SERPINA3-ANKRD11-HDAC3 pathway induced aromatase inhibitor resistance in breast cancer can be reversed by HDAC3 inhibition

Abstract

Endocrine resistance is a major challenge for breast cancer therapy. To identify the genes pivotal for endocrine-resistance progression, we screened five datasets and found 7 commonly dysregulated genes in endocrine-resistant breast cancer cells. Here we show that downregulation of serine protease inhibitor clade A member 3 (SERPINA3) which is a direct target gene of estrogen receptor α contributes to aromatase inhibitor resistance. Ankyrin repeat domain containing 11 (ANKRD11) works as a downstream effector of SERPINA3 in mediating endocrine-resistance. It induces aromatase inhibitor insensitivity by interacting with histone deacetylase 3 (HDAC3) and upregulating its activity. Our study suggests that aromatase inhibitor therapy downregulates SERPINA3 and leads to the ensuing upregulation of ANKRD11, which in turn promotes aromatase inhibitor resistance via binding to and activating HDAC3. HDAC3 inhibition may reverse the aromatase inhibitor resistance in ER-positive breast cancer with decreased SERPINA3 and increased ANKRD11 expression.

Fig. 1. Screening of candidate genes responsible for endocrine resistance in human ER+ breast cancer.

a Estrogen deprivation for 14 days reduced colony formation of MCF-7 and T47D cells but not their LTED cells. b Estrogen deprivation for 5 days induced a clear reduction in cell proliferation of MCF-7 and T47D but not the LTED cells, as analyzed by cell counting assay. c Flowchart depicts how the 9 candidate genes were selected from various sets of RNA-seq or microarray data. d, e QPCR confirmed the uniform downregulation of the 7 genes in both MCF-7 and T47D LTED cells as compared with their parent cells. E2 estradiol, OVAR ovariectomy-resistance. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test (p < 0.05*, p < 0.01**, p < 0.001***).

Fig. 2. SERPINA3 downregulation promotes AI-resistance in human ER+ breast cancer.

a The inhibit efficiencies of the siRNAs were detected by QPCR in MCF-7 cells. b MCF-7 cells were transfected with the siRNAs respectively and deprived of estrogen for 5 days. Cell proliferation was determined by counting. c T47D and MCF-7 cells were transfected with SERPINA3-targeting siRNAs and deprived of E2 or not for 7–10 days before crystal violet staining. d MCF-7 LTED and T47D LTED cells were transfected with SERPINA3-expressing plasmid and deprived of E2 or not for 7–10 days before crystal violet staining. e Downregulation of SERPINA3 in AI-resistant LTED lines were confirmed by western blot. According to the publicly available expression profiling data of clinical samples (f) GSE105777 (n = 172) and (g) GSE153470 (n = 112), SERPINA3 was downregulated in ER+ breast cancer after 2-week presurgical AI therapy. According to the data of 1057 breast cancer patients retrieved from TCGA database, low SERPINA3 expression correlates with poor survival in patients with both (h) luminal A and (i) luminal B subtypes of ER+. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***); statistical significance of microarray gene expression data of paired samples from GSE153470, GSE105777 are determined by paired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Fig. 3. SERPINA3 is an ERα target gene in human breast cancer.

