For the Data tables, Images and Projections, please contact the author directly for the Appendix- sections 1 & 2
Abstract:
Evidence of quantitative variations in overall mitochondrial DNA (mtDNA) copy numbers have been observed across various tumours. Their variations are not aligned to a common increase or decrease across all tumours, instead they are found differing in different tumours. To further understand these variations, multiple strategies attempted have been discussed in this paper to provide insights into the commonalities in the interplay between mitochondrial genes and the associated nuclear genes in tumours observed with low mtDNA copy number and tumours observed with high mtDNA copy numbers. A literature review of existing data was conducted which forms the basis of the experiments executed and the results acquired. A case control of over 37 tumour types comprising in total of 2,180 samples have been included in the study in addition to the online databank queries consisting of the TCGA databank of over 21,000 patient samples. Nucleotide specific changes in the mitochondria were used as a start point to identify tissues with high susceptibility to mitochondria mutations. Multiple nuclear genes were identified based on their normal and mutational co-expression with mitochondria encoded genes. This was followed by online databank queries using Mitochondrial Transcription Factor-A (TFAM) as a control to investigate the effects, co-expressed nuclear genes had on the mitochondrial replication machinery in different tumours. Additionally, research suggestions were provided to aid in progressing our understanding of cancer progression as a result of mitochondrial mutations.Introduction:
Background:
In the year 1890, a German pathologist, Richard Altmann published a paper that enabled researchers after him to target what is known as the powerhouse of a cell (1). The mitochondria, then known as the “Altmann’s granules” were the foundation for a new line of research, the cellular bioenergetics. Much research has gone in since then and it was only in the year 1925, when Otto Warburg identified that the mitochondria played a much larger role than just energy production in tissues that he theorized the basis for mitochondrial modulation in cancer cells which involves the shift of oxidative phosphorylation to glycolysis(2) (3) (4). The normal mitochondrial functioning includes the Krebs cycle and parts of the electron transport chain while glycolysis in the cytoplasm enables the metabolic breakdown of simple glucose sugars to pyruvate followed by the conversion of pyruvate to acetyl Co-A, which is a precursor for the cyclic process of energy production, the Kreb’s cycle (or the TriCarboxylic Acid cycle) (2). In addition to this, the mitochondria also play a role in in regulation of cellular homeostasis and apoptosis induction. The mitochondrial genome is maternally inherited, and an average mitochondrial DNA contains between 103-104 copy numbers in mammalian cells (5). The mtDNA sequence is about 16,569 base pairs long (5). According to the Warburg effect, tumourigenic cells modulate this process of converting pyruvate using pyruvate dehydrogenase to acetyl Co-A, to lactate instead through lactate dehydrogenase (3). Ideally, the TCA cycle produces a sum total of 30 molecules of ATP in addition to the 2 molecules of ATP received through aerobic glycolysis in the cytoplasm (see Fig.1); However, as in the case of all tumourigenic cells, the inhibition of the TCA cycle yields only the two ATP moleculeswhich is a drastic reduction in the overall energy produced in the mitochondria (4). The aerobic glycolysis pathway, although inefficient is a faster mode of energy production and supplies the cells with energy much quickly in comparison to the long process of Glycolysis-TCA-Electron transport chain complex (ETC pathway) (6) (7).Energy and Errors:
Later genetic studies highlighted the existence of genes that were involved in regulating key aspects of the mitochondrial respiration. Glycolysis is a cytosolic reaction sequence which is regulated by the nuclear encoded genes, mutations in the nuclear genes are bound to cause alterations in the metabolic pathways (8) (9). Similarly, the mitochondrial respiratory processes although tightly regulated are error prone as well. Mutational changes in the nuclear encoded genes have been proven to have a cascade effect on the mitochondrial respiration. The mitochondria contain most of the genes that code exclusively for the TCA cycle. Primary sites of known molecular instability induced errors in the TCA cycle are: Citrate synthase, aconitase, IDH (isocitrate dehydrogenase), SDH (succinate dehydrogenase), FH (fumarate hydratase), ME (malic enzyme) (10) (11). These mutations and copy number alterations are associated with an increased risk of disease progression (12). One such site of predominant risk of mutation is the mitochondrial Isocitrate dehydrogenase (IDH2).Citrate synthase (CS) activity and aconitase activity levels are crucial in the mitochondria as they are not only the primary sites of TCA cycle but are also for regulating the rate of the TCA cycle through the modulated conversion of pyruvate to acetyl Co-A (AcCoA) (including oxaloacetate) to citrate in the CS step. It is irreversible and downregulation of the CS activity has been observed to be linked with glycolytic bioenergetic switching, i.e. the cell re-modulates its energy production to favour quicker yield of ATP by promoting aerobic glycolysis and inhibiting the TCA cycle by downregulating pyruvate dehydrogenase activity (13). Increased EMT and tumourigenic cellular invasion has been attributed to CS deficiency (13). Increased aconitase clustering has been found to deregulate the reversible isomerization of citrate to isocitrate causing an increase in citrate oxidation and downregulating fatty acid synthesis in prostate adenocarcinomas (10) (14). It is commonly inhibited in FH deficient cells of gastric and prostate cancer (15) (16).IDH2 codes for conversion of isocitrate to α-ketoglutarate (α-OG). It is a reversible catalytic process that if reversed by mutation of IDH2, has been found to lead to oncogenic activity (10). The conversion of 2-OG to 2-HG has been attributed to its tumourigenic activity as it promotes cytokine independency and inhibits HIF1α (hypoxia inducible factor-1) stabilisation, histone demethylation and ten-eleven translocation (TET) (17) (18) (19). The HIF1α is a key regulator of cellular homeostasis, apoptosis, angiogenesis and energy metabolism. It is found to be commonly destabilised in uterine endometrial carcinomas by the inactivation of VHL (20). In healthy tissues, HIF1α expression is essential to prevent increased ROS production for maintaining cell viability and preventing DNA damage (21) (22) (20).Succinate Dehydrogenase (SDH) is an enzyme complex with specific roles in the TCA cycle that are bound to the inner mitochondrial membrane. It is encoded in its entirety by nuclear genes. The main role of SDH is to convert succinate to fumarate in a reaction that essentially reduces FAD to FADH2. It is the primary link between the electron transport chain and the TCA cycle and encoded completely by nuclear DNA and is devoid of a proton pump(23). Fumarate hydratase (FH), like SDH, behaves as a tumour suppressor in its normal state functioning and a majority of the tumours were found with an abundance of fumarate(10). Most cancers were found linked with somatic mutations on the mtDNA and are speculated to have an impact on the bioenergetic effectiveness of the cell (24).
