Characterization and impact of novel regulators of mitochondrial DNA integrity on tumourigenesis

Lead Research Organisation: Queen Mary, University of London
Department Name: Barts Cancer Institute

Abstract

Mitochondria are the known as the power houses of the cell, such that they generate the energy required for cellular processes. Consequently, aberrations in mitochondrial function or in mitochondrial DNA can have direct implications on the pathogenesis of disease including cancer. To date, although many mutations in mitochondria DNA have been associated with cancer, there is little known about the exact way in which mitochondrial DNA mutations can give rise to tumours. To further investigate this, we analysed 230 genes involved in repairing DNA to see if they could influence the levels of mitochondrial DNA, when they were silenced. Interestingly, we identified 3 genes which were previously associated with cancer, that can regulate mitochondrial DNA levels. We also observed upon depletion of these genes, changes in expression of some of the proteins involved in the energy-generating pathways in the cell. Therefore suggesting that these genes can also alter the function of mitochondria. In this proposal, we aim to investigate how these identified genes can regulate mitochondrial DNA integrity and determine how this can affect how mitochondria function. We will also look at patient tumour samples to determine the level of mitochondrial DNA in the absence of some of these genes.

Technical Summary

Mitochondrial defects and mitochondrial genomic instability have long been suspected to play an important role in the development and progression of cancer but to date little direct evidence for a functional role of these defects have been elucidated. Mutations in nuclear genes have been shown to give rise to mitochondrial DNA mutations but the mechanism behind this is unclear. To this end, we carried out an siRNA screen to identify novel regulators of mitochondrial DNA stability. We identified a novel role for the nuclear DNA repair and cancer associated genes, MLH1, PMS1 and MeCP2 in maintenance of mitochondrial DNA integrity. Loss of each of these genes results in decreased levels of the mitochondrial genes, Mt-Cot1, Mt-ND2 and Mt-ATPase6. Further analysis has revealed that deficiency in these genes increases susceptibility to oxidative stress and results in differential expression of complexes in the oxidative phosphorylation pathway. Therefore, given the increasing evidence suggesting an involvement of mtDNA stability in the transformation process, we aim to elucidate the mechanism behind this maintenance of mitochondrial DNA, determine the functional implications and elucidate its impact on carcinogenesis.

Planned Impact

This proposal investigates both basic and translational biology and therefore has obvious clinical impact.

Firstly, data generated from this proposal will provide further understanding of the role of MLH1, PMS1 and MeCP2 in cancer. Therefore in the longer term, this knowledge may impact on human health and welfare.

Secondly, a more translational impact based on this research proposal is that a reduction in mitochondrial DNA levels may prove to be a good biomarker for firstly MLH1, PMS1 and MeCP2 deficiency and secondly, potentially for a biomarker for response to mitochondria-targeted agents such as Menadione. A reduction in mtDNA levels in a patient tumour sample may suggest susceptibility to an increase in mitochondrial oxidative damage, as we observe for MLH1 deficiency. Therefore, this would have a direct impact on MLH1, PMS1 and MeCP2 deficient patients.

Lastly, in this proposal we will also analyze MeCP2 deficiency in Rett syndrome associated mutations with respect to its role in regulation of mitochondrial DNA levels. Therefore if we observe an association, this data will have translational implications for the treatment and health care of Rett syndrome patients.

Publications

10 25 50
 
Description Clinical Research Fellowship
Amount £210,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2013 
End 04/2016
 
Title MeCP2-deleted cells 
Description Using the CRISPR-cas9 gene-editing technology, we generated MeCP2-deleted HeLa cells and MeCP2-deleted human primary astrocytes. 
Type Of Material Cell line 
Provided To Others? No  
Impact These cell lines have enabled us to interrogate the function of complete MeCP2 loss in mitochondrial biology, which was not previously possible with transient siRNA transfections 
 
Title PMS1 knock-out HeLa Cell lines 
Description We have generated Zinc-finger targeted PMS1 knock-out cell lines in HeLa cells, to investigate the mechanisms of PMS1 in mitochondrial function 
Type Of Material Cell line 
Provided To Others? No  
Impact We have identified a number of novel phenotypic changes to cells upon complete loss of PMS1, including change in cell shape and reduction in mitochondrial DNA content. These phenotypic changes are currently under investigation. 
 
Description Mitochondrial ROS analysis 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided our collaborators with advice and reagents with regards to DNA damage response and DNA repair biology.
Collaborator Contribution This collaboration has enhanced our ability to analyse mitochondrial biology including analysis of mitochondrial ROS and membrane potential.
Impact We have now data on ROS generation in the absence of our genes of interest. Ultimately, this will be part of our upcoming publications based on this work.
Start Year 2013
 
Description Lab Tour 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 15 members of the public visited the lab, to discuss what research we were doing and the role of medical research in the UK
Year(s) Of Engagement Activity 2015
 
Description Lab Tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact 20 people attended a lab tour, where we discussed our research and invited people to ask questions.

Further visits are planned from other fund-raisers
Year(s) Of Engagement Activity 2013