G9a/GLP inhibitor UNC0642 induces growth arrest in colorectal cancer via induction of ROS generation, coordinated overexpression of GADD genes, and the JNK/p38 MAPK pathway

doi:10.21203/rs.3.rs-4532655/v1

Download PDF

Research Article

G9a/GLP inhibitor UNC0642 induces growth arrest in colorectal cancer via induction of ROS generation, coordinated overexpression of GADD genes, and the JNK/p38 MAPK pathway

https://doi.org/10.21203/rs.3.rs-4532655/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer death worldwide. Hence, new therapeutic agents are urgently needed to manage colorectal cancer effectively. The epigenetic abnormalities of chromatin drive the initiation and progression of human cancers. Therefore, DNA methyltransferase and histone deacetylase inhibitors have been developed for clinical use, but no inhibitors of histone methyltransferase are under development for human cancer therapy. H3K9 methyltransferase G9a/G9a-like protein (GLP) is overexpressed in various human cancers, and its knockdown inhibits cancer cell growth, suggesting the cancer-suppressing potential of G9a/GLP inhibitors. UNC0642 is a potent and selective G9a/GLP inhibitor that suppresses breast cancer cell survival and tumorigenesis. In this study, we first tested the anticancer activity of UNC0642 in CRC in vitro and in the mouse xenograft model and further explored the underlying molecular mechanism. We found that UNC0642 inhibited CRC cell proliferation, induced G2/M-phase cell cycle arrest, increased reactive oxygen species level and expression of growth arrest– and DNA damage–inducible genes (GADD), activated p38, and JNK/MAPK signaling pathway. The findings of this study provided new evidence for exploring the potential of UNC0642 for tumor suppressor drug development and might help further explore the biological functions of G9a/GLP in CRC.

Colorectal cancer

GADD genes

G9a/GLP

MAPK

ROS

UNC0642

The incidence of colorectal cancer (CRC) increases every year, makes it the third most common malignancy globally and the second leading cause of cancer-related death, accounting for about 1.36 million deaths every year. New therapeutic agents to effectively manage CRC are urgently needed. (Keum N, 2019)A critical mechanism for the initiation and progression of CRC is the accumulation of various genetic and epigenetic changes in colonic epithelial cells. Targeting epigenetic changes to develop clinical biomarkers for diagnostic, prognostic, and therapeutic applications in CRC has received increasing attention(Lao VV, 2011; Okugawa Y, 2015). DNA methylation and histone acetylation have been thoroughly researched, and the inhibitors of both DNA methyltransferase and histone deacetylases (HDACs) have been developed as cancer therapeutic agents for many years. Many of these agents are currently being tested in a clinical trial, and some have been approved for use by the authorities (Miranda Furtado CL, 2019;Park JW, 2019; Hillyar C, 2020). However, thorough knowledge of histone methylases and their inhibitors is still lacking, and no inhibitors of histone methyltransferases (HMTs) are under development for human cancer therapy. However, the abnormalities in patterns or levels of these HMTs may result in tumorigenesis and developmental defects, suggesting that HMTs are promising targets for future cancer treatment (McCabe MT, 2017; Husmann D, 2019).

The histone methyltransferase G9a (also known as KMT1C or EHMT2) and G9a-like protein (GLP/KMT1D/EHMT1) are two closely related histone lysine methyltransferases (HKMTs). They exist predominantly as a G9a/GLP heteromeric complex and participate in mono- and dimethylation of lysine 9 residue present on H3 (H3K9me1 and H3K9me2, respectively)( Saha N, 2021). Besides H3K9, they can also methylate other histones, including histones H1, H3K27, and H3K56, and a variety of nonhistone proteins, including the tumor suppressor p53, sirtuin 1, reptin, myogenic differentiation 1, chromodomain Y-like protein, and widely interspaced zinc finger motif protein(Shinkai Y, 2011; Shankar, 2013). Furthermore, they are overexpressed and promote proliferation and metastasis in various human cancers, including breast, ovarian, head and neck, gastric, colon, lung, bladder, liver, cervical, and prostate cancers, neuroendocrine tumors, hematological malignancies, and so on. Hence, targeting them and their epigenetic mechanism can be a possible therapeutic approach for cancer treatment and management (Wang, 2017; Pangeni RP, 2020; Zhang, 2018). Therefore, a variety of G9A inhibitors have been developed in recent years, including BIX-01294, UNC0224, UNC0321, E72, DCG066, A-366, E72UNC0638, and UNC0642. Among these, UNC0642 possesses excellent selectivity and improved in vivo pharmacokinetic properties, which makes it suitable for animal studies (Xiong, 2017; Rahman Z, 2021; Cao, 2019).

The anticancer activity of G9a inhibitors has been reported in various cancer types. They can inhibit cell proliferation by inducing cell cycle arrest, triggering apoptosis, or inducing autophagic cell death. BIX-01294 promoted autophagy-dependent cell death and inhibited cancer cell proliferation in CRC, breast cancer, nasopharyngeal carcinoma, liver cancer, glioma, and bladder cancer cells. UNC0638 inhibited cell proliferation in breast, cervical, lung, and liver cancers in vitro(Deng, 2020; Li, 2020; Cui, 2015). UNC0642 suppressed urinary bladder cancer cell growth and induced apoptosis in vitro and in vivo, inhibited melanoma cell growth by suppressing Notch1 and Hes1 expression, and suppressed xenograft tumor growth in vivo and sphere-forming and cell proliferation capacity in vitro in non-small-cell lung cancer (Cao, 2019; Dang, 2020).

