SLC5A8, a sodium transporter, is a tumor suppressor gene silenced by methylation in human colon aberrant crypt foci and cancers

Abstract

We identify a gene, SLC5A8, and show it is a candidate tumor suppressor gene whose silencing by aberrant methylation is a common and early event in human colon neoplasia. Aberrant DNA methylation has been implicated as a component of an epigenetic mechanism that silences genes in human cancers. Using restriction landmark genome scanning, we performed a global search to identify genes that would be aberrantly methylated at high frequency in human colon cancer. From among 1,231 genomic NotI sites assayed, site 3D41 was identified as methylated in 11 of 12 colon cancers profiled. Site 3D41 mapped to exon 1 of SLC5A8, a transcript that we assembled. In normal colon mucosa we found that SLC5A8 exon 1 is unmethylated and SLC5A8 transcript is expressed. In contrast, SLC5A8 exon 1 proved to be aberrantly methylated in 59% of primary colon cancers and 52% of colon cancer cell lines. SLC5A8 exon 1 methylated cells were uniformly silenced for SLC5A8 expression, but reactivated expression on treatment with a demethylating drug, 5-azacytidine. Transfection of SLC5A8 suppressed colony growth in each of three SLC5A8-deficient cell lines, but showed no suppressive effect in any of three SLC5A8-proficient cell lines. SLC5A8 exon 1 methylation is an early event, detectable in colon adenomas, and in even earlier microscopic colonic aberrant crypt foci. Structural homology and functional testing demonstrated that SLC5A8 is a member of the family of sodium solute symporters, which are now added as a class of candidate colon cancer suppressor genes.

Fig. 1.

Identification of SLC5A8. (A) The genomic structure of the SLC5A8 gene. Black boxes represent exons, and arrows represent the start and stop codons. (B) The nucleotide sequence of the SLC5A8 coding region.

Fig. 2.

SLC5A8 expression. (A) RT-PCR analysis demonstrating SLC5A8 transcript expression in three normal colon mucosa samples (N1, N2, and N3) but the absence of SLC5A8 transcript in most colon cancer cell lines (remaining samples). (B) RT-PCR analysis demonstrating reactivation of SLC5A8 expression in cell lines treated with 5-azacytidine (+) compared with untreated controls (-). (C) MS-PCR assay for methylated (M) or unmethylated (U) SLC5A8 exon 1 sequences detects exclusively methylated templates in SLC5A8-silenced cell lines. (D) MS-PCR detects only unmethylated SLC5A8 templates in SLC5A8-expressing cell lines. (E) MS-PCR detection of methylated SLC5A8 templates in colon cancer tumors (T) antecedent to SLC5A8-methylated cell lines (V425 and V670). Matched normal colon tissue (N) shows only unmethylated templates. Unmethylated templates in tumor tissue presumptively arise from contaminating nonmalignant cells. (F) MS-PCR analysis of colon cancer tumors (T) and matched normal colon tissues (N). Methyl-specific bands are seen in each of the tumor samples but none of the normal controls.

Fig. 3.

Real-time MS-PCR analysis of SLC5A8 methylation. Plotted is 1,000 times the ratio of measured SLC5A8-methylated product to the control MYOD1-derived product. (A) Detection of SLC5A8 methylation in primary colon cancer tissues. Column 1 displays values for normal colon tissues harvested from non-cancer resections (dark blue diamonds). Column 2 displays values for normal colon tissues harvested from colon cancer resections (red diamonds). Column 3 displays values for colon cancer tissues divided into unmethylated samples falling within the normal tissue range (pink diamonds) and methylated samples showing values greater than the normal tissue range (light blue diamonds). Adjacent green bars indicate population means. (B) Real-time MS-PCR analysis of SLC5A8 methylation in ACF. Column 1 displays values for 24 normal colon tissues harvested from colon resections of 11 individuals (red diamonds). Column 2 displays values for 15 ACF harvested from the same 11 individuals' resections. Pink diamonds indicate unmethylated samples within the normal range, and blue diamonds indicate methylated samples falling within the range previously demonstrated by methylated cancers. Adjacent green bars indicate the mean value for each group.

Fig. 4.

Real-time MS-PCR analysis of SLC5A8 methylation in DNA precipitated from the serum of colon cancer patients. Plotted is 1,000 times the ratio of measured SLC5A8-methylated product to control MYOD1-derived product. Column 1 displays an absence of detectable SLC5A8 methylation in serum of 13 individuals whose colon cancer tumors assayed as unmethylated by MS-PCR (red diamonds). Column 2 displays values of SLC5A8 methylation in the serum of 10 individuals whose colon cancer tumors assayed as methylated by MS-PCR. Pink diamonds indicate six sera without detectable methylation, and blue diamonds indicate four sera in which SLC5A8 methylation was detectable.

Fig. 5.

Analysis of SLC5A8 function in Xenopus oocytes. (A) Immunofluorescent visualization of the V5 epitope tag in mock-injected oocytes (1) or in oocytes injected with cRNA for V5-tagged SLC5A8 (2). The corresponding phase photomicrographs are shown in 3 and 4. (B) Intracellular sodium concentration [aNa_i] (mM) as measured by a sodium electrode in control water-injected and SLC5A8 cRNA-injected oocytes.

Fig. 6.

SLC5A8 suppression of colon cancer colony formation. Shown are the number of G4180-resistant colonies arising from transfection with a SLC5A8 expression vector (SLC5A8) or a control empty expression vector (pcDNA) in SLC5A8-unmethylated and -expressing V364, V457, and V9M cells (A) as compared with SLC5A8-methylated and -deficient FET, V400, and RKO cells (B).