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Original Research Article

DTT 2023; 2(2): 80-87

Published online September 30, 2023 https://doi.org/10.58502/DTT.23.0015

Copyright © The Pharmaceutical Society of Korea.

STM2457 Inhibits Breast Cancer Tumorigenesis via the Inhibition of METTL3

Pratikshya Shrestha, Hyelim Kang, Ah-yeong Song, Sun-young Jo, Poshan Yugal Bhattarai , Hong Seok Choi

College of Pharmacy, Chosun University, Gwangju, Korea

Correspondence to:Hong Seok Choi, chs@chosun.ac.kr; Poshan Yugal Bhattarai, poshanb@chosun.ac.kr

Received: April 28, 2023; Revised: June 9, 2023; Accepted: June 11, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although N6-adenosine methyltransferase (METTL3) is frequently upregulated in breast cancer patients, the anticancer effect of small-molecule inhibitors targeting METTL3 has not yet been studied. The present study aimed to investigate the anti-tumorigenic effects of STM2457, a METTL3 inhibitor, on a panel of breast cancer cells representing distinct clinical subtypes. Measurement of cell viability using MTT assay demonstrated dose- and time-dependent reduction in viability in MCF7, SKBR3, and MDA-MB-231 cells, which are representative of luminal A, HER2-positive, and triple-negative breast cancer subtypes, respectively. Immunoblotting analysis revealed that STM2457 induced pro-apoptotic effects in breast cancer cells, as evidenced by the cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3. In addition, analysis of cell cycle distribution with flow cytometry revealed that it induced cell cycle arrest at G0/G1-phase. Consistently, treatment with STM2457 increased the Annexin V-stained cell population, corroborating the pro-apoptotic effects of the compound. Moreover, analysis of colonies formed by breast cancer cells in a soft agar matrix showed that STM2457 reduced the anchorage-independent growth of breast cancer cells, suggesting its potential to inhibit colony formation. Together, these findings indicate that STM2457 exerts cytotoxic and pro-apoptotic effects in breast cancer cells. Our study demonstrates the therapeutic potential of METTL3 inhibitor STM2457 in treating breast cancer patients.

KeywordsMETTL3 inhibitor, STM2457, targeted chemotherapy, anticancer effects, breast cancer chemotherapy

Breast cancer is a malignancy that develops in the tissues of the breast, usually in the ducts that carry milk to the nipple (ductal carcinoma) or in the glandular tissue that produces milk (lobular carcinoma). It is influenced by a variety of factors (Łukasiewicz et al. 2021). According to the World Health Organization, breast cancer is the most common cancer among women worldwide, with more than 2.3 million diagnoses and 685,000 deaths yearly ((WHO 2023). Based on histopathological staining, breast cancer is generally classified into three groups: ER-positive, HER2-positive, and triple-negative breast cancer (TNBC) (Biswas et al. 1998; Lafcı et al. 2023). Breast cancer is a highly complex disease that arises from genetic and epigenetic changes in a normal mammary cell (Feng et al. 2018). Genetic changes refer to mutations or alterations in DNA sequences, while epigenetic changes refer to heritable changes in gene expression that do not involve alterations in DNA sequences but are mediated by modifications to DNA and histone proteins (Byler et al. 2014). Epigenetic changes have significantly contributed to breast cancer initiation, progression, and therapeutic response (Bhat et al. 2019). Consequently, several inhibitors of epigenetic modification, known as epidrug, such as Fluoro-2’-deoxycytidine (5-FdC), are being investigated or under clinical trials for treating breast cancer patients. 5-Fluoro-2’-deoxycytidine (5-FdC) exerts its biological effects by inhibiting DNA methylation, an epigenetic mechanism that can influence gene expression and cellular phenotype (Veselý and Čihák 1978). Emerging studies show that the chemical modification of RNA, known as epitranscriptome, also plays a crucial role in breast tumorigenesis (Kumari et al. 2021). However, the anticancer effects of the epitranscriptome-targeting drug in breast cancer remain to be explored.

N6-methyladenosine (m6A) is the most common epitranscriptomic modification present in mRNA. Increased m6A modification of oncogenic mRNA promotes breast tumorigenesis by regulating post-transcriptional events such as mRNA stability, alternative splicing, and translation efficiency (Achour et al. 2023; Bhattarai et al. 2023). The m6A modification of mRNA is mediated by a methyltransferase complex consisting of three core subunits: Methyltransferase-like 3 (METTL3), METTL14, and Wilm’s tumor-associated protein (WTAP) (Liu et al. 2014). METTL3 is the sole catalytic subunit of the methyltransferase complex, whereas METTL14 enhances the catalytic function of METTL3 (Wang et al. 2016). On the other hand, WTAP provides structural support to the METTL3-14 complex to facilitate m6A modification (Ping et al. 2014). METTL3 expression is upregulated in breast cancer cells and is associated with poor prognosis in breast cancer patients (Xu et al. 2023). For instance, METTL3-induced m6A modification of EZH2 promotes epithelial-mesenchymal transition and metastasis in breast cancer cells (Hu et al. 2022). Similarly, METTL3 promotes the mRNA stability of PD-L1 in breast cancer cells to evade tumor immune surveillance. Recently, METTL3 has been known to promote breast tumorigenesis by enhancing the translation efficiency of TAZ/EGFR mRNA and by regulating the alternative splicing of MYC mRNA (Achour et al. 2023; Bhattarai et al. 2023). Moreover, inhibition of METTL3 expression with shRNA or CRISPR/Cas9 reduces the invasion and colony formation and induces apoptosis in breast cancer cells, which suggests that the therapeutic potential of METTL3 inhibition in breast cancer (Cai et al. 2018; Achour et al. 2023; Bhattarai et al. 2023). Recently, a highly potent and selective catalytic inhibitor of METTL3, STM2457, has been studied for treating cancers induced by METTL3 overexpression (Xiao et al. 2023). However, the therapeutic efficiency of STM2457 in breast cancer cells is not yet reported.

