4SC-202 exerts an anti-tumor effect in cervical cancer by targeting PRLR signaling pathway

The aim of the present study is to investigate whether 4SC-202, a selective class I histone deacetylase inhibitor (HDACi), plays an anti-tumor role in cervical cancer (CC) by targeting prolactin receptor (PRLR). CCK-8 and colony formation assays were used to evaluate the effects of 4SC-202 on the proliferation of CC cells in vitro. Effects of 4SC-202 on the cell cycle distribution and apoptosis in SiHa cells were determined by flow cytometry and western blotting, respectively. Immunofluorescence, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the activities of PRLR-related pathways and PRLR expression in CC cells. A xenograft tumor model in nude mice was established to examine effects of 4SC-202 on the tumor growth, apoptosis and PRLR-related pathways in vivo. The biochemical analyzer and H&E staining were used to detect the serum biochemical indexes and organ toxicity. 4SC-202 inhibited the proliferation of CC cells (SiHa, HeLa, and CaSki) in vitro in a time- and dose-dependent manner. SiHa cells were treated with 1 or 5 µM 4SC-202 for 72 h and then subjected to various functional assays. The assays showed that 4SC-202 significantly induced G2/M phase arrest and apoptosis, while inhibiting the activities of PRLR-related pathways and PRLR expression. In addition, 4SC-202 reduced tumor growth and induced apoptosis in vivo. 4SC-202 down-regulated the expression of PRLR and activities of PRLR-related pathways in the mouse model, displayed no effects on serum biochemical indicators and caused no toxicity to mouse organs. This finding suggests that 4SC-202 may serve as a novel therapeutic agent for CC.


Introduction
In recent years, cervical cancer (CC) has become one of the leading malignancies that threaten the health and lives of activity of prolactin (PRL) and triggering intramembrane signal cascades, such as signal transduction and the activators of transcription (STAT) family, extracellular regulated kinase (ERK1/2), and serine threonine kinase (AKT) (Kan et al. 2016;Ramirez De Arellano et al. 2018;Lopez-Pulido et al. 2013). Studies have shown that PRLR is highly expressed in CC tissues , and signal transduction and activation of STAT3 promotes anti-apoptotic effect of PRL (Ramirez de Arellano et al. 2015). Lopez-Pulido et al. (Lopez-Pulido et al. 2013) demonstrated that PRL/PRLR signal can be used as an important survival factor for CC. In a previous study, we showed that receptor dimerization is necessary for cytokines to activate signal transduction pathways in cells (Amoutzias et al. 2008). PRLR enhances lysine acetylation by recruiting CREB-binding protein (CBP) to neutralize the positive charge of the lysine side chain, and dimerizing the receptors, thereby facilitating the acquisition of transcription factors to increase transcription (Li et al. 2010;Gallagher et al. 2015). It has been reported that histone deacetylase inhibitors (HDACi) promote PRLR dimerization and subsequent signal transduction, whereas exogenous HDACs inhibit the processes (Li et al. 2010). While several HDACi have been shown to be involved in anti-tumoral immune responses (Li and Seto 2016), the underlying molecular mechanisms have not been fully understood. Domatinostat (4SC-202), a type I HDACi (von Tresckow et al. 2019), shows potent antitumor activities in various cell lines and preclinical models. 4SC-202 has been found to markedly inhibit proliferation and survival of colorectal cancer (CRC) cells as well as the growth of colorectal tumors in vivo (Huang et al. 2016). Moreover, 4SC-202 was capable of inhibiting the cell viability in urothelial carcinoma (UC) and inducing cell cycle disturbances and cell death (Pinkerneil et al. 2016). Gruber et al. found that 4SC-202 efficiently inhibits Hedgehog (HH)/GLI signaling in SMOi-sensitive and SMOi-resistant settings, while suppressing the growth of basal cell carcinoma (BCC) cells in vivo. (Gruber et al. 2018). Given that research on anti-tumor effects of 4SC-202 on cervical cancer and the underlying mechanism is still lacking, we intended to investigate whether 4SC-202 plays an anti-cervical cancer effect through targeting the PRLR signaling pathway.
All cell culture reagents were purchased from Gibco Life Technologies (Grand Island, CA, USA).