In this study, we explored the anticancer activity of UNC0642 in CRC. UNC0642 inhibited CRC cell proliferation in vitro and suppressed the tumorigenicity of colorectal xenografts in vivo. The molecular mechanism showed that UNC0642 induced G2/M-phase arrest, increased the expression of GADD45 family proteins, increased reactive oxygen species (ROS) level, and activated p38 and JNK/MAPK signaling pathways.

2.1 Chemicals

UNC0642 with 99.92% purity was purchased from MedChemExpress ( HY-13980, Monmouth Junction, NJ, USA). For in vitro studies, the cells were dissolved in dimethyl sulfoxide (DMSO) to reach a concentration of 10 mM and diluted to the desired concentration with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, USA).

The antibodies used in this study were as follows: GADD45A (13747-1-ap, Proteintech Group, Rosemont, PA, USA), GADD45B (ab105060,Abcam, Cambridge, Mass., USA), GADD153 (15204-1-ap,Proteintech Group, Rosemont, PA, USA), p38 (8690, Cell Signaling Technology, Beverly, MA, USA), p-p38 (4511, Cell Signaling Technology, Beverly, MA, USA), H2AX (CST, 9718), ERK (4695, Cell Signaling Technology, Beverly, MA, USA), p-ERK (4370, Cell Signaling Technology, Beverly, MA, USA), cyclin B1 (ab72,Abcam, Cambridge, Mass., USA), JNK (4671, Cell Signaling Technology, Beverly, MA, USA), and p-JNK (9252, Cell Signaling Technology, Beverly, MA, USA).

2.2 Cell culture

The human CRC cell lines HT29, SW620 and HCT116 were purchased from American Type Culture Collection and cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco, Life Technologies, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (penicillin–streptomycin–glutamine; Gibco, Life Technologies, USA) at 37 ̊C in a humidified atmosphere of 5% CO₂ in a Forma Steri-Cycle CO₂ Incubator (Thermo Scientific, MA, USA).

2.3 Cell proliferation assay

MTT(3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)test was performed to assay the cell proliferation of HT29, SW620 and HCT116 cells treated with UNC0642. HT29 and SW620 cells were harvested and seeded onto a 96-well plate at a density of 3 × 10³ cells per well. Following 24-h incubation, the cells were treated with 200 µL of UNC0642-containing culture medium at the final concentrations of 1, 2.5, and 5 µmol/L and for 24, 48, 72, and 96 h. Before assay, the medium was replaced with 100 µL of the medium containing 10 µL of MTT (5 mg/mL) and then further incubated for 4 h. The absorbance of the solution was measured using a microplate reader (Varioskan Flash, Thermo Scientific, MA, USA) at 490 nm.

2.4 Plate colony formation assay

HT29, SW620 and HCT116 cells were harvested and seeded onto a six-well plate at a density of 2 × 10³ cells per well. Following 24-h incubation, the cells were treated with 2 mL of UNC0642-containing culture medium at the final concentrations of 0.25, 0.5, 1, and 2.5 µmol/L for 2 weeks, and the treatment was terminated when macroscopic colonies were found in culture dishes. Before the assay, the cells were washed with phosphate-buffered saline (PBS) and fixed with 20% cold methanol for 15 min. Then, they were stained with crystal violet dye for 20 min. After washing and air drying, the colonies were counted using a cloning counter. The colony formation rate (%) = (number of colonies/number of inoculated cells) × 100%, and the inhibition rate was calculated.

2.5 Flow cytometry

The flow cytometric analysis was conducted to assay the cell cycle arrest in HT29, SW620 and HCT116 cells treated with 5 and 10 µM of UNC0642. The cells were initially seeded onto six-well plates at a density of 3 × 10⁵ cells per well. After a 24-h incubation period, the cells were treated with UNC0642 at concentrations of 5 and 10 µM for an additional 24 h, and subsequently, the cell cycle assay was performed. The cells were harvested and washed with pre-chilled PBS twice, followed by fixation with 70% ethanol at 4℃ for 2 h. After washing with PBS, the cells were stained with FxCycle PI/RNase Staining Solution (Invitrogen, Thermo Fisher Scientific) at 4℃ for 30 min. The stained cells were analyzed using a flow cytometer (FACS Aria Ⅱ; BD Biosciences, San Jose, USA).

2.6 ROS assay

We used the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate( DCFH-DA)to measure UNC0642-induced intracellular ROS generation. After treatment with 5 and 10 µM UNC0642 for 24 h, HT29, SW620 and HCT116 cells were harvested and incubated with 10 µM DCFH-DA at 37℃ for 30 min. The fluorescence intensity was measured at excitation and emission wavelengths of 488 and 525 nm, respectively, using a flow cytometer (FACS Aria Ⅱ; BD Biosciences, San Jose, USA).

