Chaperone-mediated autophagy promotes carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells by alleviating G0/G1 arrest and maintaining cell cycle protein expression

Carboplatin inhibits tumor cell proliferation by inducing DNA damage and cell cycle arrest. Whereas abnormal regulation of the cell cycle may be involved in carboplatin resistance, the underlying regulatory mechanisms remain to be further elucidated. This study aims to investigate the effect of chaperone-mediated autophagy (CMA) on carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells and its relationship with cell cycle regulation.

① SK-BR-3 cell lines with different levels of CMA activity were established by lentiviral transduction. Transduction-associated fluorescence was observed by fluorescence microscopy, LAMP2A protein expression was detected by Western blotting, and CMA activity was evaluated in combination with LysoTracker immunofluorescence. ② Cell viability after treatment with different concentrations of carboplatin was assessed using MTT assay to determine the half-maximal inhibitory concentration (IC₅₀). Based on the IC₅₀ value, the cells in each group were continuously treated with 20 μmol/L carboplatin for 7 d to evaluate changes in cell proliferation, and cell density and attachment status were observed after treatment with different concentrations of carboplatin. ③ RNA sequencing (RNA-seq) was performed on the high-CMA-activity group and its corresponding control group. Differentially expressed genes (DEGs) were intersected with cell cycle-related genes, followed by GO and KEGG enrichment analyses. ④ Cell cycle distribution after carboplatin treatment was analyzed by flow cytometry, and the expression levels of CDK4, CyclinD1, and CyclinE1 were detected by Western blotting.

① SK-BR-3 cell models with different levels of CMA activity were successfully established. Compared with the shControl group, the shLAMP2A group showed significantly decreased LAMP2A protein expression (P=0.0003) and decreased LysoTracker Red fluorescence intensity (P<0.0001). Compared with the Control vector group, the pCMV6-LAMP2A group showed increased LAMP2A protein expression (P<0.0001) and enhanced LysoTracker Red fluorescence intensity (P=0.0002). ② MTT assay indicated that the IC₅₀ value of carboplatin against SK-BR-3 cells was approximately 20 μmol/L. After continuous treatment with 20 μmol/L carboplatin for 7 d, cell proliferation was lower in the shLAMP2A group than the shControl group, whereas it was higher in the pCMV6-LAMP2A group than in the Control vector group (P<0.0001). With the increasing of the carboplatin concentration, the number of adherent cells was generally decreased in all groups; this decrease was more pronounced in the shLAMP2A group than in the shControl group, whereas more adherent cells were retained in the pCMV6-LAMP2A group. ③ RNA-seq and enrichment analyses showed that DEGs associated with high CMA activity were mainly enriched in mitotic cell cycle transition, CDK complexes, DNA helicase activity, and cell cycle-related pathways. ④ Flow cytometry and Western blotting further revealed that, compared with the shControl group, the proportion of cells in G0/G1 phase was increased (P=0.0004) and that in S phase was decreased (P=0.0243) in the shLAMP2A group. Compared with the Control vector group, the pCMV6-LAMP2A group showed a decreased proportion of cells in G0/G1 phase (P<0.0001) and an increased proportion of cells in S phase (P=0.0003). The expression levels of CDK4, CyclinD1, and CyclinE1 were notably lower in the shLAMP2A group than the shControl group, whereas that in the pCMV6-LAMP2A group was higher than that in the Control vector group (P<0.0001).

Enhanced CMA activity attenuates the inhibitory effect of carboplatin on SK-BR-3 cell proliferation and improves cell survival and proliferative capacity under carboplatin stress. The mechanism may be related to enhanced CMA activity alleviating G0/G1 phase arrest, maintaining CDK4, CyclinD1, and CyclinE1 expression, and promoting G1/S phase transition. CMA-mediated regulation of cell cycle may be involved in carboplatin resistance in SK-BR-3 cells.