Mitochondrial genomic alterations in cholangiocarcinoma cell lines

Cholangiocarcinoma (CCA) is a diverse collection of malignant tumors that originate in the bile ducts. Mitochondria, the energy converters in eukaryotic cells, contain circular mitochondrial DNA (mtDNA) which has a greater mutation rate than nuclear DNA. Heteroplasmic variations in mtDNA may suggest an increased risk of cancer-related mortality, serving as a potential prognostic marker. In this study, we investigated the mtDNA variations of five CCA cell lines, including KKU- 023, KKU-055, KKU-100, KKU213A, and KKU-452 and compared them to the non-tumor cholangiocyte MMNK-1 cell line. We used Oxford Nanopore Technologies (ONT), a long-read sequencing technology capable of synthesizing the whole mitochondrial genome, which facilitates enhanced identification of complicated rearrangements in mitogenomics. The analysis revealed a high frequency of SNVs and INDELs, particularly in the D-loop, MT-RNR2, MT-CO1, MT-ND4, and MT-ND5 genes. Significant mutations were detected in all CCA cell lines, with particularly notable non-synonymous SNVs such as m.8462T>C in KKU-023, m.9493G>A in KKU-055, m.9172C>A in KKU-100, m.15024G>C in KKU-213A, m.12994G>A in KKU-452, and m.13406G>A in MMNK-1, which demonstrated high pathogenicity scores. The presence of these mutations suggests the potential for mitochondrial dysfunction and CCA progression. Analysis of mtDNA structural variants (SV) revealed significant variability among the cell lines. We identified 208 SVs in KKU-023, 185 SVs in KKU-055, 231 SVs in KKU-100, 69 SVs in KKU-213A, 172 SVs in KKU-452, and 217 SVs in MMNK-1. These SVs included deletions, duplications, and inversions, with the highest variability observed in KKU-100 and the lowest in KKU-213A. Our results underscore the diverse mtDNA mutation landscape in CCA cell lines, highlighting the potential impact of these mutations on mitochondrial function and CCA cell line progression. Future research is required to investigate the functional impacts of these variants, their interactions with nuclear DNA in CCA, and their potential as targets for therapeutic intervention. Author Summary Bile duct cancer has the highest incidence in northeastern Thailand. In this study, we explored the mtDNA of CCA cell lines, as mitochondria play a key role in cellular energy production. We aimed to identify mutations that could serve as therapeutic targets or biomarkers. Our analysis revealed that each CCA cell line has unique mtDNA profiles, including INDELs, SVs, and SNVs such as m.8462T>C in KKU-023, m.9493G>A in KKU-055, m.9172C>A in KKU-100, m.15024G>C in KKU-213A, m.12994G>A in KKU-452, and m.13406G>A in MMNK-1, which demonstrated high pathogenicity scores. yet they also shared some common mutations. Future research is needed to understand how these mutations affect cell function, interact with nuclear DNA, and their potential for therapeutic intervention.


Introduction
Cholangiocarcinoma (CCA) is a malignant tumor originating in the biliary tree and can be categorized into three sub-types based on anatomical location: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA).Perihilar CCA is the most prevalent form of this cancer, comprising 50-60% of CCA cases, followed by distal CCA (20-30%) and intrahepatic CCA (10-20%) (1).These tumors are the second most common primary liver malignancy, accounting for roughly 15% of all primary liver tumors and 3% of gastrointestinal malignancies worldwide (2).Notably, northeastern Thailand has the highest global incidence rate of CCA, with 85 cases per 100,000 persons annually (3).
CCA is a complicated and highly heterogeneous cancer distinguished by tumor site, etiology, genetic characteristics, and varied prognostic outcomes.These variables confound much of the analysis of CCA genetics.Cancer cell lines are critical for studying many aspects of tumor biology and therapeutic strategies (4).
Therefore, we leveraged a bioresource of CCA cell lines, including KKU-213A, KKU-100, KKU-055, KKU-452, and KKU-023, which demonstrate differential differentiation abilities to investigate the mtDNA profile of in CCA and provide insight for further prognosis biomarker development in CCA patients.
The mitochondrion serves as the energy converter in eukaryotic cells, synthesizing adenosine triphosphate (ATP) through the mitochondrial electron transport chain (ETC) within the oxidative phosphorylation (OXPHOS) system.
Mitochondria possess circular double-stranded DNA genomes, termed mitochondrial DNA (mtDNA), comprising 16,569 base pairs.The mtDNA includes a G-enriched inner light strand (L-strand) and a C-enriched heavy strand (H-strand).mtDNA encodes 2 rRNAs and 22 tRNAs for protein synthesis, as well as 13 peptides for ETC and OXPHOS (5,6).MtDNA has a greater mutation rate than nuclear DNA, estimated to be 10 to 17 times higher than the nuclear genome due to an absence of complex DNA repair mechanisms (6).Mitochondria also demonstrate high levels of heteroplasmy, defined as the coexistence of several mtDNA variants within a single cell or organism and which has significant implications for mitochondrial function and disease etiology (7).A recent study revealed that heteroplasmic variations in mtDNA, particularly single nucleotide variants (SNVs), are associated with an elevated risk of cancer-related mortality in leukemia, highlighting heteroplasmic variation as a potential prognostic indicator for cancer (8).
Oxford Nanopore Technology (ONT) provides long-read sequencing capable of generating sequences thousands of base pairs long.This enables the sequencing of the entire mitochondrial genome (~16.6kb) in a single read.Furthermore, ONT allows for a more detailed investigation of heteroplasmic deletions inside a single read as well as the identification of complicated, large rearrangements e.g., duplications, which are difficult to identify using short-read sequencing (9).Lastly, the study demonstrated the ability of long-read ONT sequencing to detect large-scale deletions and rearrangements in mtDNA, which is important in understanding and diagnosing primary mitochondrial disorders.Hence, nanopore sequencing represents a promising approach for sequencing the whole mitochondrial genome (10) and determining the presence of tumour-associated SNVs.
In this investigation, we conducted comprehensive sequencing of the entire mtDNA using ONT across five distinct CCA cell lines, alongside one non-tumor cholangiocyte cell line, MMNK-1.We then investigated the mtDNA profiles of each cell line to define whether mtDNA contained candidate pathogenic variants within CCA.

