H19-Dependent Transcriptional Regulation of β3 and β4 Integrins Upon Estrogen and Hypoxia Favors Metastatic Potential in Prostate Cancer

Abstract

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both β3 and β4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and β integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of β integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a β integrin-mediated invasion.

Keywords: H19; biomolecular analysis; epigenetic modulators; estrogen; hypoxia; lncRNA; prostate cancer; targeted therapy; tumor metastasis.

Figure 1

Response of H19 locus transcripts to estrogen and/or hypoxia in single or combined treatment. (a) Integrated Genome Viewer (IGV 2.1) screenshot showing H19 genomic region and endothelial nitric oxide synthase (eNOS) peaks (blue boxes) identified by chromatin immunoprecipitation (ChIP)-Seq in normal human primary umbilical vein endothelial cells (HUVECs) and prostate cancer C27IM cells, before and after 17 β-estradiol (10⁻⁷ M, E₂). Red circle indicates the eNOS peak nearest to the H19 transcriptional start site. (b) H19 gene locus products, H19, miR-675, HOTS and 91H, and direction of transcription showed by green (sense) and red (antisense) arrows. Specific primers for qPCR are indicated with black arrowheads. (c) H19, primiR-675, and 91H RNA levels were assessed by quantitative RT-PCR in C27IM after 6 h treatment with estrogens (E₂, 10⁻⁷ M) and 1% O₂ hypoxia (Hyp) alone or in combination (18 h for primiR-675). Data, plotted as fold induction, represent mean ± SEM of three experiments. * p < 0.05 vs. NT; $ p < 0.05 vs. E₂; # p < 0.05 vs. Hyp.

Figure 2

Transcriptional regulation of H19 upon estrogen, chemical hypoxia, or hypoxia in single or combined treatment. (a) C27IM cells were transfected for 72 h with hypoxia inducible factor (HIF)-1α or HIF-2α expression vectors. The empty vector Puc18 (empty vector) was used as control. H19, MALAT1, and GLUT-1 levels were quantified by qPCR in presence or absence of E₂ (10⁻⁷ M; 6 h). Data represent mean ± SEM of three experiments. * p < 0.05. (b) H19, MALAT1, and GLUT1 levels were quantified by qPCR in human renal cancer cell line (786-O) after 6 h treatment with E₂ (10⁻⁷ M) and CoCl₂ (100 µM) alone or in combination. Data, plotted as fold induction, represent mean ± SEM of three experiments. * p < 0.05 vs. NT; $ p < 0.05 vs. E₂; # p < 0.05 vs. CoCl₂. (c) Recruitment on H19 promoter regions, at the eNOS-peak outlined with a red circle in Figure 1a (left) and about 3500 bp from the transcriptional start site (TSS) (right), of eNOS, ERβ, and HIF-2α by ChIPs after 2 h 15 min treatment with estrogen (E₂, 10⁻⁷ M) and 1% O₂ hypoxia (Hyp), alone or in combination, in prostate cells. No antibody (NoAb) served as the negative control. Values represent mean of three independent experiments. * p < 0.05 vs. NT; $ p < 0.05 vs. E₂; # p < 0.05 vs. Hyp.

Figure 3

Cell adhesion and invasion modulation upon single or combined treatment. (a,b) Cell–cell adhesion assays were performed in the presence (Ca²⁺-dependent) or in absence (Ca²⁺-independent) of 1mM Ca²⁺ using cells cultured for 18 h with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (HYP) alone or in combination (representative images in Figure S7a). Quantification of the in vitro cell–cell adhesion assay in the presence (a) or in absence (b) of 1mM Ca²⁺ is plotted as percentage of occupied area by cell aggregates. Data represent mean ± SEM of three independent experiments. (c) E-cadherin mRNA level was assessed by quantitative RT-PCR in C27IM after 18 h treatment with E₂ (10⁻⁷ M), 1% O₂ (HYP), alone or in combination. Data, plotted as fold induction, represent mean ± SEM of three experiments. (d) ITGB3, ITGB4, ITGA2, and RUNX2 mRNA level was assessed by quantitative RT-PCR in C27IM after 18 h treatment with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (Hyp) alone or in combination (48 h for ITGB3). Data, plotted as fold induction, represent mean ± SEM of three experiments. (e) Protein level analysis of β3 and β4 integrins in C27IM after 18 h treatment with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (HYP), alone or in combination (48 h for β3 integrin), performed by Western blot. Tubulin or β Actin served as control. Molecular weight marker is indicated. Upper panels: representative experiments. Lower panels: densitometric analysis, reported as fold induction vs. NT. (f) After a pre-treatment of 18 h under normoxia (NT) or estrogen (E₂, 10⁻⁷ M) plus 1% O₂ hypoxia (HYP+E2), cells were plated on laminin-enriched matrix for 20 min. The results are expressed as percentage of adherent cells on laminin-enriched matrix. Data represent mean ± SEM of three independent experiments. (g) Cell invasion was examined by Boyden chamber after a pre-treatment of 48 h under normoxia (NT) or estrogen (E₂, 10⁻⁷ M) and hypoxia (100 µM CoCl₂) alone or in combination. Left panel: representative phase contrast microscopic images under 20× magnification (bright field) of invading cells. Right panel: number of invading cells was presented, as fold induction vs. NT, as mean ± SEM of three independent experiments. * p < 0.05 vs. NT, $ p < 0.05 vs. E₂, # p < 0.05 vs. HYP or CoCl₂.

