study, we investigated the expression of Slit2 in human gastric cancer and its correlation with the expression and subcellular localization of β-catenin. Immunohistochemistry (IHC) staining revealed that Slit2 was highly expressed in human gastric cancer tissues, while it was low or weakly expressed in normal gastric tissues. The differences in clinicopathological features between different groups were determined using Pearson's χ2 test. Slit2 levels were significantly associated with differentiation, Lauren's classification, lymph node metastasis and TNM staging. Slit2 levels were positively correlated with β-catenin level in gastric cancer tissues and cell lines. High levels of Slit2 were correlated with the membrane localization of β-catenin, and low levels of Slit2 were correlated with nuclear translocation of β-catenin in both gastric cancer tissues and cell lines assayed by IHC and immunofluorescence staining, respectively. Our data suggest that Slit2 was highly expressed in gastric cancer patients with less advanced clinicopathological features. Slit2 levels were correlated with β-catenin level and subcellular localization.

Tamm-Rosenstein K, Simm J, Suhorutshenko M, et al.
Changes in the transcriptome of the human endometrial Ishikawa cancer cell line induced by estrogen, progesterone, tamoxifen, and mifepristone (RU486) as detected by RNA-sequencing.
PLoS One. 2013; 8(7):e68907 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.
METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1.
CONCLUSIONS: Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.

Guan H, Wei G, Wu J, et al.
Down-regulation of miR-218-2 and its host gene SLIT3 cooperate to promote invasion and progression of thyroid cancer.
J Clin Endocrinol Metab. 2013; 98(8):E1334-44 [PubMed] Related Publications
CONTEXT: The functional relationships between intronic microRNAs (miRNAs) and their host genes in thyroid cancer remain unclear. miR-218, a miRNA down-regulated in several kinds of cancers and associated with multiple cancer phenotypes, is transcribed from 2 loci located on chromosomes 4p15.31 (miR-218-1) and 5q35.1 (miR-218-2) within the introns of SLIT2 and SLIT3, respectively.
OBJECTIVE: The aim of our work was to investigate the expression and the roles of miR-218-1 and miR-218-2, as well as their host genes SLIT2 and SLIT3 in thyroid carcinogenesis.
DESIGN: The expression of miR-218-1 and miR-218-2, as well as their host genes SLIT2 and SLIT3, in a panel of normal and neoplastic human thyroid tissues was assessed by quantitative RT-PCR. We restored the expression of miR-218-2 and SLIT3 in thyroid cancer cells and evaluated their effects on cell invasion, migration, and proliferation.
RESULTS: We found that miR-218-2 and its host gene SLIT3 were down-regulated concomitantly in thyroid cancer. Synergistic inhibitory effects of miR-218-2 with SLIT3 on thyroid cancer cell invasion, migration, and proliferation were observed. Moreover, the effects of miR-218-2 on thyroid cancer cells were due, at least partially, to targeting PDGFRA and PLCG1.
CONCLUSIONS: These results implicate the involvement of miR-218-2 and its host gene SLIT3 in thyroid cancer cell invasion, migration, and proliferation. Our findings highlight the functional associations of intronic miRNAs and their host genes in thyroid carcinogenesis.

Carmona FJ, Azuara D, Berenguer-Llergo A, et al.
DNA methylation biomarkers for noninvasive diagnosis of colorectal cancer.
Cancer Prev Res (Phila). 2013; 6(7):656-65 [PubMed] Related Publications
DNA methylation biomarkers for noninvasive diagnosis of colorectal cancer (CRC) and precursor lesions have been extensively studied. Different panels have been reported attempting to improve current protocols in clinical practice, although no definite biomarkers have been established. In the present study, we have examined patient biopsies starting from a comprehensive analysis of DNA methylation differences between paired normal and tumor samples in known cancer-related genes aiming to select the best performing candidates informative for CRC diagnosis in stool samples. Five selected markers were considered for subsequent analyses in independent biologic cohorts and in silico data sets. Among the five selected genes, three of them (AGTR1, WNT2 and SLIT2) were validated in stool DNA of affected patients with a detection sensitivity of 78% [95% confidence interval (CI), 56%-89%]. As a reference, DNA methylation of VIM and SEPT9 was evaluated in a subset of stool samples yielding sensitivities of 55% and 20%, respectively. Moreover, our panel may complement histologic and endoscopic diagnosis of inflammatory bowel disease (IBD)-associated neoplasia, as it was also efficient detecting aberrant DNA methylation in non-neoplastic tissue samples from affected patients. This novel panel of specific methylation markers can be useful for early diagnosis of CRC using stool DNA and may help in the follow-up of high-risk patients with IBD.

