MGMT promoter hypermethylation and K-RAS, PTEN and TP53 mutations in tamoxifen-exposed and non-exposed endometrial cancer cases

Abstract

Background: Tamoxifen has anti-oestrogenic and anti-tumour activity in the breast, but is oestrogenic and carcinogenic in the endometrium. It can induce experimental tumours by both hormonal and DNA-damaging mechanisms, but its carcinogenic mode of action in human endometrium remains unclear.

Methods: We investigated whether an epigenetic mechanism, involving promoter hypermethylation of the gene for the DNA repair enzyme MGMT (O6-methylguanine DNA methyltransferase), was associated with K-RAS, TP53 and PTEN mutations in endometrial tumours from women treated with tamoxifen (TAM, n=30) or unexposed to the drug (EC, n=38).

Results: There were significant (P<0.05) differences in tumour grade between the TAM and EC groups, with more favourable morphology in the latter. K-RAS mutations, predominantly G>A, occurred in small numbers in both groups. TP53 mutations were of mainly A>G, C>T and indel modifications in both groups, but more frequent in TAM cases. PTEN mutations dominated in EC tumours and were of the type that has large impact on protein function, such as indel or nonsense mutations. These observations alongside the mutational spectrum in PTEN suggest that the malignancies arise from different backgrounds, hence pointing to an effect of tamoxifen. Both groups displayed MGMT promoter hypermethylation. This coincided with mutations more frequently in the TAM (78%) than in the EC (50%) group, even though there were significantly (P<0.05) fewer mutations and methylations in TAM cases.

Conclusions: Although the difference in coincidence did not reach significance with the current sample size, the findings suggest that epigenetic processes may play a role in the way tamoxifen induces endometrial cancer.

Figure 1

Examples of MS-PCR analysis (A; showing the first lane of gels, including 10 patient samples of each group). ‘M' represents methylated bands and ‘U' unmethylated. Controls (B) were used at each analysis with BC-U being unmethylated human DNA that was bisulphite converted during sample workup procedures to assess the success of the conversion step. ‘C–U' and ‘C–M' were commercially available bisulphite converted unmethylated and methylated human DNA, respectively. ‘DNA' represents non-converted human DNA. The H₂O samples were lacking DNA of any sort in the PCR mixture. Note that close to the bottom line of the ladder corresponding to about 50 bp, bands can be seen. These are primer dimers. The product bands for both ‘M' and ‘U' are around 100 bp with only minor differences.

Figure 2

Mutational spectrum of K-RAS, TP53 and PTEN in tumour tissues from TAM and EC patients.