Cytological effects of mitochondrial ND5 mutation in a Cybrid cell model

Mitochondrial genomes have a higher mutation rate compared to nuclear genomes, due to oxidative damage caused by reactive oxygen species (ROS). These mutations in the mitochondrial DNA can lead to mitochondrial DNA disorders such as Leigh Syndrome (LS) and Leber Hereditary Optic Neuropathy (LHON) and other diseases such as cancer. The aim of this study is to identify the effect of mitochondrial ND5 mutations on autophagy, mitophagy, cell viability, cell proliferation, and cell oxidative stress in cancer cells. The gene encoding subunit 5 of complex I (ND5) was previously reported by our group as a hotspot for mtDNA mutations in breast cancer tissue. This prompts us to investigate the role of ND5 mutation in cell proliferation, mitochondria bioenergetics, and cellular quality control that includes autophagy and mitophagy. Initially, immunohistochemistry staining was used to determine the level of protein markers of autophagy, mitophagy, oxidative stress, and apoptosis in human breast cancer tissues. The immunohistochemistry data showed that Beclin-1 and Parkin were significantly increased in tumour tissue compared to adjacent matched other markers. In addition, a positive correlation was found between BNIP-3 and Beclin-1 as well as LC3II and cleaved caspase-3. Cybrid cell lines were developed using 3 commercial cell lines, CRL- 1739, HGT-1 and MDA-MB-231 which harbour ND5 mutations to investigate the effects of ND5 mutations. The MTT and BrdU assay on cybrid cell lines showed significantly higher cell viability and cell proliferation rate with significantly lower cell death compared to the negative control cells. The mitochondria bioenergetic assay using Seahorse XF96 Cell Mito Stress Test to measure OCR indicated that the cybrid cells showed an increase in basal respiration and proton leak while a decrease in spare respiratory capacity. This study also investigated the association between the ND5 mutation and autophagy/mitophagy activity using western blot and immunofluorescence. The results showed that Beclin-1 and LC3II protein expression and punctuation were significantly decreased, while the results on the BNIP-3 were significantly increased. The mitophagy protein marker Parkin was also significantly decreased in cybrid cells compared to the negative control. This study found that ND5 mutations resulted in increased proton leak which indicates increased ROS generation and mitochondrial dysfunction. The increased non-mitochondrial respiration is associated with altered metabolic intermediates that further promotes cell viability and proliferation rate. The reduced expression and punctuation of Parkin suggest instability in the attempt to recruit PINK1 due to increased ROS accumulation in damaged mitochondria. In summary, ND5 mutation promotes ROS formation which impairs cellular quality control that results in tumorigenesis. Overall, this study offers great potential for discovering cancer biomarkers and raising awareness of anti-autophagy therapies as a potential treatment for breast cancer.

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