Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice

Abstract

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA "de novo" SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA "de novo" SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious "de novo" SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to "heteroplasmic-shifting". A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Keywords: clonal expansion; mammary cancer; mitochondrial DNA; next-generation sequencing.

Figure 1. Mouse Mitochondrial Genome.

PCR primers were designed reference mtDNA sequence of Blab/C mouse strain. PCR primers were designed to see if the full mtDNA sequence was present (A). Mouse Mitochondrial Gel Electrophoresis. 1 kb plus ladder (Lane 1) and mtDNA PCR products of a non-tumorigenic mammary control (Lane 2–5), bands align at the expected lengths of 950 bp, 805 bp, 619 bp, and 513 bp with the exception of the first and third band of the mammary control (B). Mitochondrial Genomic ScreenTape assay. Agilent 2200 TapeStation quantifies DNA and detects integrity of DNA by using a DNA integrity number (DIN). Czech mammary epithelial tumor mtDNA was extracted using two different methods. DNA Ladder (Lane 1). MtDNA extracted using Qiagen QIAPrep Spin Miniprep Kit (Lanes 2–6 and 9). MtDNA extracted using Qiagen DNeasy Blood and Tissue Kit (Lanes 7–8). DNeasy methodology successfully extracted intact mtDNA from mammary epithelial tumors (C).

Figure 2. Phylogenetic mapping of CzechII mouse strain.

CzechII and 39 common mouse strains phylogenetically mapped, utilizing bioinformatic software and the Sanger Mouse Genome Project (A, B).

Figure 3. CzechII mammary R12 tumor mtDNA SNP variant calling via Next Generation sequencing.

Next generation sequencing was performed on R12 mammary tumor and the tumors from 7 serially transplanted CzechII mammary R12 tumor fragments, SNP variant calling was performed to analyze common somatic SNPs that were conserved across R12 tumor fragments in comparison to CzechII Lactating mammary gland, negative control and CzechII Primary R12, positive control. Samples ran in duplicates.

Figure 4. CzechII mammary R12 metastatic tumor mtDNA SNP variant calling via Next Generation sequencing.

Next generation sequencing was performed on R12 mammary tumor and 5 CzechII mammary R12 serially transplanted metastatic tumor fragments from R12 Tumor, SNP variant calling was performed to analyze common somatic SNPs that were conserved across R12 metastatic tumor fragments in comparison to CzechII Lactating mammary gland, negative control and CzechII Primary R12, positive control.

Figure 5. CzechII mammary CZN5 tumor 1 mtDNA SNP variant calling via Next Generation sequencing.

Next Generation sequencing was performed on CZN5 tumor 1 that arose from a CzechII CZN5 hyperplasia. SNP variant calling was performed to analyze common somatic SNPs that were conserved across CZN5 hyperplasia and tumor outgrowth, in comparison to CzechII lactating mammary gland control.

Figure 6. CzechII mammary CZN5 tumor 2 mtDNA SNP variant calling via Next Generation sequencing.

Next Generation sequencing was performed on CZN5 tumor 2 that arose from a CzechII CZN5 hyperplasia. SNP variant calling was performed to analyze common somatic SNPs that were conserved across CZN5 hyperplasia and tumor outgrowth, in comparison to CzechII lactating mammary gland control.

Figure 7. Gene expression of mt-Nd1 in CzechII tumor samples.

Mt-Nd1 expression was measured by performing digital droplet polymerase chain reaction (ddPCR) on CzechII R12 Tumor and CZN5 tumor 1. Mt-ATP5f1 was used as endogenous control. CzechII R12 tumor 1 and CZN5 Tumor 1 Nd1 relative gene expression was compared to CzechII liver control. Statistical analysis was performed using ANOVA and Post-Hoc student t-test, Bonferroni correction P < .05. Samples were run in triplicates. Protein expression of mt-Nd1 in Czech tumor samples. Mt-ND1 protein expression was measured by performing a western blot on CzechII R12 Tumor and CZN5 tumor 1 (A). β-actin was used as endogenous control. CzechII R12 tumor 1 and CZN5 tumor 1 ND1 protein expression was compared to CzechII liver (B).