The histone demethylase JMJD2B is regulated by estrogen receptor alpha and hypoxia, and is a key mediator of estrogen induced growth

Abstract

Estrogen receptor alpha (ERalpha) plays an important role in breast cancer. Upregulation of HIF-1alpha in ER(alpha)-positive cancers suggests that HIF-1alpha may cooperate with ERalpha to promote breast cancer progression and consequently affect breast cancer treatment. Here, we show the histone demethylase JMJD2B is regulated by both ERalpha and HIF-1alpha, drives breast cancer cell proliferation in normoxia and hypoxia, and epigenetically regulates the expression of cell cycle genes such as CCND1, CCNA1, and WEE1. We also show that JMJD2B and the hypoxia marker CA9 together stratify a subclass of breast cancer patients and predict a worse outcome of these breast cancers. Our findings provide a biological rationale to support the therapeutic targeting of histone demethylases in breast cancer patients.

Figure 1. ERα-dependent regulation of JMJD2B

A, Quantitative RT-PCR (top panel) and immunoblotting (bottom panel) show JMJD2B mRNA and protein expression, respectively, in T47D and MDA-MB-231 cells. B, After 3 days hormonal starvation, T47D cells were treated with 10 nM of E2 for 16 hours. Quantitative RT-PCR was performed to assess the expression of the indicated genes (top panel). Immunoblotting shows JMJD2B expression from above treatment (bottom panel) C, T47D cells in normal medium containing serum and phenol red i.e. estrogen present, were treated with 1 μM of ICI182780 for 16 hours. Quantitative RT-PCR was performed to assess the expression of the indicated genes. D, T47D and MCF7 cells were transfected with smartpool siRNA oligos against ERα. Immunoblotting was used to assess the JMJD2B expression. (*P<0.05; **P<0.01)

Figure 2. ERα is critical for JMJD2B induction in hypoxia

A, T47D cells were treated with 10 nM of E2 in normoxia and hypoxia for 16 hours. Immunoblotting was used to assess the JMJD2B expression. B, T47D cells were treated with 1 μM of ICI182780 in hypoxia for 16 hours. Immunoblotting was used to assess the proteins with indicated antibodies. C, T47D cells were transfected with siRNA oligos against ERα or HIF-1α. After 36 hours transfection, cells were incubated in normoxia and hypoxia for 16 hours. Immunoblot was used to assess the JMJD2B expression. D, MCF7 cells were treated the same as in (C).

Figure 3. JMJD2B regulates multiple biological processes and signaling pathways including cell cycle

A, After 48 hour depletion of JMJD2B in MCF7 cells, global gene expression profiling reveals that JMJD2B can both positively (top) and negatively (bottom) regulates gene expression in normoxia and hypoxia. B, Gene ontology analysis with Panther program shows that genes regulated by JMJD2B are involved in multiple biological processes and signaling pathways. C, Quantitative RT-PCR was performed to validate gene expression regulated by JMJD2B. (**P<0.01)

Figure 4. JMJD2B is essential for breast cancer cell proliferation

A, After 48 hour depletion of JMJD2B, the MCF7 cell proliferation was measured by cell number counting. B, Colony formation assay was used to assess cell proliferation after depletion of JMJD2B in MCF7 cells (left) and was quantified (right). C, Cell cycle profile analysis after JMJD2B depletion in normoxia and hypoxia. D, Percentage analysis of DNA histogram data (C) were derived from mathematical analysis of (C) with software called Summit Version 4.3 by gating for single-cell population with FS/Pulse Width, and analyse with “counts vs area of laser”. (**P<0.01)

Figure 5. JMJD2B is functional in hypoxia to exert its histone demethylase activity

A, Immunofluoresence shows a global loss of H3K9me3 marks in normoxia (N) and hypoxia (H) after overexpression of HA-tagged JMJD2B in MCF7 cells. B, Subcellular localization experiment demonstrates that JMJD2B is a nuclear protein in normoxia and hypoxia in MCF7 cells. (cyto =cytoplasm; Nuc = nucleus). C, JMJD2B was depleted in MCF7 cells using siRNA knockdown. The indicated histone methyl marks were analyzed by immunoblot. D, ChIP-PCR shows that JMJD2B regulates H3K9me3 mark on promoters of its target genes in normoxia and hypoxia in MCF7 cells.

Figure 6. Clinical assessment of JMJD2B

A, Immunohistochemistry shows JMJD2B expression in ER-positive and ER-negative breast cancer B, Immunohistochemistry shows JMJD2B and CA9 expression in a serial sections C, Kaplan-Meier analysis of relapse-free survival of CA9+ breast cancer with or without JMJD2B expression. D, Kaplan-Meier analysis of overall survival of CA9+ breast cancer with or without JMJD2B expression.