Figure 3

Cell adhesion and invasion modulation upon single or combined treatment. (a,b) Cell–cell adhesion assays were performed in the presence (Ca²⁺-dependent) or in absence (Ca²⁺-independent) of 1mM Ca²⁺ using cells cultured for 18 h with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (HYP) alone or in combination (representative images in Figure S7a). Quantification of the in vitro cell–cell adhesion assay in the presence (a) or in absence (b) of 1mM Ca²⁺ is plotted as percentage of occupied area by cell aggregates. Data represent mean ± SEM of three independent experiments. (c) E-cadherin mRNA level was assessed by quantitative RT-PCR in C27IM after 18 h treatment with E₂ (10⁻⁷ M), 1% O₂ (HYP), alone or in combination. Data, plotted as fold induction, represent mean ± SEM of three experiments. (d) ITGB3, ITGB4, ITGA2, and RUNX2 mRNA level was assessed by quantitative RT-PCR in C27IM after 18 h treatment with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (Hyp) alone or in combination (48 h for ITGB3). Data, plotted as fold induction, represent mean ± SEM of three experiments. (e) Protein level analysis of β3 and β4 integrins in C27IM after 18 h treatment with E₂ (10⁻⁷ M) or 1% O₂ hypoxia (HYP), alone or in combination (48 h for β3 integrin), performed by Western blot. Tubulin or β Actin served as control. Molecular weight marker is indicated. Upper panels: representative experiments. Lower panels: densitometric analysis, reported as fold induction vs. NT. (f) After a pre-treatment of 18 h under normoxia (NT) or estrogen (E₂, 10⁻⁷ M) plus 1% O₂ hypoxia (HYP+E2), cells were plated on laminin-enriched matrix for 20 min. The results are expressed as percentage of adherent cells on laminin-enriched matrix. Data represent mean ± SEM of three independent experiments. (g) Cell invasion was examined by Boyden chamber after a pre-treatment of 48 h under normoxia (NT) or estrogen (E₂, 10⁻⁷ M) and hypoxia (100 µM CoCl₂) alone or in combination. Left panel: representative phase contrast microscopic images under 20× magnification (bright field) of invading cells. Right panel: number of invading cells was presented, as fold induction vs. NT, as mean ± SEM of three independent experiments. * p < 0.05 vs. NT, $ p < 0.05 vs. E₂, # p < 0.05 vs. HYP or CoCl₂.

Figure 4

H19 mediates transcriptional repression of cell-adhesion molecules. (a–c) H19 (a); E-cadherin (CDH1) (b); ITGB3, ITGB4, ITGA2, and RUNX2 (c) mRNA levels quantified by qPCR in C27IM transfected with siRNA specific to H19 (siRNA H19) or scramble. Data, plotted as fold induction siH19 vs. scramble, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble. (d) Protein analysis of E-cadherin, β3, and β4 integrin before and after siRNA H19 by Western blot. Tubulin or β Actin served as control. Molecular weight marker is indicated. Upper panels: representative experiments. Lower panel: densitometric analysis, reported as fold induction vs. scramble, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble. (e) CDH1, ITGB3, ITGB4, and RUNX2 mRNA levels quantified by qPCR in C27IM transfected with siRNA specific to H19 (siH19) or scramble for 48 h and treated for additional 18 h with E₂ (10⁻⁷ M), 1% O₂ (HYP), alone or in combination. Data, plotted as fold induction vs. scramble NT, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble NT, ^ p < 0.05 vs. scramble.

