Repression of Fyn-related kinase in breast cancer cells is associated with promoter site-specific CpG methylation

Abstract

The triple-negative breast cancer subtype is highly aggressive and has no defined therapeutic target. Fyn-related kinase (FRK) is a non-receptor tyrosine kinase, reported to be downregulated in breast cancer and gliomas, where it is suggested to have tumor suppressor activity. We examined the expression profile of FRK in a panel of 40 breast cancer cells representing all the major subtypes, as well as in 4 non-malignant mammary epithelial cell lines. We found that FRK expression was significantly repressed in a proportion of basal B breast cancer cell lines. We then determined the mechanism of suppression of FRK in FRK-low or negative cell lines. In silico analyses of the FRK promoter region led to the identification of at least 17 CpG sites. Bisulphite sequencing of the promoter region revealed that two of these sites were consistently methylated in FRK-low/negative cell lines and especially in the basal B breast cancer subtype. We further show that treatment of these cells with histone deacetylase inhibitors, Entinostat and Mocetinostat' promoted re-expression of FRK mRNA and protein. Further, using luciferase reporter assays, we show that both GATA3-binding protein FOG1 and constitutively active STAT5A increased the activity of FRK promoter. Together, our results present the first evidence that site-specific promoter methylation contributes to the repression of FRK more so in basal B breast cancers. Our study also highlights the potential clinical significance of targeting FRK using epigenetic drugs specifically in basal B breast cancers which are usually triple negative and very aggressive.

FRK inhibits breast cancer cell migration and invasion by suppressing epithelial-mesenchymal transition

“…We examined the relationship between the expression of FRK with mesenchymal markers Vimentin ( VIM ), N-cadherin ( CDH2 ), fibronectin ( FN1 ), snail family transcriptional repressor 2 ( SNAI2 ), twist family bHLH transcription factor 1( TWIST1 ), and epithelial markers E-cadherin ( CDH1 ) and Keratin 18 ( KRT18 ), in breast cancer cell lines stratified under Basal B (BB), Basal A (BA) and luminal (LU) ( Supplementary Table 5 , Figure 10 ). We recently reported the level of FRK transcript was low in the basal B breast cancers as compared to Basal A and Luminal cells [ 40 ]. We found that the mean transcript expression of VIM , CDH2 , FN1 , and TWIST1 were higher in the basal B breast cancer cells with low FRK transcript levels as compared to Basal A and Luminal cells that harbor high FRK transcript levels (P<0.05; Figure 10B , 10D , 10F and 10H ).…”

Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2

“…Our findings are consistent with those of a previous report that Fyn-related kinase is associated with methylation at specific CpG islands of the promoter site. 39 To ascertain the association between signaling mechanisms of miR-5088-5p and Fyn, an miR-5088-5p inhibitor and Fyn-overexpressing vector were used. EMT-and stemness-related markers suppressed by the miR-5088-5p inhibitor were restored upon Fyn overexpression (Figure 6F).…”

The Effect of Myricetin Flavonoid on the Expression of Fyn Gene in Melanoma Cells (A375)

“…For instance, in breast cancer cells, the methylation of CPG sites in the promoter region of tumor suppressor was observed, and DNA methylation decreased the expression of FRK in a subset of breast cancer cells (29).…”