The novel tertiary amine LSD1 inhibitor 596 inhibits endometrial cancer through the mTOR signal transduction pathway

Highlights

• •596 is a potent inhibitor targeting LSD1 and can inhibit the growth of EC cells.
• •596 can promote autophagy in EC cells, thereby influencing the progression of tumors.
• •596 can affect the transduction of the PI3K/AKT/mTOR signaling pathway.
• •Combination therapy of 596 and mTOR inhibitor Rapamycin effectively reduced the cell viability, survival, and migration of EC cells.

Abstract

Lysine-specific demethylase 1 (LSD1) is a promising target in cancer therapy and plays an important role in the occurrence and development of tumors. However, research on LSD1 in endometrial cancer (EC) is indeed limited, and the related research on LSD1 inhibitors targeting EC is even rarer. In this study, our group developed a novel Tertiary Amine LSD1 inhibitor-596. 596 could specifically target LSD1 and inhibit the demethylation levels of H3K4me1/2 in a dose-dependent manner, thereby inhibited the proliferation of EC cells in vitro and in vivo. Moreover, in-depth studies have shown that 596 induces cell death in EC cells through the autophagy pathway, increasing the formation of autophagosomes and the expression of autophagy-related proteins. Transcriptomic sequencing revealed that 596-induced gene enrichment was related to the PI3K/AKT/mTOR pathway. The treatment group of 596 was able to attenuate the activation of the mTOR signaling cascade, and the combination treatment of 596 and the mTOR inhibitor rapamycin (RAPA) effectively reduced the survival rate, migration ability, and invasion ability of EC cells. Further studies in vitro and in vivo indicated that 596 could reduce the feedback activation of AKT mediated by the mTOR inhibitor rapamycin. In summary, our findings demonstrate that the LSD1 inhibitor 596 enhances the activity of the mTOR inhibitor by attenuating the feedback activation of AKT. LSD1 may be as a potential therapeutic target in EC, and LSD1 inhibitors represent an important therapeutic strategy in EC treatment.

Graphical abstract

Introduction

Endometrial cancer(EC) is one of the most common cancers in gynecology, accounting for the sixth highest incidence of cancer in women, and the overall incidence has increased by 132% in the past 30 years due to factors such as obesity and human aging [1]. According to statistics, the probability of women suffering from EC in their lifetime is 3%, and the average age of diagnosis of EC is 61 years old, but in recent years, the incidence of EC in women under 40 years old has been increasing year by year [2]. The number of deaths is also on the rise, with the increase in mortality over the past 10 years being twice as fast as the increase in incidence. Early treatment for EC still primarily relies on surgery. Although adjuvant chemotherapy or radiotherapy may be added based on disease staging and other factors to enhance efficacy, the treatment outcomes are still not satisfactory. Currently, various targeted therapy studies have provided more options for the treatment of advanced EC [3].

Epigenetic dysregulation plays a critical role in EC, and lysine-specific histone demethylase 1(LSD1), also known as KDM1A, was the first identified histone demethylase involved in epigenetic regulation of gene expression [4,5]. LSD1 eliminates monomethyl and dimethyl modifications of histone 3 lysine 4 (H3K4), histone 3 lysine 9 (H3K9), and histone 4 lysine 20 (H4K20) by flavin adenine dinucleotide (FAD) -dependent amine oxidase [6]and functions as a transcriptional suppressor by removing methyl groups, thereby regulating the expression of target genes [7]. LSD1 induces instability of myosin phosphatase target subunit 1 (MYPT1) protein through its demethylation activity, thereby activating RB1 phosphorylation (ser807/811) and E2F activity, thereby promoting cell cycle progression [8]. LSD1 is overexpressed in many cancers, and its overexpression is often observed to be negatively correlated with tumor prognosis, including acute myeloid leukemia (AML), breast cancer, colorectal cancer, small cell lung cancer, and prostate cancer [9,10]. Liu and colleagues studied clinical samples from 301 patients and found that LSD1 is highly expressed in EC and has a carcinogenic effect on EC. By examining the association between LSD1 expression and clinicopathological features, they found that the proportion of LSD1-positive expression in EC tissues was significantly higher than in normal endometrial tissues. Moreover, the later the stage of the EC malignancy, the higher the proportion of LSD1-positive expression. It was also found that LSD1 is associated with recurrence and poor clinical prognosis, suggesting that LSD1 may be a potential therapeutic target for EC [11]. In EC, the PI3K-AKT-mTOR pathway is the most commonly disrupted pathway that affects cell growth and survival and is associated with disease progression and a poor prognosis. Several compounds have been reported to inhibit the PI3K/Akt/mTOR pathway in cancer, including mTOR inhibitors. However, studies have found that mTOR inhibitors alone or in combination have a poor effect on EC. Although rapamycin can specifically inhibit the mTORC1-mTOR axis, however, due to feedback activation of AKT and crosstalk between the PI3K-AKT-mTOR pathway and ER [12,13], the activity of rapamycin and its analogs as single agents in cancer treatment is limited. Venkata PP et al. demonstrated that LSD1 inhibitors can enhance the therapeutic effect of rapamycin in EC treatment and inhibit the feedback activation of AKT [14].

