Kurzfassung

Mitochondria are cell organelles that are involved in many biological processes. One of their key functions is energy production generating the energy currency ATP. Mitochondria contain their own DNA, which is maternally inherited and present in multiple copies per cell. Somatic alterations of mitochondrial DNA (mtDNA), such as mutations or copy number changes, have been linked to various conditions including neurodegenerative diseases, ageing and cancer. Because many cancer cells shift their...Mitochondria are cell organelles that are involved in many biological processes. One of their key functions is energy production generating the energy currency ATP. Mitochondria contain their own DNA, which is maternally inherited and present in multiple copies per cell. Somatic alterations of mitochondrial DNA (mtDNA), such as mutations or copy number changes, have been linked to various conditions including neurodegenerative diseases, ageing and cancer. Because many cancer cells shift their energy production towards glycolysis (Warburg effect), earlier assumptions suggested that somatic mitochondrial mutations in cancer cells might lead to dysfunctional mitochondria. Recent observations show that most cancer cells depend on functional mitochondria, however, the impact of mtDNA alterations in tumourigenesis is still highly controversial.
Our aim is to study the role of mtDNA mutations and mtDNA copy number changes in colorectal cancer. To detect somatic mitochondrial DNA mutations, we will use a next-generation sequencing approach to sequence the entire mitochondrial genomes from tumour and non-tumour tissue in a cohort of patients with colorectal cancer. We already have a collection of extracted DNA samples from about 800 patients. We will first analyse 100 patients, which to our knowledge will be the largest mtDNA study in a single tumour entity. We will also determine changes in mtDNA copy number in the same patients using quantitative PCR. Furthermore, we will correlate the findings about mitochondrial DNA alterations with other tumour characteristics such as tumour grade and further clinical data.
One key component in mtDNA replication and transcription is the mitochondrial transcription factor A (TFAM). It has been shown that a reduction in the amount of TFAM leads to decreased mtDNA copy numbers. To study the effect of mtDNA copy number alterations, we have introduced heterozygeous TFAM mutations in colorectal cancer cells using CRISPR/cas9. Our preliminary results indicate that we have successfully established cell clones with decreased TFAM content. In future, we will measure the amount of mtDNA in those cells and extensively assess the impact of depleted TFAM on mitochondrial function and energy metabolism including quantification of oxygen consumption, ATP generation and glycolytic flux. We will also determine the effect on the tumourigenic potential by measuring apoptotic rate, cell proliferation and cellular motility.» weiterlesen» einklappen