Mitochondrial DNA alterations may influence the cisplatin responsiveness of oral squamous cell carcinoma

Amnani Aminuddin,Pei Yuen Ng,Eng Wee Chua,Chee-Onn Leong

doi:10.1038/s41598-020-64664-3

Amnani Aminuddin, Pei Yuen Ng + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-64664-3

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Abstract

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.

Highlights

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC)
A combination of flow cytometry, quantitative polymerase chain reaction (qPCR), Western blot, and microarray analyses showed that the tumour spheres exhibited marked stemness features, namely increased expression levels of stemness genes and proteins, common cancer stem cells (CSCs) surface markers, and genes involved in glucose and lipid metabolism
We found that SAS tumour spheres were more cisplatin-resistant than their parental cells and that they had less mitochondrial DNA (mtDNA), which is the therapeutic target of cisplatin

Summary

Introduction

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). We evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. The intrinsic or acquired resistance of cancer cells to cisplatin remains a challenge in the chemotherapy of several cancers including oral squamous cell carcinoma (OSCC)[3,6]. Cisplatin resistance in general involves reduced DNA damage due to an increase in DNA adduct repair, reduced drug uptake, or increased drug inactivation[1,3,4,6] Activation of these mechanisms depends on multiple factors including genetic changes, epigenetic alterations at both molecular and cellular levels, and heterogeneity among cancer cells[4,9,10]. Because nanopore sequencing needs only minimal pre-sequencing preparation, it can produce very long reads (sometimes > 50 kb), minimise potential nucleotide errors introduced by PCR amplification, and preserve epigenetic modifications such as DNA methylation[20]

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