Currently, several LSD1 inhibitors have been reported in the literature showing great potential in cancer therapy [9], such as in small cell lung cancer [10] and gastric cancer [15]. Although many LSD1 inhibitors have emerged, only nine have entered clinical trials, including eight irreversible inhibitors: ORY-2001, IMG-7289, TCP, ORY-1001, GSK-2879,552, INCB059872, TAK-418 and LH-1802, two reversible inhibitors: CC-90,011 and SP-2577, as well as a novel LSD1 inhibitor SYHA1807 (structure unpublished) and two dual LSD1/HDAC inhibitors: 4SC-202 and JBI-80,2[[16], [17], [18]]. Critically, the role of LSD1 inhibitors in the field of EC therapy remains poorly characterized, and no LSD1 inhibitor has been specifically developed or validated for EC treatment. So there is an urgent need to develop new LSD1 inhibitors for the treatment of EC.

Against this backdrop, we developed a novel LSD1 inhibitor, 596, and validated its antitumor potential in EC. The concentration of 596 that inhibited 50% of cathepsin activity (IC₅₀) was 129.217±2.111 nM, which inhibited the growth of EC cells both in vivo and in vitro. This article mainly elucidated the antitumor effect and mechanism of LSD1 inhibitor 596 in EC. Our findings demonstrate that 596 is a potent and selective LSD1 inhibitor with strong antitumor activity in EC. By targeting LSD1, 596 not only exerts direct antiproliferative effects but also alleviates the limitations of rapamycin monotherapy (i.e., AKT feedback activation) through synergistic inhibition of the mTOR pathway. These results highlight 596 as a promising candidate for EC treatment, particularly in combination with mTOR inhibitors, and provide a theoretical basis for further preclinical and clinical development of LSD1-targeted therapies for EC.

Materials and methods

Cell lines and reagents

The human EC cell lines ISK and HEC1A were purchased from National Cell Resource Center. All cells were identified by short tandem repeat (STR). The EC cells were cultured in DMEM medium(Viva cell, China) containing 10% fetal bovine serum (Viva cell, China) at 37°C and 5% CO₂.The main purchasing information was listed below:LSD1 (1:10,000, ab129195, Abcam, UK), H3(1:20,000, No.4499T,Cell Signaling Technology, CST, Boston, MA, USA), H3K4me1(1:2000, No.5326T, CST), H3K4me2(1:2000, No.9725S, CST), H3K4me3(1:2000, No. 9751T, CST), p62/SQSTM1(1:2000, No.5114T, CST), LC3B(1:2000, Cat# AF4650, Affinity Biosciences), Beclin-1(1:2000, Cat# AF5128, Affinity Biosciences), AKT(1:5000, YM8463, Immunoway, USK), P-AKT(Ser473)(1:2000, YM8304, Immunoway, USK), mTOR(1:5000,YM8208, Immunoway, USK), P-mTOR(Ser2448)(1:5000, YM8326, Immunoway, USK), S6(1:2000, YT4139, Immunoway, USK), P-S6(1:2000, No.F0198, Selleck.cn), p70S6K(1:2000, YT3559, Immunoway, USK), P-p70S6K(1:1000, No.F2907, Immunoway, USK), GAPDH(1:5000, Proteintech). Rapamycin, 3-MA was purchased from MCE and CC9001 was purchased from KKL Med Inc. Compound 596 was synthesized by our laboratory staff.

Cell viability assay

For preliminary screening of the concentration of EC cells acted by 596, ISK cells and HEC1A cells (3 × 10³/ well) were inoculated into 96-well plates, and cultured overnight until the cells were fully attached to the wall. Different concentrations of 596 (0.39 μM-100 μM, respectively) were added. Add 10 μL CCK8(MCE) and measure absorbance after 4 h. The semi-suppressed concentration was calculated using GraphPad Prism 8 software and XY modeling method. In order to study the sensitization effect of LSD1 inhibitor on rapamycin, ISK or HEC1A (3 × 10³/ well) was inoculated into 96 empty plates and treated with 596 or rapamycin alone or 596 and rapamycin combined, respectively, after overnight attachment. Cell viability was detected by CCK8 assay. In order to verify the effect of LSD1 knockout on EC alone or in combination with rapamycin, ISK cells (3 × 10³/ well) of LSD1 KO were inoculated into 96-well plates, treated with Vehicle or rapamycin overnight, and cell viability was detected by CCK8 assay.

Western blot

Human EC cells were lysed with RIPA lyser (Solarbio, UK) containing phosphatase inhibitors (PMSF). We used the BCA quantification kit (Solarbio, UK) to quantify protein concentrations. For the study of nuclear protein, histone extraction kit (SAIAT-BIO, China) was used for extraction. The proteins were then separated with SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (PALL Bio Trace, USA). After sealing with 5% skim milk for 2 h, the membrane was incubated with the specific antibody at 4°C overnight and then incubated with the corresponding secondary antibody at room temperature for 1 h Finally, chemiluminescence signals were detected using an ECL kit (Abbkine, China).

Cellular thermal shift assay (CETSA)

The cells were inoculated in a 100 mm petri dish and incubated in a 37°C incubator for 1 h with DMSO or 596 (12 μM) when the cell density was about 80% (1 × 10⁷ cells). The cells were then collected and washed twice with pre-cooled PBS. After re-suspension, the cells were divided into 7 equal parts, and the cell suspension was put into a PCR tube (1.5 × 10⁶ cells/tube), heated for 90 S at different temperatures (39°C-57°C), restored to room temperature 90 S, and melted rapidly at 37°C after being placed in liquid nitrogen for 3 S, repeated 3 times to break the cells. The supernatant was obtained by centrifugation at 4°C,12,000 rpm and 20 min. After quantitative denaturation, the level of LSD1 was detected by western blot.