2.7 γ-H2AX(Phosphorylation of H2AX at Ser 139) assay

After treatment with 10 and 20 µM UNC0642 for 24 h, HT29, SW620 and HCT116 cells seeded on coverslips were fixed for 20 min with 4% paraformaldehyde on ice. The permeabilization and blocking of the nonspecific binding of antibodies were performed by incubating the cells in PBS containing 10 mM HEPES, 3% BSA, and 0.3% Triton X-100 at room temperature for 60 min. The cells were incubated with γ-H2AX primary antibodies at 4˚C overnight. The incubation with secondary antibodies was performed at room temperature for 30 min. Nuclear staining was carried out by incubating the cells with DAPI at room temperature for 5 min. Between all steps, the cells were washed three times with PBS for 5 min. The stained sections were examined under an Axion Observer A1 microscope (Zeiss Axio observer A1, Carl Zeiss Micro Imaging GmbH, Goettingen, Germany) .

2.8 Real-time quantitative reverse transcription–polymerase chain reaction

Total RNA was isolated from HT29, SW620, and HCT116 CRC cells using TRIzol reagent (CWbiotech, Beijing, China), and then RNA was used to perform reverse transcription with a PrimeScript RT reagent kit (TaKaRa, Otsu, Japan). The acquired cDNAs were used as templates for real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis using the SYBR Green PCR Master Mix (Bio-Rad, California,USA). The relative RNA expression levels were calculated using the 2^-ΔΔCt method, with the levels normalized to those of β-actin mRNA.

2.9 Western blot

HT29, SW620 and HCT116 cells were seeded onto six-well plates at a density of 4 × 10⁵ cells per well. Following 24-h incubation, the cells were treated with UNC0642 at a concentration of 5 µM for 24 h. Following the treatments, the cells were harvested, washed twice with pre-chilled PBS, lysed with cell lysis buffer (Beyotime Biotechnology, Beijing,China) containing 10 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA), EDTA-free protease inhibitor cocktail (Roche Diagnostic, Manheim, Germany), and PhosSTOP (Roche Diagnostic, Manheim, Germany)for 30 min on ice, and then centrifuged at 12,000 g at 4°C for 15 min. The protein concentration in the supernatant was quantified using a BCA(Bicinchoninic acid) protein assay kit (Tiangen Biotech, Beijing, China) to ensure equal amounts of protein in each sample were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After full separation, the proteins in the gel were transferred onto polyvinylidene fluoride membranes (0.45 µm; Millipore, Billerica, Massachusetts, USA), blocked with 5% Difco skimmed milk (BD, USA) for 2 h at room temperature, and incubated with each primary antibody at 4°C overnight. The next day, the membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (pH 7.4) and incubated with the appropriate secondary antibody at room temperature for 1 h. The immunoblots were detected by enhanced chemiluminescence (Beyotime Biotechnology, China), and the gray level was analyzed using ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Pennsylvania, USA).

2.10 mRNA sequencing assay and bioinformatics analysis

SW620 and HT29 cells were treated with UNC0642 (10 µM) for 24 h, and then total RNA was isolated using TRIzol reagent following the manufacturer’s protocol. After checking quality using a Eukaryote Total RNA Nano Chip on a 2100 Bioanalyzer (Agilent, USA), the mRNA sequencing (mRNA-seq) libraries were prepared using a KAPPA stranded RNA-seq library preparation kit(Kapa Biosystems, Wilmington, MA, USA) and sequenced on an Illumina Hiseq 4000 system (Illumina, San Diego, CA,USA). The sequence reads were analyzed as follows. The sequence reads were filtered to get clean reads and aligned to the human reference genome GRCh37 (hg19) to get the mapped data. The gene expression level was analyzed by calculating fragments per kilobase of transcript per million fragments mapped. The differential expression analysis was conducted to identify true differentially expressed genes (DEGs) in the following comparisons: HT29 untreated versus SW620 untreated, HT29 untreated versus HT29 UNC0642 treated, and SW620 untreated versus SW620 UNC0642 treated. The false discovery rate (FDR) for each comparison was calculated by the Benjamini–Hochberg method using the following set of criteria: >2-fold change either upregulated or downregulated, FDR < 0.01. Then, Gene Ontology (GO) biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out for further functional annotation and functional enrichment analysis of DEGs. The P value < 0.05 was set as the cutoff criterion for both GO and KEGG functional analyses.

2.11 Mouse xenograft model

BALB/c-nu/nu mice (aged 4–6 weeks and weighed 16–20 g) were used to establish the HT29 xenograft model. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and kept in pathogen-free cages provided with standard rodent chow and sterile water ad libitum. Briefly, 5 × 10⁶ cells were suspended in 200 µL of PBS and injected subcutaneously into the right flank of the nude mice. When the tumors reached a mean diameter of 6 mm, the mice were randomly divided into two groups: control (10% DMSO) and UNC0642 (5 mg/kg), followed by intragastric administration every other day. Throughout the treatment, the mice were weighed, and their tumors were measured with a caliper every 3 days. Tumor volumes (V) were calculated using the formula: V = larger diameter × (smaller diameter)²/2 mm³. After 40 days of treatment, when the tumors reached an appropriate size, the mice were euthanized, and the tumors were excised, weighed, and either stored at − 80℃ or fixed with 4% paraformaldehyde for subsequent examination.

