Post-transcriptional regulation of O⁶-methylguanine-DNA methyltransferase MGMT in glioblastomas

Article type: Research Article

Authors: Ramakrishnan, Valya^{a; 1} | Kushwaha, Deepa^{b; 1} | Koay, Debbie C.^{b; 1} | Reddy, Hasini^c | Mao, Ying^d | Zhou, Liangfu^d | Ng, Kimberly^b | Zinn, Pascal^e | Carter, Bob^a | Chen, Clark C.^{a; *}

Affiliations: [a] Center for Theoretical and Applied Neuro-Oncology, Moores Cancer Center, Division of Neurosurgery, University of California San Diego, San Diego, CA, USA | [b] Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA | [c] Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA | [d] Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China | [e] Department of Genetics, M.D. Anderson Cancer Center, Houston, TX, USA

Correspondence: [*] Corresponding author: Clark C. Chen, Ph.D., Surgical Oncology, Stereotactic and Radiosurgery, Neurosurgery Clerkship, Medical Education, Division of Neurosurgery, University of California, San Diego, 3855 Health Science Drive #0987, La Jolla CA 92093-0987, USA. Tel.: +1 858 246 0674; Fax: +1 858 822 4715; E-mail: [email protected].

Note: [1] These authors contributed equally to the manuscript.

Abstract: Background:The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) confers therapeutic resistance to DNA alkylating agents, including temozolomide. It is largely believed that MGMT promoter methylation is associated with down regulation of MGMT transcription and corresponding protein expression, thereby predisposing tumor cells to the toxic effect of temozolomide. Here we rigorously examined this underlying assumption. Methods:We examined the correlation between MGMT promoter methylation, transcription, and protein expression using The Cancer Genome Atlas (TCGA) glioblastoma database as well as an independent collection of glioblastoma specimens. Results:In both analyses, we found that MGMT promoter methylation status correlates well with low MGMT mRNA levels (p=0.04). On the other hand, glioblastomas with unmethylated MGMT promoters exhibited a wide range of MGMT mRNA expression. Intriguingly, the MGMT mRNA levels correlated poorly with MGMT protein levels by Western blotting (R2=0.04, p=0.34) or by ImmunoHistoChemical (IHC) stain quantitation (R2=0.02, p=0.50). To exclude the possibility that the poor correlation was due to substandard specimens, we determined the mRNA and protein levels of Colony Stimulating Factor 1 (CSF1), a gene previously shown to exhibit excellent mRNA/protein correlation. In contrast to MGMT, the mRNA level of CSF1 correlated well with the protein level (R2=0.47, p=0.001). Importantly, long-term passaged glioblastoma cell lines with comparable MGMT transcript levels differed in MGMT protein levels, suggesting mechanisms of post-transcriptional regulation. Accordingly, the correlation between MGMT promoter methylation and MGMT protein expression was poor (p=0.27). In silico analysis predicted potential binding sites for several miRNA within the 3’UTR of MGMT, suggesting a mechanism for the post-transcriptional of MGMT. Conclusion:Our results suggest mechanisms such as miRNA mediated regulation for post-transcriptional regulation of MGMT. Identification of these mechanisms should enhance the value of MGMT based prognostic or predictive biomarker strategies.

Keywords: Glioblastoma, TCGA, MGMT, methylation, therapeutic resistance, microRNA

DOI: 10.3233/CBM-2012-0245

Journal: Cancer Biomarkers, vol. 10, no. 3-4, pp. 185-193, 2012