Mitochondrial DNA copy number and its influence
Mitochondrial DNA Copy numbers are collective term provided to independent circular DNA strands of the mitochondria, the numerical quantity of which is tightly regulated inside cells by a rigid transcriptional machinery. There are differing existing theories on the replication control based on the site of origin of replication, site of termination, controlled termination to maintain 650nt long DNA strands and by the regulation of mtDNA molecules available to enable concise packing of the mtDNA for initiating replication (25). The mtDNA being a circular DNA strand contains a D-loop non-coding region, a heavy strand and a light strand. The replication of mtDNA is executed through a strand displacement mechanism that is initiated by the RNA primer at the LSP and terminates at the conserved sequence block 2 (CSB2) (26). The RNA primer Pol-γ (polymerase gamma) is responsible for the initiation of replication at the origin of the heavy strand (OH).Previous studies have worked to provide insights into the impact general count of mtDNA copy numbers have in cells of individual cancer types by calculating the abundance, depletion or by calculating the ratio of the whole genome sequence (see Fig 2). Previous studies had identified an increase in overall mtDNA copy numbers in CLL, lung squamous cell carcinomas, lung adenocarcinomas, pancreatic adenocarcinomas (27); While, a decrease in overall mtDNA copy numbers is observed in kidney renal clear cell carcinomas, hepatocellular carcinomas, bladder cancers and breast invasive ductal carcinomas (28). The centralized role of HIF1α and the interplay between the normal and mutated genes leading to the destabilization of HIF1α has been given considerable mention across multiple research publications (21); This in part is assumed to be due to the dysregulation of reactive oxygen species (ROS), This is speculated to occur as a result of increased generation of hydrogen peroxide in the mitochondria as a result of mutational changes and errors in the TCA cycle. Destabilisation of HIF1α and increased ROS/NOS production are believed to increase the risks of ROS induced mutations in the genes encoded in the inner mitochondrial membrane (29). Considering the proximity of the ROS to the site of the TCA cycle and the electron transport chain, ROS induced mutations are likely to influence and affect the rate of mitochondrial DNA replication (30). Mitochondrial DNA mutations in cancer have largely been disregarded due to the abundance of data on nuclear DNA; However, the availability of new datasets as those from the Cancer Genome Atlas (TCGA) have provided fresh and improved insights into the mutational signatures of mtDNA mutations in a variety of cancers. As provided later in this paper (see figure 4), an abundance of the mutations is indicative of C>T and A>G mutations on the heavy strand (25). The susceptibilities of the ND5 region in the mitochondria are observed frequently across a variety of tumours (see Mitochondrial Co-expression and figure 5). Existing literature has provided insight into the effects upregulation or downregulation of mtDNA copy numbers (31) have in inducing or promoting increased mutational changes on regulatory subunits of the replication, cell cycle control and cell signalling pathways of the cell. (26)The scope of this investigation aims to analyse the differing expression levels of mitochondrial genes in correspondence with the co-expressing nuclear genes. From the data acquired a strong correlation was observed in tumours with low overall mtDNA copy number and their susceptibility to PI3K-mTOR signalling pathways; Similarly, tumours with high overall mtDNA copy numbers were found to show errors in TGF-β SMAD signalling (32). To validate these findings a systematic approach has been taken to correlate the existing literature to publicly available patient data on mitochondrial DNA copy number contents in individual tumour types. (10) (28) (14) (27)
Hypothesis:
Tumours detected with low mtDNA copy numbers such as kidney renal clear cell carcinomas, are likely to be more susceptible to PI3K inhibitors due to the observed coexpression of molecules associated downstream of PI3K-AkT signalling.Tumours with high mtDNA copy numbers, such as lung adenocarcinomas, are likely to be more sensitive against TGF-β mediated SMAD signalling inhibitors due to the observed increase in TFAM expression levels corresponding to reduced control.Aims and Objectives:
The aim of this investigation is to identify genes that are commonly mutated and coexpressed in tumours associated with low mtDNA copy numbers or high mtDNA copy numbers, separately. Additionally, further investigation to analyse common signalling pathways in the two quartile ends of both, high and low mtDNA copy number associated tumours. This research is intended to help formulate a hypothesis which can be used to further wet lab research for developing therapeutic approaches to inhibit cancer progression and enable early diagnosis using mtDNA as a marker for tissue malignancies.Results and Discussion:
Mitochondrial DNA copy number variationsThe Cancer Mitochondria Atlas (TCMA) webpage provided a list of databases that were found to be highly valuable for the purpose of this project. Copy numberbased queries are the foundation of the research carried over the course of this project and several cancer types were queried in order to estimate the average count of mitochondrial DNA copy numbers. From a sample size comprising over 2,189 tumour tissue samples, all samples were individually categorized, and the quartiles were calculated with the purpose of tabulation and visual presentation. The 37 tumour samples in figure 3, show a box and whisker plot that was generated by plotting the recorded count of total mtDNA (y-axis) copy numbers for the different tumour types (x-axis). Due to the absence of data on tumour adjacent normal cells in the TCMA database, no controls could be plotted along with the tumour cell readings. Existing TCGA datasheets were queried individually to identify possible mention or tests for total mtDNA copy numbers or the mtDNA coverage depth in samples tested; however, that too was unsuccessful.The results presented in figure 3 provide a basic understanding of the average mtDNA copy number variations between different tumour types. The values were uploaded on excel and a box-and-whisker plot was generated for the different quartiles. The averages count of mtDNA copy numbers for all tumours was found to be within the range of 100 to 800 per sample. The size of the plots observed was, in the case of some tumour samples, restricted by the number of samples available as in the case of breast lobular carcinomas, soft tissue lymphomas and atypical chronic myeloid leukaemia’s. On the other hand, kidney chromophobe renal clear cell carcinomas (Kidney-ChRCC) showed the least significant plot with the variations in the readings extending as high as 1500 copies per sample, to as low as less than 50. Liver cancers were one of interesting prominence as despite their large sample size, they gave consistent values, holding a range of mtDNA copy number readings between 350 to 620. The extracted data samples are of comparative value and are compared against existing literature. It would be noteworthy to mention that the purpose of this plot was to enable the visualisation of the general trend of total mtDNA copy numbers across different tumours and absence of control tissue readings restricts the scope of this experimental results.ANOVA tests concluded differing p-values for the various tumour samples are provided in table 1 comparing tumours speculated to have high overall mtDNA-CN against tumours with low overall mtDNA-CN. Tumour types that are speculated to have lower or higher mtDNA copy numbers in the respective tumours were categorised according to the closest reading and presented. A significance of p=0.09 (9%) was observed for lung SCC (n=40) against kidney RCC (n=121) which failed to reject the null hypothesis which is believed to have occurred due to an insufficient sample size. Considering the similarity in response, data associated to tumours found in the lung were grouped (n=52) and queried against kidney RCC had a p-value of 0.037 (3.7%) was observed, thus rejecting the null hypothesis due to its statistical significance. Similarly, ANOVA tests for samples of liver HCC (n=261) were queried against samples of pancreatic adenocarcinomas (n=186) due to their relatively low and high mtDNA copy numbers in tumours, respectively. As a result, the p-values acquired were at a significance level of 0.009 (0.9%) thus rejecting the null hypothesis at a high level of significance. It is noteworthy to mention that the p-value acquired could also be inaccurate as a result of error in sample acquisition.
