Estrogen receptor-beta is a potential target for triple negative breast cancer treatment

Abstract

Triple Negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2. TNBC accounts for 15-20% of all breast cancer cases but accounts for over 50% of mortality. We propose that Estrogen receptor-beta (ERβ) and IGF2 play a significant role in the pathogenesis of TNBCs, and could be important targets for future therapy. Tissue microarrays (TMAs) from over 250 TNBC patients' were analyzed for ERβ and IGF2 expression by immunohistochemistry. Expression was correlated with clinical outcomes. In addition, TNBC cell lines Caucasians (CA): MB-231/BT549 and African Americans (AAs): MB-468/HCC70/HCC1806 were used to investigate the effect of hormonal and growth factor regulation on cell proliferation. TMAs from AAs had higher expression of ERβ and IGF2 expression when compared to CA. ERβ and IGF2 were found to be upregulated in our TNBC cell lines when compared to other cell types. TNBC cells treated with ERβ agonist displayed significant increase in cell proliferation and migration when compared to controls. AA tissue samples from TNBC patients had higher expression of ERβ. African-American breast cancer TNBC tissue samples from TNBC patients have higher expression of ERβ. In addition, TNBC cell lines were also found to express high levels of ERβ. IGF2 increased transcription of ERβ in TNBC cells. Understanding the mechanisms of IGF2/ERβ axis in TNBC tumors could provide an opportunity to target this aggressive subtype of breast cancer.

Keywords: DPN; estrogen receptor-beta; estrogen receptor-beta signaling; insulin-like growth factor 2; triple negative breast cancer.

Figure 1. ERβ is associated with decrease relapse free survival in TNBC

mRNA level of ERβ in TNBC was obtained from a public database, KMPlot. Kaplan–Meier survival analysis was used to assess the association between ERβ level and relapse-free survival (RFS). (A) RFS in all TNBC between Low ERβ and High ERβ groups, (B) RFS in TNBC underwent chemotherapy (Chemo) between Low ERβ and High ERβ groups, and (C) RFS in TNBC with adjuvant chemotherapy (adj Chemo) between Low ERβ and High ERβ groups. Log-rank test was used to assess the statistical significance between the groups and P<0.05 was considered as significance.

Figure 2. ERβ is expressed in TNBC cell lines

(A) The indicated panel of breast cancer cell were maintained in growth media with 10% FBS and protein was extracted until the cells reach to 80% confluence. Western blot analyses were performed with ERα, ERβ1 and HER2 antibodies and β-actin antibody was used for loading control (A-right). The western blot of ERβ1 was repeated 3 times and the bands intensities were adjusted to β-actin. The bar graph (A-left) showed the mean intensities from three independent bots and SD for the indicated cell lines. “^*” indicated p<0.05 compared to the intensity from MCF-7 cells and determined by one-way ANOVA. (B) The indicated TNBC cells were grown in phenol red free media with 5% charcoal treated FBS until 80% confluence and RNA was extracted. RT-qPCR was performed with ERβ1 and ERβ2 primers and adjusted to GAPDH. The bar graph presented as fold changed of ERβ1 and ERβ2 levels in the indicated TNBC cells compared to MCF-7 cells. Each bar indicated mean±SD from three experiments. (C) The indicated TNBC cells were grown in phenol red media with 5% charcoal treated FBS until 80% confluence and protein was extracted. Western blot analyses with ERα and ERβ1 antibodies were performed and β-actin was used for loading control.

Figure 3. Activation of ERβ increases migration of TNBC cells

The cells were seeded in 8.5cm3 dishes with a 2 well silicone insert and treated with or without DPN or PHTPP for 24 hrs as described in methods. The wound closure was monitored and images were taken and analyzed from three different rears for each conditions. The bar graphs for MB231 (A), MB468 (B), HCC70 (C) and HCC1806 (D) presented as percentage wound closures (mean ±SD) for each conditions. P-values between the indicated conditions were determined by one-way ANOVA.

Figure 4. Activation of ERβ increases invasion of TNBC cells

The cellswere plated in top chamber of matrigel invasion chamber in phenol red free media with or without DPN or PHTPP and bottom chamber was filled with medium containing CSFBS as described in methods. After 24 hrs number of cell invasion was assessed. The total numbers of invaded cells were counted from 4 independent areas for each conditions and all experiments were independently repeated three times. The bar graphs for MB231 (A) BT549 (B) MB468 (C) and HCC70 (D), HCC-1806 (E) showed invaded cell numbers (mean ±SD) under each conditions. P-values between the indicated conditions were determined by one-way ANOVA.

