Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 30;114(22):E4482-E4491.
doi: 10.1073/pnas.1620993114. Epub 2017 May 15.

Embryonic Transcription Factor SOX9 Drives Breast Cancer Endocrine Resistance

Affiliations
Free PMC article

Embryonic Transcription Factor SOX9 Drives Breast Cancer Endocrine Resistance

Rinath Jeselsohn et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2-ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

Keywords: SOX9; breast cancer; cistrome; endocrine resistance; estrogen receptor.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TAMR cell proliferation is ERα-dependent and increased migratory capacity with the acquisition of TAM resistance. (A) Cell proliferation analyzed by cell counting on days 1, 3, and 5 after down-regulation of ERα with siRNA in TAMR and PAR (parental) cells. As controls, TAMR and PAR cells were transfected with a siCON, and ERα down-regulation was confirmed by Western blot. Western blot also confirms ERα expression in TAMR. B-actin was used as a loading control. (B) Western blot for ERα and cell proliferation curves after treatment of TAMR cells with fulvestrant (FULV) 10−7 M and 10−6 M or vehicle (Veh) control. (C) Brightfield microscopy picture of 2D culture of PAR and TAMR cells. Magnification, 20×. Shown are the radius assay and Boyden chamber assay testing PAR cell migration in FM (full medium) and TAMR in hormone-depleted white medium. *P = 0.02; **P = 0.003. Error bars denote SEM; figure represents the results of three replicates. (D) Volcano plot of the RNA-seq differential gene expression between PAR and TAMR cells using DE-seq2, log twofold change >1 or <−1, and P <0.05. The red dots are the genes that are significantly up-regulated in TAMR cells, and blue dots are the genes down-regulated. Functional annotation of genes up-regulated in TAMR cells. Top overrepresented gene categories from GO using DAVID are shown here. P values are represented by a red line and % of genes by blue bars.
Fig. S1.
Fig. S1.
TAMR proliferation counted on day 1, 3, and 5 in WM (white medium) in the presence (TAM) or absence of TAM (No TAM).
Fig. S2.
Fig. S2.
Genomic DNA samples of MCF7 cell lines were screened for ESR1 E380Q, Y537C, Y537N, Y537S, and D538G mutations by droplet digital PCR in PAR (parental), LTED (long-term estrogen deprived), and TAMR cells. Mutant plasmid DNA was serially diluted into ESR1 WT plasmid DNA (1,000, 250, and 63 mutant copies into 10,000 WT copies per reaction) as the positive control.
Fig. 2.
Fig. 2.
Redistribution of ERα chromatin binding with the acquisition of TAM resistance. (A) Venn diagram showing the overlap of ERα binding in TAMR and LTED cells without E2 stimulation and PAR (parental cells) with E2 stimulation. (B) Top motifs enriched and lost in the TAMR cells compared with parental cells. (C) Heatmap showing clustered ERα binding signals enriched in RUNX and GATA in TAMR and parental cells. The windows represent ±1-kb regions from the center of the ERα binding events. The color scale shows relative enrichment based on raw signal. (D) Relative mRNA levels of the RUNX transcription factors determined by RT-PCR calculated by ΔΔCT in parental, LTED, and TAMR cells. (E) Immunoblots for RUNX2 and GATA3 using cell lysates of PAR, LTED, and TAMR cells.
Fig. S3.
Fig. S3.
(A) Heat maps of RUNX1 and RUNX2 in TAMR and PAR cells (MDAMB415 and 600MPE) determined by RNA-seq. (B) Immunoblot for RUNX1 in PAR (parental), LTED (long-term estrogen deprived), and TAMR cells.
Fig. 3.
Fig. 3.
The ERα binding sites unique to TAMR cells and enriched in RUNX2 motifs are associated with poor outcomes in breast cancer, and endogenous RUNX2 and ERα interact to mediate transcriptional changes that promote EMT. (A) Ranked GSEA of the genes determined to be up-regulated by ERα–RUNX2 in TAMR cells based on BETA analysis. NES, normalized enrichment score; q < 0.25. Oncomine Concepts Map analysis (Compendia Bioscience) was used to compare the ER–RUNX2 gene signature in TAMR cells against published gene signatures from primary breast cancers. This analysis showed significant correlations between the ER–RUNX2-induced genes and gene expression signatures of poor outcome. The association between the molecular concepts of different gene signatures is represented as a graph using Cytoscape (www.cytoscape.org). A node represents a gene set, and significantly associated (q < 0.2) sets were connected by an edge. The node of the ER–RUNX2 gene set is in red, and the nodes of poor outcomes are in blue. The size of the nodes is proportional to the number of overlapping genes between the corresponding gene set and the ERα–RUNX2 gene set, and the thickness of the edges that connect between the nodes is proportional to the rank of the association significance. (B) Immunoprecipitation of endogenous RUNX2 using nuclear extracts and immunoblotting for endogenous ERα with IgG and input controls. FM, full medium; PAR, parental; TAMR, tamoxifen resistant; WM, white medium. Normalized protein quantification was done using ImageJ (imageJ.nih.gov). (C) RUNX2 RIME results in TAMR cells depicted in a word cloud. (D) Western blot for RUNX2 and HA confirming stable DOX-inducible expression of HA-tagged RUNX2 in MCF7 parental cells after treatment with DOX. EV, empty vector. (E) Heat map of a K-means 2 clustering of the top 1,000 genes differentially expressed between the DOX-inducible RUNX2 MCF7 cells with DOX treatment (pInd-RUNX2_DOX) or no DOX treatment (pInd-RUNX2). R1–R3, replicates 1–3. (Left) An enrichment plot from GSEA showing the Hallmark EMT gene set, which is the gene set most significantly enriched after the induction of RUNX2 expression. (F) Results of a Boyden chamber assay comparing migration and invasion in MCF7 cells with and without DOX induction of RUNX2. *P < 0.05. Error bars represent the SEM of three replicates.