Mutational variationFrom TCMA profiles datasheets, multisets of mutational variants were converted to readable XLS format and delimited to provide categorized sheets of data. The mutational variants in mitochondrial DNA from various tumourigenic samples were arranged and the recorded samples are as shown in figure 4.A bar chart is provided in figure 4(a) highlighting the total number of available patient data sets on the TCMA profile for the mutational signatures on the:- IGR (intergenic regions), LocInfo (locus of interest), ncUTR (non-coding untranslated region), nsSNP (non-synonymous single nucleotide polymorphism) and synSNP (synonymous single nucleotide polymorphisms). Each of these mutations were arranged by their frequency in each cancer type along with the reference nucleotides and variant nucleotides. The highest number of mutational changes observed were nonsynonymous single nucleotide polymorphisms that were of the missense types. The mutations observed at the highest read were of guanine missense to adenine. Upon further querying it was observed that the G>A missense mutations were predominantly linked to liver and pancreatic carcinomas at a count of 373 and 225 out of 663 and 380 samples, respectively. From these studies it is observed that the highest susceptibility for mitochondrial mutational change, G>A missense mutations were likely highest in liver hepatocellular carcinomas (Liver-HCC) (see figure 4(b)).The data was calculated to identify similarities with existing literature (32) (31) (27) (25) with regard to observed mutational signatures on the G>A nsSNPs across the 37 tumour samples acquired. The highest observed changes were observed for HCC in a total of 50.8% of the 663 samples that correspond to existing data that recorded an abundance of G>A and C>T in the mitochondria within liver cancers
In further study, the possible reason for high G>A mutations are explored. It is believed that these specific mutations are occurring due to mutational changes in the mtDNA replication machinery and play a crucial role in the promotion of tumorigenesis. It is yet to be determined if the frequency of a certain type of mutation in the mitochondrial DNA of a tissue can be associated to upregulation or downregulation of mtDNA expression in cancer cells. Acquisition of tumour types consisting of both tumour and tumour-adjacent cells would be essential for the purpose of experimentally determining the statistical probability and clinical impact of these mutations in the mitochondrial DNA. The mutations identified were found scattered across different locations of the circular mitochondrial DNA; However, an interesting consistency in site of mutation was observed in certain cancer types such as liver HCC, Kidney RCC and breast adenocarcinomas; all of which are tumours that are speculated to possess lower mtDNA copy number counts in tumourigenic cells compared to healthy cells.This project aims to elucidate the different factors involved in the expressional difference between tumours associated with low and high overall mtDNA copy numbers in cancer cells in comparison to their healthy cells. Kidney RCC and Lung adenocarcinomas are of special interest as a result in this project. Although most of the focus has been attributed to understanding the cause and effects of mtDNA copy number deregulation in kidney, for low mtDNA copy numbers, and lung, for high mtDNA copy numbers, other tumour types have not been neglected and have been added to the study to understand additional molecules and pathways involved in the process. G>A mutations although found at a high frequency, were seconded only by C>T mutations of the nsSNP missense type. There were no significant variations observed within kidney RCC’s and lung adenocarcinomas. In liver HCC samples, the mutations corresponded to sites responsible for regulation of the C1, ND4 and ND5 region of the mitochondrial genome, both of which code for the primary mitochondrial respiration phase in the TCA cycle. Mutations on the complex 3 (C3) regions of the mitochondrial DNA were frequent in KIRC samples while complex 4 (C4) and C1 mutation were consistent in LUAD tumours. The next section elaborates on the co-related nuclear genes and the impact these mutations appear to have on their functioning, both inside and outside the mitochondria.Mitochondrial co-expression:From the previous section, a brief understanding of the mutational susceptibility was derived. However, it is from the location of the mutations and the upregulated or downregulate effect in the mitochondrial genes that the sites of the mutation were correlated to respective nuclear genes. To understand the impact of mitochondrial gene mutations on nuclear genes, a co-expression analysis data was used to further investigate the correlation and impact of these mutations.Multiple tumour types were queried and the link between the mitochondrial genes and the nuclear genes at different weights of edges were observed. The edge weight is determined by the overlap in topology measured (Topology overlap measure, TOM) which considers the correlation in expression between two partner genes and how many other genes are commonly shared between the two. The weight range is measured from 0 to 1, wherein ‘0’ indicates minimal or no strength of connection while a gene at a TOM distance of ‘1’ would be considered to have the strongest correlation. Table 1 provides a brief summary of the mitochondrial genes, the associated nuclear genes and their normal cell functions.From the data observed, the start and end sites of the mutations were investigated using MitoMap.org to determine the locus and the associated nucleotide changes at the position. Each cancer type was separately queried and the frequency of mutations at a specific locus was identified. Interestingly, C-T and G-A nucleotide changes were always found associated to tumorigenesis as a result of somatic mutations; however, not all G-A nucleotide changes were associated with tumours. From figure 5, common links in low mtDNA copy number tumours showed association of ND3 to common nuclear genes such as ASXL2, RIF1, DDI2, CCNT1 and TAOK1.ASXL2 is a zinc metal binding ligand and an epigenetic regulator that binds to histone modifying enzymes that are involved in the transcriptional machinery (28). It was found commonly associated to ND3, ATP8 and ND4L activity in breast, bladder and liver hepatocellular carcinomas respectively, and mutations at the sites in the mitochondrial genome in the respective tissues are speculated to increase the risk of tumorigenesis. Considering that the liver and kidney are both tissues with a high energy demand and toxic by-products, it is noteworthy to mention that the generation of ROS is found upstream of cell signalling that code for the activation of HIF1α and NFkB. It is speculated that the mutant ASXL2 genes are co-expressed with mitochondrial nsSNP mutations in tumours associated with lower than normal mtDNA copy numbers resulting in reduced transcriptional capabilities due to the downregulation of the C1 activity. The reduced transcription prevents further copy number increase to tackle the increased energy demand in cancer cells, however, this promotes tumorigenesis as further nuclear encoded genes such as TAOK1, which codes for DNA damage response are also downregulated. It is only in the case of REST, that a transcriptional repressor is believed to be upregulated; REST gene was found to be upregulated across all tumours associated with reduced mitochondria DNA copy numbers. Similarly, the expression of CCNT1 was found to correlate with the ND3 expression in kidney renal cell carcinomas, breast invasive adenocarcinomas and a minority of the bladder cancer samples. The frequency of correlated expression of ND3 region in low mtDNA tumours is of significance as it is a respiratory subunit of C1 in the respiratory chain complex that codes for the catalysis of NADH dehydrogenase to ubiquinone (CoQ10) in the TCA cycle
Errors in aconitase caused by the depletion of Fe-S (iron-sulphur) is proteins, destabilise the CoQ10 and initiate conformational changes which prevent the supply of hydrogen ions necessary for the catalysis of NADH from CoQ10 Ubiquinone to ubiquinol, the last stage of the respiratory chain C1. Mutations in the ND3 and ND5 region are believed to be linked to upregulation of ROS generation due to its proximity to the sites of TCA cycle that correspond to the conversion of isocitrate to α-KG. Errors at this site can lead to the irreversible catalysis of isocitrate to form 2-HG (2- Hydroxyglutarate) that can increase the risk of H2O2 production.RIF1 gene was found to express at below normal levels in tumours such as bladder, breast and kidney renal clear cell carcinomas. In addition to these it was also observed in uterine endometrial carcinomas. Under steady state conditions, RIF1 functions as a regulator of TP53BP1 which play a crucial role in the double strand break repair in response to DNA damage through non homologous end joining (NHEJ). RIF1 was also found to co-express in most samples with ATM in the nucleoplasm. ATM is commonly found in cancers and was found to be a commonly mutated gene in both tumours of high and low overall mtDNA copy numbers. It is speculated that the tumours that show a co-expression of SPEN or RIF1 with ATM are markers for poor prognosis.Additionally, from the query conducted for RIF1, the absence of the NER pathway was analysed and the role of PARP and APE1 was investigated. It was further observed that a majority of the tumours associated with increased mtDNA copy numbers showed a loss of function mutation on PARP