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.