Beggs AD, Jones A, Shepherd N, et al.
Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.
PLoS Genet. 2013; 9(5):e1003488 [PubMed] Free Access to Full Article Related Publications
Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

Annaratone L, Volante M, Asioli S, et al.
Characterization of neuroendocrine tumors of the pancreas by real-time quantitative polymerase chain reaction. A methodological approach.
Endocr Pathol. 2013; 24(2):83-91 [PubMed] Related Publications
The aim of this study was to assess the suitability of using real-time quantitative PCR (RT-qPCR) to characterize neuroendocrine (NE) tumors of the pancreas. For a series of tumors, we evaluated several genes of interest, and the data were matched with the "classical" immunohistochemical (IHC) features. In 21 cases, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) blocks, and in nine cases, we also extracted RNA from fresh-frozen tissue. The RT-qPCR procedure was performed using two sets of customized arrays. The test using the first set, covering 96 genes of interest, was focused on assessing the feasibility of the procedure, and the results were used to select 18 genes indicative of NE differentiation, clinical behavior, and therapeutic responsiveness for use in the second set of arrays. Threshold cycle (Ct) values were used to calculate the fold-changes in gene expression using the 2-∆∆Ct method. Statistical procedures were used to analyze the results, which were matched with the IHC and follow-up data. Material from fresh-frozen samples performed better in terms of the level of amplification, but acceptable and concordant results were also obtained from FFPE samples. In addition, high concordance was observed between the mRNA and protein expression levels of somatostatin receptor type 2A (R = 0.52, p = 0.016). Genes associated with NE differentiation, as well as the gastrin-releasing peptide receptor and O-6-methylguanine-DNA methyltransferase genes, were underexpressed, whereas angiogenesis-associated markers (CDH13 and SLIT2) were overexpressed in tissues with malignant behavior. The RT-qPCR procedure is practical and feasible in economic terms for the characterization of NE tumors of the pancreas and can complement morphological and IHC-based evaluations. Thus, the results of the RT-qPCR procedure might offer an objective basis for therapeutic choices.

Grossmann AH, Yoo JH, Clancy J, et al.
The small GTPase ARF6 stimulates β-catenin transcriptional activity during WNT5A-mediated melanoma invasion and metastasis.
Sci Signal. 2013; 6(265):ra14 [PubMed] Free Access to Full Article Related Publications
β-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and β-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4-LRP6 (low-density lipoprotein receptor-related protein 6) receptor complex activated ARF6, which liberated β-catenin from N-cadherin, thus increasing the pool of free β-catenin, enhancing β-catenin-mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with β-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked β-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/β-catenin signaling in cancer.