2.12 Statistical analysis

The data were presented as mean values with standard error of the mean. Statistical analysis was performed with Prism GraphPad 6.0 software (GraphPad Software, Inc., San Diego, CA,USA). Differences between groups were examined by Student’s t test or one-way ANOVA followed by Bonferroni’s correction or two-way ANOVA with multiple-comparison test for comparison of means. A P value < 0.05 indicated a significant difference, and a P value < 0.01 indicated a statistically significant difference (^*P < 0.05, ^**P < 0.01).

3.1 UNC0642 inhibited the cell proliferation in human CRC cells in vitro

MTT assay was performed in three human CRC cell lines (HT29, SW620 and HCT116) 24, 48, 72, and 96 h after treatment with UNC0642 at concentrations of 1, 2.5, and 5 µM to evaluate the effect of UNC0642 on CRC cell proliferation in vitro. As shown in Fig. 1A, the proliferation of all three CRC cell lines was inhibited significantly, and the inhibition was enhanced with the increased concentrations of UNC0642. The growth of all three cell lines was completely inhibited at the concentration of 5 µM. Hence, MTT results suggested that UNC0642 exerted a potent antiproliferative effect on CRC cells in a dose- and time-dependent manner.

The plate colony formation assay was conducted to further determine the growth inhibition of CRC cells by UNC0642. As shown in Fig. 1B, the number and size of colonies formed in the cells significantly reduced after treatment with UNC0642 at concentrations of 0.25,0.5,1and 2.5 µM compared with those in the untreated control cells. Also, the reduction was enhanced with the increased concentration of UNC0642. Almost no clone formation occurred at the concentration of 2.5 µM. This suggested that UNC0642 had the potential to inhibit the colony formation of CRC cells.

3.2 UNC0642 induced G2/M cell cycle arrest in human CRC cells

Flow cytometry was carried out to determine the effect of UNC0642 on the cell cycle progression of CRC cells. The results showed that the G2/M population in HT29, SW620 and HCT116 cells significantly increased following treatment with 5 and 10 µM UNC0642 for 24 h, compared with controls. As shown in Fig. 2A, the population of HT29, SW620 and HCT116 cells in the G2/M phase after treatment with UNC0642 at the concentration of 10µM was (19.66 ± 1.84)%, (14.23 ± 1.46)%, and (15.03 ± 0.82)% versus (7.26 ± 1.07)%, (1.85 ± 0.34), and (3.09 ± 0.50)% in the control group, respectively.

The cell cycle analysis indicated that the inhibitory effect of UNC0642 on the proliferation of HT29, SW620 and HCT116 cells was correlated with G2/M cell cycle arrest. Then, the impact of UNC0642 on the expression of the proteins related to the G2/M checkpoint was examined. The cyclin B1 expression was measured using Western blot analysis following treatment with 5 µM UNC0642 for 24 h. The results showed that the cyclin B1 expression was reduced in the UNC0642-treated cells compared with the untreated cells (Fig. 2B).

3.3 UNC0642 induced significant ROS production leading to DNA strand breaks in human CRC cells

We detected DNA damage, which was reported to be highly correlated with G2/M cell cycle arrest, to further explore the molecular mechanism of UNC0642 in this arrest. We assayed the possibility of inducing DNA double-strand breaks (DSBs) in cells by γ-H2AX (phosphorylation of H2AX at Ser 139) after UNC0642 treatment. γ-H2AX is one of the major and early responses to DSBs. Immunofluorescence and Western blot analysis were conducted to assay γ-H2AX level after UNC0642 treatment. The immunofluorescence assay in human CRC cells after UNC0642 treatment (0, 10, and 20 µM for 24 h) showed a significant accumulation of γ-H2AX foci in a concentration-dependent manner (Fig. 3A). Western blot assay also showed a substantial increase in γ-H2AX level after UNC0642 treatment (Fig. 3B). Hence, these observations suggested that the induction of DNA damage inside cells could be one of the mechanisms for the observed G2/M cell cycle arrest caused by UNC0642.

Since ROS are well recognized as the mediators of DNA damage inside cells (Srinivas US, 2019), we evaluated whether the treatment with UNC0642 generated ROS inside human CRC cells. DCFH-DA staining was carried out in treated (0, 1, and 2 µM for 24 h) HT29, SW620 and HCT116 cells. The flow cytometry assay result demonstrated that the ROS-positive cell population increased after treatment with UNC0642 and the median of the histogram shifted significantly compared with that in the control cells in a concentration-dependent manner, suggesting elevated ROS production upon treatment with UNC0642 (Fig. 3C).

3.4 UNC0642 upregulated GADD protein family expression levels in CRC cells

We next performed RNA-seq analysis to identify the transcripts differentially regulated in HT-29 and SW620 CRC cells after UNC0642 (10 µM) treatment for 24 h to investigate the molecular mechanism of UNC0642-induced G2/M cell cycle arrest. The results showed that, compared with the DMSO control, the UNC0642-treated group had 2082 and 1771 DEGs in HT29 and SW620 cells, respectively, among which 498 genes were overlapping (Fig. 4A).

