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Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers.

ABSTRACT: In Drosophila, the Slit gene product, a secreted glycoprotein, acts as a midline repellent to guide axonal development during embryogenesis. Three human Slit gene orthologues have been characterised and recently we reported frequent promoter region hypermethylation and transcriptional silencing of SLIT2 in lung, breast, colorectal and glioma cell lines and primary tumours. Furthermore, re-expression of SLIT2 inhibited the growth of cancer cell lines so that SLIT2 appears to function as a novel tumour suppressor gene (TSG). We analysed the expression of SLIT3 (5q35-34) and SLIT1 (1q23.3-q24) genes in 20 normal human tissues. Similar to SLIT2 expression profile, SLIT3 is expressed strongly in many tissues, while SLIT1 expression is neuronal specific. We analysed the 5' CpG island of SLIT3 and SLIT1 genes in tumour cell lines and primary tumours for hypermethylation. SLIT3 was found to be methylated in 12 out of 29 (41%) of breast, one out of 15 (6.7%) lung, two out of six (33%) colorectal and in two out of (29%) glioma tumour cell lines. In tumour cell lines, silenced SLIT3 associated with hypermethylation and was re-expressed after treatment with 5-aza-2'-deoxycytidine. In primary tumours, SLIT3 was methylated in 16% of primary breast tumours, 35% of gliomas and 38% of colorectal tumours. Direct sequencing of bisulphite-modified DNA from methylated tumour cell lines and primary tumours demonstrated that majority of the CpG sites analysed were heavily methylated. Thus, both SLIT2 and SLIT3 are frequently methylated in gliomas and colorectal cancers, but the frequency of SLIT3 methylation in lung and breast cancer is significantly less than that for SLIT2. We also demonstrated SLIT1 promoter region hypermethylation in glioma tumour lines (five out of six; 83%), the methylation frequency in glioma tumours was much lower (two out of 20; 10%). Hence, evidence is accumulating for the involvement of members of the guidance cues molecules and their receptors in tumour development.

SUBMITTER: Dickinson RE

PROVIDER: S-EPMC2409788 | biostudies-other | 2004 Dec

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Posttranslational protein modifications are known to modulate key biological processes like proliferation and apoptosis. Accumulating evidence shows that ST6GAL1, an enzyme that catalyzes the transfer of sialic acid onto galactose-containing substrates, is aberrantly expressed in various cancers and may affect cell motility and invasion. This is the first study to describe ST6GAL1 expression and regulation in human bladder cancer. METHODS: ST6GAL1 mRNA expression levels in human cell lines (UROtsa, RT4, RT112 and J82) and tissue samples (n = 15 normal urothelium (NU), n = 13 papillary non-invasive tumors (pTa), n = 12 carcinoma in situ (CIS), n = 26 muscle invasive tumors (pT2-4)) were assessed using real-time PCR. In addition, ST6GAL1 protein expression was evaluated using immunohistochemistry. Promoter methylation analysis was performed using methylation-specific PCR (MSP) in cell lines (n = 4) and patient samples (n = 23 NU, n = 12 CIS, n = 29 pTa, n = 41 pT2-4). Epigenetic ST6GAL1 gene silencing was confirmed by in vitro demethylation of bladder cell lines. Data were validated by analysis of an independent bladder tumor data set (n = 184) based on The Cancer Genome Atlas (TCGA) portal. RESULTS: Semi-quantitative ST6GAL1 real-time PCR expression analysis showed two distinct trends: In muscle-invasive tumors ST6GAL1 expression was downregulation by 2.7-fold, while papillary non-invasive tumors showed an increased ST6GAL1 mRNA expression compared to normal urothelium. ST6GAL1 loss in muscle-invasive tumors was associated with increasing invasiveness. On the protein level, 69.2% (n = 45/65) of all tumors showed a weak ST6GAL1 protein staining (IRS ? 4) while 25.6% (16/65) exhibited a complete loss (IRS = 0) of ST6GAL1 protein. Tumor-specific DNA methylation of the ST6GAL1 promoter region was frequently found in pT2-4 tumors (53.6% (22/41)), whereas only 13.8% (4/29) of pTa tumors showed ST6GAL1 promoter methylation. Normal urothelium remained unmethylated. Importantly, we significantly revealed an inverse correlation between ST6GAL1 mRNA expression and ST6GAL1 promoter merthylation in primary bladder cancer. These findings were clearly verified by the TCGA public data set and in vitro demethylation assays functionally confirmed ST6GAL1 promoter methylation as a potential regulatory factor for ST6GAL1 gene silencing. CONCLUSIONS: Our study characterizes for the first time ST6GAL1 expression loss caused by aberrant ST6GAL1 promoter methylation potentially indicating a tumor suppressive role in bladder carcinogenesis.

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Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers.

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