
==== Front
Cancer Cell Int
Cancer Cell Int
Cancer Cell International
1475-2867
BioMed Central London

39300582
3504
10.1186/s12935-024-03504-0
Research
KDM4A promotes malignant progression of breast cancer by down-regulating BMP9 inducing consequent enhancement of glutamine metabolism
Chen Yuanxiang
Yang Shiyu
Yu Tao
Zeng Tao
Wei Lan
You Yiqing
Tang Jiafeng
Dang Tingting
Sun Haoli
Zhang Yan yanzhang@cqmu.edu.cn

https://ror.org/017z00e58 grid.203458.8 0000 0000 8653 0555 Key Laboratory of Medical Diagnostics of Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 People’s Republic of China
19 9 2024
19 9 2024
2024
24 32221 7 2024
6 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Recent studies have found that histone-modified genes play an increasingly important role in tumor progression. Lysine(K) specific demethylase 4A (KDM4A) is a histone lysine-specific demethylase highly expressed in a variety of malignant tumors, data showed that KDM4A was negatively correlated with the Bone Morphogenetic Protein 9 (BMP9) in breast cancer. And previous experiments have demonstrated that exogenous BMP9 significantly inhibits breast cancer development.

Materials and methods

We detected the expression of KDM4A in breast cancer and the relationship between KDM4A and BMP9 using real-time quantitative PCR (RT-qPCR) and Western blot, and verified the interaction between KDM4A and BMP9 by ChIP experiments. At the same time, we also detected whether KDM4A had effects on the RNA and protein stability of BMP9 using actinomycin D and cycloheximide. Measurement of alpha-ketoglutarate (α-KG) level by ELISA to observe the effect of BMP9 on glutamine metabolism in breast cancer cells. Nucleoplasmic distribution of KDM4A after exogenous BMP9 treatment in breast cancer cells were observed by immunofluorescence staining and Western blot. A subcutaneous xenograft tumor model in nude mice was used to study the therapeutic effects of exogenous BMP9 and KDM4A inhibitor (JIB-04) in breast cancer. CCK-8, conoly formation, Transwell, wound healing, and immunohistochemistry were used to monitor the growth of tumor and cell function.

Results

We found that KDM4A was abnormally highly expressed in breast cancer, and silenced BMP9 expression by removing histone methyl groups from the BMP9 gene region. Meanwhile, KDM4A could also reduce the stability of BMP9 protein. BMP9 inhibit glutamine metabolism in breast cancer, resulting in a decrease in its product α-KG, is confirmed by ELISA. Altered nucleoplasmic distribution of KDM4A due to decreased α-KG was confirmed by immunofluorescence staining and Western blot. Animal experiments confirm that the combination of exogenous BMP9 and JIB-04 shows significantly better results in breast cancer.

Conclusions

KDM4A silences BMP9 expression by removing histone methyl groups from the BMP9 gene region, leading to further enhancement of glutamine metabolism, which contributes to malignant tumor progression. In addition, using JIB-04 in combination with exogenous BMP9 could inhibit the malignant progression of breast cancer cells and the growth of tumors more significantly.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-024-03504-0.

Keywords

Breast cancer
KDM4A
BMP9
Glutamine metabolism
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81974449 81974449 81974449 81974449 81974449 81974449 81974449 81974449 81974449 81974449 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
==== Body
pmcBackground

Breast cancer (BC) is a prevalent malignant tumor that occurs in the breast tissue. According to the most recent global cancer data, the incidence of breast cancer is rising annually and has emerged as the primary cancer threat to women’s health [1]. Numerous factors, including genetic predispositions, hormone imbalances, and environmental conditions, are involved in the intricate process of breast cancer formation [2–4]. In recent years, improvements in medical technology have significantly increased the cure rate for breast cancer, but the causes of the disease are not fully understood, and treatment options are still limited [5, 6]. Therefore, the search for new effective therapeutic targets is crucial for the survival of breast cancer patients.

Histone methylation modification is a crucial epigenetic alteration. Methylation modification at various sites of different histone proteins can lead to changes in the transcriptional activity of DNA, either activating or inhibiting gene expression. For instance, methylation of histone H3 lysine 4 (H3K4), histone H3 lysine 36 (H3K36), and histone H3 lysine 79 (H3K79) can lead to activation of gene transcription [7]. Lysine(K) specific demethylase 4A (KDM4A) is a histone lysine-specific demethylase belonging to the Fe-II dependent dioxygenase family. It plays important roles in the regulation of chromatin activity, growth and development, aging, metabolism, and immunity [8]. Additionally, it can participate in the development of multiple cancers with alpha-ketoglutarate (α-KG) and molecular oxygen as cofactors [9]. However, the specific mechanism of KDM4A in breast cancer progression remains unclear.

Bone Morphogenetic Protein 9 (BMP9) is a molecular signaling factor classified as a member of the bone morphogenetic protein family. It is involved in regulating a variety of biological processes and plays an important role in embryonic development, tissue regeneration, and adult physiological processes [10–12], as well as being closely associated with tumor progression [13–15]. According to our earlier research, exogenous BMP9 not only inhibits the proliferation, migration, and invasion of human breast cancer in vitro, but it also regulates the interaction between breast cancer cells and bone marrow mesenchymal stem cells, suppresses the spread of breast cancer to the bones, and participates in autophagy, lipid metabolism and lipid utilization in breast cancer [14, 16–20]. However, BMP9 is frequently under-expressed or even de-expressed in breast cancer.