Among the 498 DEGs, we focused on a gene family closely related to the G2/M cell cycle checkpoint and proliferation, namely, growth arrest– and DNA damage (GADD)–inducible genes, including GADD153/C/EBP-homologous protein (CHOP)/DDIT3, GADD45A, GADD45B, and so on, which showed significant upregulation following UNC0642 treatment. We analyzed the changes in the expression of the three GADD family genes using real-time qRT-PCR (Fig. 4B) and Western blot analysis (Fig. 4C) to validate the accuracy of the RNA-seq analysis. The results confirmed the reliability of the RNA-seq data. All three GADD family genes were upregulated after UNC0642 treatment, among which GADD153/CHOP/DDIT3 was most significantly upregulated.

3.5 UNC0642 activated p38 and JNK/MAPK signaling pathways in human CRC cells

GO and KEGG pathway analyses were performed to investigate the major pathways mediated by UNC0642-induced DEGs so as to further investigate the molecular mechanism of UNC0642 in inhibiting CRC proliferation. The results showed that the DEGs were mainly enriched in the MAPK signaling pathway, cytokine–cytokine receptor interaction signaling pathway, NF-kappa B (NF-κB) signaling pathway, TNF signaling pathway, JAK/STAT signaling pathway, PI3K/Akt signaling pathway, and P53 signaling pathways. They were all involved in regulating metastasis and inflammation-related processes (Fig. 5A).

We performed Western blot analysis to examine the changes in the expression of the key proteins of the MAPK signaling pathway so as to validate the accuracy of the RNA-seq analysis, which was shown in the KEGG analysis to enrich the largest number of DEGs. The Western blot results indicated that the phosphorylation levels of p38 and JNK significantly increased in UNC0642-treated cells compared with the control group; however, the phosphorylation levels of ERK did not change significantly (Fig. 5B and 5C).

3.6 UNC0642 inhibited tumor growth in vivo

We examined the effect of UNC0642 on tumor growth in vivo using a mouse xenograft model after characterizing its effects on CRC cells in vitro. HT29 cells were injected into immunocompromised nude mice to evaluate the efficacy of UNC0642 in inhibiting tumor growth. As shown in Fig. 6, the oral administration of UNC0642 significantly inhibited HT29 tumor growth compared with the control treatment (P < 0.05). The tumor volumes were measured and plotted after each treatment to evaluate the effect of apatinib on the mouse xenografts of CRC cells. After 40 days, the tumor volume in the 50 mg/kg UNC0642-treated group was significantly smaller than in the negative control group (P < 0.01). In this xenograft model of CRC, UNC0642 significantly delayed tumor growth in mice. No significant differences in the body weights of the mice were observed between the groups before or after treatment (data not shown).

Abnormalities of patterns or levels of epigenetic alterations may result in tumorigenesis, suggesting that they may be one of the important targets or markers for treating this disease. To date, DNA methylation and histone acetylation have been well studied. Some of their inhibitors have been developed as clinical oncology drugs(Miranda Furtado CL, 2019;Park JW, 2019; Hillyar C, 2020). In contrast, studies on histone methylation are still lacking, although the correlations between histone methylation and cancer development have been confirmed. Notably, histone methyltransferases, especially HKMTs, hold great potential as novel promising targets for future cancer treatment. Therefore, the research on HKMTs has become a subject of great interest(McCabe MT, 2017; Husmann D, 2019). Previous studies have demonstrated that G9a/GLP, an H3K9 methyltransferase, is considerably downregulated in various tumor tissues. Additionally, inhibitors targeting G9a/GLP have exhibited anticancer activity, particularly in breast cancer(Shinkai Y, 2011). The biological properties and anticancer activity of G9a/GLP on CRC cells and the underlying mechanisms remain to be investigated. We examined CRC cells and found that the G9a/GLP inhibitor UNC0642 inhibited CRC cell growth and progression both in vitro and in vivo. We further explored the possible molecular mechanism and found that UNC0642 induced G2/M cell cycle arrest, increased ROS levels, increased the expression of the growth arrest– and DNA damage–inducible (GADD) genes, including GADD153, GADD45α, and GADD45β, p38/JNK MAPK pathways, and NF-κB pathway, providing new evidence for a comprehensive understanding of G9a/GLP.

The G2/M cell cycle checkpoint plays a key role in normal cell proliferation. When DNA is damaged, Chk2 is phosphorylated and activated, resulting in the phosphorylation and inhibition of Cdc25 and activating the Cdc2/cyclin B1 complex, which is a specific regulator of the G2/M phase (Stark GR, 2004). We found that UNC0642 induced G2/M cell cycle arrest in CRC cells and significantly upregulated the expression level of DNA damage marker γ-H2AX, which was consistent with the results reported by Zhang et al. after knocking down the G9a expression in CRC(Zhang J, 2020). However, further exploration of the molecular mechanism revealed that UNC0642 significantly upregulated the expression level of growth arrest– and DNA damage–inducible genes (GADD). Similar conclusions have not been reported earlier. The GADD gene family includes five members: GADD34, GADD45α, GADD45β, GADD45γ, and GADD153. They were initially isolated from UV radiation–treated cells and subsequently grouped according to their similar role in growth arrest and DNA damage. The upregulation of the GADD gene expression level could trigger growth inhibition, and the combined upregulation of GADD genes played a synergistic inhibitory effect on growth(Tamura RE, 2012). We found that UNC0642 induced combined upregulation of GADD genes, including GADD45A, GADD45B, and GADD153, indicating that UNC0642 had a significant inhibitory effect on the growth of CRC cells. The manner in which UNC0642 induces GADD gene expression needs further exploration. However, according to research reports, GADD genes were defined as stress response genes that could be induced by UV radiation, chemical carcinogens, starvation, oxidative stress, and apoptosis-inducing agents. We found that UNC0642 induced ROS in CRC cells. ROS-mediated oxidative stress induced GADD gene expression, especially GADD153, also known as CHOP, which was strictly correlated with endoplasmic reticulum stress evoked by ROS (Kim M, 2016; Di S, 2019). This implied that UNC0642 upregulated the expression of GADD genes by inducing ROS.