STM2457 directly binds within the SAM-binding pocket of METTL3, thereby abolishing the catalytic function of METTL3. Treatment of STM2457 in a panel of acute myeloid leukemia (AML) cells revealed significant cytotoxicity. In addition, STM2457 induced substantial apoptosis in AML cells. Examination of the underlying mechanisms revealed that STM2457 reduces the m6A-dependent translation of oncogenic mRNAs such as SP1, BRD4, and MYC in AML cells (Yankova et al. 2021). Further studies have revealed that STM2457 inhibits cholangiocarcinoma, medulloblastoma and restores chemosensitivity in small lung cell carcinoma (Xu et al. 2022; Zhang et al. 2022; Sun et al. 2023). Here, we investigated the anticancer effects of STM2457 on a panel of breast cancer cells that represent three distinct clinical subtypes. Our findings demonstrate that treatment with STM2457 significantly reduced cell viability, inhibited colony formation, and induced apoptosis. These findings support the potential use of STM2457 as a therapeutic agent for breast cancer.

Cell culture

The MCF7, SKBR3, and MDA-MB231 breast cancer cell lines were purchased from the American Type Culture Collection (ATCC). MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. SKBR3 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS. MDA-MB-231cells were maintained in (Minimum essential medium) MEM supplemented with 10% FBS. The cultures were maintained at 37℃ in a humidified atmosphere with 5% CO2.

Antibodies and reagents

Total caspase 3 (9665, western blot (WB) 1:1,000) and cleaved caspase-3 (9661, WB 1:1,000) were received from Cell Signaling Technology Inc. (Danvers, MA, USA). PARP (sc-7150, WB 1:5,000) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin (A1978, WB 1:10,000) was purchased from Sigma-Aldrich (MO, USA). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Invitrogen (Carlsbad, CA, USA). STM2457 (HY-134836) was purchased from MedChemExpress (NJ, USA). Eagle’s minimal essential medium, L-glutamine, gentamicin, and fetal bovine serum (FBS) were purchased from Invitrogen.

Cell viability assay

To determine the effects of STM2457 on cell viability, MTT assays were performed using the EZ-Cytox cell viability assay kit (Daeli Lab Service, Seoul, Republic of Korea). Briefly, MCF-7, MDA-MB-231, and SKBR3 breast cancer cells were seeded at a density of 5,000 cells per well in 96-well plates and cultured in a humidified atmosphere containing 5% CO2 at 37℃. After 24 hours of culture, the cells were treated with various concentrations of STM2457 for 24, 48, 72, or 96 hours. Subsequently, 10 µL of tetrazolium salt was added to each well, and the cells were incubated for another 4 hours. The resulting formazan crystals, generated by viable cells, were solubilized using DMSO, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). The absorbance values determined the percentage of viable cells in each treatment group.

Protein immunoblotting

For protein analysis, breast cancer cells treated with relevant doses of STM2457 were grown in monolayers and then harvested and washed with phosphate-buffered saline. Subsequently, the cells were lysed using RIPA buffer that contained 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid, 1% NP40, 1 mM NaF, 0.2 mM phenylmethylsulphonyl fluoride, 0.1 mM sodium orthovanadate, and protease/phosphatase inhibitor cocktail. First, the protein concentration of each sample was determined using a protein assay kit. Next, protein samples ranging from 10 to 30 µg were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred onto polyvinylidene fluoride membranes. The membranes were then probed with the appropriate primary antibodies against target proteins. Finally, the immunoblots were visualized using a SuperSignal West Femto chemiluminescence substrate (ThermoScientific) and imaged using an Amersham imager 680 (GE Healthcare, Chicago, IL, USA).

Cell cycle analysis

Cells were seeded and treated with the indicated chemical for 48 hours. First, the cells were washed, fixed with 70% ethanol, and then added 200 µL of MuseTM cell cycle reagent (EMD Millipore Corp. Billerica, MA, USA). Then, cells were incubated at 25℃ for 30 min in the dark. The cell cycle was analyzed using a Muse cell analyzer.

Annexin V- Propidium Iodide (PI)

The cell apoptosis was analyzed using a MuseTM Annexin V and Dead Cell kit (Merck Millipore, Billerica, MA, USA). Briefly, cells were seeded and treated with the indicated chemical for 48 hours, and then 100 µL of MuseTM Annexin V and dead cell reagent was added in a 100 µL cell suspension containing 1% BSA. Then the cells were incubated at 25℃ for 30 mins in the dark, followed by analysis with a Muse cell analyzer (EMD Millipore Corporation).