Metabolic disorders are a major feature of tumorigenesis and progression [21]. In recent decades, researchers have increasingly emphasized the role of amino acid metabolism, particularly glutamine catabolism, in carcinogenesis and the tumor microenvironment. Similar to glycolysis, cancer cells may derive energy from the breakdown of glutamine [22]. Glutamine is the most abundant amino acid in the circulation [23] and is an important metabolite that plays an important role in nucleotide, amino acid, and lipid synthesis. Through redox events mediated by glutamate aminotransferases, glutamate can produce α-KG [24]. Additionally, α-KG can function as a cofactor for a variety of chemicals, including histone demethylases, which have an impact on the progression of cancer [25]. Proliferating cancer cells rely on glutamine as a major energy source and component, this state is referred to as glutamine addiction [26].Exogenous glutamine is necessary for many tumor cells, and its absence has been shown to cause them to die [27]. Recently, a study by B.LI et al. showed that BMP9 can interact with type II glutamine aminotransferase to promote the osteogenesis of BMSCs [28], which provided a basis for the involvement of BMP9 in glutamine metabolism. Our previous studies have found that BMP9 can inhibit breast cancer progression by affecting lipid metabolism in breast cancer, etc. As an important metabolic regulator, the study of changes in glutamine metabolism mediated by BMP9 may be critical for deeper exploration of diagnostic and therapeutic targets in breast cancer.

Our analysis revealed that the promoter region of the BMP9 gene exhibited significantly lower levels of DNA methylation in breast cancer compared to normal tissue. This suggests that BMP9 expression plays a role in the control of epigenetic changes. A closer analysis revealed a trend of negative correlation between KDM4A and BMP9 expression in breast cancer. It was further confirmed that KDM4A can regulate the expression of BMP9, either depending on or independent of its histone demethylase action. Simultaneously, the expression of BMP9 has the potential to impact the altered glutamine metabolism in breast cancer, potentially leading to variations in the levels of its byproduct, α-ketoglutarate. As a coactivator of KDM4A, fluctuations in the levels of α-ketoglutarate can affect the intracellular distribution of KDM4A and modulate its function.

Materials and methods

Cell culture and reagents

The BC cell lines MCF-7, MDA-MB-231, and SK-BR-3, as well as the human immortalized normal breast epithelial cells MCF-10A, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM high glucose medium (Saimike, Chongqing, China) supplemented with 10% fetal bovine serum (FBS, Clark science, USA) and 1% penicillin/streptomycin at 37℃. The inhibitor of KDM4A (JIB-04) (HY-17559) was purchased from MCE (New Jersey, USA).

Cell transfection and infection

The KDM4A-specific siRNAs and negative control siRNA were purchased from GenePharama (Shanghai, China). The sequences are provided in Additional Table S1. The culture medium was transformed to FBS-free medium when the cell growth density reached 60%-80%. The transfection complex was then prepared and added to the culture medium according to the GP-transfect-Mate transfection reagent instruction manual. After 4–6 h, the medium was changed to one that contains FBS. The recombinant adenovirus overexpressing BMP9 (Ad-BMP9) and negative control (Ad-GFP) were infected according to the poly-transfection reagents manufacturer's instructions and replaced with fresh FBS-containing medium after 6-8 h.

Real-time quantitative PCR (RT-qPCR)

Extracting the total RNA from the cells according to the RNA-Quick Purification Kit (YiShan Biotech, Shanghai, China) and using the reverse transcription kit (TaKaRa, Shiga, Japan) to reverse RNA to cDNA. SYBR Green PCR Master Mix (TaKaRa, Shiga, Japan) was applied for RT-qPCR. Samples were standardized relative to the expression of GAPDH. The primer sequences were shown in Additional Table S2.

Western blot

Total protein was collected by lysing with RIPA lysis buffer (Beyotime, China), separated in SDS-PAGE, and transferred to a PVDF membrane. The membrane was then blocked with 5% BSA (Solarbio, China) for 2 h, followed by overnight incubation with primary antibody (Additional Table S3) at 4℃. The HRP-labeled goat anti-rabbit/mouse IgG (Zhongshan Golden Bridge Biotechnology, China, 1:5000) was added and incubated at 37℃ for 1 h the next day. The image was then developed by adding ECL chemiluminescent agent (Biosharp, China).

Conoly formation assay

SK-BR-3 or MDA-MB-231 cells treated with small interfering RNA, adenovirus, or inhibitors for 24 h were digested and counted. Cells (3 × 103 cells/well) were inoculated into 6-well plates. When the formation of cell clusters was observed under a light microscope, they were fixed with paraformaldehyde and stained with crystal violet to observe the colony generation.

CCK-8 assay

The breast cancer cells treated with siRNA (2 × 103 cells/well) were spread in 96-well plates. The OD450 nm values were measured at 0 h, 24 h, 48 h and 72 h using the CCK-8 kit.

Transwell assay

The digested cells were counted and prepared into a mixed suspension containing 1.2 × 104 cells/300 μl with FBS-free medium. For Transwell invasion experiments, 35 μl matrigel (Corning, USA) diluent was added to the chambers 1 h before the experiment. Subsequently, 300 μl of cell suspension was added to the upper chamber, and 700 μl of FBS-containing medium was added to the lower chamber. After 36 h of incubation, the cells were fixed with 4% paraformaldehyde for 20 min, and 1% crystal violet staining was performed for 15 min. Excess dye was washed away gently with PBS, and the cells were dried before being photographed ender an inverted microscope.