ROS play an essential role in the survival and proliferation of tumor cells. The tumor cells have a higher level of ROS and cannot tolerate as much exogenous oxidative stress as normal cells. Highly toxic levels of ROS in cancer cells induce cell death, which has been considered a strategic opportunity to specifically target cancer cells. Therefore, increasing attention is paid to anticancer drugs by promoting ROS generation(Perillo B., 2020; Kirtonia A, 2020; Aggarwal V, 2019). The new role of ROS in DNA damage and transcriptional regulation is worth mentioning. Perillo et al. found that the demethylation of lysine 9 in histone H3 (H3K9) induced FADH2 oxidation to produce ROS during estrogen-induced transcription, with consequent oxidation of nearby guanines (8-oxo-Gs) and recruitment of DNA repair enzymes. The generation of ROS must be controlled timely and spatially to prevent excessive damage to DNA because once ROS is overproduced, the increased oxidation of DNA overwhelms the repair device and triggers programmed cell death in a large percentage of these cells(Perillo B, 2008). This study found that the H3K9 methylase G9a/GLP inhibitor UNC0642 could induce CRC cells to produce a large amount of ROS and cause DNA damage, suggesting that inhibiting methylation could also induce ROS.

The MAPK signaling pathway has been widely recognized as one of the most important regulatory pathways, which can regulate cell proliferation and invasion, promote the EMT process, and finally promote the development of CRC(Lee S, 2020; Qi M, 2009). MAPK pathways consist of ERK1/2, JNK, and p38 pathways, of which JNK and p38 pathways are mainly activated by stresses, such as ROS, and cytokines, such as IL-1 and TNF-α, targeting NF-κB and p53 transcription factors, and are involved in regulating stress, apoptosis, and inflammation (Kim EK, 2015; Kasuya Y, 2018; Hirata Y. 2019; Zhang J, 2016). In this study, we found that UNC0642 could significantly activate the JNK and p38 MAPK pathways in CRC cells. The transcriptome sequencing results showed that the inflammation regulation pathways, such as cytokine–cytokine receptor interaction signaling pathway, NF-κB signaling pathway, and TNF signaling pathway, changed significantly. Further, ROS could phosphorylate and activate JNK and p38 kinase. These results suggested that UNC0642 could activate JNK and p38 signaling pathways by inducing stresses, such as ROS and inflammatory factors, in CRC cells. Furthermore, several studies showed that oxidative stress induced the coordinated overexpression of GADD genes through the MAPK signaling pathway(Sarkar D, 2002; Oh-Hashi K, 2001; Chen Z, 2016; Obaidi I, 2020), implying that UNC0642 inhibited CRC cell growth through ROS-p38/JNK MAPK-GADD pathway. However, further studies are needed to confirm the underlying mechanism.

In addition, our results also showed that UNC0642 exhibited cytotoxicity of different sensitivity in the three CRC cell lines, higher in HT29 and SW620 ,and lower in HCT116, which may be related to their different molecular phenotypes, although further research is needed.As HT29,SW620 and HCT116 were found to carry different molecular phenotypes,HT29 and SW620 were found to be the chromosomal instability pathway (CIN) ,and carry mutant TP53(HT29, R273H;SW620, R273H and P309S),while HCT1116 was the microsatellite instability(MSI) and carry widetype TP53(Ahmed, D., 2013).

This study was novel in elucidating the antitumor activity and potential molecular mechanism of UNC0642 in CRC cells. We found that UNC0642 could efficiently cause oxidative stress, activate p38 and JNK/MAPK signaling pathways, upregulate GADD genes, and induce growth inhibition. The findings suggested that UNC0642 had the potential to be developed as an antitumor agent that might serve as an effective therapeutic addition to the current armamentarium in managing CRC.

Ethics approval and consent to participate

The animal experiment was approved by the Institute Animal Care and Use Committee of the Beijing Academy of Science and Technology (Beijing Center for Physical and Chemical Analysis) .

Competing interests

The authors declare no conflicts of interest.

