Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (8), 857-870

ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells

Affiliations

ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells

Federica Alessandrini et al. Neoplasia.

Abstract

Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.

Figures

Figure 1
Figure 1
DNA damaging drugs promote ETV7 transcriptional activation. RT-qPCR analysis of ETV7 expression upon different chemotherapeutics treatment in breast cancer-derived MCF7 (A) and MDA-MB-231 cells (B), and in healthy donor-derived lymphocytes (C). Bars represent average Fold Changes relative to the untreated condition and standard deviations of at least three biological replicates. * = P-value <0.01.
Figure 2
Figure 2
ETV7 can trigger breast cancer resistance to Doxorubicin. A-B) MTT Assays for survival analyses upon Doxorubicin treatment in MCF7 (A) and in MDA-MB-231 (B) cells over-expressing ETV7 with respect to their empty control. C) Cell death analysis on Doxorubicin-treated (three different doses) MCF7 cells over-expressing ETV7 in comparison to the ones stably transfected with an empty vector. Percentage of dead cells was obtained through fluorescence studies (at Operetta Perkin Elmer) calculated as a ratio between the amount of Topro-3 positive cells (dead cells) and the total number of cells (Hoechst 33,342 positive cells). D) RT-qPCR analysis of ABCB1 expression in MCF7 and MDA-MB-231 cells over-expressing ETV7. E) Analysis of the ratio between the nuclear and cytoplasmic intensity of Doxorubicin in MDA-MB-231 cells over-expressing ETV7 compared with their empty control, performed through Operetta Perkin Elmer Software. F) Analysis of the cytoplasmic area of Doxorubicin efflux in MDA-MB-231 cells over-expressing ETV7 in comparison to their empty counterpart. Images are reporting one representative analyzed by Operetta PerkinElmer Software. Experiments are done in quadruplicate. * = P-value <0.01.
Figure 3
Figure 3
DNAJC15 expression is repressed by DNA damaging drugs. A) RT-qPCR analysis in MCF7 cells of the expression of a selected group of DNAJC family members repressed upon Doxorubicin treatment according to microarray analysis (GSE24065). B-C) Expression analysis of DNAJC15 mRNA upon different chemotherapeutics treatment in breast cancer-derived MCF7 (B) and MDA-MB-231 cells (C), and in healthy donor-derived lymphocytes (D). Bars represent averages Fold Changes relative to the untreated condition of at least three biological replicates and standard deviations. * = P-value <0.01.
Figure 4
Figure 4
ETV7 can repress DNAJC15 expression at the transcriptional level and DNAJC15 over-expression can rescue Doxorubicin sensitivity. A) RT-qPCR analysis of ETV7 and DNAJC15 expression in MCF7 cells transfected with pCMV6-Entry-Empty or pCMV6-Entry-ETV7 plasmids. B) Gene reporter assay of MCF7 cells transiently over-expressing pCMV6-Entry-Empty or pCMV6-Entry-ETV7 along with pGL4.26-DNAJC15 reporter plasmid or the pGL4.26-DNAJC15-BS1 or -BS2 plasmids mutated in the putative ETV7 binding sites. Data are normalized using pRL-SV40 and are shown as fold of induction relative to the empty control. C) ChIP-PCR of DNAJC15 and GAPDH (control) promoter regions in MCF7 transfected with pCMV6-ETV7. Shown is the percentage of enrichment of ETV7 or control (IgG) bound to DNAJC15 promoter region in respect to INPUT DNA. For panels A-C, bars represent averages and standard deviations of at least three biological replicates. D-E) MTT Assay of ETV7-over-expressing MCF7 (D) and MDA-MB-231 (E) cells transiently transfected with pCMV6-Entry-Empty or pCMV6-Entry-DNAJC15 plasmids and treated with Doxorubicin 1.5 μM or 3 μM for 72 hours. Experiments are done in quadruplicate. * = P-value <0.01.
Figure 5
Figure 5
ETV7 can regulate DNAJC15 expression in a methylation-dependent manner. A) Methylation status of CpGs within DNAJC15 promoter analyzed by bisulfite conversion followed by PCR and direct sequencing in MCF7 untreated, treated with Doxorubicin for 16 hours or transfected with pCMV6-Entry-Empty or pCMV6-Entry-ETV7 plasmids. Methylated CpGs are shown as black dots, whereas unmethylated CpGs as white dots. B) RT-qPCR analysis of DNAJC15 expression in MCF7 transfected with pCMV6-Entry-Empty or pCMV6-Entry-ETV7 and treated with DMSO or 5-Aza-2′-deoxycytidine for 48 hours. C) RT-qPCR analysis of DNMT1, DNMT3A and DNMT3B expression in MCF7 treated with Doxorubicin for 16 hours. D) Western blot of DNMT3A and ETV7 on the immunoprecipitation with an antibody against ETV7 or normal IgG as control and on INPUT lysates in MCF7 transfected with pCMV6-Entry-ETV7 plasmid. * = P-value <0.01. E) A graphical model for ETV7-dependent Doxorubicin resistance in breast cancer cells. In normal conditions, ETV7 and DNMT3A are maintained at basal levels (particularly low in case of ETV7) and DNAJC15 can be regularly expressed. In response to Doxorubicin treatment, ETV7 levels get elevated and DNMT3A slightly increases as well. Induced ETV7 can then accumulate into the nucleus and specifically to chromatin-enriched regions. In the nucleus, ETV7 recruits DNMT3A (through direct interactions with putative additional cofactors) on target DNA (DNAJC15 promoter in this case) that in turn it is responsible for the methylation of CpGs. This will result in DNAJC15 repression and ultimately will lead to chemoresistance, partly through the exclusion of the drug from the nucleus. EBS: ETV7 Binding Site. Methylated CpGs are shown as filled circles, whereas unmethylated CpGs as empty circles.
Figure 6
Figure 6
ETV7 and DNAJC15 levels inversely correlate with clinical status of breast cancer patients and ETV7 targeting could be exploited pharmacologically. A) ETV7, DNAJC15 and ABCB1 expression levels from microarray data (GSE76540) of MCF7 cells resistant to Adriamycin -MCF7/ADR- (e.g. Doxorubicin). Presented are the averages and standard deviations of at least three biological replicates. B) ETV7 and DNAJC15 expression levels from microarray data of Triple Negative Breast Cancer patients treated with neoadjuvant chemotherapy who were showing recurrence or not for the disease (GSE43502). C) ETV7 expression levels measured by RT-qPCR from MDA-MB-231 cells untreated (Mock) or treated with Quercetin 50μM or Genistein 30μM for 16 hours. Bars represent averages and standard deviations of at least three biological replicates. D) MTT assay in MDA-MB-231 cells over-expressing ETV7 or its empty vector and treated with increasing concentration of Quercetin. Experiments are done in quadruplicate. * = P-value <0.01.

