DNAJC12 causes breast cancer chemotherapy resistance by repressing doxorubicin-induced ferroptosis and apoptosis via activation of AKT

Abstract

Background: Chemotherapy is a primary treatment for breast cancer (BC), yet many patients develop resistance over time. This study aims to identify critical factors contributing to chemoresistance and their underlying molecular mechanisms, with a focus on reversing this resistance.

Methods: We utilized samples from the Gene Expression Omnibus (GEO) and West China Hospital to identify and validate genes associated with chemoresistance. Functional studies were conducted using MDA-MB-231 and MCF-7 cell lines, involving gain-of-function and loss-of-function approaches. RNA sequencing (RNA-seq) identified potential mechanisms. We examined interactions between DNAJC12, HSP70, and AKT using co-immunoprecipitation (Co-IP) assays and established cell line-derived xenograft (CDX) models for in vivo validations.

Results: Boruta analysis of four GEO datasets identified DNAJC12 as highly significant. Patients with high DNAJC12 expression showed an 8 % pathological complete response (pCR) rate, compared to 38 % in the low expression group. DNAJC12 inhibited doxorubicin (DOX)-induced cell death through both ferroptosis and apoptosis. Combining apoptosis and ferroptosis inhibitors completely reversed DOX resistance caused by DNAJC12 overexpression. RNA-seq suggested that DNAJC12 overexpression activated the PI3K-AKT pathway. Inhibition of AKT reversed the DOX resistance induced by DNAJC12, including reduced apoptosis and ferroptosis, restoration of cleaved caspase 3, and decreased GPX4 and SLC7A11 levels. Additionally, DNAJC12 was found to increase AKT phosphorylation in an HSP70-dependent manner, and inhibiting HSP70 also reversed the DOX resistance. In vivo studies confirmed that AKT inhibition reversed DNAJC12-induced DOX resistance in the CDX model.

Conclusion: DNAJC12 expression is closely linked to chemoresistance in BC. The DNAJC12-HSP70-AKT signaling axis is crucial in mediating resistance to chemotherapy by suppressing DOX-induced ferroptosis and apoptosis. Our findings suggest that targeting AKT and HSP70 activities may offer new therapeutic strategies to overcome chemoresistance in BC.

Keywords: AKT; Apoptosis; Breast cancer; DNAJC12; Ferroptosis; HSP70.

Fig. 1

DNAJC12 is overexpressed in non-pCR and HR+ breast cancer. A. Boruta analysis of neoadjuvant chemotherapy-related characteristics in four GEO datasets. B. DNAJC12 expression in non-pCR and pCR patients in four GEO datasets. C. RT-qPCR results from our own collection, consisting of 25 pCR patients and 90 non-pCR patients. The two vertical lines represent the median values of pCR and non-pCR, respectively. D. DNAJC12 expression in HR+ and HR- patients in four GEO datasets. E. Scatter plots indicating the correlation between DNAJC12 and ESR1 according to RT-qPCR results. F. RT-qPCR results of DNAJC12 mRNA levels in the normal breast epithelial cell line MCF 10A and 5 breast cancer cell lines with different molecular subtypes. ER: estrogen receptor; PR: progesterone receptor; HER2: Human epidermal growth factor receptor. G. Western blot measuring DNAJC12 protein levels in the normal breast epithelial cell line MCF 10A and 5 breast cancer cell lines with different molecular subtypes. ***, P < 0.01.

Fig. 2

DNAJC12 promotes cell proliferation and DOX resistance. A. RT-qPCR and B. Western blot results showing DNAJC12 levels in MDA-MB-231 cells transfected with DNAJC12v, and MCF-7 cells transfected with shDNAJC12. C. Cell growth measured by CCK-8. D. Colony formation assay of MDA-MB-231 and E. MCF-7, along with the corresponding statistics of colony numbers. F. Flow cytometry assay of cell apoptosis without any treatment after 48 h and G. the corresponding statistics of apoptosis. H. mRNA and I. protein levels of DNAJC12 in BT-474, SK-BR-3, and MCF-7 cells interfered with siDNAJC12. J. Drug sensitivity assays of doxorubicin and K. docetaxel in different breast cancer cells. ***, P < 0.01; *, P < 0.05; N·S., P > 0.05.