Immunofluorescence

ISK and HEC1A cells (1 × 10⁴/ well) were inoculated on a 24-well plate, treated with 596 (12.5 μM) after overnight adhesion, fixed with 4% paraformaldehyde after 48 h, and permeated with 0.5% Triton X-100 (Beyotime, China). The cells were incubated in 10% goat serum (Solarbio, UK) for 30 min, then incubated at 4°C overnight with the primary antibody (LC3B, p62), then incubated at room temperature for 1 h with the secondary antibody. The nuclei were stained using DAPI (Beyotime, China), and the images were taken by 20x objective lens. Fiji Is Just ImageJ for post-processing.

Immunohistochemistry

The endometrial cancer tissue samples used in this study were obtained from the Department of Pathology of the Third Affiliated Hospital of Zhengzhou University and were approved by the Ethics Committee. The continuous sections (thickness: 4 μm) were attached to polyline-coated slides and dried in a 60 °C oven for 3 h. The sections were dewaxed with xylene, hydrated with gradient ethanol (100%, 95%, 85%, 75%) to distilled water, and repaired in a microwave oven with EDTA antigen remediation solution for 20 min. Endogenous peroxidase was blocked in 3% hydrogen peroxide methanol solution for 20 min, and blocked in 5% goat serum for 30 min. Incubate the antibody LSD1(1:200, ab129195, Abcam, UK) at 4°C overnight. The negative control was replaced with PBS instead of the primary antibody. The secondary antibody labeled with HRP (1:500, M25AP98, ZENBIO, China) was used at room temperature for 3 h. DAB chromogenic reaction (Beyotime, China), control the color development time under a microscope. Hematoxylin staining followed sequential dehydration with 75%, 85%, 95%, 100% ethanol and xylene, and mounting with neutral gum, followed by analysis with AT2.

Cell proliferation experiment

In order to study the effect of LSD1 inhibitor on EC, EC cells ISK or HEC1A were inoculated into a 6-well plate (500/ well) and treated with 596 (4 μM, 8 μM, 12 μM, 16 μM) overnight for 1 week, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet to observe the colony status. To investigate the sensitization effect of LSD1 inhibitor 596 on rapamycin, cells were inoculated in 6-well plates and incubated overnight, and then cells were treated with 596 and rapamycin alone or in combination for 7 days, respectively, and stained as above. Colony area was quantitatively analyzed using NIH ImageJ software and used for analysis.

Wound-healing assay

EC cells ISK and HEC1A were plated in 6-well plates. When the cells grew into fused single-cell layer, a horizontal line was drawn in the middle with the tip of a 200 μL pipetting gun, the floating cells were cleaned with PBS, and then treated with LSD1 inhibitor 596 or rapamycin alone or in combination. After 0 h, 24 h and 48 h, the scratched area was photographed under the microscope and the mobility was calculated using NIH ImageJ software, and the inhibition rate curves were graphed by GraphPad Prism 8 software.

Transwell

For the Transwell invasion experiment Matrigengel was thawed on the ice and diluted at an appropriate ratio (1:8), pipette 60 μL of the diluted Matrigengel vertically into the Transwell upper chamber and allow it to be evenly tiled on the bottom. Matrigengel was placed in a 37°C, 5% CO₂ incubator for 1 h to allow Matrigengel to form a thin film. The excess blood in the upper chamber was then washed off, 100 μL of serum-free medium was added to each well, and the basement membrane was hydrated in the incubator for 30 min. The liquid in the upper chamber was then aspirated, 600 μL of medium containing 10% serum was added to the lower chamber of the Transwell plate, 1 × 10⁴ cells and 400 μL of serum-free medium were added to the upper chamber, and the culture was continued in the incubator for 48 h. For Transwell migration experiments, direct plating of cells is sufficient. After the culture, it was washed with PBS for 3 times, fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 20 min, and observed with a microscope. Count the number of cells penetrated with NIH ImageJ software and draw the graphs with Graphpad Prism 8.

Subcutaneous xenograft models

All mouse experiments were approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University and conducted in strict accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. Female BALB/c-nu athymic nude mice (5–6 weeks old) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) and housed under specific pathogen-free (SPF) conditions at our facility. A total of 3 × 10⁶ ISK tumor cells were suspended in an equal-volume mixture of phosphate-buffered saline (PBS) and Matrigel® (Corning, NY, USA; Cat. #356,237) and then subcutaneously injected into the left flank of each mouse. Once tumors reached a volume of approximately 100 mm³, mice were enrolled in the in vivo efficacy study.

Mice were randomly assigned to four treatment groups (n = 5 per group): (i) negative control (vehicle only), (ii) low-dose 596 compound (10 mg/kg), (iii) medium-dose 596 compound (20 mg/kg), and (iv) high-dose 596 compound (30 mg/kg). In parallel, to evaluate the combinatorial effect of 596 and RAPA, an additional cohort of mice was randomized into four groups (n = 5 per group): (i) vehicle control, (ii) 596 compound alone (20 mg/kg), (iii) RAPA alone (5 mg/kg), and (iv) 596 + RAPA combination. All compounds were administered via daily intraperitoneal injection for 15 consecutive days. Body weight and tumor dimensions were measured every two days. Tumor volume (V) was calculated using the formula: V = (length × width²) / 2 (in mm³). Mice were humanely euthanized if body weight loss exceeded 20% of the initial weight or if tumor volume surpassed 1500–2000 mm³. At the end of the 21-day experimental period, tumors were excised, weighed, and fixed in 4% paraformaldehyde for downstream histological and molecular analyses.