Anchorage-independent cell transformation (soft agar assay)

To investigate the effects of the indicated drugs on cell colony formation, 8,000 cells were treated with STM2457 dose-dependently and cultured in 1 mL of 0.3% Eagle’s basal medium supplemented with 10% FBS. The cultures were maintained at 37℃ in a humidified atmosphere containing 5% CO2 for 14 days. The resulting cell colonies were then assessed by microscopy using an Axiovert 200 M microscope and Axio Vision software (Carl Zeiss, Thornwood, NY, USA). A total of six images were captured for each experimental group. ImageJ software (NIH, Bethesda, MD, USA) was used to be determined the number of colonies.

STM2457 induces cytotoxicity in breast cancer cells

A previous study has identified STM2457 as a potent inhibitor of METTL3 and 14 methyltransferase activity (Fig. 1A) (Yankova et al. 2021). To further investigate the potential of STM2457 as a therapeutic agent for breast cancer, we performed MTT assays to study the effect of STM2457 on the viability of MCF7, SKBR3, and MDA-MB-231 breast cancer cells by varying the dosage and duration of treatment. The results showed that STM2457 treatment resulted in decreased cell viability dependent on the dose and duration of treatment (Fig. 1B-1D). At the highest concentration of STM2457 (20 µM), cell viability was reduced to 50% compared to the control cells after 96 hours of treatment (Fig. 1B-1D). Notably, cytotoxic effects of STM2457 proportionally increased with the duration of treatment, and significant reductions in cell viability at the concentration of 7 µM, 10 µM, and 20 µM were observed after 72 and 96 hours of treatment. Our results suggest that STM2457 has potent cytotoxic effects on breast cancer cells and that these effects are consistent across all three cell lines tested.

Figure 1.Effects of STM2457 on cell viability of MCF7, SKBR3, and MDA- MB-231 cells. (A) Chemical structure of N-((6-((cyclohexyl methyl)amino)methyl)imidazole[1,2-a] pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide, STM2457. Half maximal inhibitory concentration (IC50) value against methyltransferase activity of METTL3-METTL14 complex is shown below structure. (B–D) MCF7, SKBR3, and MDA-MB-231 cells were treated with different concentrations of STM2457 for 24 h, 48 h, 72 h, and 96 h, respectively. The cell viability was measured using EZ-Cytox reagent as described in materials and methods. Values represent mean ± standard deviation (S.D.), N = 3. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

STM2457 induces apoptosis in breast cancer cells via caspase 3 and PARP activation Following our initial assessment of the cytotoxic effects of STM2457 on breast cancer cell viability. Next, we investigated the effects of STM2457 treatment on apoptotic cell death by examining the expression of cleaved caspase 3 and cleaved PARP, which are markers of apoptosis, in MCF7, SKBR3, and MDA-MB-231 breast cancer cells. The western blotting results showed STM2457-induced cleavage of PARP in all three cell lines and cleavage of caspase 3 in SKBR3 and MDA-MB-231 cells (Fig. 2A). In agreement with a previous report, we didn’t observe the expression of caspase 3 in MCF7 cells (Jänicke 2009). We performed a cell cycle analysis to further investigate the effects of STM2457 treatment on cell cycle progression. Consistently, cell cycle analysis revealed that treatment of STM2457 causes cell cycle arrest in G0/G1 phase in these breast cancer cell lines (Fig. 2B). Annexin V-FITC/PI double labeling was used to further analyze the degree of cell apoptosis. STM2457 treatment raised the proportion of late-stage apoptotic cells (Annexin V-FITC and PI-positive cells) in the previously mentioned breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231) in comparison to the control group dose-dependently raised (Fig. 3). Together, these data suggest that STM2457 induces apoptosis and cell cycle arrest in breast cancer cells.

Figure 2.Effects of STM2457 on apoptotic signaling pathway and cell cycle arrest in MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with the indicated doses of STM2457 for 48 h and harvested. The cleaved and total proteins of PARP and caspase 3 in whole-cell lysates were determined by immunoblotting analysis using indicated antibodies. (B) Cells were treated with the indicated doses of STM2457 for 48 h, and cell cycle distribution was analyzed using a MuseTM cell analyzer. The percentage of cells in the G0/G1, S, and G2/M phases are shown in the inset.

Figure 3.Effects of STM2457 on cell apoptosis in MCF7, SKBR3, and MDA-MB-231 cells. (A–C) Cells were treated with either DMSO or 5 µM, 10 µM, and 20 µM STM2457 for 48 h. The apoptosis profile was detected using MuseTM Annexin V–7AAD dead cell assay. The percentage of live, early apoptosis (Apop), late Apop, and dead cells are shown within each figure.

STM2457 exhibits an inhibitory effect on colony formation ability in breast cancer cells

The tumorigenic potential of breast cancer cells was evaluated by the soft agar assay using MCF7, SKBR3, and MDA-MB-231 cells (Fig. 4A). Our result demonstrates that the treatment with STM2457 dose-dependently led to a significant reduction in both the size and the number of colonies formed by all three cells (Fig. 4B). These findings indicate that STM2457 may have the therapeutic potential for breast cancer treatment.