and an amplification of fumarate hydratase along with an increase in AKT3 which is a key regulator in the PI3K-AKT-mTOR pathway (33) (34). The normal function of the AKT3 involves the regulation of cell cycle control and the activation of the DNA repair mechanism; However, in the absence of the nucleotide excision repair in the mitochondria (35), it utilizes an interdependent method of mismatch repair with BER. The MMR lacks the specificity of BER and given that PARP is mutated with a loss of function (36), it further influences the DNA damage repair by incorrect repair of the damaged DNA sequence. Further investigation is necessary to verify and validate this claim as the speculations made are based out of existing and published literature and data and not from independent results.Similarly, within tumours identified with an overall high mtDNA copy number with respect to their corresponding normal tissues, the co-expression of genes RPS7, PFDN5, RPL27, SPEN, SNRPG, UBL5 and UQCR11/10 are frequent and consistent. The consistency was researched using existing literature on tumours associated with higher than normal mtDNA copy number content as the preliminary data (27). This data was further investigated using TCMA data profile on the respective tumours, such as lung adenocarcinoma (LUAD), lung squamous cell carcinomas (LUSC) and pancreatic adenocarcinomas (PACA).SPEN showed the highest weighted score (at w >= 0.05) against ATP8, ATP6 and ND5. At lower weighted scores, it was found to be frequently associated to CYTB, ND1, ND2, ND6 and ND4/4L. Similarly, RPL27 and RPL27A showed a high level of coexpression against ATP8 activity. ATP8 and ATP6 activity has been found to be dysregulated in cardiomyopathy and infantile hypertrophy and considering its role in ADP to ATP conversion in the mitochondria is essential in maintaining cellular homeostasis in tumourigenic cells. The upregulation of ATP6 and ATP8 is consistent across all tumours with high mtDNA copy numbers according to the co-expression data, however its combined activity with the ND5 region is speculated to be due to an increased catalysis of NADH and thus to produce ATP.SNRPG was observed (at w >= 0.05) to be co-expressed with ND5 and ND6 which are the end points of the C1 subunit. Mutations at ND5 and ND6 were found with a co-expression of SNRPG with ATP8 at lower weighted score of 0.04. It is speculated that these mutations in combination affect the mitochondrial biogenesis by providing an abundance of energy subunits that increase the rate of the replication process. Simultaneously, as the gene goes unrepaired, the replication includes the defective gene and the newly replicated gene consequently continue to demand more energy recruitment and ATP generation. Mutations of C1, in specific at the ND5 and ND6 correspond to the functioning near the Cis-aconitase phase in the TCA cycle. Inactivating effect of the ubiquinone causes an increase of Fe-S enzyme clusters that can promote oncogenesis by deregulating citrate availability for conversion to IDH which is essential for HIF stabilisation and DNA methylation; This in turn is likely to result in the destabilisation of HIF1α causing a reprogramming of the glucose metabolic pathway in cancer cells (37). The specific role of iron metabolism in cancer is poorly understood, however, existing data validates the presence of increased iron retention in tumorigenic cells in comparison to the tumour adjacent cells (38). Regulator of telomere length (RTEL1) is an Fe-S cluster protein that functions to maintain the integrity of the telomeric ends and unwinds various DNA structures which are also likely to exhibit SNPs that result in liver HCC’s (39). The metabolic switch is consistent in both tumours of low mtDNA copy numbers and high mtDNA copy numbers, however, the site of mutation and energy demands of that respective tissue determine the mtDNA copy number alterations. The increase in mtDNA copy number is a likely occurrence in lung cancers due to the tissue being an O2 rich region and thus being able to generate more energy locally but due to the slow pace of the mitochondrial respiratory chain complex, the metabolic switch is easily triggered and could cause irreversible damage at sites such as SDH and IDH2. The same is not the case in tumours associated with low mtDNA copy number tumours as the mutations link nuclear encoded genes exclusively to either ATP8 or ND3 or ND4 subunits. The high energy requirement in kidney, liver and ovarian cancer having not been met, generates a similar metabolic switch that results in reduced mtDNA replication. Copy number alterations in variants of SDH were frequent in tumours associated to low mtDNA copy number; in specific these variants were SDHD and SDHC. While SDHD was homo-deleted and frequent in the lower quartiles of low mtDNA copy number tumours, SDHC was amplified and frequent in the upper quartiles of breast invasive ductal carcinomas and kidney renal clear cell carcinomas.UBL5 gene was found to at an expression level of above 5% in liver HCC. Upon further investigation with existing patient data it was observed that the UBL5 gene was present in samples of liver HCC associated with the upper quartile of high mtDNA copy number tumours. The UBL5 gene is identified as a positive regulator of mitochondrial biogenesis and protein modification as well as a regulator of mitochondrial protein targeting. It was observed that the UBL5 gene regulates mitochondrial biogenesis through a combined effect of NER and homologous recombination (40). The UBL5 being a nuclear encoded gene can carry out NER however in the absence of this repair mechanism in the mitochondria, the role of UBL5 in regulating proper mRNA splicing and protein modifications is essential to prevent mismatch and mutational changes from occurring. Mutation of the UBL5 is speculated to dysregulate the kinase activation for cell cycle regulation and ubiquitin-like modifier processes, activation and conjugation/deconjugation. The dysregulation of the ubiquitin pathway affects the conversion of pyruvate to acetyl-CoA and can thus cause the cell to modulate the metabolic pathway from proceeding into the TCA cycle. The UBL5 gene was found to co-express at an edge weight of >=0.5 with ATP8. At readings lower than 0.5 it was found associated with ATP6, ND5, ND4 and ND4L. The coexpression was able to elucidate the interference of these mutations on the functioning of the other mitochondrial genes that were not directly found to be associated to UBL5, such as: COX2, COX3, CYTB, ND1, ND2 and ND6.UQCR11 (Ubiquinol-cytochrome C Reductase, Complex 3 subunit 11) is a part of the mitochondrial ETC that encodes three subunit complexes, namely, SDH, Cytochrome c oxidoreductase and cytochrome c oxidase. It functions as a binding factor for the Fe-S proteins in the mitochondrial membrane and is a crucial driver of the ATP synthase and transmembrane transport. From the TCMA analysis, UQCR11 was found to be co-expressed with ATP8 and ATP6 at edge weights greater than 0.5. further investigation is needed to ascertain its role as a regulator of Fe-S protein binding.From the list of samples observed and the genes that were queried, an interesting link between tumourigenic samples of high mtDNA copy numbers and low mtDNA copy numbers was identified. Tumours associated with low overall mtDNAcopy number in tumours was found to express proteins that were responsible for cell cycle regulation and transcription machinery.
Multiple and consistent levels of proteins associated with the mTOR-AKT-PI3K signalling pathways were observed to be dysfunctional. Similarly, tumours associated with high overall mtDNA copy numbers were found to show a correlated expression of proteins linked with the TGF-β SMAD signalling. Myc mutations were frequent in nearly all cancer types associated with a relatively high mtDNA copy number content in cell.Comparing associated nuclear gene expressions relative to mitochondrial replication factorAs a result of the co-expression analysis and the tabulated data for the various associated nuclear genes found to co-express relative to specific mitochondrial genes, it was felt necessary to elucidate what effects these mutational changes had on the mitochondrial DNA replication and vice versa. To ascertain this, multiple samples of tumours were screened, and the associated genes mentioned in table 2 were queried for their putative copy number readings against TFAM expression levels.The plots in each type of tissue was labelled and highlighted only if they were recognized as drivers of tumorigenesis and if apparent TFAM expression changes were observed with the respective nuclear DNA variations. Figure 6 highlights the number of nuclear genes that are co-expressed with increased or decreased TFAM expression in cancerous samples. A control sample is introduced to indicate ordinary TFAM expression under each of the categories of mutational expression against their putative log2 readings (see figure 6(a)). It is important to emphasize that figure 6(a) does not provide any data that signifies TFAM expression changes as a driver of oncogenesis in this review. Figure 6(b) on the other hand shows many of theoncogenic drivers associated with relatively increased TFAM expression levels with loss of function mutations of ASXL2 dominating with one deep deletion. Since ASXL2 mutations are found predominantly mutated in tumour tissues associated with low mtDNA copy numbers, it is speculated that the mutation triggers reduced histone modification which is likely a source of reduced transcriptional activity thus forcing the remodulation of cellular metabolism.