Statistical analysis

Data analysis was conducted using GraphPad Prism 8 statistical software. Comparisons between two groups were performed using the t-test, and homogeneity of variance was evaluated by the Levene test. Differences among multiple groups were analyzed by one-way ANOVA. Results were considered statistically significant when P < 0.05.

Result

LSD1 is highly expressed in EC, and inhibition of LSD1 exerts suppressive effects on EC

We analyzed the expression of LSD1 in various human tumors using the TCGA database and discovered that LSD1 is significantly upregulated in multiple tumors, including EC (Fig. 1A-B). Survival analysis revealed that high expression of LSD1 in EC is negatively correlated with overall survival, indicating a potential association with poor prognosis (Fig. 1C). Immunohistochemical analysis further demonstrated that LSD1 protein levels are markedly elevated in EC tissues compared to normal endometrial tissues (Fig. 1D), which was consistent with the public database. Next, to explore the functional role of LSD1 in EC cells, used the CRISPR-Cas9 method to generate LSD1-knockout ISK cells and observed that LSD1 knockout significantly reduced the proliferation and colony-forming ability of ISK cells (Fig. 1E-G). In conclusion, these data indicated that LSD1 is highly expressed in human EC tissues and is closely related to the progression of EC. Inhibition of LSD1 exerts significant suppressive effects on EC cell growth.

Fig. 1.

Elevated expression of LSD1 in EC. (A). The expression levels of LSD1 in different tumor types from TCGA. (B). The expression level of LSD1 in EC patients and normal patients was analyzed from the TCGA database. (C). The Kaplan-Meier plot shows the overall survival of tumors with high expression (red) or low expression (black) of LSD1 in EC patients (log-rank test). (D). The relative expression of LSD1 in EC and normal tissues was detected using Immunohistochemistry. Scale bars=200 μm. (E). The growth of ISK cells with LSD1 gene knockout and those in the control group was detected by the CCK8 assay. (F). Representative images of colony formation experiments in EC cells with LSD1 knockout.

Compound 596 interacted with LSD1 and could inhibit cell proliferation

To address the current paucity of effective LSD1 inhibitors for EC therapy, our research group developed a novel and potent LSD1 inhibitor, designated 596, the molecular structure was shown in Fig. 2A. The IC₅₀ of compound 596 against LSD1 enzymatic activity was 129.217±2.111 nM. Due to the demethylation of LSD1 with H3K4me1/2, we examined the effect of 596 on LSD1 catalytic function in EC cells by measuring histone H3K4 methylation levels via Western blot. After treating EC cells with gradient concentrations of compounds (0–16 μM) for 48 h, we observed that 596 could significantly increase the levels of H3K4me1 and H3K4me2 in EC cells, while there was no significant change in H3K4me3. These results indicated that compound 596 can eliminate the LSD1-mediated demethylation of H3K4me1 and H3K4me2 in EC cells. (Fig. 2B). To further validate the targeted effect of the compound on LSD1 within the EC cell, we employed the CETSA method to assess whether 596 could bind to LSD1 and improve the thermal stability of LSD1. The results showed that in both ISK and HEC1A cell lines, the thermal stability of LSD1 was enhanced after 596 (12.5 μM) treatment compared to the control group. 596 treatments increased the melt temperature of LSD1 in ISK and HEC1A cells by approximately 3°C, respectively. This demonstrated the ability of compound 596 to target LSD1 in cells, thereby enhanced the thermal stability of LSD1 in EC cells (Fig. 2C-D).

Fig. 2.

596 interacts with LSD1 in EC Cells. (A). The molecular structure of 596. (B). Western blot detection of H3K4me1, H3K4me2, and H3K4me3 after 596(0,4,8,12,16 μM) treatment(n = 3). (C-D). After treating ISK and HEC1A cells with DMSO and 596 (12 μM) for 2 h, the thermal stability of LSD1 was detected by CETSA method. The quantitation (right) of western blot results. (E). In ISK and HEC1A cells, detect the proliferation of compound 596 after 48–96 h of different concentrations by CCK8. (F-G). GraphPad Prism 9 was used to calculate IC50 of 596 in ISK and HEC1A cells. (H). ISK cells and HEC1A cells were treated with different concentrations of 596 respectively, and the proliferation of the cells was detected by colony formation assay.

Next, the cell viability of EC cell lines ISK and HEC1A treated with different concentrations of 596 intervention, the effect of 596 on EC cell viability were investigated. The results showed that 596 had a strong inhibitory effect on the growth of EC cell lines ISK and HEC1A, and was time- and dose-dependent (Fig. 2E). When compound 596 was treated for 48 h, the IC₅₀ values for ISK and HEC1A were 12.31 μM and 15.69 μM, the IC₅₀ values for 72 h were 10.61 μM and 14.2 μM, and the IC₅₀ values for 96 h were 7.536 μM and 12.48 μM, respectively (Fig. 2F-G). Notably, the normal endometrial stromal cell line T-HESC was less sensitive to 596, with an IC₅₀ of 58.19 μM after 48 h, indicating a favorable selectivity toward EC cells (Supplementary Fig. 1A). In addition, the colony formation experiment further demonstrated that the LSD1 inhibitor 596 could inhibit the proliferation of EC cells (Fig. 2.H). The above results indicated that the novel LSD1 inhibitor 596 directly binds to and stabilizes LSD1, inhibited its demethylase activity toward H3K4me1/2, and can reduce the growth of EC cells. Therefore, compound 596 could interact with LSD1 to inhibit the proliferation of ISK and HEC1A in EC cells.

