The Jumonji Domain-Containing Histone Demethylase Homolog 1D/lysine Demethylase 7A (JHDM1D/KDM7A) Is an Epigenetic Activator of RHOJ Transcription in Breast Cancer Cells

Abstract

The small GTPase RHOJ is a key regulator of breast cancer metastasis by promoting cell migration and invasion. The prometastatic stimulus TGF-β activates RHOJ transcription via megakaryocytic leukemia 1 (MKL1). The underlying epigenetic mechanism is not clear. Here, we report that MKL1 deficiency led to disrupted assembly of the RNA polymerase II preinitiation complex on the RHOJ promoter in breast cancer cells. This could be partially explained by histone H3K9/H3K27 methylation status. Further analysis confirmed that the H3K9/H3K27 dual demethylase JHDM1D/KDM7A was essential for TGF-β-induced RHOJ transcription in breast cancer cells. MKL1 interacted with and recruited KDM7A to the RHOJ promoter to cooperatively activate RHOJ transcription. KDM7A knockdown attenuated migration and invasion of breast cancer cells in vitro and mitigated the growth and metastasis of breast cancer cells in nude mice. KDM7A expression level, either singularly or in combination with that of RHOJ, could be used to predict prognosis in breast cancer patients. Of interest, KDM7A appeared to be a direct transcriptional target of TGF-β signaling. A SMAD2/SMAD4 complex bound to the KDM7A promoter and mediated TGF-β-induced KDM7A transcription. In conclusion, our data unveil a novel epigenetic mechanism whereby TGF-β regulates the transcription of the prometastatic small GTPase RHOJ. Screening for small-molecule inhibitors of KDM7A may yield effective therapeutic solutions to treat malignant breast cancers.

Keywords: breast cancer cell; epigenetics; histone demethylase; histone methylation; transcriptional regulation.

FIGURE 1

MKL1 deficiency impedes the assembly of the preinitiation complex. (A–D) MCF7 (left panel) and T47D (right panel) cells were transfected with indicated siRNAs followed by treatment with TGF-β for 48 h. RHOJ expression was examined by qPCR (A). ChIP assays were performed with anti-RNA polymerase II (B), anti-TBP (C), and anti-TFIID (D). All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test. MKL1 depletion attenuated RHOJ expression and dampened the recruitment of RNA polymerase II, TBP, and TFIID to the RHOJ promoter.

FIGURE 2

H3K9 dimethylation and H3K27 dimethylation explain differential RHOJ expression. (A–D) MCF7 (left panel) and T47D (left panel) cells were transfected with indicated siRNAs followed by treatment with TGF-β for 48 h. ChIP assays were performed with anti-H3Ac (A), anti-H3K4Me3 (B), anti-H3K9Me2 (C), and anti-H3K27Me2 (D). All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test. MKL1 depletion partially restored H3K9/H3K27 dimethylation without altering H3 acetylation or H3K4 trimethylation on the RHOJ promoter.

FIGURE 3

JHDM1D/KDM7A mediates TGF-β-induced RHOJ transcription. (A–H) MCF7 (left panel) and T47D cells (left panel) were transfected with indicated siRNAs followed by treatment with TGF-β for 48 h. Knockdown efficiencies were validated by Western blotting (A). RHOJ expression was examined by qPCR (B) and Western blotting (C). ChIP assays were performed with anti-H3K9Me2 (D), anti-H3K27Me2 (E), anti-RNA polymerase II (F), anti-TBP (G), and anti-TFIID (H). All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test. KDM7A mediated RHOJ trans-activation by removing H3K9/H3K27 methylation to facilitate the recruitment of RNA polymerase II, TBP, and TFIID to the RHOJ promoter. (I) Expression data were extracted from the public database to draw the scatter plot. Pearson correlation coefficient was calculated. Positive correlation between KDM7A expression and RHOJ expression was identified in human breast cancer tissues.

