Estrogen receptor α-NOTCH1 axis enhances basal stem-like cells and epithelial-mesenchymal transition phenotypes in prostate cancer

Abstract

Background: Prostate cancer (PCa) is the second leading cause of mortality and a leading cause of malignant tumors in males. Prostate cancer stem cells (PCSCs) are likely the responsible cell types for cancer initiation, clinical treatment failure, tumor relapse, and metastasis. Estrogen receptor alpha (ERα) is mainly expressed in the basal layer cells of the normal prostate gland and has key roles in coordinating stem cells to control prostate organ development. Here, we investigated the roles of the estrogen-ERα signaling pathway in regulating PCSCs.

Methods: Correlation of CD49f and ERα/NOTCH1 was analyzed in human clinical datasets and tissue samples. Flow cytometry was used to sort CD49f^Hi and CD49f^Low cells. EZH2 recruitment by ERα and facilitation of ERα binding to the NOTCH1 promoter was validated by Co-IP and ChIP. Primary tumor growth, tumor metastasis and sensitivity to 17β-estradiol (E2) inhibitor (tamoxifen) were evaluated in castrated mice.

Results: ERα expression was significantly higher in CD49f^Hi prostate cancer basal stem-like cells (PCBSLCs), which showed basal and EMT features with susceptibility to E2 treatment. ERα-induced estrogen effects were suggested to drive the NOTCH1 signaling pathway activity via binding to the NOTCH1 promoter. Moreover, EZH2 was recruited by ERα and acted as a cofactor to assist ERα-induced estrogen effects in regulating NOTCH1 in PCa. In vivo, E2 promoted tumor formation and metastasis, which were inhibited by tamoxifen.

Conclusions: Our results implicated CD49f+/ERα + prostate cancer cells associated with basal stem-like and EMT features, named EMT-PCBSLCs, in heightened potential for promoting metastasis. NOTCH1 was regulated by E2 in CD49f^Hi EMT-PCBSLCs. These results contribute to insights into the metastatic mechanisms of EMT-PCBSLCs in PCa.

Keywords: CD49f; EMT; ERα; Estrogen; NOTCH1; Prostate cancer.

Fig. 1

The expression of CD49f and ERα. a, The correlation of CD49f and ERα in normal prostate and PRAD patients from TCGA (num (Normal) = 52), num (PRAD) = 498)) and GTEx (num (Normal) = 100) datasets. b, Heat map analysis of the differentially expressed genes in PRAD patients. The TCGA consortium devised a subclassification of prostate cancers (num(N) = 497) into four distinct groups (1–4) based upon mRNA-Seq clustering and z-score analysis in Morpheus with a P ≤ 0.05 criterion, Rows: samples; columns: the indicated genes. c, IHC staining of CD49f and ERα in normal prostate tissues and cancer tissues with different Gleason score. Scale bar, 200 μm. d, Flow cytometry analysis of the expression of CD49f in LNCaP-abl and PC3 treated with either DMSO or 10 nM E2 for 72 h (n = 3). e, Analysis of enriching stem cell spheres treated with E2 for 2 weeks. Scale bar, 100 μm. f, Sphere-forming and diameter analysis of e. g, Flow cytometry sorting of CD49f^Hi and CD49f^Low cells in LNCaP-abl and PC3, qRT-PCR analysis showing expression changes of the indicated genes. The data are presented as the means±SD (n = 3). *, P < 0.05 vs. CD49f^Low PCBSLCs. Abbreviate: PRAD: Prostate Adenocarcinoma

Fig. 2

E2 promotes EMT in CD49f^Hi PCBSLCs. a, qRT-PCR analysis showing expression changes of the indicated genes in the sorted CD49f^Hi and CD49f^Low PCBSLCs. The data are presented as the mean ± SD (n = 3). *, P < 0.05 vs. CD49f^Low PCBSLCs. b, Flow cytometry analysis of the coexpression of CD49f and Vimentin in LNCaP-abl and PC3 cells treated with either DMSO or 10 nM E2 for 72 h (n = 3). c and d, Western blot analysis the indicated proteins in (c) LNCaP-abl or PC3 treated with DMSO or 10 nM E2 treatments for 72 h; (d) LNCaP-abl or PC3 with ERα knockdown (n = 3). e and f, qRT-PCR analysis showing expression changes of EMT markers in LNCaP-abl and enriched stem cell spheres of LNCaP-abl (PCSCs) (e), and treated with 10 nM E2 or DMSO (f). The data are presented as the means±SD (n = 3). *, P < 0.05 vs. DMSO