Cell culture
Cervical cancer cell lines SiHa, Hela, and CaSki were obtained from the American Type Culture Collection (Manasas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cell culture was conducted in a humidified atmosphere of 5% CO 2 at 37 °C. Cells were grown to 80% confluence for further assays. All culture reagents were obtained from Gibco (Carlsbad, CA).

CCK-8 assay
SiHa, Hela, and CaSki cells were plated in 96-well plates and incubated with various concentrations of 4SC-202 (0, 0.01, 0.1, 1, 5, and 10 µM) for a certain period of time (24 h, 48 h, or 72 h). CCK-8 reagent (10 µL) was added and the mixture was incubated for 2 h in the dark. The optical density (OD) value for each well was measured at a wavelength of 450 nm. The half maximal inhibitory concentration (IC50) was calculated by using SPSS software.

Colony formation assay
Colony formation assay was carried out as described previously (Liao et al. 2020). SiHa, Hela, and CaSki cells (5 × 10 3 cells/well) were plated in 6-well plates and incubated with various concentrations of 4SC-202 (0, 1, and 5 µM) for 72 h. Culture media were refreshed every 2 days. The cells were stained with crystal violet, and the colonies were photographed and manually counted on day 10.

Cell cycle analysis and apoptosis detection
For cell cycle analysis, SiHa cells were incubated with various concentrations of 4SC-202 (0, 1, and 5 µM) for 72 h, digested with 0.25% trypsin and resuspended in phosphatebuffered saline (PBS) at a density of 1 × 10 6 cells/mL. Cell cycle distribution was detected by flow cytometry (BD Biosciences, CA, USA) and analyzed using Cell Quest software. For apoptosis detection, the cell suspension was gently mixed with 5 µL Annexin V-FITC and 10 µL PI, followed by an incubation at room temperature for 15 min in the dark. Apoptosis rate was determined by using the flow cytometry.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from the cell lines using the TRIzol kit (Thermo Fisher Scientific, Inc.). Afterward, total RNA was then reverse transcribed to cDNA by the SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific, Inc.) using the manufacturer's protocol. The gene expression was then detected using an ABI Stepone plus real-time PCR instrument (illumina eco, USA). The 2-ΔΔCq method was used to analyze the relative expression of mRNA. The primer sets are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control. The thermal cycling conditions were 95 °C for 3 min, followed by 45 cycles of 95 °C for 7 s, 57 °C for 10 s and 72 °C for 15 s.

Immunofluorescence
Cultured cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were blocked with 1% BSA at room temperature for 30 min. After the pretreatments, the cells were incubated with the primary antibodies (PRLR 1:20) at 4 °C overnight, followed by an incubation with Alexa Flour-conjugated secondary antibody (Invitrogen) at room temperature for 1 h in the dark. Nuclear DNA was counterstained with 10 ng/ml DAPI at 4 °C for 30 min in the dark. Stained cells were observed and photographed under a fluorescence microscope (200 ×).

Xenograft assay
Five-week-old female C57BL/6 nude mice were obtained from Bioscience (Beijing, China) and housed in a laboratory animal room under standard conditions (12-h light/dark cycle, 20-25 °C, and 60-85% humidity), with ad libitum access to sterilized food and water. The mice were randomly divided into three groups (n = 5 per group): control, 50 mg/ kg 4SC-202, and 100 mg/kg 4SC-202. SiHa cells were injected into the upper right flank of 15 nude mice. 4SC-202 treatment was started on day 6 post-injection when the tumors were palpable. 4SC-202 was dissolved in DMSO and administered by gavage every two days. The tumor size was measured every 3 days, and tumor volumes were calculated as follows: (V) = width 2 × length × 0.5. In the meantime, body weights of the mice were recorded every week. All the animals were euthanized, and the xenografts were weighted after 30 days.
Subcutaneous tumors were dissected, removed and kept for immunohistochemical, western blot and qRT-PCR Moreover, we observed that while 4SC-202 exerted cytotoxic effect on SiHa cells in a time-dependent manner, the effect was the most obvious at 72 h. Strikingly, 4SC-202 exhibited the similar effect on both Hela (Fig. 1B) and CaSki cells (Fig. 1 C). Among the three cell lines, SiHa cells treated with 4SC-202 showed the most significant decrease in the cell viability ( Fig. 1 A). Meanwhile, the IC50 values of 4SC-202 in all three cell lines decreased with increasing length of incubation. Collectively, these data indicated that 4SC-202 had anti-proliferative activity on cervical cancer cells. Besides, we performed colony formation assay and found that colony-forming ability of SiHa cells treated with 4SC-202 was most significantly inhibited (Fig. 1D), indicative of a suppressive effect of 4SC-202 on the growth of cervical cancer cells. The SiHa cells showed greater sensitivity to 4SC-202 than HeLa and CaSki cells. We, therefore, chose SiHa cells for subsequent experiments.

