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By Jamshed Arslan Pharm.D.
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. NSCLC cells show increased expression of HIF-1alpha and HIF-2 alpha, which help cells overcome hypoxia. However, NSCLC samples have shown decreased mRNA levels of HIF-2 alpha-encoding gene called EPAS1. The mechanism of downregulation of EPAS1 transcription in NSCLC is unknown. A team of researchers from various institutes in Shanghai and Yangzhou, China, suspected transcriptional inactivation through DNA methylation as a plausible explanation behind EPAS1 downregulation in NSCLC. Their findings supported their hypothesis: EPAS1 promoter was hypermethylated in lung cancer cells, negatively correlating with EPAS1 mRNA levels. They found that hypoxic lung cells had stabilized EPAS1 proteins that transactivated DNMT1 (an EPAS1 target gene), which in turn further decreased the EPAS1 mRNA levels by regulating DNA methylation.
The researchers first evaluated the EPAS1 mRNA levels in 120 NSCLC samples and four lung cancer cell lines. The qPCR analysis showed that 99 of the 120 NSCLC samples and all the tested lung cancer cell lines had significantly reduced levels of EPAS1 mRNA relative to the controls. To determine if EPAS1 mRNA downregulation was due to hypermethylation, the team analyzed a 296bp CpG rich area in lung cancer cells relative to healthy lung cells through bisulfite sequencing PCR. As expected, the EPAS1 promoter region in lung cancer cells was hypermethylated, and demethylating EPAS1 promoter enhanced the EPAS1 mRNA levels.
This indicated that promoter hypermethylation is a possible mechanism behind epigenetic silencing of EPAS1 transcription. The next step was to find out which DNMT was responsible for this phenomenon.
Methylation of DNA by DNMTs leads to gene silencing via different mechanisms including:
Methylated CpGs prevent binding of specific transcription factors to gene regulatory regions thereby inhibiting transactivation. Transcriptional repressors bind preferentially to methylated CpGs impeding access to gene regulatory regions.
The team evaluated DNMTs’ expression in NSCLC samples by qPCR and immunohistochemistry. Although the mRNA and protein levels of DNMT1, DNMT3a and DNMT3b were significantly increased in lung cancer tissues, only reducing DNMT1 and DNMT3b (not DNMT3a) by siRNAs could restore EPAS1 levels.
To see if EPAS1 proteins could regulate DNMTs, researchers analyzed hypoxia-response elements (HREs) in DNMTs’ promoters. HREs in a gene, are regions where HIF alpha-HIF beta dimers bind and mediate adaptive response to hypoxia. Sequence blasts suggested that the promoters of DNMT1, DNMT3a and DNMT3b had 7, 2, and 1 HREs, respectively. ChIP-qPCR analysis showed that two of the 7 HREs in the DNMT1 promoter were tentative binding sites for EPAS1. Luciferase reporter assay further confirmed the notion that EPAS1 can activate DNMT1 transcription.
The researchers then focused on how EPAS1 gene is regulated under hypoxia, because hypoxia-stabilized EPAS1 proteins, in association with HIF-beta, can transactivate DNMT1.
The team found that hypoxia increased EPAS1 protein levels in lung cells, and as a result, transcription of DNMT1 (but not DNMT3a or DNMT3b) was induced. ChIP-qPCR confirmed the DNMT1-EPAS1 association. Silencing EPAS1 through siRNA under hypoxia decreased DNMT1 mRNA and protein levels. Increased methylation of EPAS1 promoter and the consequent decrease in EPAS1 mRNA levels under hypoxia further validated a negative feedback regulation of EPAS1 in hypoxia.
This study highlights the importance of epigenetic modulation in tumorigenesis. The aberrant gene expression and negative feedback regulation of EPAS1 described in this paper has implications beyond lung cancer, because EPAS1 protein is implicated in many types of cancers.
Learn more about Epigenetics resources
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
References
Xu, Xiang-Hong, et al. "Hypoxic-Stabilized EPAS1 Proteins Transactivate DNMT1 and Cause Promoter Hypermethylation and Transcription Inhibition of EPAS1 in Non-Small Cell Lung Cancer." FASEB Journal, vol. 32, 2018, n.pag. doi: 10.1096/fj.201700715