Figure 5. Activation of ERβ increases cell proliferation

The cells were treated with or without DPN, PHTPP, or DPN+PHTPP for 3 days and cell proliferation was measured by The CellTiter-Glo® Luminescent Cell Viability Assay as described in methods. Proliferation rate of different TNBC cells without treatment (A), MB231 (B), BT549 (C) MB468 (D) HCC70 (E) and HCC1806 (F) cells with and without treated with DPN, PHTPP and DPN+PHTPP were determined by compared to MCF7 cells. The bar graphs indicated the mean fold-changes with SD from 3 independent measurements and each measurements contained 6 repeated values for each condition. The p-values were determined by One-Way ANOVA (^*P < 0.01, ^**P < 0.001, ^***P < 0.0009, ^****P < 0.0001). (G) HCC70 cells were treated with DNP for the indicated time and protein was extracted. Western blot analysis was performed with p21 and p27^kip antibodies and β-actin was used as loading control.

Figure 6. Activation of ERβ upregulates IGF2 and activates the IR/IGF1R/MAPK pathways

(A) The indicated cells were treated with or without DPN. mRNA level of IGF2 was determined by RT-qPCR and adjusted for GAPDH. The bar graph presented fold changes of IGF2 level in cells treated with DPN compared to non-treated cells (control). Each bar indicated mean ±SD from 3 measurements and “^*” indicated p<0.05 determined by one-way ANOVA. (B) Total protein was extracted from the indicated cells treated with or without DPN. Western blot analysis was performed with antibodies for IGF2, insulin receptor (IR) and IGFIR. β-actin was used for loading control. (C) The cells were treated with or without DPN, PHTPP or DPN+PHTPP as descripted in methods and total protein was extracted. Protein levels of ERβ1, IGF2 and IGFIR were determined by Western Blot analysis and β-actin was used as loading control. (D) The cells were maintained in serum free media and treated with or without DPN or PHTPP. Secreted protein level of IGF2 in the media was measured by IGF2 ELISA kit according to manufacture instruction as described in methods. The bars indicated mean level of IGF2 with SD from duplicated measurements and “^*” indicated P<0.05 for the differences in level of IGF2 between the indicated cells and MCF7 cells determined by one-way ANOVA. (E) The cells were treated with DPN from 0 to 30 min and protein was extracted. Western blot analysis was performed with antibodies for P-ERK1/2, ERK1/2, P-p38, p38 and p27^kip. β-actin was used as loading control. The level of P-ERK1/2 was further quantified with β-actin and showed under membrane of P-ERK1/2. (F) T47D cells were treated with 3 different siRNA sequences of ERβ and ERβ1 expression was determined Western Blot analysis with antibody for ERβ1 was used to assess efficiency of the siRNAs. (G) The cells were treated with pooled siRNA sequences for ERβ and the level of ERβ was determined by Western blot. The protein levels of cyclin D1 (CD1) and EGFR were also determined by Western blot analysis. β-actin was used as loading control. (H) The cells were treated with or without siRNA for ERβ and cell proliferation was determined by The CellTiter-Glo® Luminescent Cell Viability Assay as described in methods. The bars indicated fold changes (mena ±SD) of cells treated with siRNA compared to cells without treating with siRNA (control). “^**” indicated P<0.001 between the indicated groups determined by one-way ANOVA.

Figure 7. Knockdown of ERβ results in increased apoptosis

TNBC cells were knockdown for ERβ and then subjected to flow cytometer as described in methods Representative images of flow cytometry data were presented. Percentage of apoptotic cells in (A) MB231, (B) BT-549, (C) MB-468, (D) HCC-70, and (E) HCC-1806 were showed in bar graphs as mean ±SD. The statistical differences between the indicated groups were determined by paired t-test.

Figure 8. ERβ is expressed in breast cancer patients' tissue microarray

(A) Immunohistochemical (IHC) analysis was used to determine ERβ1 and IGF2 expression in breast cancer primary tissues as described in methods. Represented images from IHC of H&E, ERβ1 and IGF2 in TNBC from Asian (AS), African American (AA), and Hispanic/Latina (His) patients were shown. (B and C) Bar graphs showed quantified IHC data of ERβ1 (B) and IGF2 (C) expression in different subtypes of breast cancer. The bars indicated percentage of positive staining for ERβ1 or IGF2 (mean ±SD) in the indicated subtype of breast cancers. (D) IHC data of ERβ1 was quantified and the bars indicated percentage positive expression of ERβ1 in the indicated racial ethnicities. (E) The correlation between expressing ERβ and IGF2 in patients was evaluated by Bivariate Correlations with two tailed test, performed with SPSS v.22.