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.

Main

Epigenetic inactivation of tumour suppressor genes (TSGs) by promoter region CpG island hypermethylation is now well documented and several TSGs have been demonstrated to be inactivated by this mechanism (reviewed in Jones and Baylin, 2002; Herman and Baylin, 2003). In more recent years, a novel class of TSGs has been identified where epigenetic inactivation plays the predominant role, while somatic mutations are rare. This class of genes is exemplified by the 3p21.3 TSG, Ras association domain family 1A gene (RASSF1A) (Dammann et al, 2000; Lerman and Minna, 2000; Agathanggelou et al, 2001; Burbee et al, 2001). The CpG island in the promoter region of isoform A is frequently and heavily methylated in many types of cancers, including lung, breast, kidney, NPC, gastric, bladder, neuroblastoma, testicular, etc. (reviewed in Pfeifer et al, 2002; Dammann et al, 2003), while somatic inactivating mutations are absent or rare.

SLITs, ROBOs and Semaphorins belong to families of proteins that play important roles in axon guidance and cell migration in Drosophila and vertebrates (reviewed in Brose and Tessier-Lavigne, 2000; Wong et al, 2002). These proteins are widely expressed in mammalian tissues and the expression is not confined to neurons. Hence, they may have other yet unidentified roles. Slits are secreted proteins that are ligands for the Robo receptors (Brose et al, 1999; Kidd et al, 1999; Li et al, 1999). Recently, Slit was shown to inhibit leucocyte chemotaxis, this inhibition appears to be mediated by Robo (Wu et al, 2001). In mammals, four Robo genes have so far been identified, Robo1, Robo2, Rig-1 (Robo3) and magic Robo (Robo4) (Kidd et al, 1998; Girard et al, 2000; Goel et al, 2003). We have now demonstrated that SLIT3 5′ CpG island similar to SLIT2 is frequently hypermethylated in colorectal and glioma tumours and less so in breast tumours. And loss of SLIT3 expression can be reversed by treatment with a demethylating agent. While SLIT1 gene is frequently methylated in glioma tumour lines but at low frequencies in glioma tumours, hence SLIT1 may play a role in late gliomagenesis.

Slits, netrins, semaphorins and the ephrins constitute conserved families of axonal guidance cues that have prominent developmental effects. Recently, SEMA3B was also demonstrated to be inactivated in lung cancer by promoter region hypermethylation (Tomizawa et al, 2001; Kuroki et al, 2003). Re-expression of SEMA3B inhibited lung cancer cell growth and induced apoptosis.

The finding of epigenetic inactivation in various human cancers of ROBO1, SEMA3B, SLIT2 and now SLIT3 and to a lesser extent SLIT1, all of which are involved in axon and cell migration in Drosophila and vertebrates, suggests a novel, and common underlying theme for these molecules in tumour suppression.