Analysis
The mitoverse results encompass a QC report, haplogroup analysis, and mean depth coverage (S14 Table ) along

KKU-055 Cell Line
The  2).Most SNVs occurred in the MT-ND5 and MT-ATP6 genes, located in Complex I and V, respectively, indicating these may be specific pathogenic genes of this poorly differentiated cell line.The SVs in KKU-055 included 59 deletions, 55 duplications, and 71 inversions (Fig 2) . These findings underscore the potential functional impact of mtDNA mutations in this cell line.
Among these, mutations m.8160A>G, m.13406G>A, and m.13970G>A showed high MutPred, Selection, and CI scores.Additionally, mutations m.5442T>C, m.8701A>G, m.8860A>G, m.10398A>G, m.14766C>T, and m.15326A>G had heteroplasmy levels exceeding 95% (Table 6).There was no high mutation concentration in any specific gene.For SVs, there were 74 deletions, 58 duplications, and 85 inversions (Fig 2).These mutation patterns highlight the distinct mtDNA mutational landscape observed in the CCA cell lines.Across all cell lines, we found one SNV and seven INDELs were shared among five CCA cell lines (Table 7).Most mutations were concentrated in Complex with the annotated variants.The number of mtDNA SNVs, insertions, and deletions identified in each cell line showed no significant differences among the cell lines (Fig 1A).However, the number of INDELs within each gene varied, with the D-loop, MT-RNR2, MT-CO1, MT-ND4, and MT-ND5 genes exhibiting a high frequency of INDELs across all cell lines (Fig 1C and S6-S11 Tables).The number of SNVs per gene revealed that the D-loop, MT-CO1, MT-ND4, MT-ND5, and MT-CYB genes had a higher number of SNVs in CCA cell lines compared to MMNK-1, particularly in KKU-213A and KKU-452.Interestingly, five CCA cell lines had SNVs in the MT-ND6 gene, while the MMNK-1 cell line had none (Fig 1D).

Fig 1 .
Fig 1.The number of mtDNA alterations of each cell line.

Fig 2 .
Fig 2. The circos plots of each cell line.The circos plots depict the positions of SNVs and INDELs, marked by red dots along the circumference.Structural deletions are indicated by violet lines, structural duplications by blue-green lines, and structural inversions by red-wine lines.

I
genes and the D-loop region.The number of non-synonymous SNVs ranged from 9 in MMNK-1 to 21 in KKU-213A, with common SNVs, including m.1787G>T and m.4334A>C, identified in all six cell lines and noted for their potential pathogenicity and conservation across species.Common mutations such as m.8860A>G, m.8701A>G, and m.15326A>G consistently presented with high heteroplasmy levels, suggesting a fundamental role in the mitochondrial dysfunction observed in CCA.Additionally, the SNV m.750A>G and seven common INDELs were observed across the CCA cell lines.The high heteroplasmy levels associated with several SNVs, such as m.8462T>C in KKU-023, m.9493G>A in KKU-055, m.9172C>A in KKU-100, m.15024G>C in KKU-213A, m.12994G>A in KKU-452, and m.13406G>A in MMNK-1, suggest a need for further investigation into their potential role in carcinogenesis.The diversity in SVs among the cell lines, ranging from 69 in KKU-213A to 231 in KKU-100, highlights differences in genetic alterations, suggesting distinct underlying mechanisms of disease progression and potential therapeutic targets, necessitating further research into the functional consequences of these SVs and their role in cholangiocarcinoma (CCA) development.

Table 3
).Most SNVs occurred in the MT-CYB and MT-ATP6 genes, located in Complex III and V, respectively, suggesting these may be specific pathogenic genes of this poorly differentiated cell line.This cell line had 72 deletions, 74 duplications, and 85 inversions (Fig2).The high prevalence of these mutations suggests a potential role in the mitochondrial dysfunction and oncogenic behavior of KKU-100 cells.