Hypermethylation of mitochondrial DNA facilitates bone metastasis of renal cell carcinoma

Abstract

Kidney cancers including clear cell carcinoma (RCC) are identified with very vulnerable mitochondria DNA (mtDNA) and frequent epigenetic aberrations. Bone metastasis from RCC is prevalent and destructive. Bone marrow contains a quite hypoxic microenvironment that usually insitigate 50% of hypermethylation events in conferring a selective advantage for tumor growth. We hypothesized that hypermethylation of mtDNA in RCC cells would significantly contribute to bone metastatic tumor progression. Methylation-specific polymerase chain reaction assay (MSP) was adopted to measure the methylation status of D-loop region of mtDNA in 15 pairs of bone metastatic and primary RCC as well as tumor adjescent normal kidney tissues. mtDNA copy number was examined by the real-time quantitative polymerase chain reaction (qPCR). Western blotting analysis was used to measure the accumulation of several DNA methyltransferases (DNMTs) in the mitochondria and nucleus fractions of bone metastatic RCC cells. mRNA expression of mitochondria encoded genes was examined by RT-PCR. Reactive oxygen species (ROS), mitochondrial membrane potential and ATP content were measured using in vitro cells treated with de-methylation drug 5-Azacytidine (5-Aza). Non-invasive bioluminescent imaging was performed to monitor tumor occurrence in skeleton in mice. Our results showed that the D-loop region in bone metastatic tumor cells was markedly hypermethylated than those in primary RCC tumor cells, that is associated with a decreased mtDNA copy number and accumulation of DNMT1 in the mitochondria. The bone-tropism tumor colonization and progression of RCC cells was significantly suppressed by demethylating the D-loop region of mtDNA and reducing the intracellular level of ROS and ATP by 5-Aza treatment. In conclusion, our study provided a direct association between hypermethylation of mtDNA in RCC with bone metastastic tumor growth.

Role of DNA Methyltransferases (DNMTs) in metastasis

“…Total DNMT1 levels remained unchanged in bone metastatic RCC cells (16). However, there was an evident increase in the accumulation of DNMT1 in mitochondria.…”

“…Collectively, these results clearly indicated that hypermethylation of the mtDNA in bone metastatic tumor cells is because of the accumulation of DNMT1 in mitochondria. 5-Aza pre-treated bone metastatic RCC cells did not induce skeleton metastases when injected through the left cardiac ventricle (16). Ecotropic viral integration site-1 (EVI1) is a zincfinger transcription factor (17).…”

Mitochondrial Genetic and Epigenetic Regulations in Cancer: Therapeutic Potential

“…It was shown that hypoxia not only activates HIF-1α but also triggers approximately 50% of all hypermethylation events in solid tumours [ 168 ]. Nevertheless, direct regulation of mtDNA gene expression by mtDNA methylation remains an open question [ 169 ].…”

“…Several studies point to higher methylation rates in the D-loop region and more frequent hypermethylation events in cancer compared to non-cancerous samples, as well as in metastatic compared to primary tumour samples. This confers a selective advantage for tumour growth and metastatic potential [156,[168][169][170][171][172]. A study by Liu et al (2022) on kidney clear cell carcinoma (RCC) bone metastasis provided evidence of a higher methylation rate at the D-loop region of mtDNA in bone metastases than in primary RCC tumour cells.…”

“…This confers a selective advantage for tumour growth and metastatic potential [156,[168][169][170][171][172]. A study by Liu et al (2022) on kidney clear cell carcinoma (RCC) bone metastasis provided evidence of a higher methylation rate at the D-loop region of mtDNA in bone metastases than in primary RCC tumour cells. These authors observed a significant increase in gene expression for many mitochondrial genes after demethylation with the DNMT inhibitor 5-azacitidine.…”