Suzuki M, Shiraishi K, Eguchi A, et al.
Aberrant methylation of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer.
Oncol Rep. 2013; 29(4):1308-14 [PubMed] Free Access to Full Article Related Publications
Genome-wide DNA hypomethylation and gene hypermethylation play important roles in instability and carcino-genesis. Methylation in long interspersed nucleotide element 1 (LINE-1) is a good indicator of the global DNA methylation level within a cell. Slit homolog 2 (SLIT2), myelin and lymphocyte protein gene (MAL) and insulin-like growth factor binding protein 7 (IGFBP7) are known to be hypermethylated in various malignancies. The aim of the present study was to assess the precise methylation levels of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer (NSCLC) using a pyrosequencing assay. Methylation of all regions was examined in 56 primary NSCLCs using a pyrosequencing assay. Changes in mRNA expression levels of SLIT2, MAL and IGFBP7 were measured before and after treatment with a demethylating agent. Methylation of these genes was also examined in 9 lung cancer cell lines using RT-PCR and a pyrosequencing assay. Frequencies of hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7, defined by predetermined cut off values, were 55, 64, 46 and 54% in NSCLCs, respectively and exhibited tumor-specific features. The hypermethylation of all genes was well correlated with changes in expression. The methylation level and frequency of MAL were significantly higher in smokers and in patients without EGFR mutations. Through accurate measurement of methylation levels using pyrosequencing, hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7 were frequently detected in NSCLCs and associated with various clinical features. Analysis of the methylation profiles of these genes may, therefore, provide novel opportunities for the therapy of NSCLCs.

Richter GH, Fasan A, Hauer K, et al.
G-Protein coupled receptor 64 promotes invasiveness and metastasis in Ewing sarcomas through PGF and MMP1.
J Pathol. 2013; 230(1):70-81 [PubMed] Related Publications
Metastatic spread in Ewing sarcomas (ES) is frequent and haematogenous. G-protein coupled receptor 64 (GPR64), an orphan receptor with normal expression restricted to human epididymis is specifically over-expressed in ES among sarcoma, but also up-regulated in a number of carcinomas derived from prostate, kidney or lung. Inhibition of GPR64 expression in ES by RNA interference impaired colony formation in vitro and suppressed local tumour growth and metastasis in Rag2(-/-) γC (-/-) mice. Microarray analysis after GPR64 knock down revealed a GPR64-mediated repression of genes involved in neuronal development like SLIT, drosophila, homolog of, 2 (SLIT2), and genes regulating transcription including pre-B cell leukemia homeobox 2 (PBX2). Concurrently, the suppression of GPR64 increased ES susceptibility to TRAIL induced apoptosis. Moreover, a GPR64-mediated induction of placental growth factor (PGF) in ES was observed. PGF suppression by RNA interference resulted in a reduction of metastatic growth similar to that observed after GPR64 knock down. Importantly, inhibition of GPR64 as well as PGF expression was associated with a reduced expression of matrix metalloproteinase (MMP) 1 and invasiveness in vitro. Furthermore, MMP1 knock down abrogated lung metastasis in Rag2(-/-) γC (-/-) mice. Thus, GPR64 expression in ES maintains an immature phenotype that is less sensitive to TRAIL-induced apoptosis and via its up-regulation of PGF and MMP1 orchestrates and promotes invasiveness and metastatic spread.

Dallol A, Al-Maghrabi J, Buhmeida A, et al.
Methylation of the polycomb group target genes is a possible biomarker for favorable prognosis in colorectal cancer.
Cancer Epidemiol Biomarkers Prev. 2012; 21(11):2069-75 [PubMed] Related Publications
BACKGROUND: Colorectal cancer (CRC) is the second most common cancer in the Kingdom of Saudi Arabia with ever increasing incidence rates. DNA methylation is a common event in CRC where it is now considered an important phenomenon in CRC carcinogenesis and useful for the classification and prognosis of CRC.
METHODS: To gain insight into the molecular mechanisms underpinning CRC in Saudi Arabian patients, we profiled the DNA methylation frequency of key genes (MLH1, MSH2, RASSF1A, SLIT2, HIC1, MGMT, SFRP1, MYOD1, APC, CDKN2A, as well as five CIMP markers) in 120 sporadic CRC cases. CRC tumors originating from the rectum, left, and right colons are represented in this cohort of formalin-fixed paraffin-embedded tissues.
RESULTS: The most common methylation frequency was detected in the polycomb group target genes (PCGT) including SFRP1 (70%), MYOD1 (60.8%), HIC1 (61.7%), and SLIT2 (56.7%). In addition, MGMT methylation was detected at a high frequency (68.3%). RASSF1A, APC, and CDKN2A methylation frequencies were 42.5%, 25%, and 32.8%, respectively. K-means clustering analysis of the methylation events results in the clustering of the CRC samples into three groups depending on the level of methylation detected.
CONCLUSION: Group II (PCGT methylation and CIMP-negative) methylation signature carried a favorable prognosis for male patients, whereas older patients with group I rare methylation signature have a potentially poorer clinical outcome.
IMPACT: Methylation of the PCGT genes along with RASSF1A, APC, and MGMT can be potentially used as a new biomarker for the classification and prognosis of CRC tumors and independently of where the tumor has originated.

