Methylation of the PTEN Promoter Leads to PTEN Inactivation in Multiple Myeloma

Aberrant methylation of CpG island of the promoter regions of genes is an important epigenetic mechanism, alternative to deletions or mutations, that cause the silencing of genes involved in carcinogenesis. We therefore investigated epigenetic events involved in multiple myeloma by screening 6 genes of interest for evidence of promoter hypermethylation.

PATIENTS AND METHODS: The methylation patterns of the tumor suppressor genes p16^INK4a (p16), E-cadherin (ECAD), FHIT and PTEN (negative regulator of AKT/PKB signalling pathway), Wnt signalling pathway antagonist genes WIF-1 and DKK-3 were determined in the bone marrow aspirates of 50 patients with Multiple Myeloma (MM) (37 at diagnosis; male 22; female 28; 68.8 median age. ISS stage: 47% stage I, 8% stage II and 45% stage III.), using the methylation-specific polymerase chain reaction after DNA bisulphite modification. Ten patients with monoclonal gammopathy of undetermined significance (MGUS) and 4 peripheral blood from controls were also evaluated. For statistical analysis Student t and Chi squared or Fisher exact tests were used.

RESULTS: We found at least one hypermethylated gene in all MM patients. Gene methylation frequencies varied from 6% to 74%. ECAD, FHIT and p16 genes demonstrated a relatively high frequency of aberrant methylation with frequencies of 74%, 69% and 42%, respectively. WIF-1, PTEN, DKK-3 genes showed a low frequency of methylation with values of 28%, 14%, 6%, respectively. The methylation frequencies detected in MGUS were 91.6% for ECAD and FHIT, 41.6% for WIF-1, 16,6% for p16 and DKK3. The difference in methylation profile between MM and MGUS was statistically significant (X²= 37, df=5; p=0.0000). PTEN was found hypermethylated in 7/50 (14%) MM but none of MGUS or control samples were methylated. Of these 6 patients had IgG K isotype, 4 had abnormal cariotype with 13q deletion, one hyperdiploidia, one IgH rearrangements. Two patients were simultaneously hypermethylated in p16 gene promoter. No statistically significant correlation between methylation data and clinical parameters: gender, age, isotype of M component, type of light chain, haemoglobin, serum albumin level, calcium, β2 microglobulin, serum creatinine level, LDH, lytic bone lesions were found for any of examined genes. When correlation with Durie-Salmon Stage disease was tested hypermethylation of p16 and WIF-1 resulted more frequently associated with stage IIIA/B (p values 0.006 and 0.000, respectively). When gene methylation status and cytogenetics abnormalities were compared, we found that aberrant methylation of p16 and ECAD were significantly more frequent in MM patients with chromosome 13 abnormalities as sole entity or with 13q deletion associated to IgH translocations. By contrast, promoter hypermethylation of Wnt signalling antagonist genes was associated with 13q deletion co-occurring with a hyperdiploide cariotype. (X²=17; df=8 p value: 0.000). According to the number of methylated genes observed in each sample 48% MM had 3–4 hypermethylated genes. When differences between methylator phenotype and cytogenetics abnormalities were analyzed we found that 3–4 hypermethylated genes have the tendency to be associated with chromosome 13 abnormalities more often than with hyperdiploide cariotype (p value 0.05).

CONCLUSION: Our results indicate that the accumulation of epigenetic events affecting genes regulating cell cycle control, cell adhesion and apoptosis is a common phenomenon in plasma cell disorders and may further contribute to the phenotype of MM cells. This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MM patients. Since talidomide seems to have a role in the PTEN/PI3K/AKT pathway, the PTEN function is worthy to be further investigated in Multiple Myeloma.