LSD1 inhibitor 596 reduced the transduction of the mTOR signaling pathway and the rapamycin -mediated feedback activation of AKT

In order to explore the molecular mechanism and action pathways of 596 in inhibiting EC, we treated ISK cells with 596 for 48 h and then conducted RNA-seq analysis. The RNA-seq results showed that 596 treatment significantly affected the gene expression profile of ISK cells, with a total of 2776 genes were significantly altered. The principal component analysis (PCA) analysis revealed a clear division into two separate cell populations, 596 and Control (Supplementary Fig. 2A). Further analysis indicated that the PI3K-AKT signaling pathway in the genes regulated by the 596 treatment group and the control group was significantly changed. Moreover, among these changes, it was found that the 596 treatment group up-regulated the expression of the key negative regulatory factor DDIT4 in the mTOR signaling pathway and down-regulated the expression of the positive regulatory factor IRS2 in the mTOR pathway, revealed that 596 play an important role in modulating the PI3K/AKT/mTOR pathway (Fig. 3A-C). To further investigate the clinical correlation between LSD1 and mTOR pathway genes, we analyzed the correlation between LSD1 and the expression levels of mTOR, RICTOR, MAPKAP1, RPIK3CA, and IRS2 in EC patient data from the TCGA database. As shown in Supplementary Fig. 2B, LSD1 was positively correlated with mTOR, RICTOR, MAPKAP1, and IRS2 expression levels.

Fig. 3.

596 alters the expression of genes involved in mTOR signaling. (A). Volcano plot of differentially expressed genes in ISK cells after 596(12 μM) treatment (from RNA-seq data). Blue dots represent significantly downregulated genes, red dots represent significantly up-regulated genes, gray dots represent no meaningful changed genes. (B). KEGG enrichment pathway diagrams of RNA-seq differential genes in the ISK 596 and Control groups. (C). Heatmap of differential genes between 596 and control based on their RNA-seq data. (D). ISK and HEC1A cells were serum starved for 24 h and pretreated with 596 (12 μM) for 1 h following stimulation with 10% FBS for 0, 10, 30, and 60 min. Western blotting was used to analyze the activation of Akt/mTOR signaling components. (E). ISK and HEC1A cells were treated with rapamycin or rapamycin + 596 for 0, 24, 48, and 72 h, respectively, and the phosphorylation level of AKT was detected by Western Blot.

To determine the active role of LSD1in mTOR signaling pathway, we measured the protein expression levels of mTOR signaling pathway-related markers and confirmed that the 596 treatment significantly reduced the serum-induced phosphorylation levels of Akt, mTOR and their downstream components S6 and p70S6K in ISK and HEC1A cells compared with the control group (Fig. 3D). In ISK cells with LSD1 knockout, we also observed significantly reduced phosphorylation levels of serum-induced AKT, mTOR, S6 and p70S6K compared to vehicle treatment (Supplementary Fig. 3A). The TOR inhibitor rapamycin has been extensively studied for its role in long-term mTOR inhibition, but rapamycin partially induces feedback activation of AKT through the mTORC2 complex, limiting the clinical use of this class of drugs as monotherapy [19]. In order to explore whether 596 could enhance the anti-tumor effect of rapamycin, we combined rapamycin with 596 in EC cells, and the results showed that the mTOR inhibitor rapamycin increased the phosphorylation level of AKT in ISK and HEC1A cells, while the combination of 596 with rapamycin inhibited rapamycin-induced feedback activation of AKT (Fig. 3E). The above results suggested that LSD1 inhibition can weaken the mTOR signaling pathway and eliminate rapamycin mediated feedback activation of AKT in EC cells.

596 induces cell death via autophagy activation

Cell death can be mediated by apoptosis, necrosis, ferroptosis, autophagy and other mechanisms, and a number of studies have proved the relationship between LSD1 and autophagy. For example, Angel Chao.etc. et al. proved that overexpression of LSD1 can reduce the stability of p62 protein in gynecological malignancies, thereby promoting tumor occurrence and inhibiting autophagy. LSD1 knockdown can increase the level of p62 protein and autophagic flux, which can reduce the occurrence of tumors in vivo [[20], [21], [22]]. Our previous studies have proved that 596 attenuates mTOR pathway activation a key regulator of autophagy. Based on this finding, we hypothesized that compound 596 may inhibit proliferation through autophagy of EC cells. Firstly, the expression levels of autophagy markers LC3B, Beclin-1 and p62 were detected when treated with compound 596 in EC cells. Western blot analysis revealed d that after treatment of EC cells with 596 for 48 h, LC3BI was significantly induced to LC3BII, and the expression of p62 also increased, the Beclin-1 level remained unchanged (Fig. 4A). Quantify the band intensities of protein blot images using the ImageJ software (Supplementary Fig. 4A-B).

Fig. 4.