Author Contribution

Xiao-yan Cheng wrote the main manuscript text All authors reviewed the manuscript

Acknowledgments

This study was supported by the Beijing Municipal Reform and Development Project (No.2021ZL0107), as well as by the Scientific Research Program of BJAST(No.23CA005-01,23CA005-02)

Ahmed D, Eide P, Eilertsen I, et al. Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis. 2013;2:e71. https://doi.org/10.1038/oncsis.
Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K, Varol M, Jain A, Khan MA, Sethi G. Role of Reactive Oxygen Species in Cancer Progression: Molecular Mechanisms and Recent Advancements. Biomolecules. 2019;9:735. 10.3390/biom9110735.
Cui J, Sun W, Hao X, Wei M, Su X, Zhang Y, Su L, Liu X. EHMT2 inhibitor BIX-01294 induces apoptosis through PMAIP1-USP9X-MCL1 axis in human bladder cancer cells. Cancer Cell Int. 2015;15(4). 10.1186/s12935-014-0149-x.
Chen Z, Wan X, Hou Q, Shi S, Wang L, Chen P, Zhu X, Zeng C, Qin W, Zhou W, Liu Z. (2016). GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway. Cell Death Dis, 7,e2068. 10.1038/cddis.2015.300.
Cao YP, Sun JY, Li MQ, et al. Inhibition of G9a by a small molecule inhibitor, UNC0642, induces apoptosis of human bladder cancer cells. Acta Pharmacol Sin. 2019;40:1076–84. 10.1038/s41401-018-0205-5.
Cao H, Li L, Yang D, Zeng L, Yewei X, Yu B, Liao G, Chen J. Recent progress in histone methyltransferase (G9a) inhibitors as anticancer agents. Eur J Med Chem. 2019;179:537–46. 10.1016/j.ejmech.2019.06.072.
Di S, Fan C, Ma Z, Li M, Guo K, Han D, Li X, Mu D, Yan X. PERK/eIF-2α/CHOP Pathway Dependent ROS Generation Mediates Butein-induced Non-small-cell Lung Cancer Apoptosis and G2/M Phase Arrest. Int J Biol Sci. 2019;15:1637–53. 10.7150/ijbs.33790.
Deng BB, Jiao BP, Liu YJ, Li YR, Wang GJ. BIX-01294 enhanced chemotherapy effect in gastric cancer by inducing GSDME-mediated pyroptosis. Cell Biol Int. 2020;44:1890–9. 10.1002/cbin.11395.
Dang NN, Jiao J, Meng X, An Y, Han C, Huang S. Abnormal overexpression of G9a in melanoma cells promotes cancer progression via upregulation of the Notch1 signaling pathway. Aging. 2020;12:2393–407.
Hirata Y. Reactive Oxygen Species (ROS) Signaling: Regulatory Mechanisms and Pathophysiological Roles. Yakugaku Zasshi. 2019;139:1235–41. 10.1248/yakushi.19-00141.
Husmann D, Gozani O. Histone lysine methyltransferases in biology and disease. Nat Struct Mol Biol. 2019;26:880–9. 10.1038/s41594-019-0298-7.
Hillyar C, Rallis KS, Varghese J. Adv Epigenetic Cancer Ther Cureus. 2020;12:e11725. 10.7759/cureus.11725.
Kim EK, Choi EJ. (2015). Compromised MAPK signaling in human diseases: an update. Arch Toxicol, 89,867 – 82. 10.1007/s00204-015-1472-2.
Kim M, Baek HS, Lee M, Park H, Shin SS, Choi DW, Lim KM. (2016). Rhododenol and raspberry ketone impair the normal proliferation of melanocytes through reactive oxygen species-dependent activation of GADD45. Toxicol In Vitro, 32,339 – 46. 10.1016/j.tiv.2016.02.003.
Kasuya Y, Umezawa H, Hatano M. Stress-Activated Protein Kinases in Spinal Cord Injury: Focus on Roles of p38. Int J Mol Sci. 2018;19:867. 10.3390/ijms19030867.
Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol. 2019. https://doi.org/10.1038/s41575-019-0189-8.
Kirtonia A, Sethi G, Garg M. The multifaceted role of reactive oxygen species in tumorigenesis. Cell Mol Life Sci. 2020. https://doi.org/10.1007/s00018-020-03536-5.
Lao VV, Grady WM. Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol. 2011;8:686–700. 10.1038/nrgastro.2011.173.
Lee S, Rauch J, Kolch W. Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity. Int J Mol Sci. 2020;21:1102. 10.3390/ijms21031102.
Li Q, Wang M, Zhang Y, Wang L, Yu W, Bao X, Zhang B, Xiang Y, Deng A. BIX-01294-enhanced chemosensitivity in nasopharyngeal carcinoma depends on autophagy-induced pyroptosis. Acta Biochim Biophys Sin (Shanghai). 2020;521131–1139. 10.1093/abbs/gmaa097.
McCabe MT, Mohammad HP, Barbash O, Kruger RG. Targeting Histone Methylation in Cancer. Cancer J. 2017;23:292–301. 10.1097/PPO.0000000000000283.
Miranda Furtado CL, Dos Santos Luciano MC, Silva Santos RD, Furtado GP, Moraes MO, Pessoa C. Epidrugs: targeting epigenetic marks in cancer treatment. Epigenetics. 2019;14:1164–76. 10.1080/15592294.2019.1640546.
Oh-Hashi K, Maruyama W, Isobe K. (2001). Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells. Free Radic Biol Med, 30,213 – 21. 10.1016/s0891-5849(00)00461-5.
Okugawa Y, Grady WM, Goel A. Epigenetic Alterations in Colorectal Cancer: Emerging Biomarkers. Gastroenterology. 2015;149:1204–e122512. 10.1053/j.gastro.2015.07.011. Epub 2015 Jul 26.
Obaidi I, Cassidy H, Gaspar VI, McCaul J, Higgins M, Halász M, Reynolds AL, Kennedy BN, McMorrow T. Curcumin Sensitizes Kidney Cancer Cells to TRAIL-Induced Apoptosis via ROS Mediated Activation of JNK-CHOP Pathway and Upregulation of DR4. Biology Basel. 2020;9:92. 10.3390/biology9050092.
Perillo B, Ombra MN, Bertoni A, Cuozzo C, Sacchetti S, Sasso A, Chiariotti L, Malorni A, Abbondanza C, Avvedimento EV. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science. 2008;319:202–6.
Park JW, Han JW. Targeting epigenetics for cancer therapy. Arch Pharm Res. 2019;42:159–70. 10.1007/s12272-019-01126-z.
Pangeni RP, Yang L, Zhang K, et al. G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer. Clin Epigenetics. 2020;12:88. 10.1186/s13148-020-00879-5.
Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. ROS in cancer therapy: The bright side of the moon. Exp Mol Med. 2020;52:192–203. 10.1038/s12276-020-0384-2.
Qi M, Elion EA. MAP kinase pathways. J Cell Sci. 2005;118:3569–72.
Wagner E, Nebreda Á. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer, 9, 537–49.
Rahman Z, Bazaz MR, Devabattula G, Khan MA, Godugu C, Su ZZ, Lebedeva IV et al. (2002). mda-7 (IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci U S A, 99,10054–10059. 10.1073/pnas.152327199.
Stark GR, Taylor WR. (2004). Analyzing the G2/M checkpoint. Methods Mol Biol, 280:51–82. 10.1385/1-59259-788-2:051. PMID: 15187249.
Shankar SR, Bahirvani AG, Rao VK, Bharathy N, Ow JR, Taneja R. (2013) G9a, a multipotent regulator of gene expression.Epigenetics, 8, 16–22.
Srinivas US, Tan BWQ, Vellayappan BA, Jeyasekharan AD. (2019). ROS and the DNA damage response in cancer. Redox Biol, 25,101084.10.1016/j.redox.2018.101084.
Saha N, Muntean AG. Insight into the multi-faceted role of the SUV family of H3K9 methyltransferases in carcinogenesis and cancer progression. Biochim Biophys Acta Rev Cancer. 2021;1875:188498. 10.1016/j.bbcan.2020.188498.
Shinkai Y, Tachibana M. H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 2011;25:781–8. 10.1101/gad.2027411.
Tamura RE, de Vasconcellos JF, Sarkar D, Libermann TA, Fisher PB, Zerbini LF. (2012). GADD45 proteins: central players in tumorigenesis. Curr Mol Med, 12,634 – 51. 10.2174/156652412800619978.
Wang YF, Zhang J, Su Y, et al. G9a regulates breast cancer growth by modulating iron homeostasis through the repression of ferroxidase hephaestin. Nat Commun. 2017;8:274. 10.1038/s41467-017-00350-9.
Xiong Y, Li F, Babault N, et al. Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase. J Med Chem. 2017;60:1876–91. 10.1021/acs.jmedchem.6b01645.
Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W. (2016). ROS and ROS-Mediated Cellular Signaling. Oxid Med Cell Longev, 2016,4350965. 10.1155/2016/4350965.
Zhang K, Wang J, Yang L, Yuan YC, Tong TR, Wu J, Yun X, Bonner M, Pangeni R, Liu Z, Yuchi T, Kim JY, Raz DJ. (2018). Targeting histone methyltransferase G9a inhibits growth and Wnt signaling pathway by epigenetically regulating HP1α and APC2 gene expression in non-small cell lung cancer. Mol Cancer, 17,153. 10.1186/s12943-018-0896-8.
Zhang J, He P, Xi Y, Geng M, Chen Y, Ding J. Correction: Down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation. Oncotarget. 2020;11:302–3. 10.18632/oncotarget.27441.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