Fig. 3

DNAJC12 causes DOX resistance in BC cells via blocking both ferroptosis and apoptosis. A. Pathway enrichment of RNA-seq in MDA-MB-231 cells overexpressing DNAJC12 compared with the negative control. B. GSEA of RNA-seq showed enrichment in the ferroptosis pathway. C. Flow cytometry assay stained with a Fe²⁺ fluorescent probe in MDA-MB-231 cells treated with 50 nM DOX and MCF-7 cells treated with 1 μM DOX. D. Flow cytometry assay stained with BODIPY 581/591 C11 in MDA-MB-231 cells treated with 50 nM DOX and MCF-7 cells treated with 1 μM DOX to detect lipid peroxidation levels. E. Concentrations of MDA were detected in the presence of 50 nM DOX in MDA-MB-231 and 1 μM DOX in MCF-7. F. Western blot results of the ferroptosis inhibitor proteins GPX4 and SLC7A11 without any treatment. G. Flow cytometry assay and H. Western blot results of the apoptosis-related protein cleaved caspase 3 were used to analyze apoptosis in the presence of 50 nM DOX in MDA-MB-231 and 1 μM DOX in MCF-7. I. Cell viability measured by CCK-8 after cells were treated with 2 μM Fer, 5 μM zVAD, or a combination of Fer and zVAD in the presence or absence of 50 nM DOX in MDA-MB-231. J. Cell viability measured by CCK-8 after cells were treated with 2 μM Fer, 5 μM zVAD, or a combination of Fer and zVAD in the presence or absence of 1 μM DOX in MCF-7. ***, P < 0.01; *, P < 0.05.

Fig. 4

DNAJC12 promotes ferroptosis and apoptosis by upregulating phosphorylation of AKT. A. Western blot measuring the phosphorylation level of AKT. B. Western blot measuring the inhibitory effect of AKT inhibitor at different concentrations. C. Cell viability measured by CCK-8 after cells were treated with or without 0.1 μM CAPI in the presence or absence of 50 nM DOX in MDA-MB-231. D. Cell viability measured by CCK-8 after cells were treated with or without 0.1 μM CAPI in the presence or absence of 1 μM DOX in MCF-7. E. Cell viability measured by CCK-8 after cells were treated with or without 0.1 μM CAPI in the presence or absence of 2 μM RSL3 in MDA-MB-231. F. Cell viability measured by CCK-8 after cells were treated with or without 0.1 μM CAPI in the presence or absence of 10 μM RSL3 in MCF-7. G. BODIPY 581/591 C11 and H. MDA concentrations were used to detect lipid peroxidation levels after treatment with or without 0.1 μM CAPI in the presence or absence of 50 nM DOX in MDA-MB-231, and 1 μM DOX in MCF-7. I. Western blot results of the ferroptosis inhibitor proteins GPX4 and SLC7A11 after treatment with CAPI at different concentrations. J. Flow cytometry assay and K. Western blot results of the apoptosis-related protein cleaved caspase 3 were used to analyze apoptosis in cells treated with or without 0.1 μM CAPI in the presence or absence of 50 nM DOX in MDA-MB-231, and 1 μM DOX in MCF-7. ***, P < 0.01; *, P < 0.05; N·S., P > 0.05.

Fig. 5

DNAJC12 up-regulates phosphorylation of AKT via activation of HSP70. A. Western blot measuring the phosphorylation level of HSP70. B. Co-IP results of MCF-7 using an HSP70 antibody. C. Western blot measuring the phosphorylation level of AKT after treatment with HSP70 inhibitors at different concentrations. D. Cell viability measured by CCK-8 after cells were treated with or without 2 μM APOP, VER in the presence or absence of 50 nM DOX in MDA-MB-231. E. Cell viability measured by CCK-8 after cells were treated with or without 2 μM APOP, VER in the presence or absence of 1 μM DOX in MCF-7. F. Cell viability measured by CCK-8 after cells were treated with or without 2 μM APOP, VER in the presence or absence of 2 μM RSL3 in MDA-MB-231. G. Cell viability measured by CCK-8 after cells were treated with or without 2 μM APOP, VER in the presence or absence of 10 μM RSL3 in MCF-7. H. BODIPY 581/591 C11 was used to detect lipid peroxidation levels after treatment with or without 2 μM APOP, VER in the presence or absence of 50 nM DOX in MDA-MB-231, and 1 μM DOX in MCF-7. I. Western blot results of ferroptosis inhibitor proteins GPX4 and SLC7A11 after treatment with 2 μM or 4 μM APOP or VER. J. Flow cytometry assay and K. Western blot results of cleaved caspase 3 were used to analyze apoptosis in cells treated with or without 2 μM APOP, VER in the presence or absence of 50 nM DOX in MDA-MB-231, and 1 μM DOX in MCF-7. ***, P < 0.01; *, P < 0.05; N·S., P > 0.05.

Fig. 6

AKT inhibitor reverses DNAJC12-induced DOX-resistance in vivo. A. Schematic representation of the drug administration schedule for the xenograft tumor model. B. Image of resected tumors from mice in different treated groups. C. Volume analysis of primary mice tumors in different treated groups. D. DNAJC12, p-AKT, GPX4, and SLC7A11 expressions were determined by IHC in the tumor sections of MDA-MB-231 OE and NC groups without any treatment. Scale bars: 200 μm. E. Model depicting the DNAJC12/HSP70/AKT axis induced doxorubicin resistance via inhibiting ferroptosis and apoptosis.

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