Wound healing assay

Cells were seeded in 6-well plates after 24 h of different treatments. When the cells were full grown, a 10 μl sterile lance tip was used to create a wound in the middle of the 6-well plate. The floating cells were removed using PBS, and then added with FBS-free medium. A photo was taken under the microscope and counted as 0 h, and then photos were taken at the same position when 24 h and 48 h. CorelDRAW software was used to analyze the wound width at each time point and to calculate the average wound healing rate.

Chromatin immunoprecipitation (ChIP)

The ChIP kit (Beyotime, China, P2078) was used to examine the enrichment of H3K36me3 and H3K4me3 in the promoter region of BMP9 gene. Logarithmic growth phase cells (about 1 × 106 cells) were fixed using 1% formaldehyde at room temperature for 10 min to crosslink DNA and protein. Subsequently, sonication was performed to cleave DNA into 200–800 bp chromatin fragments, and immunoprecipitation was carried out by overnight incubation at 4 °C with IgG as a negative control. The precipitated chromatin DNA was then obtained and analyzed using ChIP-PCR agarose gel electrophoresis. The primer sequences for ChIP-PCR are shown in Additional Table S4.

RNA stability assay

To investigate the impact of KDM4A on the RNA stability of BMP9, cells were inoculated in 60 mm dishes. Small interfering RNA targeting KDM4A (siKDM4A) and negative control (siNC) were transfected into the cells when the growth density reached 60% ~ 80%. 24 h later, the solution was changed and actinomycin D (Selleck, USA) was added according to different time gradients to make the final concentration of 5 μg/ml. Total RNA was extracted after treating the cells for 0, 30, 60, 90 and 120 min and the mRNA level of BMP9 was quantified.

CHX protein stability assay

To investigate the impact of KDM4A on the protein stability of BMP9, cells were inoculated in 100 mm dishes. Small interfering RNA targeting KDM4A (siKDM4A) and negative control (siNC) were transfected into the cells when the growth density reached 60%–80%. After 48 h, the solution was changed and Cycloheximide (Selleck, USA) was added according to different time gradients to make the final concentration of 100 μg/ml. Total protein was extracted after treating the cells for 0, 30, 60, 90 and 120 min and the protein level of BMP9 was examined to analyze its degradation.

ELISA

Cells from different treatment groups were collected and lysed, and the resulting supernatant was collected after high-speed centrifugation to isolate intracellular fluid. The level of α-ketoglutarate was then quantified using an ELISA kit (Jingmei, China) according to the manufacturer's guidelines.

Immunofluorescence (IF)

The pre-treated cells were spread in 24-well plates, washed with PBS and fixed with 4% paraformaldehyde for 30 min, followed by blocking with 1% goat antiserum for 1 h, then immersed in primary antibody overnight at 4℃. On the next day, the cells were washed with PBS and then incubated with an Alexa Fluor 647 fluorescent secondary antibody for 1 h. The nuclei were stained with a DAPI staining solution. Images were captured using a confocal microscope (Leica TCS SP8, Germany).

Animal models

Well-conditioned MDA-MB-231 cells were digested and counted after treatment with adenovirus or KDM4A inhibitor (JIB-04) for 24 h. 100 μl of cell suspension was injected subcutaneously into 5-week-old female nude mice at an amount of 6 × 106/100 μl, and the mice were necropsied after 39 days (no more than 1.5 cm3), the tumors were peeled off, and tumor volume was calculated.

Immunohistochemical staining (IHC)

The stripped tumors were embedded, sectioned, dewaxed and hydrated, antigenically repaired and blocked, and then incubated overnight at 4 °C with a primary antibody (E-cadherin: 1:5000, N-cadherin: 1:5000, Vimentin: 1:2500, Snail: 1:50), followed by incubation with an enhanced enzyme-labeled goat anti-rabbit IgG secondary antibody for 30 min at room temperature, and images were captured under a microscope after color development with a chromogenic solution.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 software, and the experiment was repeated three times for each group. The Student's t-test and one-way ANOVA were used to compare intra- and inter-group differences. All data were expressed as mean ± SD. P < 0.05 was considered statistically significant.

Results

KDM4A is abnormally highly expressed in breast cancer and promotes cancer progression

By analyzing the UALCAN database, the mRNA and protein expression of KDM4A in breast cancer was determined to be higher than in normal tissues (Fig. 1A, B), and further analysis revealed that there was no statistically significant variation in KDM4A expression across the different stages of breast cancer (Fig. 1C, D), suggesting that KDM4A is involved in the entire process of breast cancer occurrence and development. We also analyzed the expression of KDM4A in different breast cancer subtypes in our database, but our results showed that there was no significant difference in the expression of KDM4A in different subtypes of breast cancer. This may suggest that targeting KDM4A may be a critical approach to inhibit breast cancer progression. Results from RT-qPCR and Western blot analysis confirmed that the expression of KDM4A was significantly higher than that of MCF-10A in multiple breast cancer cell lines (Fig. 1E, F). To clarify the biological role of KDM4A in breast cancer, we conducted a series of experiments by silencing KDM4A using small interfering RNA in SK-BR-3 and MDA-MB-231cell lines. Colony formation and CCK-8 assays confirmed that the proliferation of breast cancer cells was significantly inhibited after KDM4A knockdown (Fig. 1G, H). The same as the transwell and wound healing assays verified that KDM4A knockdown significantly reduced the ability of breast cancer cells to migrate and invade (Fig. 1I, J). Furthermore, knockdown of KDM4A significantly reduced the epithelial-mesenchymal transition in breast cancer cells, as demonstrated by RT-qPCR (Fig. 1K) and Western blot (Fig. 1L). These findings show that KDM4A contributes to breast cancer by acting as a pro-carcinogen.Fig. 1 KDM4A is highly expressed in breast cancer and promotes malignant progression of breast cancer. A: The mRNA expression of KDM4A in TCGA dataset. B: The protein expression of KDM4A in CPTAC dataset. C: The mRNA expression of KDM4A in various stages of breast cancer in TCGA dataset. D: The protein expression of KDM4A in various stages of breast cancer in CPTAC dataset. E–F: The mRNA and protein expression of KDM4A in MCF-10A, MCF-7, SK-BR-3 and MDA-MB-231 were detected by RT-qPCR and Western blot. G: Knockdown of KDM4A inhibited colony formation in breast cancer cells. H: Effect of knockdown of KDM4A on cell proliferative capacity was detected by CCK-8 assay. I: Changes in cell migration and invasion were detected by transwell assay. J: Alterations in the lateral migration capacity of cells were detected by wound healing assay. K: The related indexes of cell proliferation, migration, apoptosis and EMT were detected by RT-qPCR. L: Changes in EMT-related indexes were detected by Western blot. **P < 0.01, ***P < 0.001