Figure 4.Inhibitory effects of STM2457 on anchorage-independent growth of MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with DMSO or indicated concentrations of STM2457 and maintained for 2 weeks in soft agar. Representative figures from six different experiments are shown. (B) The colonies formed by MCF7, SKBR3, and MDA-MB-231 cells were counted using Fiji software. Values represent mean ± S.D, N = 6. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Breast cancer represents a complex malignancy that exhibits significant heterogeneity and can be further characterized into distinct molecular subtypes based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). Specifically, hormone receptor-positive tumors express either ER or PR, which are classified as luminal A or luminal B subtypes. On the other hand, HER2-positive tumors are characterized by the amplification of the HER2 gene and belong to the HER2-positive subtype. Finally, tumors that lack ER, PR, and HER2 expression are classified as triple-negative breast cancer (Biswas et al. 1998; Johnson et al. 2020; Lafcı et al. 2023). Breast cancer is difficult to treat due to its heterogeneity and different molecular subtypes (Harbeck and Gnant 2017). Recent studies have shown that the deregulation of post-transcriptional events, such as mRNA translation and stability, is responsible for the heterogeneity of different types of cancers. For instance, aberrant mRNA translation is crucial in breast cancer heterogeneity (Polyak 2011). Interestingly, dysregulation of translation initiation factors, such as eIF4E, has been linked to increased cell proliferation, survival, and invasiveness in various types of cancer (de la Parra et al. 2018). Similarly, the mammalian target of the rapamycin (mTOR) system, which controls mRNA translation, has been linked to the increased growth of breast cancer and has a poor prognosis (Ueng et al. 2012). Dysregulation of mRNA stability can lead to gene expression changes in various aspects of cancer biology (Griseri and Pagès 2014). Emerging studies show that m6A modification of mRNA mediated by METTL3 plays a crucial role in regulating post-transcriptional events (Zhao et al. 2017; Yang et al. 2018; Shi et al. 2019). m6A increases the stability and translation of keratin 7 (KRT7), which promotes breast cancer lung metastasis (Chen et al. 2021). METTL3 promotes proliferation and inhibits apoptosis in breast cancer cells by targeting BCL2 and EZH2 mRNA (Wang et al. 2020; Hu et al. 2022). Also, METTL3 controls alternative splicing of MYC mRNA to promote breast tumorigenesis (Achour et al. 2023). METTL3 plays a crucial role in breast tumorigenesis; therefore, inhibiting METTL3 can have important therapeutic implications. Recently a promising METTL3 inhibitor STM2457 has been developed, which can be a therapeutic drug. The present study aimed to investigate the cytotoxic effects of STM2457 on MCF7, SKBR3, and MDA-MB-231 breast cancer cells. It is already reported that STM2457 significantly reduces the cell viability in the AML cell (Yankova et al. 2021). This finding suggests that STM2457 has potent cytotoxic effects against cancer cells. Our results indicate that STM2457 treatment led to a dose- and time-dependent reduction in cell viability in all three breast cancer cell lines. Moreover, the observed cytotoxic effects were more prominent with the increasing treatment of STM2457.

Cleaved caspase-3, cleaved PARP-1, and cleaved caspase-12 cause osteoblast apoptosis (Kong et al. 2022). However, the effect of STM2457 on the expression or stability of the apoptosis-related protein in breast cancer has not been studied yet. Our results suggest that STM2457 induced apoptosis in breast cancer cells, as evidenced by the activation of caspase 3 and PARP cleavage in all three cell lines tested. Additionally, STM2457 treatment led to cell cycle arrest in the G0/G1 phase, further supporting its ability to induce apoptosis in breast cancer cells. A recent study has shown that the knockout of METTL3 suppresses the colony formation capacity in breast cancer cells (Xu et al. 2023). In our research, the METTL3 inhibitor STM2457 was used to assess the impact on the anchorage-independent growth of breast cancer cells in three subtypes of breast cancer cells. In agreement with METTL3 knockdown studies, STM2457 inhibited colony formation ability in breast cancer cells, as evidenced by the significant reduction in both the size and the number of colonies formed by MCF7, SKBR3, and MDA-MB-231 cells treated with STM2457. Together, these results suggest that STM2457 may have the potential as a therapeutic agent for breast cancer treatment.

In conclusion, the present study demonstrates that STM2457 exhibits potent cytotoxic effects on breast cancer cells, induces cell apoptosis, and impedes clonogenicity. Based on these results, STM2457 may be proposed as a novel anticancer agent for breast cancer treatment. However, further studies are required to investigate its efficacy and safety in vivo before clinical translation.

The authors declare that they have no conflict of interest.

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Article

Original Research Article

DTT 2023; 2(2): 80-87

Published online September 30, 2023 https://doi.org/10.58502/DTT.23.0015

Copyright © The Pharmaceutical Society of Korea.

STM2457 Inhibits Breast Cancer Tumorigenesis via the Inhibition of METTL3

Pratikshya Shrestha, Hyelim Kang, Ah-yeong Song, Sun-young Jo, Poshan Yugal Bhattarai , Hong Seok Choi

College of Pharmacy, Chosun University, Gwangju, Korea

Correspondence to:Hong Seok Choi, chs@chosun.ac.kr; Poshan Yugal Bhattarai, poshanb@chosun.ac.kr

Received: April 28, 2023; Revised: June 9, 2023; Accepted: June 11, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Although N6-adenosine methyltransferase (METTL3) is frequently upregulated in breast cancer patients, the anticancer effect of small-molecule inhibitors targeting METTL3 has not yet been studied. The present study aimed to investigate the anti-tumorigenic effects of STM2457, a METTL3 inhibitor, on a panel of breast cancer cells representing distinct clinical subtypes. Measurement of cell viability using MTT assay demonstrated dose- and time-dependent reduction in viability in MCF7, SKBR3, and MDA-MB-231 cells, which are representative of luminal A, HER2-positive, and triple-negative breast cancer subtypes, respectively. Immunoblotting analysis revealed that STM2457 induced pro-apoptotic effects in breast cancer cells, as evidenced by the cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3. In addition, analysis of cell cycle distribution with flow cytometry revealed that it induced cell cycle arrest at G0/G1-phase. Consistently, treatment with STM2457 increased the Annexin V-stained cell population, corroborating the pro-apoptotic effects of the compound. Moreover, analysis of colonies formed by breast cancer cells in a soft agar matrix showed that STM2457 reduced the anchorage-independent growth of breast cancer cells, suggesting its potential to inhibit colony formation. Together, these findings indicate that STM2457 exerts cytotoxic and pro-apoptotic effects in breast cancer cells. Our study demonstrates the therapeutic potential of METTL3 inhibitor STM2457 in treating breast cancer patients.

