Downregulated ALDH2 Contributes to Tumor Progression and Targeted Therapy Resistance in Human Metastatic Melanoma Cells

Abstract

Aldehyde dehydrogenase 2 (ALDH2) is a crucial detoxifying enzyme that eliminates toxic aldehydes. ALDH2 deficiency has been linked to various human diseases, including certain cancers. We have previously reported ALDH2 downregulation in human melanoma tissues. Here, we further investigated the biological significance of ALDH2 downregulation in this malignancy. Analysis of TCGA dataset revealed that low ALDH2 expression correlates with poorer survival in metastatic melanoma. Examination of human metastatic melanoma cell lines confirmed that most had ALDH2 downregulation (ALDH2-low) compared to primary melanocytes. In contrast, a small subset of metastatic melanoma cell lines exhibited normal ALDH2 levels (ALDH2-normal). CRISPR/Cas9-mediated ALDH2 knockout in ALDH2-normal A375 cells promoted tumor growth and MAPK/ERK activation. Given the pivotal role of MAPK/ERK signaling in melanoma and cellular response to acetaldehyde, we compared A375 with ALDH2-low SK-MEL-28 and 1205Lu cells. ALDH2-low cells were intrinsically resistant to BRAF and MEK inhibitors, whereas A375 cells were not. However, A375 cells acquired resistance upon ALDH2 knockout. Furthermore, melanoma cells with acquired resistance to these inhibitors displayed further ALDH2 downregulation. Our findings indicate that ALDH2 downregulation contributes to melanoma progression and therapy resistance in BRAF-mutated human metastatic melanoma cells, highlighting ALDH2 as a potential prognostic marker and therapeutic target in metastatic melanoma.

Keywords: MAPK/ERK; acetaldehyde; aldehyde dehydrogenase 2; drug resistance; melanoma; targeted therapy.

Figure 1

Relationship between aldehyde dehydrogenase 2 (ALDH2) gene expression and metastatic melanoma patient survival. Kaplan-Meier survival curves of TCGA (a) and Swedish dataset GSE65904 (b) metastatic melanoma patients. Patients with complete clinicopathological information were dichotomized into ALDH2-high and ALDH2-low expression groups based on their normalized RNA-seq expression levels of the ALDH2 gene. * p < 0.05 and ** p < 0.01, log-rank test.

Figure 2

ALDH2 is expressed at low levels in most human metastatic melanoma cell lines. (a) ALDH2 gene expression data of Cancer Cell Line Encyclopedia (CCLE) human melanoma cell lines were obtained from the Gene Expression and Mutations in Cancer Cell Lines (GEMiCCL) database. (b) qRT-PCR analysis of ALDH2 mRNA levels in 12 human metastatic melanoma cell lines and 5 normal skin cell types. GAPDH served as an internal control. Data are presented as mean ± SD (n = 3). (c) Western blot analysis of ALDH2 protein levels in the 12 human metastatic melanoma cell lines shown in (b). Cyclophilin A (CyPA) was used as a loading control.

Figure 3

Effects of knocking out ALDH2 on acetaldehyde (AcAH) and reactive oxygen species (ROS) production and cellular energy metabolism in A375 cells. (a) ALDH2 knockout (KO) was confirmed by Western blot in three single-cell clones. WT, wild-type. (b) Mitochondrial ALDH2 activity. (c) AcAH levels in the culture medium of cells. (d) Intracellular ROS levels were detected using a DCFDA cellular ROS assay kit. (e) Cellular oxygen consumption rate (OCR) was measured with the Seahorse XF Cell Mito Stress Test kit. Left: respiratory flux profile; right: quantification of respiratory parameters. (f) The extracellular acidification rate (ECAR) was measured with the Seahorse assay. The data are representative of 2–4 experiments and expressed as the mean ± SD (n = 3 (b), 7 (c), 12 (d) or 19 or 22 (e,f)). * p < 0.05 and *** p < 0.001.

Figure 4

Effects of knocking out ALDH2 on A375 tumor growth, ERK activation, and mRNA expression of ATF4, IL1B, MITF, and MYC. (a) Tumor growth curves of A375 cells injected subcutaneously in 8 female nude mice (4 for WT and 4 for ALDH2-KO). (b) Western blot of p-ERK in A375 cells treated with 0–100 μM AcAH for 1 h. Band densities of phosphorylated ERK were quantified and adjusted by those of total ERK at the same dose of AcAH. (c) Western blot of ERK phosphorylation in three single clones of WT and ALDH2-KO A375 cells (left panel). Band densities of phosphorylated ERK were quantified and adjusted by those of total ERK (right panel). (d) qRT-PCR of ATF4 mRNA. (e) qRT-PCR of IL1B mRNA. (f) qRT-PCR of MITF and MITF-M mRNA. (g) qRT-PCR of MYC mRNA. The data are expressed as the mean ± SD (n = 8 (tumors, (a)) or 3 (c–g)). * p < 0.05 and *** p < 0.001.

Figure 5

Effects of overexpressing ALDH2 on AcAH and ROS production, mitochondrial respiration, IL-1β secretion, and MITF/MYC expression in 1205Lu cells. (a) Confirmation of ALDH2 overexpression (OE) by Western blot. (b) Mitochondrial ALDH2 activity. (c) AcAH levels in culture medium. (d) Intracellular ROS levels were detected using a cellular ROS assay kit (Red). (e) Measurement of OCR. (f) Measurement of ECAR. (g) ATF4 mRNA. (h) IL-1β secretion by ELISA. (i) MITF and MITF-M mRNA. (j) MYC mRNA. The data are representative or expressed as the mean ± SD (n = 5 (b), 4 (c), 10 (d), 4–6 (e,f), 3 (g), 9 (h) or 6 (i,j). * p < 0.05, ** p < 0. 01, and *** p < 0.001.

Figure 6

Relation of ALDH2 expression levels to sensitivity to BRAF and MEK inhibitors. (a) MTS proliferation assay of three BRAF-mutated melanoma cell lines treated with a single dose of vemurafenib (VEM) at the indicated doses for 72 h. (b) MTS proliferation assay of three melanoma cell lines treated with a single trametinib (TRA) dose at the indicated doses for 72 h. (c) Western blot analysis of ALDH2 levels in A375, SK-MEL-28, and 1205Lu cells (3 cell lysate samples each). (d) MTS proliferation assay of WT and ALDH2-KO A375 cells treated with one dose of 1 µM VEM and/or 0.1 µM TRA for 48 h. (e) ALDH2-OE in SK-MEL-28 cells was confirmed by Western blot. (f) MTS proliferation assay of WT and ALDH2-OE SK-MEL-28 cells treated with one dose of VEM and/or TRA for 48 h. The data are expressed as the mean ± SD (n = 3 (a,b) or 4 (d,f). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 7

ALDH2 expression in VEM- and TRA-resistant melanoma cells. (a) Western blot analysis of ERK phosphorylation and ALDH2 expression in parental (Par), VEM-resistant (VEM-R), and TRA-resistant (TRA-R) A375 cells. The band densities of p-ERK and ALDH2 were quantitated and normalized. (b) Western blot analysis of ERK phosphorylation and ALDH2 expression in Par, VEM-R, and TRA-R SK-MEL-28 cells. The data are representative of 2–3 experiments.