KDM4A promotes malignant progression of breast cancer by downregulating BMP9

Our previous studies have shown that BMP9 is crucial in breast cancer development, but the reason for its low expression remains unclear, so we proposed to excavate why BMP9 is lowly expressed in breast cancer. First, the low expression of BMP9 in breast cancer cells was confirmed by RT-qPCR (Fig. 2A) and Western blot (Fig. 2B). A search of the GEPIA (http://gepia.cancer-pku.cn/) database to analyze the correlation between KDM4A and BMP9 revealed that KDM4A was negatively correlated with BMP9 in different cancers, and it also showed a trend of negative correlation in breast cancer (Fig. 2C). RT-qPCR and Western blot results showed that mRNA and protein levels of BMP9 were significantly up-regulated after knockdown of KDM4A in MDA-MB-231 and SK-BR-3 cells, furthermore, the expression of BMP9 increased in a linear fashion with KDM4A, meaning that the lower the KDM4A, the higher the expression of BMP9(Fig. 2D, E). However, the expression of KDM4A was not significantly affected by BMP9 overexpression (Fig. 2F). These results suggested that KDM4A acts as an upstream regulator of BMP9 to regulate the expression of BMP9. Of the aforementioned breast cancer cells, only SK-BR-3 cells expressed BMP9 endogenously. After knockdown of BMP9 in SK-BR-3 cells along with knockdown of KDM4A, cell proliferation, migration, invasion, and EMT ability enhancing and apoptosis decreasing caused by reduced BMP9 were reversed to different degrees (Fig. 2G-K), which implied that KDM4A could influence the malignant progression of breast cancer by regulating BMP9 expression.Fig. 2 KDM4A promotes malignant progression of breast cancer by downregulating BMP9. A-B: The expression of BMP9 in different breast cancer cell lines was detected by RT-qPCR and Western blot. C: The expression of KDM4A is negatively correlated with the expression of BMP9 in HNSC, KIRC and BRCA. D-E: The expression of BMP9 after knockdown of KDM4A were detected by RT-qPCR and Western blot. F: The expression of KDM4A was detected by RT-qPCR after overexpression of BMP9. G: Colony formation experiment. H: Transwell migration and invasion experiments. I: Wound healing test. J: RT-qPCR was used to detect the related malignant indexes of cancer. K: The related indexes of EMT were detected by Western blot. **P < 0.01, ***P < 0.001

KDM4A exerts demethylation to silence BMP9 expression and can affect BMP9 stability

Considering that KDM4A is a histone demethylase, we proposed to explore whether it exerts its enzymatic activity to affect the expression of BMP9 in breast cancer. Western blot results showed that the methylation levels of histone H3K4 and H3K36, which can cause gene activation, were significantly increased after knockdown of KDM4A (Fig. 3A), indicating that the knockdown of KDM4A can affect the overall methylation level. To clarify the site of action of KDM4A on BMP9, we used chromatin immunoprecipitation to explore the specific sites where KDM4A protein has interaction with the BMP9 gene. The results showed that the enrichment of H3K36 trimethylation in the promoter region of BMP9 gene was significantly higher than that of H3K4 trimethylation, and the enrichment increased significantly after knockdown of KDM4A (Fig. 3B), suggesting that KDM4A mainly acts at the H3K36 position rather than the H3K4 position in breast cancer. Since it has been reported that histone demethylases can act on non-histone substrates, we also explored the effect of KDM4A on BMP9 mRNA and protein stability. After treating MDA-MB-231 with actinomycin D for different gradient times, the mRNA expression level of BMP9 was assessed. The results showed that reduction of KDM4A had no significant impact on the RNA stability of BMP9 (Fig. 3C). Similarly, the protein expression level of BMP9 was detected after treating MDA-MB-231 for different gradient times with the protein synthase inhibitor CHX, and it was found that knockdown of KDM4A significantly improved the stability of BMP9 protein and prolonged its half-life (Fig. 3D). These results showed that KDM4A can silence the expression of BMP9 by removing the methyl of histone H3K36, and at the same time can shorten the half-life of BMP9 and accelerate its degradation.Fig. 3 Mechanism of BMP9 downregulation by mediated KDM4A. A: Sites with altered histone methylation levels after knockdown of KDM4A. B: The BMP9 gene promoter’s exact locations where KDM4A activates were confirmed by ChIP. C: The effect of knockdown of KDM4A on mRNA stability of BMP9 was detected by RT-qPCR. D: The effect of knockdown of KDM4A on protein stability of BMP9 was detected by Western blot. **P < 0.01, ***P < 0.001, ns: no significant