FIGURE 4

MKL1 recruits JHDM1D/KDM7A to the RHOJ promoter. (A) FLAG-tagged KDM7A and Myc-tagged MKL1 were transfected into HEK293 cells. Immunoprecipitation was performed with anti-Myc or IgG. (B) MCF7 whole-cell lysates were immunoprecipitated with anti-MKL1 or IgG. (C) MCF7 (left panel) and T47D cells (left panel) were treated with or without TGF-β for 48 h. Re-ChIP was performed with indicated antibodies. (D) MCF7 (left panel) and T47D cells (left panel) were transfected with indicated siRNAs followed by treatment with TGF-β for 48 h. ChIP assay was performed with anti-KDM7A. (E) MCF7 (left panel) and T47D cells (left panel) were treated with TGF-β in the presence or absence of CCG-1423 (10 μM) for 48 h. ChIP assay was performed with anti-KDM7A. All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test. MKL1 interacted with KDM7A and depletion/inhibition of MKL1 reduced KDM7A recruitment to the RHOJ promoter.

FIGURE 5

KDM7A promotes breast cell migration and invasion in vitro and in vivo. (A,B) MCF7 cells (A) and T47D cells (B) were transfected with indicated siRNAs followed by TGF-β treatment for 48 h. When the cells reached confluence, scratch wound was created by using a sterile micropipette tip. Photos were taken at 0 and 24 h after wound creation. The changes in side-to-side wound area were measured by Image-Pro. Data were expressed as % migration compared with control arbitrarily set as 100%. (C,D) MCF7 cells (C) and T47D cells (D) were transfected with indicated siRNAs. Cell invasion was examined by transwell assay. All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. (E,F) Heterotopic xenograft assay was performed as described in the section “Materials and Methods.” N = 10 mice for each group. (G,H) In vivo metastasis was performed as described in the section “Materials and Methods.” N = 8 mice for each group. Data represent mean ± SD. *p < 0.05, two-tailed t-test. KDM7A depletion attenuated migration/invasion of breast cancer cells in vitro and partially blockaded metastasis of breast cancer cells in vivo. (I) Kaplan–Meier plot of survival in breast cancer patients with high and low KDM7A expression either singularly or in combination of RHOJ expression. Breast cancer patients with higher KDM7A/RHOJ expression displayed poorer survival.

FIGURE 6

JHDM1D/KDM7A is transcriptionally activated by TGF-β in breast cancer cells. (A,B) MCF7 (left panel) and T47D cells (left panel) were treated with TGF-β and harvested at indicated time points. KDM7A expression was examined by qPCR and Western blotting. (C) Human KDM7A promoter constructs were transfected into MCF7 cells followed by treatment with TGF-β. Luciferase activities were normalized by protein concentration and GFP fluorescence. Data are expressed relative luciferase activities compared with the control group. (D) Wild-type and mutant human KDM7A promoter constructs were transfected into MCF7 cells followed by treatment with TGF-β. Luciferase activities were normalized by protein concentration and GFP fluorescence. Data are expressed as relative luciferase activities compared with the control group. All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test.

FIGURE 7

SMAD2/SAMD4 complex mediates transcriptional activation of JHDM1D/KDM7A in breast cancer cells. (A) MCF7 (left panel) and T47D cells (left panel) were treated with TGF-β and harvested at indicated time points. KDM7A expression was examined by qPCR and Western blotting. ChIP assay was performed with anti-SMAD2, anti-SMAD3, anti-SMAD4, or IgG. (B) MCF7 (left panel) and T47D cells (left panel) were treated with TGF-β and harvested at indicated time points. KDM7A expression was examined by qPCR and Western blotting. Re-ChIP assay was performed with indicated antibodies. (C,D) MCF7 (left panel) and T47D cells (left panel) were transfected with siRNAs targeting SMAD or scrambled siRNA followed by treatment with TGF-β. KDM7A expression was examined by qPCR and Western blotting. All experiments were performed in triplicate wells and repeated three times, and one representative experiment is shown. Data represent mean ± SD. *p < 0.05, two-tailed t-test. (E) Expression data were extracted from the public database to draw the scatter plot. Pearson correlation coefficient was calculated. Positive correlation between KDM7A expression and SMAD2/3/4 expression was identified in human breast cancer tissues.