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Cagel M, Grotz E, Bernabeu E, Moretton MA, Chiappetta DA. Doxorubicin: nanotechnological overviews from bench to bedside. Drug Discov Today. 2017;22(2):270–281. - PubMed
    1. Bodley A, Liu LF, Israel M, Seshadri R, Koseki Y, Giuliani FC, Kirschenbaum S, Silber R, Potmesil M. DNA topoisomerase II-mediated interaction of doxorubicin and daunorubicin congeners with DNA. Cancer Res. 1989;49:5969–5978. - PubMed
    1. De Angelis A, Urbanek K, Cappetta D, Piegari E, Ciuffreda LP, Rivellino A, Russo R, Esposito G, Rossi F, Berrino L. Doxorubicin cardiotoxicity and target cells: a broader perspective. Cardio-Oncol. 2016;2(1):1–8.
    1. Bottero V, Busuttil V, Loubat A, Magne N, Fischel JL, Milano G, Peyron JF. Activation of nuclear factor kappaB through the IKK complex by the topoisomerase poisons SN38 and doxorubicin: a brake to apoptosis in HeLa human carcinoma cells. Cancer Res. 2001;61:7785–7791. - PubMed
    1. Wang CY, Mayo MW, Baldwin AS., Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science. 1996;274:784–787. - PubMed

Publication types

MeSH terms

Feedback