4SC-202 induces cell cycle arrest and apoptosis
Effects of 4SC-202 on the cell cycle distribution of SiHa cells were analyzed by flow cytometry. As illustrated in We next performed western blotting to analyze the expression of several apoptosis-related proteins. As shown in Figs. 2B and 4SC-202 down-regulated the expression of BCL-2 (26 kDa), while up-regulating the expression of Cleaved-caspase3 (17 kDa) and Bax (21 kDa). In addition, we evaluated the effects of 4SC-202 on cell apoptosis by flow cytometry and found that 4SC-202 treatment led to a dose-dependent increase in the apoptosis rate of SiHa cells (Fig. 2 C).

4SC-202 reduces the expression of PRLR in cervical cancer cells by inhibiting the PRLR pathway
We further examined the expression of PRLR pathwayrelated proteins by western blot. As presented in Fig. 3 A-C, compared with the control group, the phosphorylation levels of STAT5a, JAK2, AKT and PI3K were decreased after 4SC-202 treatment of Siha cells, and the phosphorylation analyses. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by AU680 Chemistry System (Beckman Coulter, Brea, CA, USA). Organ tissues including lung, liver, kidney, and heart were removed, sectioned and subjected to H&E staining. Animal experiments were approved by the Ethics Committee of Medical Laboratory Animal Center of Chinese People's Liberation Army General Hospital and performed in accordance with National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals.

Immunohistochemical analysis
Paraffin-embedded sections were dewaxed with dimethyl benzene and rehydrated in dH 2 O. The slides were immersed in citrate buffer (0.01 M, pH 6.0) with high-pressure heating for 2 min. After being blocked with 1% BSA (Sigma, USA) in TBST buffer for 5 min at room temperature, the slides were incubated with primary antibodies against Ki-67 (Proteintech, Wuhan, China) at a dilution of 1:16000 overnight at 4 °C, followed by an incubation with goat anti-rabbit IgG secondary antibody (Thermo Fisher, 1:5000 dilution) and DAB staining. Finally, the slides were counterstained with hematoxylin, dehydrated and sealed with neutral gum. Organ tissues were stained with H&E for histological analysis. The images were captured under a Nikon microscope.

Statistical analysis
Results were presented as the mean ± standard deviation (SD). At least three experimental replicates were evaluated for each sample. The significance of the experimental data was analyzed by using PRISM® GraphPad 8.0 software, one-way or two-way analysis of variance (ANOVA) followed by Tukey's post-hoc tests. The significance level was set at P < 0.05, and p-values were indicated accordingly.

