Although mutation(s) in the POLG gene are shown to result in decreased OXPHOS, decreased mtDNA content and the pathogenesis of human mitochondrial diseases, its role in the pathogenesis of cancer is unclear. Therefore, we screened all coding exons and intron/splice junctions of POLG for mutations in breast tumors. Our analysis identified novel mutations in POLG. We also identified previously described mutations that are known to be involved in the pathogenesis of many mitochondrial diseases. Mutations were found in all three domains of the POLG protein. We identified a mutation in the exonuclease domain (C752T) of the breast tumor that is associated with PEO and infantile hepatocerebral syndrome 52-54
Several mutations in the POLG linker region that lead to neuromuscular diseases, including Alpers's disease and Parkinson's disease have been described 35,55
. However, we identified two novel linker region mutations in breast tumors. These include: (a) a missense mutation in the evolutionarily conserved (R628Q) linker region and (b) a silent linker region mutation (A703A). Previous functional analysis of the linker region mutants shows decreased enzyme activity, DNA binding and processivity of the polymerase 56
. The mutants in the linker region of the fruit fly enzyme also affect its enzyme activity, processivity and DNA-binding affinity 57
. The codon usage analysis for human POLG suggest that 56/103 Alanines use the GCC codon, but only 13/103 alanines use the GCA codon. This is important in the context of identified c.2109C>A (A703A) substitution in the Linker region. It is conceivable that base substitution causes ribosome stalling because Alanyl-tRNAs don't recognize the GCA codon so well which may slow the synthesis of protein. In some proteins, this type of substitution results in improper folding of protein leading to reductions in activity.
Breast tumors also harbored mutations in the polymerase domain (Y831C and E1143G) of POLG. Previous studies suggest that these mutations inhibit mtDNA polymerase activity and, hence, may lead to mtDNA depletion 58
. Targeting POLG polymerase mutations in mice hearts also provides in vivo
evidence for the depletion of mtDNA 59
One of the common features associated with mitochondrial diseases is the co-occurrence of mutations in POLG. The mutation T251I is found to occur in cis
with P587L in many mitochondrial diseases 34
. Likewise, T251I was found in cis
with P587L in two breast tumors. However, the E1143G mutation, frequently found in conjunction with W748S in ataxia 60
, was uniquely present in breast tumors. POLG contains trinuleotide repeats (CAG) in the coding region 37
. CAG trinucleotide repeat sequences are highly unstable, leading to the expansion or contraction of the repeat sequence, and are known to be involved in the pathogenesis of many human diseases 61
. Our study revealed that the expansion of CAG repeats in more than 20% of breast tumors analyzed.
We also identified novel intron/splice junction variants in conjunction with CAG repeats. Mutations in the intron/splice junctions of other genes are known to induce exon skipping, activation of the cryptic splice sites or alteration of the balance of the alternative spliced isoforms 62
. Variants in the splice junctions, particularly the GTAG insertion into intron 17, are predicted to alter splicing and POLG activity, as is also observed in PEO patients 63,64
. The CAG in 43-55Q was found to co-occur with seven variants in the intron/splice junction in two breast cancer cases. Interestingly, all breast tumors with CAG repeat expansion contained at least one splice site variant c.2734+39 insGTAG. POLG repeat expansion is reported to be associated with testicular cancer 65
. The POLG CAG repeats variation is also a predisposing genetic factor in idiopathic sporadic Parkinson's disease 55
. The expansion of CAG located in number of genes has been shown to cause many dominantly inherited neurodegenerative diseases, described as polyglutamine diseases 66
. The CAG repeats variation in other genes, such as androgen and estrogen receptors, plays an important role in breast and other cancers 67-69
. The contraction of CAG repeats in POLG affects its expression 58
. However, it is unknown at this time whether the expansion of CAG repeats in the POLG gene described in this paper affects its expression. An expanded CAG tract seems to affect the function of the host protein through protein-protein interaction 66
. It is conceivable that CAG expansion in POLG affects its function and may contribute to tumorigenesis. However, further studies are required to identify the exact role of POLG CAG expansion in cancer.
Mutations in POLG are known to deplete mtDNA in multiple tissues of mitochondrial disease patients 70
. Interestingly, our analysis also revealed 1) decreased mtDNA content in primary breast tumors and 2) when mutant POLG was expressed in breast cancer cells it led to depletion of mtDNA. Furthermore we identified mutations that were predominantly present in the D-loop control region of mtDNA. An increased incidence of novel mtDNA point mutations has been demonstrated in patients with POLG mutations 71,72
. The highest incidence of the mtDNA D-loop mutations could be due to the mutations affecting exonuclease and the polymerase domains of POLG. These findings suggest that reduced mtDNA content in breast tumors may arise due to 1) inefficient enzyme activity associated with POLG mutations and/or 2) mutations in the D-loop region affecting the binding of nuclear factors involved in mtDNA replication. Irrespective of POLG-induced depletion, our studies 11,73
and those of others 74,75
suggest that mtDNA depletion leads to tumorigenicity. Indeed, we recently demonstrated that depletion of mtDNA in breast epithelial cells lead to neoplastic transformation, and that this process is mediated by p53 9
. These studies led us to ask whether POLG mutations, particularly the one in the polymerase domain that causes mtDNA depletion play a role in tumorigenesis. Studies presented in this paper demonstrate that D1135A polymerase domain mutant when expressed in MCF7 cells functions as dominant negative and promote tumorigenesis in vitro.
We also show that expression of mutant protein results in decreased mtDNA content, decreased OXPHOS, decreased mitochondrial membrane potential and increased oxidative stress which together contribute to increased tumorigenic phenotype. We also asked whether other POLG mutations play a role in tumorigenesis. The data presented in this paper show that with the exception of linker domain mutation (P587L), all other mutants (Polymerase domain E1143G; and exonuclease domain T251I) show increased tumorigenicity in breast cancer cells. Since mutations P587L and T251I are often found in cis
in many mitochondrial diseases we also determined the effect of double mutant on Matrigel invasion. Our results show lack of synergistic effects on tumorigenicity in double mutants. The single T251I mutant was as invasive as the double P587L/T251I mutant. These studies suggest that P587L is not a significant player towards increased invasive property of MCF7 cells.
Apart from depletion, breast tumors contained mutations in mtDNA. Mutations in POLG are known to cause mutations in mtDNA. The mtDNA mutator mice that harbor the mutation in the exonuclease domain (that abolishes the POLG proof reading activity) show a marked reduction in lifespan due to the increased rate of mtDNA mutation 46, 76
. To date, there is no published report that describes the incidence of tumor development in these mice. It is possible that mtDNA mutations observed in these mice do not initiate tumorigenesis, i.e., transform normal cells, but rather are involved in the promoting tumoringenesis (as described in this paper) once cells are transformed. This argument is substantiated by our report which demonstrates that mtDNA mutations in normal cells do not confer tumorigenicity. In contrast, mutant mtDNA from breast tumors when transferred to transformed cells show metastasis 77
. In summary, our studies described in this paper provide the first comprehensive analyses of POLG gene mutations in human cancer that suggest a role for POLG in human tumorigenesis.