a The expression of SERPINA3 is positively correlated with ESR1 in breast cancer (retrieved from cBioPortal). b SERPINA3 exhibits significant higher expression in ER+ breast cancer (n = 7247) than ER− subtype (n = 2710) (retrieved from bc-GenExMiner v4.9). c QPCR and d Western blot results revealed a decrease of ERα expression in LTED cells, when compared with their parental cells. e The expression of ESR1 could be effectively inhibited by the siRNAs in MCF-7 and T47D cells. f QPCR and g western blot results showed that siRNA knockdown of ESR1 in MCF-7 and T47D effectively downregulated SERPINA3 expression. GAPDH and SERPINA3 were blotted from different gels for their close molecular weight. h SERPINA3 mRNA expression decreased with extended estrogen deprivation incubation in MCF-7 and T47D. i The details of the predicted ERα binding sequence in SERPINA3 promoter obtained from JASPAR. j Comparison of ERα binding motif and the predicted ERα binding sequence in SERPINA3 promoter. k Dual luciferase reporter assay verified the physical interaction of ERα and SERPINA3 promoter at −462 to −446 bp. The promoter plasmid or its mutant type were co-transfected with pCDH empty vector or ESR1-expressing plasmid as indicated. Luciferase assays were then performed. pCDH-Vector: pCDH-CMV-MCS-EF1-puro; pCDH-ESR1: pCDH-CMV-MCS-EF1-puro-ESR1. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Fig. 4. SERPINA3 downregulation drives AI resistance by ANKRD11 upregulation.

a Flowchart depicting work steps to identify SERPINA3 downstream effectors in inducing AI-resistance. SERPINA3 correlated genes in breast cancer were retrieved from cBioPortal. The genes indicating poor prognosis in ER+ breast cancer were screened by using bc-GenExMiner v4.9. The expressions of the 5 genes were detected by QPCR after siRNA inhibition of SERPINA3 in (b) MCF-7 and (c) T47D. LTED cells of (d) MCF-7 and (e) T47D were transfected with the siRNAs of ANKRD11 and DYSF respectively and deprived of estrogen or not for 5 days, then cell proliferation was examined by cell counting. ANKRD11 inhibition restored sensitivity to estrogen deprivation in SERPINA3 knock-down (f) MCF-7 and (g) T47D cells. h Western blot showed that ANKRD11 was upregulated by SERPINA3 knockdown in MCF-7 and T47D. ANKRD11, SERPINA3 and GAPDH were blotted from 3 different gels. i QPCR and j western blot showed that ANKRD11 is overexpressed in LTED cells. ANKRD11 and GAPDH were blotted from different gels. k Expressions of SERPINA3 and ANKRD11 are negatively related in breast cancer (retrieved from cBioPortal). l ANKRD11 exhibits significant lower expression in ER+ than ER− subtype (retrieved from bc-GenExMiner v4.9). According to clinical samples’ gene expression profiling data in GSE147271 (n = 28), the expression of (m) SERPINA3 was significantly downregulated and (n) ANKRD11 was significantly upregulated after presurgical tamoxifen therapy for 20.7 (±9.6) days. o High expression of ANKRD11 correlates with poor survival in patients with luminal A ER+ breast cancer according to TCGA database. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***); statistical significance of microarray gene expression data of paired samples from GSE147271 is determined by paired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Fig. 5. ANKRD11-enhanced HDAC3 activity endows AI-resistance to breast cancer.

a Western blot showed a decreased acetylation of H3K9, a HDAC3 specific target, in LTED cells. b Inhibiting ANKRD11 by siRNA significantly suppressed H3K9 acetylation without changing HDAC3 expression in LTED cells. H3K9Ac and GAPDH were separated and blotted from one gel, Histone H3 and HDAC3 were separated and blotted from another gel, and ANKRD11 were separated individually from one gel. c Immunoprecipitation confirmed the physical interaction between ANKRD11 and HDAC3 in MCF-7 LTED. IP immunoprecipitation, IgG immunoglobulin G. RGFP966 showed much higher anti-proliferation effect in (d) MCF-7 LTED and (e) T47D LTED cells than their parent cells. f More specific inhibition of HDAC3 using siRNA showed similar results as RGFP966. RGFP966 re-sensitized SERPINA3-knockdown- (g) MCF-7 and (h) T47D cells to estrogen-deprivation. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Fig. 6. Schematic illustration of ERα-SERPINA3-ANKRD11-HDAC3 pathway.

Schematic showing the proposed mechanism of anti-estrogen-therapy-induced SERPINA3 expression loss driving endocrine resistance in human ER+ breast cancer.