The role of the ALKBH5 RNA demethylase in invasive breast cancer

Abstract

Background: N6-methyladenosine (m⁶A) is the most common internal RNA modification and is involved in regulation of RNA and protein expression. AlkB family member 5 (ALKBH5) is a m⁶A demethylase. Given the important role of m⁶A in biological mechanisms, m⁶A and its regulators, have been implicated in many disease processes, including cancer. However, the contribution of ALKBH5 to invasive breast cancer (BC) remains poorly understood. The aim of this study was to evaluate the clinicopathological value of ALKBH5 in BC.

Methods: Publicly available data were used to investigate ALKBH5 mRNA alterations, prognostic significance, and association with clinical parameters at the genomic and transcriptomic level. Differentially expressed genes (DEGs) and enriched pathways with low or high ALKBH5 expression were investigated. Immunohistochemistry (IHC) was used to assess ALKBH5 protein expression in a large well-characterised BC series (n = 1327) to determine the clinical significance and association of ALKBH5 expression.

Results: Reduced ALKBH5 mRNA expression was significantly associated with poor prognosis and unfavourable clinical parameters. ALKBH5 gene harboured few mutations and/or copy number alternations, but low ALKBH5 mRNA expression was seen. Patients with low ALKBH5 mRNA expression had a number of differentially expressed genes and enriched pathways, including the cytokine-cytokine receptor interaction pathway. Low ALKBH5 protein expression was significantly associated with unfavourable clinical parameters associated with tumour progression including larger tumour size and worse Nottingham Prognostic Index group.

Conclusion: This study implicates ALKBH5 in BC and highlights the need for further functional studies to decipher the role of ALKBH5 and RNA m⁶A methylation in BC progression.

Keywords: Breast cancer; Epitranscriptomics; N6-methyladenosine; Prognosis; m6A.

Fig. 1

ALKBH5 basal expression in breast cell lines. A ALKBH5 mRNA expression in primary human mammary epithelial cells (HMEC), non-malignant MCF10A and breast cancer MCF7, T-47D, MDA-MB-436, and MDA-MB-231 cell lines (n = 5/6). B Western blot showing ALKBH5 protein is expressed across the breast cell lines, β-actin was used as a loading control (n = 3). *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 by t-test

Fig. 2

Bioinformatic analysis of ALKBH5 in breast cancer datasets. The cBioPortal was used to investigate ALKBH5 mRNA and copy number alterations (A-D) in breast cancer patients from the TCGA (Firehose Legacy) and METABRIC datasets

Fig. 3

Kaplan–Meier plots was used to investigate ALKBH5 mRNA expression and (A) overall survival (n = 626), (B) relapse free survival (n = 1764), and (C) distant metastasis free survival (n = 664)

Fig. 4

ALKBH5 immunohistochemical staining in the Nottingham Invasive BC TMA. A range of staining in the nuclear and cytoplasmic compartments was observed (A–F). Examples of weakly stained (A, B), moderately stained (C, D), and strongly stained (E, F) tumour samples are shown

Fig. 5

Kaplan–Meier plots was used to investigate ALKBH5 protein expression and (A) breast cancer specific survival, (B) distant metastasis free survival, and (C) disease free interval (n = 1318)

Fig. 6

Forest plots showing the hazard ratios and 95% confidence interval of the multivariate survival analyses for ALKBH5 protein expression in the patient cohort for (A) breast cancer specific survival, (B) distant metastasis free survival, and (C) disease free interval. ALKBH5 protein expression was an independent prognostic factor

Fig. 7

The TCGA RNA-seq dataset was stratified into low and high ALKBH5 expression by quartile, and the differentially expressed genes analysed using DeSeq2. Genes with significantly higher expression in low ALKBH5 are coloured red and genes significantly lower in low ALKBH5 are coloured green. Non-significantly differentially expressed genes are plotted in black. Significant gene expression: FC ± 2 and FDR < 0.05