Keywords: METTL3 inhibitor, STM2457, targeted chemotherapy, anticancer effects, breast cancer chemotherapy

Introduction

Breast cancer is a malignancy that develops in the tissues of the breast, usually in the ducts that carry milk to the nipple (ductal carcinoma) or in the glandular tissue that produces milk (lobular carcinoma). It is influenced by a variety of factors (Łukasiewicz et al. 2021). According to the World Health Organization, breast cancer is the most common cancer among women worldwide, with more than 2.3 million diagnoses and 685,000 deaths yearly ((WHO 2023). Based on histopathological staining, breast cancer is generally classified into three groups: ER-positive, HER2-positive, and triple-negative breast cancer (TNBC) (Biswas et al. 1998; Lafcı et al. 2023). Breast cancer is a highly complex disease that arises from genetic and epigenetic changes in a normal mammary cell (Feng et al. 2018). Genetic changes refer to mutations or alterations in DNA sequences, while epigenetic changes refer to heritable changes in gene expression that do not involve alterations in DNA sequences but are mediated by modifications to DNA and histone proteins (Byler et al. 2014). Epigenetic changes have significantly contributed to breast cancer initiation, progression, and therapeutic response (Bhat et al. 2019). Consequently, several inhibitors of epigenetic modification, known as epidrug, such as Fluoro-2’-deoxycytidine (5-FdC), are being investigated or under clinical trials for treating breast cancer patients. 5-Fluoro-2’-deoxycytidine (5-FdC) exerts its biological effects by inhibiting DNA methylation, an epigenetic mechanism that can influence gene expression and cellular phenotype (Veselý and Čihák 1978). Emerging studies show that the chemical modification of RNA, known as epitranscriptome, also plays a crucial role in breast tumorigenesis (Kumari et al. 2021). However, the anticancer effects of the epitranscriptome-targeting drug in breast cancer remain to be explored.

N6-methyladenosine (m6A) is the most common epitranscriptomic modification present in mRNA. Increased m6A modification of oncogenic mRNA promotes breast tumorigenesis by regulating post-transcriptional events such as mRNA stability, alternative splicing, and translation efficiency (Achour et al. 2023; Bhattarai et al. 2023). The m6A modification of mRNA is mediated by a methyltransferase complex consisting of three core subunits: Methyltransferase-like 3 (METTL3), METTL14, and Wilm’s tumor-associated protein (WTAP) (Liu et al. 2014). METTL3 is the sole catalytic subunit of the methyltransferase complex, whereas METTL14 enhances the catalytic function of METTL3 (Wang et al. 2016). On the other hand, WTAP provides structural support to the METTL3-14 complex to facilitate m6A modification (Ping et al. 2014). METTL3 expression is upregulated in breast cancer cells and is associated with poor prognosis in breast cancer patients (Xu et al. 2023). For instance, METTL3-induced m6A modification of EZH2 promotes epithelial-mesenchymal transition and metastasis in breast cancer cells (Hu et al. 2022). Similarly, METTL3 promotes the mRNA stability of PD-L1 in breast cancer cells to evade tumor immune surveillance. Recently, METTL3 has been known to promote breast tumorigenesis by enhancing the translation efficiency of TAZ/EGFR mRNA and by regulating the alternative splicing of MYC mRNA (Achour et al. 2023; Bhattarai et al. 2023). Moreover, inhibition of METTL3 expression with shRNA or CRISPR/Cas9 reduces the invasion and colony formation and induces apoptosis in breast cancer cells, which suggests that the therapeutic potential of METTL3 inhibition in breast cancer (Cai et al. 2018; Achour et al. 2023; Bhattarai et al. 2023). Recently, a highly potent and selective catalytic inhibitor of METTL3, STM2457, has been studied for treating cancers induced by METTL3 overexpression (Xiao et al. 2023). However, the therapeutic efficiency of STM2457 in breast cancer cells is not yet reported.

STM2457 directly binds within the SAM-binding pocket of METTL3, thereby abolishing the catalytic function of METTL3. Treatment of STM2457 in a panel of acute myeloid leukemia (AML) cells revealed significant cytotoxicity. In addition, STM2457 induced substantial apoptosis in AML cells. Examination of the underlying mechanisms revealed that STM2457 reduces the m6A-dependent translation of oncogenic mRNAs such as SP1, BRD4, and MYC in AML cells (Yankova et al. 2021). Further studies have revealed that STM2457 inhibits cholangiocarcinoma, medulloblastoma and restores chemosensitivity in small lung cell carcinoma (Xu et al. 2022; Zhang et al. 2022; Sun et al. 2023). Here, we investigated the anticancer effects of STM2457 on a panel of breast cancer cells that represent three distinct clinical subtypes. Our findings demonstrate that treatment with STM2457 significantly reduced cell viability, inhibited colony formation, and induced apoptosis. These findings support the potential use of STM2457 as a therapeutic agent for breast cancer.