4SC-202 suppresses the proliferation of cervical cancer cells
To study the effect of 4SC-202 on the proliferation of cervical cancer cells, we performed CCK-8 assay to detect the cell proliferation in selected three human cervical cancer cell lines SiHa, Hela and CaSki. As depicted in Fig. 1 A, 4SC-202 displayed a dose-dependent cytotoxicity against SiHa cells, while SiHa cells treated with 4SC-202 at a concentration of 1-10 µM showed significantly reduced cell viability.
the expression of PRLR in SiHa cells compared with the control group (Fig. 3E-F). Together, these results indicated that 4SC-202 reduced the expression of PRLR in CC cells through down-regulating PRLR pathway-related proteins. levels of MEK1/2 and ERK1/2 increased. qRT-PCR showed that 4SC-202 resulted in a significant decrease in STAT5a, AKT and PI3K mRNA expression, while a significant increase in MEK mRNA expression. However, the mRNA expression of JAK2 and ERK1/2 only changed to a certain extent, and there was no significant difference. Moreover, we performed an immunofluorescence assay and qRT-PCR and observed that 4SC-202 significantly down-regulated  (Fig. 4E), and PRLR mRNA was also significantly inhibited ( Fig. 4 F). In addition, we observed a consistency in the expression of PRLR pathway related proteins and mRNAs between in vivo and in vitro studies (Fig. 5).
Finally, H&E staining was performed to histologically analyze major organs such as lung, liver, spleen and heart. As shown in Fig. 6 A, no significant histological changes were detected in the tissue sections of mice treated with 4SC-202. We found that 4SC-202 had no significant effects on the serum biochemical function in the nude mice (Fig. 6B).

Discussion
HDAC is essential for epigenetic control of cancer gene expression and functions, while being involved in the regulation of cell cycle progression, cell proliferation and differentiation, apoptosis, metastasis as well as angiogenesis (Fu et al. 2016;Li and Seto 2016;Falkenberg and Johnstone 2014). HDACi can restore the homeostasis of cell

4SC-202 inhibits tumor growth via the PRLR pathway
To investigate effects of 4SC-202 on the growth of cervical cancer cells in vivo, the nude mice were injected subcutaneously with SiHa cells and then administered with 50 or 100 mg/kg 4SC-202 by gavage every two days for 30 days. As depicted in Fig. 4 A-C, both tumor volume and tumor weight were significantly reduced in the mice treated with 4SC-202 as compared to the control group. Furthermore, we performed Ki67 immunostaining on tumor sections to assess tumor proliferation in vivo. The assay showed that compared with the control, 4SC-202 treatment led to a significant decrease in the percentage of Ki-67 positive cells in the tumor tissue (Fig. 4D).
We next evaluated the expression of apoptotic proteins and PRLR mRNA in the nude mice treated with 4SC-202. Consistent with the in vitro data, we found that 4SC-202 remarkably down-regulates BCL-2 expression and up-regulated the expression of cleaved-caspase3 and Bax in the  Immunofluorescence was performed to detect the expression of PRLR. The immunofluorescence intensity was quantified. Scale bar = 30 μm. (F) The PRLR mRNA expression was determined by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group anti-tumor and anti-apoptotic effects in xenograft mouse model, dramatically decreased the percentage of Ki-67-positive cells and inhibited the PRLR pathway in tumor tissues. Meanwhile, we observed no significant alterations in serum biochemical function and histological morphology of organ tissues in the nude mice treated with 4SC-202, indicating that 4SC-202 was nontoxic in vivo. All these results suggested that 4SC-202 could serve as a potential therapeutic agent for CC.
HDACi can cause cell cycle arrest in G1/S or G2/M phase, highlighting HDACi as a promising target for cancer treatment (Li and Seto 2016). In this study, we showed that 4SC-202 treatment led to cell cycle arrest of SiHa cells in G2/M phase (Fig. 2 A). This observation is consistent with the previous study showing a G2/M phase arrest in colorectal cancer (CRC) cell lines and primary human CRC tumor cells following 4SC-202 treatment (Huang et al. 2016). In addition, HDACi can regulate apoptosis in cancer cells processes of cancer. And HDACi has been applied in some clinical trials (Lu et al. 2016;Iyer and Foss 2015;Lee et al. 2015). DNA methylation and covalent histone modification have been identified as the most important epigenetic changes in cervical cancer (Fang et al. 2014). To date, HDACi such as valproic acid (VPA) (de la Cruz-Hernández et al. 2007), suberoylanilide hydroxamic acid (SAHA) (Zuo 2010), and trichostatin A (TSA) (Liu et al. 2012) have been used as anti-tumor agents in cervical cancer. However, the research on anti-tumor effects of HDACi 4SC-202 on CC is still lacking.
In this study, we showed that 4SC-202, a novel oral class I specific HDACi of benzamide compounds (Henning et al. 2010), can effectively inhibit the growth, proliferation, and cell cycle progression of CC cells and promote the apoptosis. Moreover, we found that 4SC-202 treatment led to inhibition of the PRLR pathway as well as down-regulation of PRLR expression in CC cells. Notably, 4SC-202 displayed PRL had a protective effect on etoposide-induced apoptosis in CC cells (Lopez-Pulido et al. 2013). A study on melanoma cells showed that HDACi mainly suppressed the expression of anti-apoptotic proteins by inhibiting transcription (Gallagher et al. 2015). Cytotoxicity of 4SC-202 against CRC cells was enhanced by AKT inhibition or AKT1 knockdown (Huang et al. 2016). Herein, we observed that the expression of PRLR and PRLR-related pathway components (STAT5a, JAK2, MEK1/2, ERK1/2, AKT, and PI3K) was significantly inhibited in SiHa cells treated with 4SC-202 (Fig. 3). Furthermore, 4SC-202 markedly decreased the proliferation of CC cells (SiHa, HeLa, and CaSki) in a doseand time-dependent manner (Fig. 1). Combined with the results of apoptosis, we speculated that 4SC-202 may promote apoptosis in CC cells by down-regulating the expression of PRLR and inhibiting the PRLR pathway, thereby reducing the cell proliferation.
The present study provided evidence that tumor growth was inhibited in the nude mice administered with 4SC-202, while no toxicity was detected in major organs of the mice. through the mitochondrial pathway and the expression of some Bcl family proteins (Wen et al. 2012). Zhang et al. demonstrated that 4SC-202 induced apoptosis in hepatocellular carcinoma (Fu et al. 2016). Clearly, the present study confirmed that 4SC-202 promoted apoptosis in CC cells by up-regulating the expression of caspase3 and Bax and inhibiting Bcl-2 expression (Fig. 2B). In the meantime, flow cytometry analysis revealed that 4SC-202 significantly increased the apoptosis rate in CC cells (Fig. 2 C).
Previous studies have shown that PRLR signaling pathways play an important role in initiating CC (Ascencio-Cedillo et al. 2015). PRLR dimerization activates intracellular signaling cascades including downstream signaling pathways such as JAK/STAT5a, P13K/AKT, and ERK1/2, thereby promoting cell proliferation, differentiation and survival (Kan et al. 2016;Gorvin et al. 2019). It has been reported that PRL induced an increased expression of anti-apoptotic genes in CC cell lines by activating the STAT pathway, which was linked to cell survival (Ramirez de Arellano et al. 2015). In a previous study, we found that The relative mRNA expression of STAT5a, JAK2, MEK1/2, ERK1/2, AKT and PI3K were measured by qRT-PCR. *P < 0.01, **P < 0.01, ***P < 0.  Data availability All data generated or analyzed during this study are included in this published article.

