mtDNA mutations increase tumorigenicity in prostate cancer

Abstract

Mutations in the mtDNA have been found to fulfill all of the criteria expected for pathogenic mutations causing prostate cancer. Focusing on the cytochrome oxidase subunit I (COI) gene, we found that 11-12% of all prostate cancer patients harbored COI mutations that altered conserved amino acids (mean conservation index=83%), whereas <2% of no-cancer controls and 7.8% of the general population had COI mutations, the latter altering less conserved amino acids (conservation index=71%). Four conserved prostate cancer COI mutations were found in multiple independent patients on different mtDNA backgrounds. Three other tumors contained heteroplasmic COI mutations, one of which created a stop codon. This latter tumor also contained a germ-line ATP6 mutation. Thus, both germ-line and somatic mtDNA mutations contribute to prostate cancer. Many tumors have been found to produce increased reactive oxygen species (ROS), and mtDNA mutations that inhibit oxidative phosphorylation can increase ROS production and thus contribute to tumorigenicity. To determine whether mutant tumors had increased ROS and tumor growth rates, we introduced the pathogenic mtDNA ATP6 T8993G mutation into the PC3 prostate cancer cell line through cybrid transfer and tested for tumor growth in nude mice. The resulting mutant (T8993G) cybrids were found to generate tumors that were 7 times larger than the wild-type (T8993T) cybrids, whereas the wild-type cybrids barely grew in the mice. The mutant tumors also generated significantly more ROS. Therefore, mtDNA mutations do play an important role in the etiology of prostate cancer.

Fig. 1.

Tumor 18 COI G16X mutation in LCM-isolated prostate cancer epithelium. (A) mtDNA sequencing chromatograms of LCM-collected pure prostate cancer epithelium (upper chromatogram) reveals only the mutant 5949T (G5949A, opposite strand, black letters above), mtDNAs and pure normal epithelium (lower chromatogram) reveals only the wild-type 5949C (G5949G, opposite strand, black letters above) mtDNAs. (B) RFLP analysis of the G5949A mutation detected through the creation of an intentionally introduced DdeI restriction site. Lanes 1–4, DdeI digests. Lanes: 1, pure cancer epithelium (pure mutant); 2, pure normal epithelium (pure wild-type); 3 and 4, wild-type controls; 5, molecular weight markers. (C) Immunohistochemical staining of a section from tumor 18 stained with a COI-specific antibody. M, malignant glands; B, benign glands.

Fig. 2.

Increased tumor growth of PC3(mtDNA T8993G versus T8993T) cybrids. This graph is a composite of four independent experiments in which nude mice were injected s.c. with six different mutant PC3 cybrid clones [PC3(mtDNA T8993G)] and four different wild-type cybrid clones [PC3(mtDNA T8993T)]. All six mutant and four wild-type clones were tested at an early passage (at about passage 6), and two of the mutant clones and one wild-type clone were again tested at a later passage (at about passage 25). A total of 91 animals were injected with the mutant clones, 71 with the early passage clones and 20 with the late-passage clones; whereas 55 mice were injected with the wild-type clones, 45 with the early passage and 10 with the late-passage. Each injected mouse was measure for tumor volume every 10 days, with the final data set encompassing 11 time points. However, in experiments where the animals receiving the mutant clones developed debilitating tumors, the mice had to be euthanized before the maximum experimental time point. This decision resulted in a final total of 615 determinations of tumor volume for mice harboring the mutant clones and 378 determinations for those harboring the wild-type clones.

Fig. 3.

Dihydroethidium evaluation of PC3(mtDNA T8993T) versus PC3(mtDNA T8993G) tumor ROS production. (A) Relative fluorescence level seen in PC3(mtDNA T8993T) tumor sections, representative of wild-type clones 3 and 10. (B) Relative fluorescence level seen in PC3(mtDNA T8993G) tumor sections, representative of mutant clones 5, 8, and 20.