Materials and Methods

Cell culture

The MCF7, SKBR3, and MDA-MB231 breast cancer cell lines were purchased from the American Type Culture Collection (ATCC). MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. SKBR3 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS. MDA-MB-231cells were maintained in (Minimum essential medium) MEM supplemented with 10% FBS. The cultures were maintained at 37℃ in a humidified atmosphere with 5% CO2.

Antibodies and reagents

Total caspase 3 (9665, western blot (WB) 1:1,000) and cleaved caspase-3 (9661, WB 1:1,000) were received from Cell Signaling Technology Inc. (Danvers, MA, USA). PARP (sc-7150, WB 1:5,000) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin (A1978, WB 1:10,000) was purchased from Sigma-Aldrich (MO, USA). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Invitrogen (Carlsbad, CA, USA). STM2457 (HY-134836) was purchased from MedChemExpress (NJ, USA). Eagle’s minimal essential medium, L-glutamine, gentamicin, and fetal bovine serum (FBS) were purchased from Invitrogen.

Cell viability assay

To determine the effects of STM2457 on cell viability, MTT assays were performed using the EZ-Cytox cell viability assay kit (Daeli Lab Service, Seoul, Republic of Korea). Briefly, MCF-7, MDA-MB-231, and SKBR3 breast cancer cells were seeded at a density of 5,000 cells per well in 96-well plates and cultured in a humidified atmosphere containing 5% CO2 at 37℃. After 24 hours of culture, the cells were treated with various concentrations of STM2457 for 24, 48, 72, or 96 hours. Subsequently, 10 µL of tetrazolium salt was added to each well, and the cells were incubated for another 4 hours. The resulting formazan crystals, generated by viable cells, were solubilized using DMSO, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). The absorbance values determined the percentage of viable cells in each treatment group.

Protein immunoblotting

For protein analysis, breast cancer cells treated with relevant doses of STM2457 were grown in monolayers and then harvested and washed with phosphate-buffered saline. Subsequently, the cells were lysed using RIPA buffer that contained 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid, 1% NP40, 1 mM NaF, 0.2 mM phenylmethylsulphonyl fluoride, 0.1 mM sodium orthovanadate, and protease/phosphatase inhibitor cocktail. First, the protein concentration of each sample was determined using a protein assay kit. Next, protein samples ranging from 10 to 30 µg were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred onto polyvinylidene fluoride membranes. The membranes were then probed with the appropriate primary antibodies against target proteins. Finally, the immunoblots were visualized using a SuperSignal West Femto chemiluminescence substrate (ThermoScientific) and imaged using an Amersham imager 680 (GE Healthcare, Chicago, IL, USA).

Cell cycle analysis

Cells were seeded and treated with the indicated chemical for 48 hours. First, the cells were washed, fixed with 70% ethanol, and then added 200 µL of MuseTM cell cycle reagent (EMD Millipore Corp. Billerica, MA, USA). Then, cells were incubated at 25℃ for 30 min in the dark. The cell cycle was analyzed using a Muse cell analyzer.

Annexin V- Propidium Iodide (PI)

The cell apoptosis was analyzed using a MuseTM Annexin V and Dead Cell kit (Merck Millipore, Billerica, MA, USA). Briefly, cells were seeded and treated with the indicated chemical for 48 hours, and then 100 µL of MuseTM Annexin V and dead cell reagent was added in a 100 µL cell suspension containing 1% BSA. Then the cells were incubated at 25℃ for 30 mins in the dark, followed by analysis with a Muse cell analyzer (EMD Millipore Corporation).

Anchorage-independent cell transformation (soft agar assay)

To investigate the effects of the indicated drugs on cell colony formation, 8,000 cells were treated with STM2457 dose-dependently and cultured in 1 mL of 0.3% Eagle’s basal medium supplemented with 10% FBS. The cultures were maintained at 37℃ in a humidified atmosphere containing 5% CO2 for 14 days. The resulting cell colonies were then assessed by microscopy using an Axiovert 200 M microscope and Axio Vision software (Carl Zeiss, Thornwood, NY, USA). A total of six images were captured for each experimental group. ImageJ software (NIH, Bethesda, MD, USA) was used to be determined the number of colonies.

Results

STM2457 induces cytotoxicity in breast cancer cells

A previous study has identified STM2457 as a potent inhibitor of METTL3 and 14 methyltransferase activity (Fig. 1A) (Yankova et al. 2021). To further investigate the potential of STM2457 as a therapeutic agent for breast cancer, we performed MTT assays to study the effect of STM2457 on the viability of MCF7, SKBR3, and MDA-MB-231 breast cancer cells by varying the dosage and duration of treatment. The results showed that STM2457 treatment resulted in decreased cell viability dependent on the dose and duration of treatment (Fig. 1B-1D). At the highest concentration of STM2457 (20 µM), cell viability was reduced to 50% compared to the control cells after 96 hours of treatment (Fig. 1B-1D). Notably, cytotoxic effects of STM2457 proportionally increased with the duration of treatment, and significant reductions in cell viability at the concentration of 7 µM, 10 µM, and 20 µM were observed after 72 and 96 hours of treatment. Our results suggest that STM2457 has potent cytotoxic effects on breast cancer cells and that these effects are consistent across all three cell lines tested.