Fig. 3

The expression of CD49f and NOTCH1. a, Heat map analysis of the differentially expressed genes in PRAD patients from TCGA. b, Mean mRNA analysis of NOTCH1 and NOTCH4 in PRAD of TCGA. Significance was assessed using Student’s paired t-test. The scatter dot plot is presented as the median. ***, P < 0.001. c, The correlation of CD49f or ERα and NOTCH1 in normal prostate and PRAD from TCGA (num (Normal) = 52), num (PRAD) = 498)) and GTEx (num (Normal) = 100)). d, IHC staining of CD49f and NOTCH1 in normal prostate tissues and cancer tissues with different Gleason score. Scale bar, 200 μm. e, Flow cytometry analysis of the coexpression of CD49f and NOTCH1 in LNCaP-abl and PC3 treated with either DMSO or 10 nM E2 for 72 h (n = 3). f, Western blot analysis of the indicated proteins in LNCaP-abl or PC3 cells treated with 10 nM E2 treatments for 72 h (n = 3). g, qRT-PCR analysis showing expression changes of the indicated genes in LNCaP-abl treated with either DMSO or 10 nM E2 for 72 h. The data are presented as the means±SD (n = 3). *, P < 0.05 vs. siNC

Fig. 4

The mechanism of E2 regulated NOTCH1. a, Western blot analysis of the indicated proteins in LNCaP-abl or PC3 cells with ERα knockdown (n = 3). b, Western blot analysis of the indicated proteins in LNCaP-abl or PC3 with or without ERα knockdown after treatment with either DMSO or 10 nM E2 for 72 h (n = 3). c, Diagram of the EZH2 promoter regions analyzed for EREs in the ChIP assays. d-f, Anti-ERα antibodies were used for ChIP assays regarding the EREs (ERE1-ERE4) of the NOTCH1 promoter in LNCaP-abl (d), LNCaP-abl with ERα knockdown (e), and LNCaP-abl treated with DMSO or E2 for 30 min before being harvested (f). qRT-PCR amplification was performed using a series of primers targeting EREs. The data are presented as the mean ± SD (n = 3). *, P < 0.05 vs. IgG (d), siNC (e), or DMSO (f). g, Anti-EZH2 antibodies were used for ChIP assays regarding the ERE2 and ERE3 of the NOTCH1 promoter in LNCaP-abl. The data are presented as the mean ± SD (n = 3). *, P < 0.05 vs. IgG. h, Co-IP analysis of the interaction between ERα and EZH2 in LNCaP-abl or PC3 cells following treatment with either DMSO or 10 nM E2 for 72 h (n = 3). Abbreviation: abl: LNCaP-abl. i and j, ChIP-re-ChIP qPCR analysis of ERE3 of NOTCH1 promoter in LNCaP-abl (i) and ChIP qPCR analysis of LNCaP-abl with ERα or EZH2 knockdown (j). The data are plotted as the mean ± SD (n = 3). *P < 0.05 vs. IgG (i) and siNC (j). k and l, Western blot analysis of the indicated proteins in LNCaP-abl with ERα or/and EZH2 knockdown (n = 3)

Fig. 5

Tamoxifen inhibits the growth and metastasis of prostate tumors in vivo. a, Schematic illustration of the experimental strategy. b, LNCaP-abl-Green cells were implanted into nude mice, with 9 castrated mice in each group. Tumor growth and metastasis were detected with an IVIS system. The data are presented as the mean ± SD. c, Changes in body weight over time (n = 3). d and e, Three primary tumors were selected randomly to be photographed with a digital camera (d), and the volume and weight of the primary tumors were measured (e). The scatter dot plot is presented as the median. *P < 0.05; **P < 0.01 vs. Vehicle+PBS. f, IF analysis of CD49f and ERα or NOTCH1 co-expression in prostate primary tumors tissues of the E2 group. g, Western blot analysis of the indicated proteins in prostate primary tumors tissues (n = 3). h, Number of surviving and metastatic mice injected with LNCaP-abl-Green cells in different groups. Abbreviation: TAM: tamoxifen

Fig. 6

The total signaling pathway. ERα-induced estrogen effects promoted the expression of NOTCH1 by binding NOTCH1 promoter at − 1364 and − 1188 bp, EZH2 was recruited by ERα and acts as a cofactor to assist ERα-induced estrogen effects in regulating NOTCH1 in PCa. The ERα-induced estrogen effects may promote CD49f^Hi EMT-PCBSLCs metastasis to other organs