Figure 5

EZH2 recruitment and epigenetic modifications onto promoter of cell-adhesion molecules upon combined treatment or H19 silencing. (a–c) Recruitment of EZH2 and H3K27me3 level on the promoter region of CDH1 (a), ITGB3 (b), and ITGB4 (c) by ChIPs after 18 h treatment with estrogen (E₂, 10⁻⁷ M), 1% O₂ hypoxia, alone or in combination in PCa cells (C27IM). No antibody (NoAb) served as negative control. Values represent mean ± SEM of three independent experiments. * p < 0.05 vs. NT, $ p < 0.05 vs. E₂, # p < 0.05 vs. Hyp. (d) In vivo H19 interaction with polycomb subunit EZH2 before and after Hyp+E2 (18 h) detected by RNA-ChIP assays. IgG was used as control. Immunoprecipitated RNA was recovered and analyzed by qRT-PCR. The results are expressed as mean ± SEM of three independent experiments. * p < 0.05 EZH2 vs. IgG, $ p < 0.05 Hyp+E2 vs. NT. (e) Recruitment of EZH2 and H3K27me3 level on the promoter region of CDH1, ITGB3, ITGB4, and RUNX2 by ChIPs upon H19 silencing (siH19) compared with control (scamble, scr) in PCa cells (C27IM). Non-specific immunoglobulin (IgG) served as negative control. Values represent mean ± SEM of three independent experiments. * p < 0.05 vs. scramble.

Figure 6

H19 and integrin modulation on ex vivo PCa-derived organotypic slice cultures (OSCs) upon single or combined treatment. (a) H19 levels were assessed by qPCR in OSCs from five different PCa patients (OSC A–E) after 6 h treatment with E₂ (10⁻⁷ M) and CoCl₂ (300 µM), alone or in combination. Data are plotted as fold induction vs. NT. (b) ITGB3 and ITGB4 levels were assessed by qPCR in OSCs from three different PCa patients after 48 h treatment with E₂ (10⁻⁷ M) and CoCl₂ (300 µM), alone or in combination. Data are plotted as fold induction vs. NT. * p < 0.05 vs. NT, $ p < 0.05 vs. E₂, # p < 0.05 vs. CoCl₂.

Figure 7

Effects of epigenetic drugs on H19/integrin pathway. (a) ITGB3 (left) and ITGB4 (right) mRNA levels were assessed by qPCR in C27IM after 18 h treatment with estrogen (E₂; 10⁻⁷ M), 1% O₂ hypoxia (HYP), alone or in combination, in the presence or absence of inhibitor specific to EZH2 (GSK-126, 1 µM) or JMJD3 (GSK-J4, 1 µM) added 30 min before E₂ and/or HYP. Data, plotted as fold induction vs. Ctr/NT, represent mean ± SEM of three experiments. *p < 0.05 vs. NT, $ p < 0.05 vs. E₂, # p < 0.05 vs. HYP, § p < 0.05 vs. HYP+ E₂. (b) H3K27me3 level on the promoter region of ITGB3 and ITGB4 by ChIPs in C27IM cells treated as in panel a. Data represent mean ± SEM of three experiments. IgG served as negative control. * p < 0.05 vs. NT, # p < 0.05 vs. Hyp. (c) Cell invasion was examined by Boyden chamber after a pre-treatment of 48 h under normoxia (NT) or combination of estrogen (E₂, 10⁻⁷ M) and hypoxia (100 µM CoCl₂), in the presence or absence of EZH2 or JMJD3 inhibitor added as in (a). Vehicle (DMSO) was used as control. Left panel: representative phase contrast microscopic images under 20× magnification (bright field) of invading cells. Right panel: number of invading cells, presented as fold induction vs. NT DMSO, was mean ± SEM of three independent experiments. * p < 0.05 vs. NT DMSO, # p < 0.05 vs. CoCl₂ + E₂ DMSO.

Figure 8

Cartoon of H19-dependent metastasis dissemination under combined treatment. Cartoon illustrating different mechanisms of metastasis dissemination, in which H19 is involved in aggressive prostate cancer under single or combined estrogen plus hypoxia stimuli.