Figure 1. Effects of STM2457 on cell viability of MCF7, SKBR3, and MDA- MB-231 cells. (A) Chemical structure of N-((6-((cyclohexyl methyl)amino)methyl)imidazole[1,2-a] pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide, STM2457. Half maximal inhibitory concentration (IC50) value against methyltransferase activity of METTL3-METTL14 complex is shown below structure. (B–D) MCF7, SKBR3, and MDA-MB-231 cells were treated with different concentrations of STM2457 for 24 h, 48 h, 72 h, and 96 h, respectively. The cell viability was measured using EZ-Cytox reagent as described in materials and methods. Values represent mean ± standard deviation (S.D.), N = 3. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

STM2457 induces apoptosis in breast cancer cells via caspase 3 and PARP activation Following our initial assessment of the cytotoxic effects of STM2457 on breast cancer cell viability. Next, we investigated the effects of STM2457 treatment on apoptotic cell death by examining the expression of cleaved caspase 3 and cleaved PARP, which are markers of apoptosis, in MCF7, SKBR3, and MDA-MB-231 breast cancer cells. The western blotting results showed STM2457-induced cleavage of PARP in all three cell lines and cleavage of caspase 3 in SKBR3 and MDA-MB-231 cells (Fig. 2A). In agreement with a previous report, we didn’t observe the expression of caspase 3 in MCF7 cells (Jänicke 2009). We performed a cell cycle analysis to further investigate the effects of STM2457 treatment on cell cycle progression. Consistently, cell cycle analysis revealed that treatment of STM2457 causes cell cycle arrest in G0/G1 phase in these breast cancer cell lines (Fig. 2B). Annexin V-FITC/PI double labeling was used to further analyze the degree of cell apoptosis. STM2457 treatment raised the proportion of late-stage apoptotic cells (Annexin V-FITC and PI-positive cells) in the previously mentioned breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231) in comparison to the control group dose-dependently raised (Fig. 3). Together, these data suggest that STM2457 induces apoptosis and cell cycle arrest in breast cancer cells.

Figure 2. Effects of STM2457 on apoptotic signaling pathway and cell cycle arrest in MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with the indicated doses of STM2457 for 48 h and harvested. The cleaved and total proteins of PARP and caspase 3 in whole-cell lysates were determined by immunoblotting analysis using indicated antibodies. (B) Cells were treated with the indicated doses of STM2457 for 48 h, and cell cycle distribution was analyzed using a MuseTM cell analyzer. The percentage of cells in the G0/G1, S, and G2/M phases are shown in the inset.

Figure 3. Effects of STM2457 on cell apoptosis in MCF7, SKBR3, and MDA-MB-231 cells. (A–C) Cells were treated with either DMSO or 5 µM, 10 µM, and 20 µM STM2457 for 48 h. The apoptosis profile was detected using MuseTM Annexin V–7AAD dead cell assay. The percentage of live, early apoptosis (Apop), late Apop, and dead cells are shown within each figure.

STM2457 exhibits an inhibitory effect on colony formation ability in breast cancer cells

The tumorigenic potential of breast cancer cells was evaluated by the soft agar assay using MCF7, SKBR3, and MDA-MB-231 cells (Fig. 4A). Our result demonstrates that the treatment with STM2457 dose-dependently led to a significant reduction in both the size and the number of colonies formed by all three cells (Fig. 4B). These findings indicate that STM2457 may have the therapeutic potential for breast cancer treatment.

Figure 4. Inhibitory effects of STM2457 on anchorage-independent growth of MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with DMSO or indicated concentrations of STM2457 and maintained for 2 weeks in soft agar. Representative figures from six different experiments are shown. (B) The colonies formed by MCF7, SKBR3, and MDA-MB-231 cells were counted using Fiji software. Values represent mean ± S.D, N = 6. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

Breast cancer represents a complex malignancy that exhibits significant heterogeneity and can be further characterized into distinct molecular subtypes based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). Specifically, hormone receptor-positive tumors express either ER or PR, which are classified as luminal A or luminal B subtypes. On the other hand, HER2-positive tumors are characterized by the amplification of the HER2 gene and belong to the HER2-positive subtype. Finally, tumors that lack ER, PR, and HER2 expression are classified as triple-negative breast cancer (Biswas et al. 1998; Johnson et al. 2020; Lafcı et al. 2023). Breast cancer is difficult to treat due to its heterogeneity and different molecular subtypes (Harbeck and Gnant 2017). Recent studies have shown that the deregulation of post-transcriptional events, such as mRNA translation and stability, is responsible for the heterogeneity of different types of cancers. For instance, aberrant mRNA translation is crucial in breast cancer heterogeneity (Polyak 2011). Interestingly, dysregulation of translation initiation factors, such as eIF4E, has been linked to increased cell proliferation, survival, and invasiveness in various types of cancer (de la Parra et al. 2018). Similarly, the mammalian target of the rapamycin (mTOR) system, which controls mRNA translation, has been linked to the increased growth of breast cancer and has a poor prognosis (Ueng et al. 2012). Dysregulation of mRNA stability can lead to gene expression changes in various aspects of cancer biology (Griseri and Pagès 2014). Emerging studies show that m6A modification of mRNA mediated by METTL3 plays a crucial role in regulating post-transcriptional events (Zhao et al. 2017; Yang et al. 2018; Shi et al. 2019). m6A increases the stability and translation of keratin 7 (KRT7), which promotes breast cancer lung metastasis (Chen et al. 2021). METTL3 promotes proliferation and inhibits apoptosis in breast cancer cells by targeting BCL2 and EZH2 mRNA (Wang et al. 2020; Hu et al. 2022). Also, METTL3 controls alternative splicing of MYC mRNA to promote breast tumorigenesis (Achour et al. 2023). METTL3 plays a crucial role in breast tumorigenesis; therefore, inhibiting METTL3 can have important therapeutic implications. Recently a promising METTL3 inhibitor STM2457 has been developed, which can be a therapeutic drug. The present study aimed to investigate the cytotoxic effects of STM2457 on MCF7, SKBR3, and MDA-MB-231 breast cancer cells. It is already reported that STM2457 significantly reduces the cell viability in the AML cell (Yankova et al. 2021). This finding suggests that STM2457 has potent cytotoxic effects against cancer cells. Our results indicate that STM2457 treatment led to a dose- and time-dependent reduction in cell viability in all three breast cancer cell lines. Moreover, the observed cytotoxic effects were more prominent with the increasing treatment of STM2457.