BMP9 can affect KDM4A distribution by inhibiting glutamine metabolism

As mentioned previously, KDM4A requires the small molecule metabolite α-ketoglutarate as its coactivator, glutamine metabolism is a major source of α-KG, and the role of BMP9 in glutamine metabolism in breast cancer is unclear. Therefore, it was next investigated whether BMP9 affects glutamine metabolism and α-KG-mediated KDM4A activation. ELISA results showed that overexpression of BMP9 resulted in a significant decrease in α-ketoglutarate, while knockdown of BMP9 resulted in the opposite (Fig. 4A). After overexpression of BMP9, a decrease in glutaminase expression, a key enzyme in glutamine metabolism, was detected by Western blot, while the opposite was observed with knockdown of BMP9 (Fig. 4B). These findings indicated that BMP9 could inhibit glutamine metabolism by inhibiting glutaminase, which lead to a decrease in α-KG production. Overexpression of BMP9 decreased the distribution of KDM4A in the nucleus and increased its content in the cytoplasm were detected by Western blot (Fig. 4C). The same result was confirmed by immunofluorescence staining. Overexpression of BMP9 decreased the distribution of KDM4A in the nucleus and increased its content in the cytoplasm, whereas the opposite was found in the nucleoplasmic distribution of KDM4A after knockdown of BMP9 (Fig. 4D). All of the above results suggested that BMP9 affects KDM4A nucleoplasmic distribution through the glutamine metabolism pathway, thus affecting the function of KDM4A.Fig. 4 BMP9 affects KDM4A nucleoplasmic distribution through the glutamine metabolic pathway. A: Intracellular α-KG levels were detected by ELISA. B: The expression of GLS, a key enzyme in glutamine metabolism was detected by Western blot. C: The nucleoplasmic distribution of KDM4A was detected by Western blot. D: The nucleoplasmic distribution of KDM4A was detected by immunofluorescence staining. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant

Combination of BMP9 and KDM4A inhibitor significantly suppresses breast cancer progression

Many inhibitors of histone methyltransferases have been developed and many clinical trials have been conducted for different cancers, in which the use of histone methyltransferase inhibitors in combination with other therapeutic agents has also received widespread attention. Therefore, we also evaluated the utility of KDM4A for the treatment of breast cancer to observe its potential as a therapeutic agent for breast cancer. The use of BMP9 and the KDM4A inhibitor JIB-04 alone or in combination inhibited the proliferation, migration, and invasion of breast cancer cells, but the effect was more pronounced when used in combination, which was confirmed by the colony formation assay (Fig. 5A), wound healing assay (Fig. 5B), and Transwell assay (Fig. 5C). The same effect was obtained by targeting EMT-related indicators using Western blot (Fig. 5D). Subcutaneous tumor formation experiments in nude mice showed that the use of BMP9 alone or simultaneously with the KDM4A inhibitor JIB-04 inhibited tumor growth, but its effect was more significant when used together (Fig. 5E, F). Immunohistochemical staining gave the same results (Fig. 5G).Fig. 5 Overexpression of BMP9 combined with inhibition of KDM4A suppresses breast cancer progression more significantly. A: Cell proliferative capacity was detected by colony formation assay. B: Cell migration capacity were examined by wound healing assay. C: Cell migration and invasion capacity were measured by Transwell assays. D: EMT-related indicators were detected by Western blot. E: Changes in body weight of mice. F: Comparison of tumor volume in mice. G: Breast cancer progression related indicators were detected by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

Treatment of breast cancer involves a multidisciplinary approach, including surgery, radiotherapy, neoadjuvant chemotherapy and adjuvant chemotherapy combined with endocrine therapy [29], Although the cure rate of breast cancer has improved significantly thanks to these technologies, there are still problems such as recurrence and metastasis, drug resistance, and poor prognosis. Therefore, there is an urgent need to find new breast cancer treatment options to prolong patient survival.

In recent years, epigenetic modification has emerged as a promising target for cancer therapy, and histone methylation modification, as an important component of epigenetic modification, is involved in the regulation of a variety of cancers. JIB-04 is a potential small molecule inhibitor targeting the Jumonji structural domain of histone demethylases. Vaupel P et al. found that the intensity of JIB-04 inhibition was enhanced in a hypoxic environment, suggesting a role for JIB-04 in the hypoxic environment of tumor cells [30]. Cascella B et al. also found that JIB-04 interacts with Lys 241 and Tyr 177 via hydrogen bonding, thereby disrupting the binding of KDM4A active site O2 to histone substrates [31]. Numerous studies have shown that JIB-04 is an effective anticancer agent against a variety of cancers, such as lung cancer, prostate cancer, and Ewing's sarcoma [32, 33]. A recent study by Kim MS et al. also reported that JIB-04 can selectively target colorectal CSC [34]. However, there is no experimental evidence that JIB-04 has a similar effect on breast cancer.

JIB-04 has been used as an inhibitor of H3K9/ K36 demethylase activity of KDM4A in leukemia [35]. In the study by Kim MS et al., although the concentration of JIB04 was lower than that of salinomycin (a known anticolorectal CSC drug), the effect of JIB-04 was equal to or greater than that of salinomycin [36]. These findings strongly suggest that JIB-04 is a potential promising therapeutic agent for cancer evidence and provide a rationale for its development as a clinical drug for cancer treatment. Therefore, studying its effectiveness against cancer in breast cancer may provide a new approach to the treatment of breast cancer. Anyway, although many inhibitors targeting histone methyl modification have been developed and many phase I/II clinical trials have been conducted for different cancers[35, 37, 38], the combination of histone methyltransferases with other therapeutic agents is crucial in these clinical trials; therefore, investigating the efficacy of KDM4A inhibitors in breast cancer while searching for new combination therapies may be the key to breast cancer treatment.

