N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression

The mRNA modification N6-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However , it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs) , as compared with their primary cell predecessor. Furthermore , the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines , providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line , overexpression of METTL3 and METTL14 , or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably , these treatments had little effect on the immortalized cells. Together , these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally , we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3 , METTL14 or ALKBH5 expression , suggesting a novel pathway for regulation of m6A levels under stress