Cleaved caspase-3, cleaved PARP-1, and cleaved caspase-12 cause osteoblast apoptosis (Kong et al. 2022). However, the effect of STM2457 on the expression or stability of the apoptosis-related protein in breast cancer has not been studied yet. Our results suggest that STM2457 induced apoptosis in breast cancer cells, as evidenced by the activation of caspase 3 and PARP cleavage in all three cell lines tested. Additionally, STM2457 treatment led to cell cycle arrest in the G0/G1 phase, further supporting its ability to induce apoptosis in breast cancer cells. A recent study has shown that the knockout of METTL3 suppresses the colony formation capacity in breast cancer cells (Xu et al. 2023). In our research, the METTL3 inhibitor STM2457 was used to assess the impact on the anchorage-independent growth of breast cancer cells in three subtypes of breast cancer cells. In agreement with METTL3 knockdown studies, STM2457 inhibited colony formation ability in breast cancer cells, as evidenced by the significant reduction in both the size and the number of colonies formed by MCF7, SKBR3, and MDA-MB-231 cells treated with STM2457. Together, these results suggest that STM2457 may have the potential as a therapeutic agent for breast cancer treatment.

In conclusion, the present study demonstrates that STM2457 exhibits potent cytotoxic effects on breast cancer cells, induces cell apoptosis, and impedes clonogenicity. Based on these results, STM2457 may be proposed as a novel anticancer agent for breast cancer treatment. However, further studies are required to investigate its efficacy and safety in vivo before clinical translation.

Acknowledgements

None.

Conflict of interest

The authors declare that they have no conflict of interest.

Fig 1.

Figure 1.Effects of STM2457 on cell viability of MCF7, SKBR3, and MDA- MB-231 cells. (A) Chemical structure of N-((6-((cyclohexyl methyl)amino)methyl)imidazole[1,2-a] pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide, STM2457. Half maximal inhibitory concentration (IC50) value against methyltransferase activity of METTL3-METTL14 complex is shown below structure. (B–D) MCF7, SKBR3, and MDA-MB-231 cells were treated with different concentrations of STM2457 for 24 h, 48 h, 72 h, and 96 h, respectively. The cell viability was measured using EZ-Cytox reagent as described in materials and methods. Values represent mean ± standard deviation (S.D.), N = 3. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.
Drug Targets and Therapeutics 2023; 2: 80-87https://doi.org/10.58502/DTT.23.0015

Fig 2.

Figure 2.Effects of STM2457 on apoptotic signaling pathway and cell cycle arrest in MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with the indicated doses of STM2457 for 48 h and harvested. The cleaved and total proteins of PARP and caspase 3 in whole-cell lysates were determined by immunoblotting analysis using indicated antibodies. (B) Cells were treated with the indicated doses of STM2457 for 48 h, and cell cycle distribution was analyzed using a MuseTM cell analyzer. The percentage of cells in the G0/G1, S, and G2/M phases are shown in the inset.
Drug Targets and Therapeutics 2023; 2: 80-87https://doi.org/10.58502/DTT.23.0015

Fig 3.

Figure 3.Effects of STM2457 on cell apoptosis in MCF7, SKBR3, and MDA-MB-231 cells. (A–C) Cells were treated with either DMSO or 5 µM, 10 µM, and 20 µM STM2457 for 48 h. The apoptosis profile was detected using MuseTM Annexin V–7AAD dead cell assay. The percentage of live, early apoptosis (Apop), late Apop, and dead cells are shown within each figure.
Drug Targets and Therapeutics 2023; 2: 80-87https://doi.org/10.58502/DTT.23.0015

Fig 4.

Figure 4.Inhibitory effects of STM2457 on anchorage-independent growth of MCF7, SKBR3, and MDA-MB-231 cells. (A) Cells were treated with DMSO or indicated concentrations of STM2457 and maintained for 2 weeks in soft agar. Representative figures from six different experiments are shown. (B) The colonies formed by MCF7, SKBR3, and MDA-MB-231 cells were counted using Fiji software. Values represent mean ± S.D, N = 6. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.
Drug Targets and Therapeutics 2023; 2: 80-87https://doi.org/10.58502/DTT.23.0015

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