Figure 6(c) shows most of the oncogenic drivers correlating to the gain of function mutations for SPEN with a high read between 0 to -0.3 log RNA for tumours associated with high mtDNA copy numbers. Within diploid copy number changes of SPEN, most of the oncogenic drivers are between -1 and 2 mRNA TFAM expression levels with less than while shallow deletions are found in larger numbers against gain of function mutations. Insignificant gain of function is observed with the bulk of the samples showing a diploidic nature of copy number changes; however, within diploid changes, the mRNA expression levels are observed above the median 0 and less than 2 providing proof of possible increased activity in the SPEN gene thus promoting increase FA oxidation to support tumourigenic growth.A similar investigation was done for various genes identified and tabulated in Table 2. While most of the malignancies were identified in Lung and prostate cancers expressed SPEN, SNRPG and UBL5, tumours identified with SPEN showed the highest frequency of tumourigenic status. Interestingly, the mutations observed in tumour samples with the expression of UBL5 and SPEN were found with increased expression of TGFBRAP1 and SMAD7. Considering the roles of UBL5 and SPEN are linked with mitochondrial biogenesis and Fatty Acid (FA) oxidation respectively, the increased mRNA expression indicates that a rise in mtDNA copy number causes an increase in the rate of FA oxidation and thereby promotes enhanced metabolic output through glycolysis in tumourigenic cells. The role of TGFBRAP1 and SMAD7 links the expression of most of the genes found co-expressed with ATP8, ATP6 and ND3 to a speculated role of the TGF-β SMAD dependant signalling. ASXL2, RIF1, CCNT1, TAOK1 and REST were found at lower frequencies in the respective tumours. It is important to note that RIF1, CCNT1 and REST are regulators of transcription and cell cycle regulation,
frequent and consistent missense and truncating mutations are all shown to reduce the overall TFAM expression at a minor level.Associated pathwaysFrom the quartile-based observations of mtDNA copy number expression levels and the associated nuclear genes, a contrast can be drawn regarding the pathways involved. This is made easier as the genes expressed are exclusive in some cases to specific tissue types and in some are expressed only during tumorigenesis. PTEN inactivation and the increased AKT signalling is observed in all tumours associated with low mtDNA copy numbers (9). The mutations are found to link PTEN, mTOR, AKT and PIK3CA to reduced cell proliferation and cell survival. The frequent mutations are speculated to be the product of linked mitochondrial mutations that cause the dysregulation of the nuclear encoded genes. VHL and PBRM1 mutations were consistent in most of the tumours associated with mtDNA copy numbers such as kidney RCC, Liver HCC and bladder cancers. The mutations are believed to be initiation level events that cause the elimination of the VHL control on HIF1α expression thereby leading to increased H2O2 production and damaged mitochondrial respiration. These mutations were consistent across tumours associated with low mtDNA copy numbers and in the lower quartiles in tumours with high mtDNA copy number. Similarly, for the tumours showing an abundance of mtDNA copy numbers, as in the case of lung adenocarcinomas and the upper quartiles of mtDNA copy number associated with liver HCC’s, the abundant expression of TGF-β mediated SMAD and Snail signalling enabled EMT transition (epithelial to mesenchymal transition) to promote metastasis (41). Metastasis is generally regarded feasible in cancer cells once the metabolic regulation within the cell has been re-modulated to meet the increased energy demand. The downstream ligands of the TGF/SMAD/Snail signalling pathway include a most important intermediary gene; The GLI2 and SOD2 are speculated to be a feasible target despite the lack of adequate information on the same.DNA damage repair and mitochondrial susceptibilitiesIn addition to understanding the variations in the behaviour of tumours affiliated with low or high mtDNA copy numbers in cell, it is essential to understand the possible effects mitochondrial deregulation and mutations can have on DNA damage repair on the mitochondrial DNA sequences. Certain mutations in the mitochondria were found to show a co-expression of nuclear encoded genes, PARP1 and APE1 (or APEX1), both of which are crucial regulators of the base excision repair mechanism were found to be homo-deleted and amplified across different tumour types, differently (42). PARP1 was found to be amplified in tumours associated with increased mtDNA along with amplifications in the FH (Fumarate hydratase) and mutation of PIK3CA, SPEN, CDH1, FAT1 and TGFBRAF1, all of which were observed to be co-expressed with ATP8, ATP6 and ND5. PARP1 homo-deletion on the other hand was linked with SDHD and ARID4- A and B homo-deletion along with mutations to the genes: RUNX1T1, KMT2D, HLF, SETD2, ERBB3, FGFR2 and NF2; all of which were found correlated previously among tumours showing decreased mtDNA copy numbers. PARP1 sensitivity to complex 2 and complex 4 in the mitochondria was hypothesised as a result of ROS and RNS induced DNA double strand break mediated PARP activation; This is speculated to cause reduced cellular NAD levels and abnormalities in the oxidative phosphorylation (42).
Future Investigation:A list of genes was observed but could not be queried further as they are not fully understood and available across all platforms used for querying in this research project.Further investigations are suggested to analyse the susceptibility of tumours associated with low mtDNA copy numbers to inhibitors of downstream signalling of PI3 kinases in cell cycle regulation. Considering the nuclear genes found co-expressed were linked to the C1 subunits ND1 and ND3. The nuclear genes mentioned previously were found to have functions linked with cell cycle and cell proliferation regulation. More research into the effects of inhibitors such as PI-103 and LY-294002 is recommended to analyse if they influence recovery in mtDNA copy number depleted cells. Research results from studies on PTEN/mTOR/PI3K inhibitors, BEZ235 showed tumourigenic cells developed resistance to PTEN inactivation (43). BEZ235 is an existing drug that is in the process of completing its stage 3 clinical trial approval in patients diagnosed with advanced kidney clear cell carcinomas.Similarly, the sensitivity of tumours to TGF-bet mediated SMAD signalling inhibitors and the subsequent mtDNA copy number changes are to be investigated. Decrease, in the mtDNA copy number count, upon treatment of tumours associated with high mtDNA copy numbers to TGF-β mediated SMAD signalling inhibitors would be a positive outcome in the treatment of the respective tumours. AP12009 (Trabederson) is an existing drug currently in the process of gaining approval for stage I/II clinical trial in patients diagnosed with advanced pancreatic/colorectal cancers/ malignant melanomas/ lung adenocarcinomas (32)Conclusion:From the analysis executed in this research, abundant evidence has been provided linking the expression of certain genes in certain types of tumours more than others, i.e. tumours with high mtDNA copy numbers overall were observed to show a co-expression of the ATP8, ATP6 and ND5 regions of the mitochondria against recurrent nDNA co-expression of SPEN, UQCR10, UQCR11 and SNRPG which were found to exist in tumour samples alongside mutations on BARD1, TGFBRAF1, PIK3CA, FAT1, ATM, CDH1, CDK8 and ROS1. Similarly, tumours associated with low mtDNA copy numbers were found to co-express mitochondrial genes COX1, ND3 and ND4L against ASXL2, ASXL3, RIF1, REST, mTOR, CCNT1 and RPL27A. The mutations were consistent across their respective tumour’s types and a systematic chart of mutational expressions observed has been provided. Additionally, taking note of the respective nuclear genes involved and their normal cell functioning, a distinct link was observed within the two tumour categories created, i.e. high mtDNA tumours and low mtDNA tumours. Tumours observed with an overall low mtDNA copy number were found linked with dysregulation of the cell cycle regulation machinery and DNA damage response. Specifically, a parallel was drawn between the genes observed and the link with the PI3K-AKT-mTOR signalling pathway. Tumours observed with overall high mtDNA copy numbers in comparison to their normal tissue counterparts were observed to show a distinct deregulation of genes associated with TGF-β SMAD signalling. The activation of the TGF- β signalling in the presence of SMAD activation was found common across all tumours in this category. Both categories of tumours were analysed and are recommended for further investigation to enable further insights into the difference in response and the possibility of designing improved drug-based inhibitors for common targets across various tumour types. The susceptibilities of certain tumours to specific signalling pathways with an accurate correlation to the mtDNA copy number deregulation allows the use of mtDNA copy numbers as a prognostic marker enabling future research to investigate the deregulation as a sign of better or worse survival.
