RASSF2 can suppress the growth of breast cancer cell lines and is epigenetically inactivated in breast tumours
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KeywordsBreast Tumour Tumour Suppressor Gene Nuclear Localisation Signal Bisulphite Tumour Suppressive Activity
RASSF2 is located at 20p13, a region frequently lost in human cancers. RASSF2 is a recently identified member of the ras association domain of family tumour suppressor genes, and many other members of this family are inactivated in human tumours by promoter methylation.
Methylation-specific PCR and combined bisulphite and restriction analysis were used to analyse the methylation status of the RASSF2 promoter CpG island in a series of breast tumours and cell lines. Bioinformatic approaches were used to study RASSF2 and a highly conserved putative bipartite nuclear localisation signal (NLS) was identified. Colony formation, growth in soft agar and growth in immunocompromised mouse assays were used to assess the tumour suppressive activities of RASSF2.
RASSF2 was frequently methylated in breast tumour cell lines, 65% (13/20), and in primary breast tumours, 38% (15/40). In the 10 samples for which corresponding normal DNA was available this methylation was tumour specific. RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC. Endogenous RASSF2 localised to the nucleus and mutation of the putative nuclear localisation signal abolished the nuclear localisation. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished.
These data indicate that RASSF2 is frequently methylated in breast tumours, and thus RASSF2 is a novel methylation marker that has the potential to be developed into a valuable epigenetic marker for screening. We also demonstrate that RASSF2 acts as a tumour suppressor gene and that it contains a functional NLS that is important for its tumour suppressor gene function.
Supported by Breast Cancer Campaign.