Zhao L, Gu F, Ma YJ
[Association between Slit/Robo signal pathway and the genesis, progression, invasion and metastasis of malignant tumors].
Zhonghua Zhong Liu Za Zhi. 2012; 34(6):405-8 [PubMed] Related Publications

Peifer M, Fernández-Cuesta L, Sos ML, et al.
Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer.
Nat Genet. 2012; 44(10):1104-10 [PubMed] Related Publications
Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Yang L, Li Q, Wang Q, et al.
Silencing of miRNA-218 promotes migration and invasion of breast cancer via Slit2-Robo1 pathway.
Biomed Pharmacother. 2012; 66(7):535-40 [PubMed] Related Publications
MiRNAs play an important role in regulating tumor migration and invasion, and abnormal expression of miRNAs occurs in various kinds of human cancers. In this essay, it is reported that the level of miRNA-218 decreases in metastatic breast cancer cells, moreover, miRNA-218 suppresses breast cancer cells migration and invasion through binding Robo1 (one of Slit receptors) to its 3'UTR. MiRNA-218 restoration suppresses Robo1 expression and inhibits breast cancer cells invasion and migration. What the results describe is that the function of Robo1 regulated by miRNA-218 may provide a new strategy for inhibiting migration and invasion of breast cancer cells.

Mitra S, Mazumder-Indra D, Mondal RK, et al.
Inactivation of SLIT2-ROBO1/2 pathway in premalignant lesions of uterine cervix: clinical and prognostic significances.
PLoS One. 2012; 7(6):e38342 [PubMed] Free Access to Full Article Related Publications
The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.

Dong R, Yu J, Pu H, et al.
Frequent SLIT2 promoter methylation in the serum of patients with ovarian cancer.
J Int Med Res. 2012; 40(2):681-6 [PubMed] Related Publications
OBJECTIVE: The slit homologue 2 (SLIT2) gene is a tumour suppressor gene. This study investigated the methylation status of the SLIT2 promoter in women with ovarian cancer in terms of identifying whether this might aid the early diagnosis of ovarian cancer.
METHODS: Using methylation-specific polymerase chain reaction analysis, the methylation status of the SLIT2 promoter was measured in tumour tissue and serum samples from 36 patients with ovarian cancer and in matched serum samples from 25 controls without cancer.
RESULTS: Aberrant methylation of the SLIT2 promoter was present in ovarian tissue from 29/36 (80.6%) ovarian cancer patients, but not in the 25 healthy controls. Among the cases with hypermethylation in their ovarian tissue, 27/29 (93.1%) of the case-matched serum DNA samples, including all four cases of early-stage ovarian cancer, showed hypermethylation of the SLIT2 promoter.
CONCLUSIONS: Hypermethylation of the SLIT2 promoter may be a relatively early event in ovarian cancer; thus, its detection may be an effective approach to improve early diagnosis.