The mutations observed were queried by cancer type and the copy number alterations were investigated against TFAM expression levels to verify and validate the effects of mutations in specific cancer, on the mitochondrial replication machinery. TFAM deletions was linked with PTEN inactivation in certain tumours while amplification of TFAM expression levels was linked with hyperactivation of the SMAD signalling. RPL27A was found in the lower quartiles of tumours found with low, as well as tumours found with high overall mtDNA copy numbers. The function of RPL27A was queried and the mutational effect is speculated to respond differently based on the tissue type and its average energy consumption, i.e. tissues with high O2 consumption and diffusion such as lung and uterus were found to link the G-A nsSNP mutations to incorrect ligand binding and instability in the genome. This is believed to occur due to RPL27A genes affinity of binding to ATP binding ligands at G-rich sites in the mitochondrial genome.PARP1 and APE1 (42) mutations were observed inconsistently between different tumour types with no specific bias. Further investigations are recommended as the scope of this project was unable to investigate and elucidate the possible deregulatory effects of mtDNA copy number variations on the base excision repair pathway (BER).Acknowledgement:I would like to give my sincerest thanks to Dr Payam Gammage for guiding me through the course of my research and to Ms Mahnoor Mahmood for providing me valuable insights and advice. Additionally, I would also like to give my thanks to Mr Ananya Naithani for helping me better understand ANOVA and the statistics presented in this paper.
References:1. Die Elementarorganismen und ihre Beziehungen zu den Zellen. Altmann, Richard. 1890, Veit & comp, p. 145.2. The mitochondrion-Molecular Biology of the Cell . Alberts B, Johnson A, Lewis J, et al. 2002, Garland Science, Vol. IV.3. The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles? Sugden, Mary C and Mark J. Holness. 2011, Islets, pp. 302-319.4. Understanding the warburg effect: The metabolic requirement of cell proliferation. Vander Heiden MG, Cantley LC. 2009, Science, pp. 324(5930): 1029-1033.5. The mitochondrial genome: Structure, transcription, translation and replicaiton. Taanman, JanWillem. 2, London : Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1999, Vol. 1410.6. Locasale JW, Cantley LC, Thompson CB. Metabloc flux and the regulation of mammalian cell growth. Cell metabolism. 2011, pp. 14(4):443-451.7. Crabtree, Herbert G. Observations of carbohydrate metabolism in tumours. Imperial cancer research. 22nd April 1929, pp. 536-545.8. Regulation of pyruvate metabolism and human disease. Gray LR, Tompkins SC, Taylor EB. 14, 2014, Springer: Cellular and molecular life sciences, Vol. LXXI, pp. 2577-2604.9. Mitochondria and cancer. Wallace, Douglas C. 10, 2012, Nature reviews, Vol. XII, pp. 685-698.10. Defects in mitochondrial metabolism and cancer. Gaude, Eduardo. 2014, eCollection, p. 2:10.11. The emerging role and targetability of the TCA cycle in cancer metabolism. Anderson, Nicole M. 2, 2018, Protein & cell, Vol. IX, pp. 216-237.12. Hieronymus, Haley. Tumour copy number alteration burden is a pan-cancer prognostic factor associated with recurrence and death. eLife. 4th September 2018, Vol. 7, p. 7:37294.13. Loss of the respiratory enzyme citrate synthase directly links the warburg effect to tumour malignancy. Lin CC, Cheng TL, Tsai WH, Hu KH, Chang HC, Yeh CW, Chen YC, Liao CC, Chang WT. s.l. : Sci, 2012, Sci-Rep, p. 2:785.14. Mitochondria and Cancer. Zong, W.X., Rabinowitz, J.D., & White, E. 2016, Molecular cell 61(5), pp. 667-676.15. Inhibition of mitochondrial aconitase by succination in fumerate hydratase deficiency. Ternette N, Yang M, Laroyia M, Kitagawa M, O'Flaherty L, Wolhulter K, Igarashi K, Saito K, Kato K, Fischer R, Berquand A, Kessler BM, Lappin T, Frizzell N, Soga T, Adam J, Pollard PJ. 2013, Cell Rep, Vol. 3, pp. 689-700.16. Decreased expression of the mitochondrial metabolic enzyme aconitase (ACO2) is associated with poor prognosis in gastric cancer. Wang P, Mai C, Wei YL, Zhao JJ, Hu YM, Zheng ZL, Yang J, Lu WH, Xu RH, Huang P. 30, 2013, Med Oncol, p. 552.17. (R)-2-hydroxyglutarate is sufficient to promote leukimogenesis and its effects are reversivble. Losman JA, Looper RE, Koivunen P, Lee S, Schneider RK, McMahon C, Cowley GS, Root DE, Ebert BL, Kaelin WG Jr. 339, 2013, Science, pp. 1621-1625.18. The oncometabolite 2-hydroxygluterate inhibits histone lysine demethylases. Chowdhury R, Yeoh KK, Tian YM, Hillringhaus L, Bagg EA, Rose NR, Leung IK, Li XS, Woon EC, Yang M, McDonough MA, King ON, Clifton IJ, Klose RJ, Claridge TD, Ratcliffe PJ, Schofield CJ, Kawamura A. 12, s.l. : EMBO, 2011, EMBO, Vol. I, pp. 463-469.19. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate dependant dioxygenases. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, Ito S, Yang C, Xiao MT, Liu LX, Jiang WQ, Liu J, Zhang JY, Wang B, Frye S, Zhang Y, Xu YH, Lei QY, Guan KL, Zhao SM, Xiong Y. 19, 2011, Cancer cell, Vol. 1, pp. 17-30.20. HIF and reactive oxygen species regulate oxidative phosphorylation in cancer. Hervouet E, Cízková A, Demont J, et al. 29, 2008, Carcinogenesis, Vol. 8, pp. 1528-1537.21. Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway. Kung-Chun Chiu, D Pui-Wah Tse, A Law,. 2019, Cell Death Dis, Vol. 10, p. 934.22. Hypoxia induces rapid, STAT3 and ROS dependent, mitochondrial translocation of RelA(p65) and IκBα. Ivanova IG, Perkins ND. 2019, Biosci Rep, Vol. 9, p. 39.23. Mutations in SDHD, a mitochondrial complex II gene in hereditary paraganglioma. Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC, Myssiorek D, Bosch A, van der Mey A, Taschner PE, Rubinstein WS, Myers EN, Richard CW 3rd, Cornelisse CJ, Devilee P, Devlin B. 287, 2000, Science, Vol. III, pp. 848-851.24. Mitochondrial DNA mutations in human cancer. Chatterjee A, Mambo E, Sidransky D. 25, 2006, Oncogene, Vol. V, pp. 4663-4674.25. Maintainance and expression of mammalian mitochondrial DNA. Gustafsson CM, Falkenberg M, Larsson N-G. 1, s.l. : Annu Rev Biochem, 2016, Vol. 85, pp. 60-133.26. Mitochondrial DNA: the overlooked oncogenome? Gammage P, Frezza C. 53, s.l. : BMC biology, 2019, BMC Biology, Vol. 17, pp. 17-53.27. Comprehensive and molecular characterization of mitochondrial genomes in human cancers. Yuan, Y., Ju, Y.S., Kim, Y. et al. 2020, Nature Genetics, Vol. 52, pp. 342-352.28. Reznik E, Miller ML, Şenbabaoğlu Y, et al. Mitochondrial DNA copy number variation across human cancers. 