596 induces autophagy in EC Cells. (A). Western blot detection of LC3B-Ⅰ/Ⅱ, p62, and Beclin-1 in ISK and HEC1A cells after 596(0,4,8,12,16 μM) treatment. (B). The autophagosome structure in ISK and HEC1A cells was observed by transmission electron microscopy (TEM), Scale bar, 500 nm. (C-D). Representative images of LC3B and p62 detected by immunofluorescence, nuclei are labeled with DAPI. Scale bar, 100 μm. (E). Western blot detection of p62, Beclin-1, LC3B with 596(12 μM) and RAPA (200 nM) treatment. (F). Western blot detection of LC3B with 596(12 μM) and 3MA (5 mM) treatment.

Then transmission electron microscopy (TEM) was performed to visualize the autophagosomes formation of cells. Consistent with the biochemical data, TEM images showed a marked increase in double-membrane autophagic vacuoles in the 596 treatment group compared with the control group (Fig. 4B). Immunofluorescence staining further corroborated these findings: the LC3B and p62 levels in the 596 treatment group were significantly higher than those in the control group (Fig. 4C-D). These results preliminarily demonstrated that compound 596 can induce EC cell death through the mechanism of autophagy. To demonstrate that 596 can specifically induce autophagy, we co-treated cells with the well-characterized autophagy inducer rapamycin and the early-stage autophagy inhibitor 3-methyladenine (3MA). As expected, rapamycin synergistically enhanced the 596-mediated LC3II accumulation. 3MA, as an upstream inhibitor of autophagy, significantly suppressed the formation of LC3BII induced by the 596 treatment group (Fig. 4E-F). Quantify the band intensities of protein blot images using the ImageJ software (Supplementary Fig. 5A-C). Immunofluorescence analysis yielded were also consistent with the above results, rapamycin could enhance the LC3B fluorescence expression in the 596 treatment group (Supplementary Fig. 6). Taken together, these results indicated that LSD1 functions as a negative regulator of autophagy activation and modulates mTOR signaling. Consequently, pharmacological inhibition of LSD1 by compound 596 potentiates autophagy, including that triggered by mTOR inhibition.

596 inhibits tumor growth in vivo

The above in vitro experiments have confirmed that 596 has significant anti-tumor activity. Based on this, we selected the ISK cells that were the most sensitive to 596 to construct a subcutaneous xenograft tumor model in nude mice to further evaluate its anti-tumor effect in vivo. The experiment used BALB/c nude mice as the research subjects, and intraperitoneal injection of different concentrations of 596 was performed daily for 15 consecutive days. The results showed that 596 significantly inhibited the growth of the transplanted tumors in a dose-dependent manner (Fig. 5A-C). Notably, during the entire administration period, the mice did not show significant weight loss, and no obvious damage to the heart, liver, spleen, lung, or kidney was observed during dissection, suggesting that 596 has no significant systemic toxicity at the effective anti-tumor dose (Fig. 5D). The molecular detection results of the transplanted tumor tissues were highly consistent with the in vitro experiments: the positive rate of proliferation marker Ki67 was significantly reduced, and the expression level of autophagy-related protein LC3B was significantly upregulated (Fig. 5E). These results fully confirmed that 596 regulates tumor cell autophagy in vivo and exerts anti-tumor effects.

Fig. 5.

The antitumor effect of 596 against EC in vivo.

A-C. (A) The tumor images, (B) tumor volumes, and (C) tumor weights for ISK xenograft model treated with vehicle, 596(10 mg/kg, 20 mg/kg, 30 mg/kg), respectively (n = 5 per group). The data are presented as the mean±SEM. *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test. (D). The body weights of ISK xenograft model mice during treatment with 596 (n = 5 per group). The data are presented as the means±SEM. (E). Representative images of the Ki67, and LC3B staining of tumors after 596 treatment. Scale bar, 50 μm.

Compound 596 combined with rapamycin reduced the proliferation, migration and invasion of EC cells

It has been proved that LSD1 knockdown in EC cells can enhance the sensitivity to rapamycin, and thus enhanced the inhibition effect on EC cells. Our previous study further showed that compound 596 synergizes with rapamycin can enhance autophagy in EC cells. Based on these findings, we hypothesized that compound 596 may similarly enhance rapamycin sensitivity, leading to co-inhibiting EC cell survival. To determine if 596 could sensitize EC cells to rapamycin treatment. Cell viability assays were performed and showed that combined treatment with 596 and rapamycin reduced EC cell viability compared with either agent alone. Clonogenic survival assay also confirmed that 596 combined with rapamycin treatment significantly reduced the colony-forming capacity compared with either agent alone, and this finding was also demonstrated in ISK cells with LSD1 KO compared to the control group (Fig. 6A-C, Supplementary Fig, 7A). These results suggested that inhibition of LSD1 enhanced the efficacy of rapamycin in EC cells and also illustrated the combined effect of compound 596 with rapamycin. Scratch wound healing assays were conducted to evaluate the efficacy of the combination of 596 and rapamycin on EC cell migration ability compared to the effect alone (Fig. 6D-E). The combination treatment significantly reduced the migration ability of EC cells compared to monotherapy alone. and most importantly, rapamycin was highly effective in reducing the migration of LSD1 KO cells compared to the control group when treated with rapamycin alone or with LSD1 KO cells alone (Supplementary Fig, 7B). Finally, transwell assays demonstrated that the combined effect of 596 and rapamycin significantly reduced the migration and invasion ability of EC compare with 596 or rapamycin alone (Fig. 6F). Collectively, these results suggested that the combination of compound 596 and rapamycin can reduce the proliferation, migration and invasion of EC cells.