G9a/GLP inhibitor UNC0642 induces growth arrest in colorectal cancer via induction of ROS generation, coordinated overexpression of GADD genes, and the JNK/p38 MAPK pathway

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Chemicals

2.2 Cell culture

2.3 Cell proliferation assay

2.4 Plate colony formation assay

2.5 Flow cytometry

2.6 ROS assay

2.7 γ-H2AX(Phosphorylation of H2AX at Ser 139) assay

2.8 Real-time quantitative reverse transcription–polymerase chain reaction

2.9 Western blot

2.10 mRNA sequencing assay and bioinformatics analysis

2.11 Mouse xenograft model

2.12 Statistical analysis

3. Results

3.1 UNC0642 inhibited the cell proliferation in human CRC cells in vitro

3.2 UNC0642 induced G2/M cell cycle arrest in human CRC cells

3.3 UNC0642 induced significant ROS production leading to DNA strand breaks in human CRC cells

3.4 UNC0642 upregulated GADD protein family expression levels in CRC cells

3.5 UNC0642 activated p38 and JNK/MAPK signaling pathways in human CRC cells

3.6 UNC0642 inhibited tumor growth in vivo

4. Discussion

5. Conclusions

Declarations

Ethics approval and consent to participate

Author Contribution

Acknowledgments

References

Additional Declarations

Status:

Version 1