Conflict of interest
The authors declare that they have no financial and non-financial conflict of interest.
In addition, immunohistochemical assays revealed that the expression of Ki67, a nuclear and nucleolar protein related to cell proliferation and severity of cervical lesions (Aslani et al. 2013;Riera-Leal et al. 2018), decreased significantly with the increasing concentration of 4SC-202 in the tumor tissues. The expression of apoptotic protein and PRLR pathway components decreased in the tumor tissues, and 4SC-202 displayed no effect on the levels of ALT and AST in serum. All these findings indicated that 4SC-202 may affect the proliferation and activity of CC cells through inhibiting the expression of PRLR-related pathway components as well as induce cycle phase arrest in vivo and was non-toxic to organs.
In conclusion, the present study showed that 4SC-202 caused cell cycle arrest of CC cells at G2/M phase through regulating the expression of PRLR-related pathway components (STAT5a, JAK2, MEK1/2, ERK1/2, AKT, and PI3K), induced apoptosis and inhibited CC cell proliferation. Moreover, 4SC-202 significantly suppressed the growth of CC in vivo, while exerting no toxicity to major organs in the nude mice. These findings suggest that 4SC-202 may potentially serve as a novel and efficacious therapeutic agent for CC.

CC
cervical cancer PRL prolactin PRLR prolactin receptor