Fig. 6.

Combination of 596 and mTOR inhibition reduced EC cell survival and migration and invasion. (A). In ISK and HEC1A cells, the viability in 596 group, RAPA group, and 596 +RAPA group was detected by CCK8 method. (B). ISK cells and HEC1A cells were treated with Control, 596, RAPA, 596+RAPA, and the proliferation of the cells was detected by colony formation assay, quantitation of colony area is shown. (C) is shown relative migration of cells was quantified via ImageJ software. (D-E). The effect on cell migration of ISK and HEC1A cells treated with 596 and RAPA alone or in combination was determined. (F). Representative images of ISK and HEC1A migration and invasion assays. Scale bars=200 μm. Data are represented as mean ± SE. P*<0.05; **P < 0.01; ****P < 0.0001.

The combination therapy of compound 596 and mTOR inhibitors significantly suppresses tumor progression in EC xenograft models

We evaluated the in vivo antitumor efficacy of compound 596 in combination with rapamycin using mouse ISK xenograft model. Tumor-bearing mice were randomly assigned to four groups (n = 5 per group): (i) vehicle control, (ii) compound 596 monotherapy (10 mg/kg, intraperitoneally), (iii) rapamycin monotherapy (5 mg/kg, intraperitoneally), and (iv) combination therapy (compound 596 at 10 mg/kg + rapamycin at 5 mg/kg, both administered intraperitoneally). As shown in Fig. 7A-C, the combination treatment markedly delayed tumor growth compared with either vehicle control or either monotherapy. No overt signs of systemic toxicity were observed in the combination group, and body weight remained stable throughout the study period (Fig. 7D). To assess tumor cell proliferation, we performed immunohistochemical staining for Ki67 on formalin-fixed, paraffin-embedded xenograft tissue sections. Consistent with the tumor growth inhibition, the combination-treated tumors exhibited significantly reduced Ki-67 labeling indices relative to those in the vehicle control or monotherapy groups (Fig. 7E).

Fig. 7.

Combination of 596 and rapamycin treatment inhibited in vivo xenograft tumor growth.

BALB/c-nu mice were implanted subcutaneously with ISK cells. After tumor establishment, mice were randomly assigned to four groups (n = 5 per group): (i) vehicle control, (ii) compound 596 monotherapy (10 mg/k), (iii) rapamycin monotherapy (5 mg/kg), and (iv) combination therapy (compound 596 at 10 mg/kg + rapamycin at 5 mg/kg), both administered intraperitoneally. (A). The tumor images. (B). Tumor volumes are shown. (C). Tumor weights are shown. (D). Body weights of the mice are shown. (E-F). Representative images of the Ki67 and p-AKT staining of tumors. Scale bar, 50 μm Data represented as mean ± SE. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Given that mTOR inhibition can trigger feedback activation of the PI3K/AKT pathway, we further examined whether compound 596 mitigates rapamycin-induced phosphorylation of AKT. Immunohistochemical analysis revealed that p-AKT (Ser473) levels were substantially elevated in rapamycin-treated tumors compared with vehicle control; however, this compensatory activation was significantly attenuated in the combination group (Fig. 7F). Collectively, these findings demonstrate that co-administration of compound 596 and rapamycin synergistically inhibits EC growth in vivo partly by counteracting rapamycin-induced AKT hyperactivation.

Discussion

In recent years, LSD1 has attracted significant attention as an epigenetic therapeutic target in various cancers [23,24], Several inhibitors (such as ORY-100,1, IMG-7289, GSK2879552, and CC-900,1[27],etc.) have entered clinical trials, and it has demonstrated the potential to regulate cell growth, differentiation, and migration in tumors such as breast cancer and small cell lung cancer [26,28,29]. However, the role of LSD1 inhibitors in EC remains largely unexplored. In this paper, our study proved that: (1) the novel tertiary amine LSD1 inhibitor 596 developed by our research group could inhibit the proliferation of EC cells, and compound 596 could reduce the demethylation of LSD1, resulting in increased expression of H3K4me1 and H3K4me2. (2) Compound 596 can downward adjustment mTOR signal transduction and reduce AKT feedback activation. (3) The combination of 596 and mTOR inhibitors can inhibit the migration and invasion of EC cells. This work fills a critical gap in LSD1-targeted therapy for EC and provides a promising chemical entity for precision treatment of this malignancy.

Abnormal activation of the PI3K/AKT/mTOR signaling pathway is present in 24–39% of patients with EC, mTOR is a serine/threonine protein kinase in the PI3K-associated kinase family, divided into two distinct parts (mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTOR2)) [[30], [31], [32]], the activation of mTOR signal can promote the growth, proliferation and invasion of EC cells. Our RNA-seq results are consistent with these findings, and the expression of the upstream regulators PIK3CA and PIK3CG of AKT was down-regulated, as were the expressions of AKT-related genes AKT3, AKTIP, and IRS2 after treatment of EC cells with 596. Importantly, the direct upstream activators of mTORC1, such as RHEB and SLC38A9, were also significantly down-regulated. And inhibition or knockdown of LSD1 reduces the cascade activation of PI3K/AKT/mTOR signals. mTOR inhibitor rapamycin reduces the phosphorylation levels of key downstream targets of the mTOR signaling pathway [33,34]. Rapamycin binds to FKBP12 and specifically inhibits the mTORC1-mTOR axis, but long-term inhibition of mTOR can induce feedback activation of AKT via the mTORC2 complex, limiting the clinical use of the mTOR inhibitor rapamycin as monotherapy [14,35]. A pivotal observation in our study is that 596 effectively reversed rapamycin‑induced AKT feedback activation, as demonstrated at the protein level both in vitro and in vivo. In addition, LSD1 inhibitor 596 combined with rapamycin can significantly reduce the proliferation, migration and invasion ability of EC cells.