Azuara D, Rodriguez-Moranta F, de Oca J, et al.
Novel methylation panel for the early detection of neoplasia in high-risk ulcerative colitis and Crohn's colitis patients.
Inflamm Bowel Dis. 2013; 19(1):165-73 [PubMed] Related Publications
BACKGROUND: Patients with ulcerative colitis and Crohn's colonic disease are at increased risk of developing colorectal cancer (CRC). The aim of the study was to analyze the methylation status of selected genes as a risk marker for CRC in inflammatory bowel disease (IBD) patients.
METHODS: We evaluated the methylation status of four genes (TGFB2, SLIT2, HS3ST2, and TMEFF2) in biopsies of four groups of patients: 60 patients with sporadic CRC, 32 patients with IBD-associated neoplasia, 85 patients with IBD without associated neoplasia (20 at high risk and 65 at low risk), and 28 healthy controls. Methylation-specific melting curve analysis (MS-MCA) was used. Methylation status of these genes was also assessed in stool DNA from 60 IBD patients without neoplasia.
RESULTS: Methylation of the panel of genes analyzed was a very common phenomenon (78%) in IBD-associated neoplasia. The prevalence of methylation in adjacent nonneoplastic mucosa was also high (12/30). This prevalence was higher than in mucosa from healthy controls (2/28;7.1%; P < 0.05). Methylation of SLIT2 and TMEFF2 was more frequently detected in the mucosa of IBD patients at high risk of dysplasia or cancer (15/20) than patients at low risk (32/63) (P = 0.05 and P = 0.03, respectively). When stool samples were assessed, only SLIT2 gene methylation was more frequently methylated in the group of patients at high risk of dysplasia or cancer (4/16) compared to low risk (0/37) (P = 0.006).
CONCLUSIONS: Analysis of a panel of methylation markers may help in the early identification of colorectal dysplasia or cancer in high-risk IBD patients.

Alvarez C, Tapia T, Cornejo V, et al.
Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer.
Mol Carcinog. 2013; 52(6):475-87 [PubMed] Related Publications
Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressor genes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array-CGH analysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breast cancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene promoters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally, the presence of methylated PCR products was associated with absence of protein expression for the three genes and statistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6 grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found. These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, and could be a marker for pre-malignant phenotypes.

Zhu J, Jiang Z, Gao F, et al.
A systematic analysis on DNA methylation and the expression of both mRNA and microRNA in bladder cancer.
PLoS One. 2011; 6(11):e28223 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported.
METHODOLOGY/PRINCIPAL FINDINGS: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples.
CONCLUSIONS/SIGNIFICANCE: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

Dickinson RE, Fegan KS, Ren X, et al.
Glucocorticoid regulation of SLIT/ROBO tumour suppressor genes in the ovarian surface epithelium and ovarian cancer cells.
PLoS One. 2011; 6(11):e27792 [PubMed] Free Access to Full Article Related Publications
The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.

Kwon YJ, Lee SJ, Koh JS, et al.
Genome-wide analysis of DNA methylation and the gene expression change in lung cancer.
J Thorac Oncol. 2012; 7(1):20-33 [PubMed] Related Publications
INTRODUCTION: The recent DNA methylation studies on cancers have revealed the necessity of profiling an entire human genome and not to restrict the profiling to specific regions of the human genome. It has been suggested that genome-wide DNA methylation analysis enables us to identify the genes that are regulated by DNA methylation in carcinogenesis.
METHODS: So, we performed whole-genome DNA methylation analysis for human lung squamous cell carcinoma (SCC), which is strongly related with smoking. We also performed microarrays using 21 pairs of normal lung tissues and tumors from patients with SCC. By combining these data, 30 hypermethylated and down-regulated genes, and 22 hypomethylated and up-regulated genes were selected. The gene expression level and DNA methylation pattern were confirmed by semiquantitative reverse-transcriptase polymerase chain reaction and pyrosequencing, respectively.
RESULTS: By these validations, we selected five hypermethylated and down-regulated genes and one hypomethylated and up-regulated gene. Moreover, these six genes were proven to be actually regulated by DNA methylation by confirming the recovery of their DNA methylation pattern and gene expression level using a demethylating agent. The DNA methylation pattern of the CYTL1 promoter region was significantly different between early and advanced stages of SCC.
CONCLUSION: In conclusion, by combining the whole-genome DNA methylation pattern and the gene expression profile, we identified the six genes (CCDC37, CYTL1, CDO1, SLIT2, LMO3, and SERPINB5) that are regulated by DNA methylation, and we suggest their value as target molecules for further study of SCC.