BMP9 has been found to play an anti-cancer role in a variety of cancers, Study has previously found that BMP9 also plays an important oncogenic role in breast cancer [14, 16–20]. Ouarné M et al. also found that in the mouse E0771 breast cancer model, BMP9 deletion resulted in decreased vascular perfusion and increased size and number of lung metastases, suggesting that BMP9 has a positive role in inhibiting tumor growth, metastasis, and vascular normalization [21]. These findings provide a hopeful therapeutic approach for the development of effective targeted therapies for breast cancer, namely the development of new activated BMP9-based therapies.

This study have demonstrated that KDM4A can down-regulate BMP9 expression dependent or independent of its demethylation, and that down-regulation of BMP9 causes enhanced glutamine metabolism and elevated α-KG levels, further activating KDM4A. Based on the effective cancer inhibitory effects of JIB-04 and BMP9, this study combined BMP9 with KDM4A inhibitor in breast cancer to observe its effects on breast cancer progression in vitro and in vivo, the results showed that BMP9 overexpression combined with inhibition of KDM4A not only inhibited the proliferation, migration and invasion of breast cancer better in vitro, but also showed better tumor suppression effect. These results confirmed that the combination of BMP9 and KDM4A inhibitors is expected to be an alternative option for the treatment of breast cancer.

Our findings provide a new perspective for targeting KDM4A and its downstream BMP9 for breast cancer therapy, but as a histone demethylase, whether there are other potential targets for KDM4A in breast cancer still needs to be explored further.

Conclusions

This article identified the role of KDM4A in silencing BMP9 expression in breast cancer by removing the methyl group at position H3K36 in the promoter region of the BMP9 gene and down-regulating the stability of the BMP9 protein, resulting in a subsequent enhancement of glutamine metabolism due to changes in BMP9 levels, causing alterations in the nucleoplasmic distribution of KDM4A and leading to cancer progression. In addition, this study also found that exogenous BMP9 combined with KDM4A inhibition can effectively inhibit the growth of breast tumors, which provides new perspectives for the clinical development of therapeutic drugs for breast cancer (Fig. 6).Fig. 6 Summary mechanism

Supplementary Information

Additional file1

Author contributions

YX C, SY Y: Conceptualization, Methodology, Writing Original Draft. TY, LW: Investigation, Software, Validation. TZ: Investigation, Resources. YQ Y, JF T: Visualization. TT D, HL S: Software, Data Curation. YZ: Conceptualization, Writing—Review & Editing, Funding acquisition. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NO. 81974449).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing of interest

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yuanxiang Chen and Shiyu Yang have contributed equally.
==== Refs
References