22 February 2016.29. Marco Tafani, Luigi Sansone, Federica Limana, Tania Arcangeli, Elena De Santis, Milena Polese, Massimo Fini, Matteo A. Russo. Marco Tafani, Luigi Sansone, Federica Limana, Tania Arcangeli, Elena De Santis, Milena Polese, Massimo Fini, Matteo A. Russo. 2016, 2016, Oxidative Medicine and Cellular Longevity, p. 3907147.30. Mitochondrial ROS-Modulated mtDNA: A Potential Target for Cardiac Aging. Yue Quan, Yanguo Xin, Geer Tian, Junteng Zhou, Xiaojing Liu. 20, 2020, Oxidative Medicine and Cellular Longevity, p. 11.31. Simultaneous DNA and RNA mapping of somatic mutations across diverse human cancers. Stewart JB, Alaei-Mahabadi B, Sabarinathan R, Samuelsson T, Gorodkin J,. s.l. : PLoS Genet., 2015.32. Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer. Young Seok Ju, Ludmil B Alexandrov, Moritz Gerstung, Inigo Martincorena, Serena Nik-Zainal. s.l. : eLife digest, 2014.33. Akt3 knockdown induces mitochondrial dysfunction in human cancer cells. Kim, M., Kim & Yun, J. 2016, Acta biochimica et biophysica Sinica, Vol. 48(5), pp. 447–453.34. The PI3K–AKT network at the interface of oncogenic signalling and cancer metabolism. Hoxhaj, G., Manning, B.D. 2020, Nat Rev Cancer, Vol. 20, pp. 74-88.35. Mitochondrial DNA damage and its consequences for mitochondrial gene expression. Cline, Susan D. 9-10, 2012, Biochimica et biophysica acta, Vol. 1819, pp. 979-91.36. Principal Aspects Regarding the Maintenance of Mammalian Mitochondrial Genome Integrity. Vasileiou, P., Mourouzis, I., & Pantos, C. 18(8), 2017, International journal of molecular sciences, Vol. 1821.37. HIF-1-Dependent Reprogramming of Glucose Metabolic Pathway of Cancer Cells and Its Therapeutic Significance. Nagao, A., Kobayashi, M., Koyasu, S., Chow, C., & Harada, H. 2019, International journal of molecular sciences, pp. 20(2), 238.38. Iron and cancer: recent insights. Manz DH, Blanchette NL, Paul BT, Torti FM, Torti SV. 2016, Ann N Y Acad Sci., pp. 149-161.39. RTEL1: functions of a disease associated helicase. Vannier JB, Sarek G, Boulton SJ. 2014, Trends Cell Biol, pp. 416-25.40. Ubiquitin-like proteins in the DNA damage response: the next generation. Da Costa, Isabelle C, Schmidt, Christine K. 2016, Essays Biochem EBC20190095.41. Mitochondrial dysfunction induces EMT through the TGF-beta/SMAD/Snail Signalling pathway in Hep3B hepatocellular carcinoma cells. Yi, E., Park S., Jung S., Jang, W., Kim, Y.,. 47, 2015, Internation journal of Oncology, Vol. V, pp. 1845-1853.42. Mitochondrial function in spesis. Arulkumaran, N. 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frequent and consistent missense and truncating mutations are all shown to reduce the overall TFAM expression at a minor level.Associated pathwaysFrom the quartile-based observations of mtDNA copy number expression levels and the associated nuclear genes, a contrast can be drawn regarding the pathways involved. This is made easier as the genes expressed are exclusive in some cases to specific tissue types and in some are expressed only during tumorigenesis. PTEN inactivation and the increased AKT signalling is observed in all tumours associated with low mtDNA copy numbers (9). The mutations are found to link PTEN, mTOR, AKT and PIK3CA to reduced cell proliferation and cell survival. The frequent mutations are speculated to be the product of linked mitochondrial mutations that cause the dysregulation of the nuclear encoded genes. VHL and PBRM1 mutations were consistent in most of the tumours associated with mtDNA copy numbers such as kidney RCC, Liver HCC and bladder cancers. The mutations are believed to be initiation level events that cause the elimination of the VHL control on HIF1α expression thereby leading to increased H2O2 production and damaged mitochondrial respiration. These mutations were consistent across tumours associated with low mtDNA copy numbers and in the lower quartiles in tumours with high mtDNA copy number. Similarly, for the tumours showing an abundance of mtDNA copy numbers, as in the case of lung adenocarcinomas and the upper quartiles of mtDNA copy number associated with liver HCC’s, the abundant expression of TGF-β mediated SMAD and Snail signalling enabled EMT transition (epithelial to mesenchymal transition) to promote metastasis (41). Metastasis is generally regarded feasible in cancer cells once the metabolic regulation within the cell has been re-modulated to meet the increased energy demand. The downstream ligands of the TGF/SMAD/Snail signalling pathway include a most important intermediary gene; The GLI2 and SOD2 are speculated to be a feasible target despite the lack of adequate information on the same.DNA damage repair and mitochondrial susceptibilitiesIn addition to understanding the variations in the behaviour of tumours affiliated with low or high mtDNA copy numbers in cell, it is essential to understand the possible effects mitochondrial deregulation and mutations can have on DNA damage repair on the mitochondrial DNA sequences. Certain mutations in the mitochondria were found to show a co-expression of nuclear encoded genes, PARP1 and APE1 (or APEX1), both of which are crucial regulators of the base excision repair mechanism were found to be homo-deleted and amplified across different tumour types, differently (42). PARP1 was found to be amplified in tumours associated with increased mtDNA along with amplifications in the FH (Fumarate hydratase) and mutation of PIK3CA, SPEN, CDH1, FAT1 and TGFBRAF1, all of which were observed to be co-expressed with ATP8, ATP6 and ND5. PARP1 homo-deletion on the other hand was linked with SDHD and ARID4- A and B homo-deletion along with mutations to the genes: RUNX1T1, KMT2D, HLF, SETD2, ERBB3, FGFR2 and NF2; all of which were found correlated previously among tumours showing decreased mtDNA copy numbers. PARP1 sensitivity to complex 2 and complex 4 in the mitochondria was hypothesised as a result of ROS and RNS induced DNA double strand break mediated PARP activation; This is speculated to cause reduced cellular NAD levels and abnormalities in the oxidative phosphorylation (42).
