Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion

Abstract

Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (ρ 0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ρ 0 epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in ρ 0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in ρ 0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in ρ 0 breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.

Cancer as a Metabolic Disease

“…The recent studies of the Singh and the Jazwinski groups provide compelling evidence that mitochondrial dysfunction, operating largely through the RTG response (mitochondrial stress signaling), can underlie the mutator phenotype of tumor cells [71,113,115,117,138]. Chromosomal instability, expression of gene mutations, and the tumorigenic phenotype were significantly greater in human cells with mtDNA depletion than in cells with normal mtDNA.…”

“…The RTG response would be essential for maintaining a stable ΔG' ATP for cell viability during periods when respiration is impaired. A prolonged RTG response, however, would leave the nuclear genome vulnerable to instability and mutability [112,117,119]. Mitochondrial dysfunction also increases levels of cytoplasmic calcium, the multi-drug resistance phenotype, production of reactive oxygen species, and abnormalities in iron-sulfur complexes, which together would further accelerate aberrant RTG signaling and genome mutability [85,106,107,110,111,[120][121][122].…”

“…In the on state, cytoplasmic Rtg2 disengages the Rtg1/Rtg-3 complex through a dephosphorylation of Rtg3 [110]. The Rtg1 and Rtg3 proteins then individually enter the nucleus where Rtg3 binds to R box sites, Rtg1 reengages Rtg3, and transcription and signaling commences for multiple energy and anti-apoptotic related genes and proteins to include MYC, TOR, p53, Ras, CREB, NFkB, and CHOP [110,112,113,[116][117][118]. The RTG response also involves the participation of multiple negative and positive regulators, which facilitate the bioenergetic transition from respiration to substrate level phosphorylation [110].…”

Mitochondrial DNA in Tumor Initiation, Progression, and Metastasis: Role of Horizontal mtDNA Transfer

“…In addition, stem cells and cancer stem cells that divide infrequently yet have high proliferative potential preferentially use glycolysis that is thought to be a safer form of energy metabolism that protects DNA integrity (21). Nevertheless, many tumor cell lines have very high respiration rates, and increasing evidence suggests a complex relationship between energy metabolism of tumor cells and tumor formation and progression (20)(21)(22)(23)(24)(25)(26). Indeed, one of the hallmarks of cancer appears to be metabolic flexibility as well as remodeling toward glycolytic metabolism as originally postulated by Warburg (20).…”

“…Alterations in mtDNA occur in most cancers due to high mutational rate relative to nuclear DNA and poor repair mechanisms, but the contribution of these mutations to cancer development has been established in only a modest number of cancers (26)(27)(28). Other mitochondrial damage, e.g., due to oxidative damage resulting from sustained inefficient respiration and oxygen radical production, not directly related to genome damage may also occur, but how this damage plays out in terms of cancer initiation and progression is unclear at present.…”