Kim GE, Lee KH, Choi YD, et al.
Detection of Slit2 promoter hypermethylation in tissue and serum samples from breast cancer patients.
Virchows Arch. 2011; 459(4):383-90 [PubMed] Related Publications
Promoter hypermethylation has been shown to be a common mechanism for inactivation of tumor suppressor genes in breast cancer. The aim of this study was to investigate the prevalence of Slit2 promoter hypermethylation in both the tumor and serum samples of breast cancer patients with ductal carcinoma in situ (DCIS) or invasive breast carcinoma (IBC). The methylation status of Slit2 was investigated in 210 tissue samples (15 breast with no pathological findings, 26 DCIS, and 169 IBC samples) and 123 corresponding serum samples (15 breast with no pathological findings, 26 DCIS, and 82 IBC samples) using methylation-specific polymerase chain reaction. Immunohistochemical staining for Slit2 was also performed using tissue microarray blocks to determine whether Slit2 promoter hypermethylation correlated with loss of Slit2 expression. Slit2 promoter hypermethylation was not detected in breast tissue and serum samples from patients with no pathological findings. DCIS or IBC showed a statistically higher frequency of Slit2 promoter hypermethylation compared to breast with no pathological findings in both the tissue and serum samples; however, there were no statistically significant differences between DCIS and IBC samples. Similar Slit2 promoter hypermethylation patterns were seen in the tissue samples and corresponding serum specimens (p < 0.001). Slit2 promoter hypermethylation was associated with loss of Slit2 expression. These results suggest that Slit2 promoter hypermethylation appears to be responsible for functionally silencing Slit2 expression. Slit2 promoter hypermethylation may be considered as a possible serum marker for early detection of breast cancer.

Buhmeida A, Merdad A, Al-Maghrabi J, et al.
RASSF1A methylation is predictive of poor prognosis in female breast cancer in a background of overall low methylation frequency.
Anticancer Res. 2011; 31(9):2975-81 [PubMed] Related Publications
Breast cancer (BC) is the most common cancer worldwide. The Kingdom of Saudi Arabia is no exception, with ever increasing incidence rates. An interesting feature of this disease is the relatively young age of the affected women. The average age in the present cohort of 100 sporadic cases of invasive ductal carcinomas was 45 years, with a median of 46 years (range between 19-81 years). In an effort to understand the molecular signature of BC in the Saudi population, we undertook this study to profile the methylation events in a series of key genes including Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A), hypermethylated in cancer 1 (HIC1), cyclin-dependent kinase inhibitor 2A (CDKN2A), retinoic acid receptor beta (RARB2), estrogen receptor 1 (ESR1), progesterone receptor (PGR), paired-like homeodomain 2 (PITX2), secreted frizzled-related protein 1 (SFRP1), myogenic differentiation 1 (MYOD1), and slit homolog 2 (SLIT2), using MethyLight analysis in archival tumour samples. Interestingly, the overall methylation levels were low in this cohort, with only 84% of the cases displaying methylation in one or more of the analysed genes. The frequency of RASSF1A methylation was the highest (65%), while there was almost complete absence of methylation of the ESR1 and the CDH1 genes (1% and 3%, respectively). Several statistically significant correlations were identified between specific methylation events and clinical parameters which gained more significance when analysis was limited to the estrogen receptor positive samples. Although there was no significant correlations between any methylation event and disease-specific survival, methylation of MYOD1 or RASSF1A was associated with lower disease-free survival and increased chance of disease recurrence. Furthermore, multivariate (Cox) regression analysis identified RASSF1A as an independent predictor of poor prognosis in terms of disease-free survival in this cohort. Our findings provide further evidence on the usefulness of RASSF1A methylation status as an informative prognostic biomarker in BC in a Saudi population.

Vasiljević N, Wu K, Brentnall AR, et al.
Absolute quantitation of DNA methylation of 28 candidate genes in prostate cancer using pyrosequencing.
Dis Markers. 2011; 30(4):151-61 [PubMed] Free Access to Full Article Related Publications
Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001).Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.

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