1. Hyuna Sung, Jacques Ferlay, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA CANCER J CLIN, 2021, 71:209–249.
2. Michaels E Worthington RO Rusiecki J Breast Cancer: Risk Assessment, Screening, and Primary Prevention[J] Med Clin North Am 2023 107 2 271 284 10.1016/j.mcna.2022.10.007 36759097
Michaels E, Worthington RO, Rusiecki J. Breast Cancer: Risk Assessment, Screening, and Primary Prevention[J]. Med Clin North Am. 2023;107(2):271–84.36759097
3. Houghton SC Hankinson SE Cancer Progress and Priorities: Breast Cancer[J] Cancer Epidemiol Biomarkers Prev 2021 30 5 822 844 10.1158/1055-9965.EPI-20-1193 33947744
Houghton SC, Hankinson SE. Cancer Progress and Priorities: Breast Cancer[J]. Cancer Epidemiol Biomarkers Prev. 2021;30(5):822–44.33947744
4. Barzaman K Karami J Zarei Z Breast cancer: Biology, biomarkers, and treatments[J] Int Immunopharmacol 2020 84 106535 10.1016/j.intimp.2020.106535 32361569
Barzaman K, Karami J, Zarei Z, et al. Breast cancer: Biology, biomarkers, and treatments[J]. Int Immunopharmacol. 2020;84: 106535.32361569
5. Chopra S Khosla M Vidya R Innovations and Challenges in Breast Cancer Care: A Review[J] Medicina (Kaunas) 2023 59 5 957 10.3390/medicina59050957 37241189
Chopra S, Khosla M, Vidya R. Innovations and Challenges in Breast Cancer Care: A Review[J]. Medicina (Kaunas). 2023;59(5):957.37241189
6. Neves Rebello Alves L, Dummer Meira D, Poppe Merigueti L, et al. Biomarkers in Breast Cancer: An Old Story with a New End[J]. Genes (Basel), 2023, 14(7):1364.
7. Martin C Zhang Y The diverse functions of histone lysine methylation[J] Nat Rev Mol Cell Biol 2005 6 838 849 10.1038/nrm1761 16261189
Martin C, Zhang Y. The diverse functions of histone lysine methylation[J]. Nat Rev Mol Cell Biol. 2005;6:838–49.16261189
8. Tsukada Y Fang J Erdjument-Bromage H Histone demethylation by a family of JmjC domain-containing proteins[J] Nature 2006 439 7078 811 816 10.1038/nature04433 16362057
Tsukada Y, Fang J, Erdjument-Bromage H, et al. Histone demethylation by a family of JmjC domain-containing proteins[J]. Nature. 2006;439(7078):811–6.16362057
9. Young NL Dere R Mechanistic insights into KDM4A driven genomic instability[J] Biochem Soc Trans 2021 49 1 93 105 10.1042/BST20191219 33492339
Young NL, Dere R. Mechanistic insights into KDM4A driven genomic instability[J]. Biochem Soc Trans. 2021;49(1):93–105.33492339
10. Desroches-Castan A Tillet E Bouvard C BMP9 and BMP10: Two close vascular quiescence partners that stand out[J] Dev Dyn 2022 251 1 178 197 10.1002/dvdy.395 34240497
Desroches-Castan A, Tillet E, Bouvard C, et al. BMP9 and BMP10: Two close vascular quiescence partners that stand out[J]. Dev Dyn. 2022;251(1):178–97.34240497
11. Xu JZ Zhou YM Zhang LL BMP9 reduces age-related bone loss in mice by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis[J] Cell Death Discov 2022 8 1 254 10.1038/s41420-022-01048-8 35523787
Xu JZ, Zhou YM, Zhang LL, et al. BMP9 reduces age-related bone loss in mice by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis[J]. Cell Death Discov. 2022;8(1):254.35523787
12. Jiang Q Li Q Liu B BMP9 promotes methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis in non-obese mice by enhancing NF-κB dependent macrophage polarization[J] Int Immunopharmacol 2021 96 107591 10.1016/j.intimp.2021.107591 33812253
Jiang Q, Li Q, Liu B, et al. BMP9 promotes methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis in non-obese mice by enhancing NF-κB dependent macrophage polarization[J]. Int Immunopharmacol. 2021;96: 107591.33812253
13. Chen H Nio K Tang H BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma[J] Int J Mol Sci 2022 23 3 1475 10.3390/ijms23031475 35163396
Chen H, Nio K, Tang H, et al. BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma[J]. Int J Mol Sci. 2022;23(3):1475.35163396
14. Yu H Chen Y Lang L BMP9 promotes autophagy and inhibits migration and invasion in breast cancer cells through the c-Myc/SNHG3/mTOR signaling axis[J] Tissue Cell 2023 82 102073 10.1016/j.tice.2023.102073 36963166
Yu H, Chen Y, Lang L, et al. BMP9 promotes autophagy and inhibits migration and invasion in breast cancer cells through the c-Myc/SNHG3/mTOR signaling axis[J]. Tissue Cell. 2023;82: 102073.36963166
15. Chen H Nio K Yamashita T BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma[J] Mol Oncol 2021 15 8 2203 2218 10.1002/1878-0261.12963 33834612
Chen H, Nio K, Yamashita T, et al. BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma[J]. Mol Oncol. 2021;15(8):2203–18.33834612
16. Wang W Weng Y Ren W Biological roles of human bone morphogenetic protein 9 in the bone microenvironment of human breast cancer MDA-MB-231 cells[J] Am J Transl Res 2015 7 9 1660 1674 26550465
Wang W, Weng Y, Ren W, et al. Biological roles of human bone morphogenetic protein 9 in the bone microenvironment of human breast cancer MDA-MB-231 cells[J]. Am J Transl Res. 2015;7(9):1660–74.26550465
17. Wang T Zhang Z Wang K Inhibitory effects of BMP9 on breast cancer cells by regulating their interaction with pre-adipocytes/adipocytes[J] Oncotarget 2017 8 22 35890 35901 10.18632/oncotarget.16271 28415788
Wang T, Zhang Z, Wang K, et al. Inhibitory effects of BMP9 on breast cancer cells by regulating their interaction with pre-adipocytes/adipocytes[J]. Oncotarget. 2017;8(22):35890–901.28415788
18. Ren W Sun X Wang K BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression[J] Mol Biol Rep 2014 41 3 1373 1383 10.1007/s11033-013-2982-8 24413988
Ren W, Sun X, Wang K, et al. BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression[J]. Mol Biol Rep. 2014;41(3):1373–83.24413988
19. Li S Dai H He Y BMP9 inhibits the growth of breast cancer cells by downregulation of the PI3K/Akt signaling pathway[J] Oncol Rep 2018 40 3 1743 1751 30015950