Autophagy plays a very important role in the development and progression of tumors [36]. mTOR signal transduction pathway is the core signaling pathway of autophagy. Under normal conditions, mTOR activity is high and autophagy is inhibited. Under starvation or stress conditions, mTOR activity is inhibited, which in turn activates autophagy. Microtubule-associated protein-1 light chain 3 (LC3), divided into LC3-Ⅰ (cytoplasmic form) and LC3-Ⅱ (membrane-bound form), is a key regulator of autophagy [37]. LC3-Ⅱis a signature protein of autophagy, involved in the formation of autophagy and substrate recognition [[36], [37], [38]].Wei et al. found that LSD1 has the role of an autophagy inhibitor in ovarian cancer, and they found that inhibition of LSD1 or knockdown of LSD1 leads to the formation of autophagosomes and the activation of autophagy, and that LSD1 can promote rapamycin-induced autophagy [36,38]. In this study, we found that LSD1 inhibitor 596 resulted in the accumulation of LC3-II in a dose-dependent manner while increasing the level of H3K4me2 in a dose-dependent manner, and that the level of Beclin-1 remained unchanged. Immunofluorescence and transmission electron microscopy also demonstrated the promoting effect of LSD1 inhibitor 596 on autophagy. Functionally, 596 suppressed phosphorylation of the mTORC1 effector p70S6K, thereby relieving mTOR‑mediated repression of autophagy. This resulted in accumulation of the autophagosome marker LC3B‑II and degradation of p62, observations further validated by immunofluorescence and transmission electron microscopy. Notably, autophagy induction was independent of Beclin‑1 upregulation but was directly linked to mTOR pathway suppression. These findings establish a clear mechanistic axis linking LSD1 inhibition to mTOR signaling and the autophagic process, elucidating a central mode of action for 596.

Although our newly developed novel LSD1 inhibitor is one of the few studies on EC at present, there is still a lack of selective LSD1 inhibitor development for EC-specific subtypes, and further studies are needed to elucidate the specific mechanism by which LSD1 inhibitor 596 enhances the effect of rapamycin. Since autophagy is a complex process, further studies are needed to reveal the autophagy process involved in LSD1. In summary, we report the first comprehensive study of a novel LSD1 inhibitor 596 in EC. We delineate a dual mechanism of action: (i) induction of a cytotoxic form of autophagy via mTOR pathway inhibition, and (ii) epigenetic reprogramming that blunts AKT feedback activation triggered by mTOR inhibitors, thereby enabling potent synergistic therapy. These findings establish LSD1 as a compelling therapeutic target in EC and provide a mechanistic rationale for combining LSD1 inhibitors with mTOR inhibitors as a promising treatment strategy. This work establishes a foundational framework for further preclinical development and subsequent clinical assessment of this combination therapy in EC [25].

Abbreviations

LSD1: Histone lysine-specific demethylase 1; EC: Endometrial cancer; RAPA: rapamycin. KO: Knockout; DAPI: 4′,6-Diamidino-2-phenylindole; TCGA The Cancer Genome Atlasm; TORC1: mTOR Complex 1; mTORC2: mTOR Complex 2; BC: Breast cancer. LincRNA: Long non-coding RNA; LC3: Light chain 3.

Ethics approval and consent to participate

Endometrial cancer tissue and adjacent tissues were taken from the Third Affiliated Hospital of Zhengzhou University. All human tissues were collected using the protocol approved by the Ethics Committee of Research on the Molecular Mechanism of LSD1 Regulating the Immunosuppressive Microenvironment of Endometrial Cancer of The Third Clinical Medical College of Zhengzhou university (protocol code 2024-Y151 and date of 2025.01.01).

Availability of data and materials

All of the data obtained and/or analyzed during the current study are available from the corresponding authors upon reasonable request. The raw transcriptome sequencing data generated in this study have been deposited in the [NCBI Sequence Read Archive (SRA) under the accession number [PRJNA1302302].

Funding

This work was supported by the funding from Youth Fund of National Natural Science Foundation of China (82303741); Youth Science and Technology Health Innovation Talent Training Project of Henan Province (YXKC2022019);Lipid metabolism reprogramming is studied in the pathogenesis of official neck cancer(2025-ZZLC-0513-007); Henan Provincial Department of Science and Technology, Natural Science Foundation of Henan Province (242300420082); Science and Technology Research Project of Henan Province (232102310092).

CRediT authorship contribution statement

Chunli Wang: Writing – review & editing, Writing – original draft, Data curation. Dandan Shen: Writing – review & editing, Writing – original draft, Methodology. Nanshan Lin: Writing – review & editing, Methodology. Panjie Wang: Visualization, Methodology. Yaping Bi: Resources, Methodology. Huina Hu: Writing – review & editing, Visualization, Investigation. Yujia Shang: Supervision, Software, Project administration. Chenchen Ren: Methodology, Investigation, Funding acquisition, Conceptualization. Li Yang: Supervision, Software, Resources, Funding acquisition. Qisheng Ma: Supervision, Resources, Methodology.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.