Dysregulation of microRNA expression has been correlated with outcome or prognostic factors for many different cancer types.4, 6, 20–30, 32
Aberrant promoter methylation is one mechanism through which microRNA expression can be suppressed. There have been recent reports in the literature of associations of promoter methylation of individual microRNAs and cancer prognosis,25, 34, 35
including correlation of promoter methylation of miR-124a
, another microRNA that targets Cdk6, with poorer overall and disease-free survival in acute lymphocytic leukemia patients.34
Here, we report the detection of miR-137
promoter methylation in tumor tissue of 16.4% of SCCHN patients, occurring in oral, pharyngeal and laryngeal cancers; and describe an association of miR-137
promoter methylation with poorer overall survival among SCCHN patients. To our knowledge, this is the first study to evaluate the potential prognostic value of promoter methylation of a microRNA in SCCHN.
The observed association between miR-137
promoter methylation and overall survival among SCCHN patients may relate, in part, to the involvement of miR-137 in cell-cycle control through regulation of Cdk6. Loss of miR-137 expression results in reduced ability of the cell to arrest at the G1 phase, increasing proliferation,36,16
which may lead to accumulation of DNA damage thus enhancing genomic instability. Another potential contributor is the possible role of miR-137 in cellular differentiation or a switch from proliferation to differentiation.41
Expression levels of miR-137 are reported to be elevated in neuronal differentiation and decreased in poorly differentiated gliomas,16
although it is presently unknown if this generalizes to other histologies. Contrary to this point, we found no association of miR-137
promoter methylation with tumor grade, although low statistical power limits our ability to detect such an association. In spite of its purported involvement in cell cycle control, we also found no significant associations of miR-137
promoter methylation with other SCCHN prognostic factors, including stage, tumor size, nodal involvement or surgical tumor margin positivity.
Strengths of this study include high-quality prospective data collection; employment of methylation-specific PCR, a relatively inexpensive and sensitive method to detect DNA methylation for ascertainment of miR-137
methylation status; and the use of surgical tumor tissue taken prior to initiation of radiation and/or chemotherapy, which precludes potential bias relating to treatment effects. Another strength is our use of exact logistic regression modeling to obtain more accurate inferences for small sample sizes.51
This study also has several limitations. The relatively small sample size and proportion of miR-137
promoter methylation-positive samples limits our power to detect associations with prognostic factors. The present study has power ranging from 10%–18% to detect an association with an OR of 2.0. Another limitation is the relatively short follow-up time; future studies with longer follow-up time are needed to confirm our observed association between miR-137
promoter methylation and overall survival and further assess the association with disease-free survival. Another aspect that needs to be addressed in future studies is the correlation of promoter methylation with miR-137 expression with larger sample sizes. However, we did find that all the available samples that presented with miR-137
promoter methylation (8) had little or no expression of miR-137 whereas four of the 32 tumor tissue samples available for analysis had elevated expression of miR-137, yet none of these samples had miR-137
promoter methylation. Complicated modes of gene regulation such as this have been observed previously. For example, MAD2
is down-regulated in the absence of promoter methylation54
regulation requires both promoter methylation and histone acetylation.55
Despite methylation-specific PCR being a tried-and-true sensitive method for detection of promoter methylation,56
it is not quantitative. MicroRNA-137
methylation-positive tumors, as detected by methylation-specific PCR, may contain a low percentage of methylated alleles, which may not be sufficient to produce a substantial reduction of miR-137 expression, and this could dilute the magnitude of an association between methylation and prognosis/outcome.
The results of this study suggest that miR-137 promoter methylation is a relatively common occurrence in SCCHN, occurs across all sites, and may have value as a prognostic biomarker for the disease. However, future research efforts should focus on quantitative methylation analysis; further evaluation of the prognostic value of miR-137 promoter methylation in expanded cohorts with longer follow-up; and assessment of the combined effect of loss of multiple tumor suppressors involved in regulation of the G1/S phase checkpoint. Continued efforts to identify such novel prognostic biomarkers or biomarker panels are crucial in reaching the greater goal of understanding the biology behind aggressive tumor behavior, and ultimately improving survival and reducing mortality from head and neck cancer.