Li S, Dai H, He Y, et al. BMP9 inhibits the growth of breast cancer cells by downregulation of the PI3K/Akt signaling pathway[J]. Oncol Rep. 2018;40(3):1743–51.30015950
20. Ouarné M Bouvard C Boneva G BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer[J] J Exp Clin Cancer Res 2018 37 1 209 10.1186/s13046-018-0885-1 30165893
Ouarné M, Bouvard C, Boneva G, et al. BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer[J]. J Exp Clin Cancer Res. 2018;37(1):209.30165893
21. Hanahan D Hallmarks of Cancer: New Dimensions[J] Cancer Discov 2022 12 1 31 46 10.1158/2159-8290.CD-21-1059 35022204
Hanahan D. Hallmarks of Cancer: New Dimensions[J]. Cancer Discov. 2022;12(1):31–46.35022204
22. Hensley CT Wasti AT DeBerardinis RJ Glutamine and cancer: cell biology, physiology, and clinical opportunities[J] J Clin Invest 2013 123 9 3678 3684 10.1172/JCI69600 23999442
Hensley CT, Wasti AT, DeBerardinis RJ. Glutamine and cancer: cell biology, physiology, and clinical opportunities[J]. J Clin Invest. 2013;123(9):3678–84.23999442
23. Scriver CR Rosenberg LE Amino acid metabolism and its disorders[J] Major Probl Clin Pediatr 1973 10 1 478 4768607
Scriver CR, Rosenberg LE. Amino acid metabolism and its disorders[J]. Major Probl Clin Pediatr. 1973;10:1–478.4768607
24. Altman BJ Stine ZE Dang CV From Krebs to clinic: glutamine metabolism to cancer therapy[J] Nat Rev Cancer 2016 16 10 619 634 10.1038/nrc.2016.71 27492215
Altman BJ, Stine ZE, Dang CV. From Krebs to clinic: glutamine metabolism to cancer therapy[J]. Nat Rev Cancer. 2016;16(10):619–34.27492215
25. Lane AN Fan TW Regulation of mammalian nucleotide metabolism and biosynthesis[J] Nucleic Acids Res 2015 43 4 2466 2485 10.1093/nar/gkv047 25628363
Lane AN, Fan TW. Regulation of mammalian nucleotide metabolism and biosynthesis[J]. Nucleic Acids Res. 2015;43(4):2466–85.25628363
26. DeBerardinis RJ Cheng T Q's next: the diverse functions of glutamine in metabolism, cell biology and cancer[J] Oncogene 2010 29 3 313 324 10.1038/onc.2009.358 19881548
DeBerardinis RJ, Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer[J]. Oncogene. 2010;29(3):313–24.19881548
27. Still ER Yuneva MO Hopefully devoted to Q: targeting glutamine addiction in cancer[J] Br J Cancer 2017 116 11 1375 1381 10.1038/bjc.2017.113 28441384
Still ER, Yuneva MO. Hopefully devoted to Q: targeting glutamine addiction in cancer[J]. Br J Cancer. 2017;116(11):1375–81.28441384
28. Li B Tian XB Hu RY Mechanism of BMP and TG2 in mesenchymal stem cell osteogenesis[J] Eur Rev Med Pharmacol Sci 2015 19 22 4214 4219 26636505
Li B, Tian XB, Hu RY, et al. Mechanism of BMP and TG2 in mesenchymal stem cell osteogenesis[J]. Eur Rev Med Pharmacol Sci. 2015;19(22):4214–9.26636505
29. Fisusi FA Akala EO Drug Combinations in Breast Cancer Therapy[J] Pharm Nanotechnol 2019 7 1 3 23 10.2174/2211738507666190122111224 30666921
Fisusi FA, Akala EO. Drug Combinations in Breast Cancer Therapy[J]. Pharm Nanotechnol. 2019;7(1):3–23.30666921
30. Vaupel P Mayer A Höckel M Tumor hypoxia and malignant progression[J] Methods Enzymol 2004 381 335 354 10.1016/S0076-6879(04)81023-1 15063685
Vaupel P, Mayer A, Höckel M. Tumor hypoxia and malignant progression[J]. Methods Enzymol. 2004;381:335–54.15063685
31. Cascella B Lee SG Singh S The small molecule JIB-04 disrupts O2 binding in the Fe-dependent histone demethylase KDM4A/JMJD2A[J] Chem Commun (Camb) 2017 53 13 2174 2177 10.1039/C6CC09882G 28144654
Cascella B, Lee SG, Singh S, et al. The small molecule JIB-04 disrupts O2 binding in the Fe-dependent histone demethylase KDM4A/JMJD2A[J]. Chem Commun (Camb). 2017;53(13):2174–7.28144654
32. Wang L Chang J Varghese D A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth[J] Nat Commun 2013 4 2035 10.1038/ncomms3035 23792809
Wang L, Chang J, Varghese D, et al. A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth[J]. Nat Commun. 2013;4:2035.23792809
33. Parrish JK McCann TS Sechler M The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth[J] Oncotarget 2018 9 69 33110 33123 10.18632/oncotarget.26011 30237855
Parrish JK, McCann TS, Sechler M, et al. The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth[J]. Oncotarget. 2018;9(69):33110–23.30237855
34. Mar BG Chu SH Kahn JD SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia[J] Blood 2017 130 24 2631 2641 10.1182/blood-2017-03-775569 29018079
Mar BG, Chu SH, Kahn JD, et al. SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia[J]. Blood. 2017;130(24):2631–41.29018079
35. Song Y Wu F Wu J Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives[J] J Hematol Oncol 2016 9 1 49 10.1186/s13045-016-0279-9 27316347
Song Y, Wu F, Wu J. Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives[J]. J Hematol Oncol. 2016;9(1):49.27316347
36. Kim MS Cho HI Yoon HJ JIB-04, A Small Molecule Histone Demethylase Inhibitor, Selectively Targets Colorectal Cancer Stem Cells by Inhibiting the Wnt/β-Catenin Signaling Pathway[J] Sci Rep 2018 8 1 6611 10.1038/s41598-018-24903-0 29700375
Kim MS, Cho HI, Yoon HJ, et al. JIB-04, A Small Molecule Histone Demethylase Inhibitor, Selectively Targets Colorectal Cancer Stem Cells by Inhibiting the Wnt/β-Catenin Signaling Pathway[J]. Sci Rep. 2018;8(1):6611.29700375
37. Duan R Du W Guo W EZH2: a novel target for cancer treatment[J] J Hematol Oncol 2020 13 1 104 10.1186/s13045-020-00937-8 32723346
Duan R, Du W, Guo W. EZH2: a novel target for cancer treatment[J]. J Hematol Oncol. 2020;13(1):104.32723346
38. Kaniskan HÜ Martini ML Jin J Inhibitors of Protein Methyltransferases and Demethylases[J] Chem Rev 2018 118 3 989 1068 10.1021/acs.chemrev.6b00801 28338320
Kaniskan HÜ, Martini ML, Jin J. Inhibitors of Protein Methyltransferases and Demethylases[J]. Chem Rev. 2018;118(3):989–1068.28338320