Future Investigation:A list of genes was observed but could not be queried further as they are not fully understood and available across all platforms used for querying in this research project.Further investigations are suggested to analyse the susceptibility of tumours associated with low mtDNA copy numbers to inhibitors of downstream signalling of PI3 kinases in cell cycle regulation. Considering the nuclear genes found co-expressed were linked to the C1 subunits ND1 and ND3. The nuclear genes mentioned previously were found to have functions linked with cell cycle and cell proliferation regulation. More research into the effects of inhibitors such as PI-103 and LY-294002 is recommended to analyse if they influence recovery in mtDNA copy number depleted cells. Research results from studies on PTEN/mTOR/PI3K inhibitors, BEZ235 showed tumourigenic cells developed resistance to PTEN inactivation (43). BEZ235 is an existing drug that is in the process of completing its stage 3 clinical trial approval in patients diagnosed with advanced kidney clear cell carcinomas.Similarly, the sensitivity of tumours to TGF-bet mediated SMAD signalling inhibitors and the subsequent mtDNA copy number changes are to be investigated. Decrease, in the mtDNA copy number count, upon treatment of tumours associated with high mtDNA copy numbers to TGF-β mediated SMAD signalling inhibitors would be a positive outcome in the treatment of the respective tumours. AP12009 (Trabederson) is an existing drug currently in the process of gaining approval for stage I/II clinical trial in patients diagnosed with advanced pancreatic/colorectal cancers/ malignant melanomas/ lung adenocarcinomas (32)Conclusion:From the analysis executed in this research, abundant evidence has been provided linking the expression of certain genes in certain types of tumours more than others, i.e. tumours with high mtDNA copy numbers overall were observed to show a co-expression of the ATP8, ATP6 and ND5 regions of the mitochondria against recurrent nDNA co-expression of SPEN, UQCR10, UQCR11 and SNRPG which were found to exist in tumour samples alongside mutations on BARD1, TGFBRAF1, PIK3CA, FAT1, ATM, CDH1, CDK8 and ROS1. Similarly, tumours associated with low mtDNA copy numbers were found to co-express mitochondrial genes COX1, ND3 and ND4L against ASXL2, ASXL3, RIF1, REST, mTOR, CCNT1 and RPL27A. The mutations were consistent across their respective tumour’s types and a systematic chart of mutational expressions observed has been provided. Additionally, taking note of the respective nuclear genes involved and their normal cell functioning, a distinct link was observed within the two tumour categories created, i.e. high mtDNA tumours and low mtDNA tumours. Tumours observed with an overall low mtDNA copy number were found linked with dysregulation of the cell cycle regulation machinery and DNA damage response. Specifically, a parallel was drawn between the genes observed and the link with the PI3K-AKT-mTOR signalling pathway. Tumours observed with overall high mtDNA copy numbers in comparison to their normal tissue counterparts were observed to show a distinct deregulation of genes associated with TGF-β SMAD signalling. The activation of the TGF- β signalling in the presence of SMAD activation was found common across all tumours in this category. Both categories of tumours were analysed and are recommended for further investigation to enable further insights into the difference in response and the possibility of designing improved drug-based inhibitors for common targets across various tumour types. The susceptibilities of certain tumours to specific signalling pathways with an accurate correlation to the mtDNA copy number deregulation allows the use of mtDNA copy numbers as a prognostic marker enabling future research to investigate the deregulation as a sign of better or worse survival.
The mutations observed were queried by cancer type and the copy number alterations were investigated against TFAM expression levels to verify and validate the effects of mutations in specific cancer, on the mitochondrial replication machinery. TFAM deletions was linked with PTEN inactivation in certain tumours while amplification of TFAM expression levels was linked with hyperactivation of the SMAD signalling. RPL27A was found in the lower quartiles of tumours found with low, as well as tumours found with high overall mtDNA copy numbers. The function of RPL27A was queried and the mutational effect is speculated to respond differently based on the tissue type and its average energy consumption, i.e. tissues with high O2 consumption and diffusion such as lung and uterus were found to link the G-A nsSNP mutations to incorrect ligand binding and instability in the genome. This is believed to occur due to RPL27A genes affinity of binding to ATP binding ligands at G-rich sites in the mitochondrial genome.PARP1 and APE1 (42) mutations were observed inconsistently between different tumour types with no specific bias. Further investigations are recommended as the scope of this project was unable to investigate and elucidate the possible deregulatory effects of mtDNA copy number variations on the base excision repair pathway (BER).Acknowledgement:I would like to give my sincerest thanks to Dr Payam Gammage for guiding me through the course of my research and to Ms Mahnoor Mahmood for providing me valuable insights and advice. Additionally, I would also like to give my thanks to Mr Ananya Naithani for helping me better understand ANOVA and the statistics presented in this paper.
References:1. Die Elementarorganismen und ihre Beziehungen zu den Zellen. Altmann, Richard. 1890, Veit & comp, p. 145.2. The mitochondrion-Molecular Biology of the Cell . Alberts B, Johnson A, Lewis J, et al. 2002, Garland Science, Vol. IV.3. The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles? Sugden, Mary C and Mark J. Holness. 2011, Islets, pp. 302-319.4. Understanding the warburg effect: The metabolic requirement of cell proliferation. Vander Heiden MG, Cantley LC. 2009, Science, pp. 324(5930): 1029-1033.5. The mitochondrial genome: Structure, transcription, translation and replicaiton. Taanman, JanWillem. 2, London : Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1999, Vol. 1410.6. Locasale JW, Cantley LC, Thompson CB. Metabloc flux and the regulation of mammalian cell growth. Cell metabolism. 2011, pp. 14(4):443-451.7. Crabtree, Herbert G. Observations of carbohydrate metabolism in tumours. Imperial cancer research. 22nd April 1929, pp. 536-545.8. Regulation of pyruvate metabolism and human disease. Gray LR, Tompkins SC, Taylor EB. 14, 2014, Springer: Cellular and molecular life sciences, Vol. LXXI, pp. 2577-2604.9. Mitochondria and cancer. Wallace, Douglas C. 10, 2012, Nature reviews, Vol. XII, pp. 685-698.10. Defects in mitochondrial metabolism and cancer. Gaude, Eduardo. 2014, eCollection, p. 2:10.11. The emerging role and targetability of the TCA cycle in cancer metabolism. Anderson, Nicole M. 2, 2018, Protein & cell, Vol. IX, pp. 216-237.12. Hieronymus, Haley. Tumour copy number alteration burden is a pan-cancer prognostic factor associated with recurrence and death. eLife. 4th September 2018, Vol. 7, p. 7:37294.13. Loss of the respiratory enzyme citrate synthase directly links the warburg effect to tumour malignancy. Lin CC, Cheng TL, Tsai WH, Hu KH, Chang HC, Yeh CW, Chen YC, Liao CC, Chang WT. s.l. : Sci, 2012, Sci-Rep, p. 2:785.14. Mitochondria and Cancer. 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