Fig. S4.
Fig. S4.
(A) Immunofluorescence staining of cells isolated from a malignant pleural effusion. Staining is for epcam and DAPI (Left) and ERα and DAPI (Right). Magnification, 20×. Venn diagram showing the overlap between the ER–RUNX2 binding sites enriched in the TAMR cells and the pleural effusion metastatic cells in ER binding sites with a RUNX motif within ±1 kb from the summit. (B) Comparison of overlap between the ERα–RUNX2 binding sites in TAMR cells with published ERα binding in ER+ breast cancer tissue samples including primary breast cancers of good prognosis and poor prognosis and metastatic samples. Unpaired Student’s t test was used to calculate the P values. *P < 0.05.
Fig. S5.
Fig. S5.
(A) Heat map of unsupervised analysis of RNA-seq from control MCF7 parental cells (EV, empty vector) and MCF7 parental cells stably overexpressing SOX9. (B) Graph depicting the genes up-regulated in the JAK–STAT pathway with up-regulation of SOX9 in MCF7 cells. Graph was rendered by Pathview (82).
Fig. 4.
Fig. 4.
The RUNX2 cistrome highly overlaps with the ER–RUNX2 cistrome. (A) Venn diagram (Left) depicts the overlap between the ERα binding sites with a RUNX2 motif in TAMR cells and RUNX2 binding sites in MCF7 cells with DOX-inducible expression of RUNX2. Venn diagram (Right) showing the overlap between the genes up-regulated by the ERα–RUNX2 complex in TAMR cells and genes up-regulated by RUNX2 in MCF7. Both gene sets were determined by integrating ChIP-seq and RNA-seq applying BETA (45). (B) Relative mRNA levels of genes regulated by the ERα–RUNX2 complex after transfection of TAMR cells with either siERα, siRUNX2, or siControl (siCON) and extraction of RNA on day 3 after transfection. Shown here are the relative mRNA levels after siERα knockdown or siRUNX2 knockdown compared with siControl. (C) Cell proliferation analyzed by cell counting on days 1, 3, and 5 after down-regulation of RUNX2 with siRNA. (D) Cell proliferation analyzed by cell counting on days 1, 3, and 5 after down-regulation of RUNX1 with siRUNX1 in TAMR cells. As controls, TAMR cells were transfected with a siCON. (E) Relative mRNA levels of RUNX2 (Left) and RUNX1 (Right) in control MCF7 cells and after down-regulation of RUNX1 and RUNX2.
Fig. 5.
Fig. 5.
SOX9 is up-regulated in TAMR and mediates resistance to estrogen deprivation and TAM. (A) Western blot for SOX9 in parental (PAR) and TAMR MCF7 cells (Left) and in MCF7 cells infected with an EV control and a DOX-inducible RUNX2 expression vector with and without DOX treatment. (B) Heat maps of mRNA levels of ESR1, RUNX2, and SOX9 in parental (PAR) and TAMR cell lines derived from 600MPE, MDAMB415, and T47D cell lines. (C) Cell proliferation analyzed by cell counting on days 1, 3, and 5 in TAMR cells after SOX9 silencing with two different siRNAs or with an siRNA control (si-CON). Western blot for SOX9 in TAMR cells showing successful knockdown of SOX9. (D, Top) Western blot of SOX9 in parental MCF7 cells after stable expression of SOX9 and an EV. Western blot shows stable ERα expression after expression of SOX9 in MCF7 parental cells. Cell proliferation analyzed by cell counting on days 1, 3, and 5 in MCF7 parental cells after SOX9 expression or an EV control in (Top Left) full medium or (Top Right) white medium. Dose–response curves for TAM treatment in MCF7 cells expressing SOX9 and an EV as control. TAM IC50 in SOX9-overexpressing cells is 1.4 × 10−9 M and in control cells expressing an EV 6 × 10−10 M.
Fig. 6.
Fig. 6.
TAM resistance in clinical samples is associated with SOX9 expression. (A) Representative figures of immunohistochemistry staining for SOX9 showing an increase in nuclear staining in the recurrent TAMR samples. Magnification, 40×. (B) SOX9 expression in ER+ primary and recurrent breast cancer samples. The scatter plot shows that in a number of cases there was a decrease in SOX9 expression after the development of TAM resistance, but in the majority of the cases (67%) there was an increase and overall there was a significant increase. (C) SOX9 expression levels in the recurrent tumors that remained ER+ and the recurrent tumors that changed to ER– and their matched primary tumors. (D) Scheme of the ERα–RUNX2 model in TAM resistance.

Similar articles

See all similar articles

Cited by 24 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback