Background
With over half a million cases occurring annually worldwide, cervical cancer remains the second most common cancer and third leading cause of cancer-related death among females in developing countries [
1‐
3]. Although human papillomavirus (HPV) vaccines are available and are effective in reducing the incidence and mortality, improved treatment for cervical cancer remains an urgent need [
4]. In particular, patients with advanced cervical cancer have extremely poor prognosis and low survival rate [
5]. According to National Comprehensive Cancer Network (NCCN) 2016, radiotherapy can be performed in all stages of patients [
6]. However, increased dose of radiation inevitably leads to adverse reactions, and accumulating resistance to radiation therapy needs to be taken into account [
7]. Therefore, questions remain as to which factors contribute to the radioresistance and poor outcomes of cervical cancer.
As a family of evolutionarily conserved enzymes, (mitogen-activated protein kinase) MAP kinases play a critical role in signaling cascades, which mediate a wide range of extracellular stimuli and result in different cellular responses, including cell proliferation, cell motility, cell cycle, cytokine biosynthesis, and chromatin remodeling and so on [
8]. A total of 13 human MAP kinases have been identified, including ERK1/2 (MAPK3/1), P38α/β/γ/δ (MAPK14/11/12/13) and JNK1/2/3 (MAPK8/9/10) [
9]. MAPK4, also known as ERK4, p63 MAPK, ERK3β and Prkm4, has been mapped to chromosome 18q12–21 following the identification of ERK1, ERK2 and ERK3 [
10]. As an atypical MAPK, MAPK4 lacks TXY and APE motifs, which are highly conserved in other MAPKs. Instead, MAPK4 consists of a SEG sequence and SPR motif. Thus, MAPK4 can not be phosphorylated by the dual Ser/Thr and Tyr MAPK kinase (MAPKK) [
11,
12]. Transcriptomic profiling data provided by The Cancer Genome Atlas (TCGA) [
13] show that MAPK4 expression is correlated with the survival rates in patients with lung cancer, bladder cancer and glioma. A recent report demonstrated that MAPK4 activates protein kinase B (AKT) in a phosphatidylinositol 3-hydroxykinase (PI3K)-independent manner and promotes cell proliferation and xenograft tumor growth [
14]. After exposure to ionizing radiation, AKT phosphorylation plays a pivotal role in the stimulation of DNA-PKcs and recruitment of AKT1/DNA-PKcs complex as an initiating step of DNA double-strand break (DNA-DSB) repair [
15]. A single gene may play different roles depending on the tissue context. In this study, the expression and function of MAPK4 were determined in cells of lung cancer, colon cancer and prostate cancer. Nevertheless, the functions of MAPK4 in radiation and its involvement in diseases, including cervical cancer, requires further investigation.
As an abundant nuclear protein implicated in DNA single-strand break (DNA-SSB) repair, PARP1 plays a vital role in catalyzing poly ADP-ribose formation and maintaining genome integrity [
16]. Suppression of PARP1 leads to defective DNA-SSB repair and results in accumulation of DNA-DSB [
17]. Given the regulatory roles of MAPK4/AKT and PARP1 in DNA-DSB and DNA-SSB repair, respectively, the possible synergistic effects between MAPK4 deletion and PARP1 inhibition requires further elucidation. Previous study showed that PARP1 hyperactivation leads to therapeutic resistance, and the therapeutic potential of PARP inhibition in combination with cisplatin has shown profound anti-cancer effect in cervical cancer [
18]. In addition, high PARylation activity is correlated with sensitivity to olaparib in cervical cancer cells, and represents a biomarker for the identification of patients likely to benefit from PARP1 inhibition [
19]. However, the synergistic role of PARP1 inhibition in combination with MAPK4 intervention for cervical cancer therapy is yet to be elucidated.
Here we report that high MAPK4 expression is associated with significantly decreased overall survival rate in cervical cancer patient. Cervical cancer cells were more sensitive to radiation treatment after MAPK4 knockout, as determined by colony formation, immunofluorescence and western blotting. Based on the results from the protein analysis, we found that MAPK4 specifically activated AKT phosphorylation and further affected DNA repair. In addition, MAPK4 knockout enhanced the sensitivity of cervical cancer cells to PARP1 inhibitors, olaparib and veliparib. The synergistic lethality of MAPK4 deletion in combination with PARP1 inhibition was further evaluated in a xenograft mouse model. Together, these results demonstrated that MAPK4 knockout alongside with PARP1 inhibition may improve the therapy of cervical cancer.
Methods
Patient tissue samples
Paired cervical cancer tissues (tumor, n = 60) and normal adjacent tissues (non-tumor, n = 60) were obtained from the Henan Cancer Hospital. All patients were pathologically and clinically diagnosed as cervical cancer and received no chemotherapy or radiotherapy before section. The collection and experiments were performed under the permission of the Ethics Committee of Henan Cancer Hospital (Approval no.2016HC047). Written informed consents were obtained from all participants.
Quantitative real-time PCR (qRT-PCR)
First, total RNA was extracted from tissues and cells using the TRIzol reagent (TaKaRa, Otsu, Japan) according to the manufacturerʼs instructions. Total RNA was then converted into complementary DNA (cDNA) using a PrimerScript™ RT Reagent Kit (TaKaRa), following standard procedures. qRT-PCR was performed using a One-Step SYBR PrimeScript RT-PCR Kit (TaKaRa) on a 7500 Real-Time PCR System (Applied Biosystems, Lincoln Centre Drive Foster City, CA). β-Actin was used as a housekeeping gene to normalize the gene expression. The primer sequences for MAPK4 and RAD51 recombinase (RAD51) are listed in Table S
1.
Overall survival analysis
After qRT-PCR analysis, mean MAPK4 mRNA level was calculated and used as a criterion to estimate the expression of MAPK4. If the MAPK4 mRNA level was higher than the mean value, the corresponding patient was identified as “MAPK4 high”, whereas an MAPK4 mRNA level lower than the mean value was denoted as “MAPK4 low”. After surgery combined with radiotherapy or postoperative radiotherapy, follow-up data was collected and the overall survival rate of cervical cancer patients in “MAPK4 high” and “MAPK4 low” group was analyzed using the Kaplan-Meier method.
Cell culture
In the present study, human cervical cancer cells, SiHa and caSki, were obtained from the ATCC cell lines (Manassas, USA). Cells were recently authenticated by STR profiling. Eagle’s Minimum Essential Medium (EMEM) and RPMI-1640 medium were used to culture SiHa and caSki cells, respectively. Cells were cultured in medium supplied with 10% FBS (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO2 at 37 °C.
Generation of MAPK4 knockout cell lines
SiHa and caSki cells with MAPK4 deletion were established by CRISPR-Cas9 technique as previously reported [
14]. Briefly, lentiviral packaging vectors pMD2.G and psPAX2 along with sgRNA-expressing vector, CRISPR-v2 vector, were transfected into HEK293T cells using the Lipo2000 transfection reagent (Thermo Fisher Scientific). The sgRNA sequence for MAPK4 used in study is ACTTCACTGTTCACTTCAGGGAG. Viruses were collected 72 h after transfection and infected SiHa and caSki cells. Then, MAPK4-deleted stable cell lines were selected by puromycin and were continuously monitored by western blotting to avoid potential reversal effects.
Western blotting
Western blotting was performed as previously reported [
20]. Briefly, we collected protein lysates from cells and tissues using RIPA lysis buffer (Sigma-Aldrich, 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mM β-glycerophosphate, 1% Triton X-100, 5 mM EDTA, 0.2 mM Na
3VO
4, 2 μg/mL leupeptin, 2 μg/mL pepstatin A). Protein lysates were quantified by a BCA protein assay kit (Thermo Fisher Scientific) and loaded onto the sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) for electrophoresis. Next, proteins were transferred onto a PVDF membrane. Membranes were blocked with 5% BSA, and incubated with primary antibodies at 4 °C overnight. After that, HRP (horseradish peroxidase)-conjugated secondary antibodies were used to incubate the membrane. Immunoreactivity was determined by an immobilon ECL western HRP substrate and (Millipore, Billerica, MA, USA) and a chemiluminescence imaging system. β-actin protein level was used as a control. Antibodies used in this study are as follows: MAPK4, phosphorylated-DNA-dependent protein kinase (p-DNA-PK), RAD51, p-AKT T308, p-AKT s473, AKT, AKT1, AKT2, p-AKT and phosphorylated histone H2AX (γH2AX). Western blot bands were quantified by ImageJ and the quantification is shown in histogram. Antibody details are listed in Table S
2.
Irradiation was performed by X-rays at a dose rate of 5 Gy/min using a linear accelerator (PRIMUS-M, Siemens) for 2 h. After radiation treatment, SiHa or caSki cells were primarily blended into top agar, and the mixture was then added onto base agar. After three weeks, colonies were stained with crystal violet and counted by dissection microscope (Nikon, Tokyo, Japan).
Immunofluorescence
Cells were seeded and treated with 5 Gy irradiation following cell adhesion. After fixation with 4% paraformaldehyde, SiHa or caSki cells were permeabilized in 0.1% Triton X-100. Then, cells were blocked with 5% BSA and incubated with anti-53BP1 and anti-γH2AX primary antibodies overnight at 4 °C. Cy5-conjugated and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were used. To visualize nuclear DNA, cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich, St. Louis, MO, USA). Cells were observed and photographed using a confocal microscope (Nikon).
Cell transfection and cell treatment
SiHa or caSki cells were transfected with MAPK4 expressing plasmid (MAPK4), shRNA for MAPK4 (sh-MAPK), siRNA for AKT1 and AKT2 (si-AKT1, si-AKT2), plasmid with constitutively activated AKT (CA-AKT) or the corresponding controls (Ctrl or si-NC) using Lipofectamine 2000 (Thermo Fisher Scientific). Three shRNAs for MAPK4, three siRNAs for AKT1 and AKT2 were tested. The siRNA sequences are listed in Table S
3. Oligos with highest efficiency were used for subsequent experiments. To construct the MAPK4-overexpressing vector, a One Step Cloning Kit (Vazyme Biotech, Nanjing) was used according to the manufacturer’s instructions. The coding sequence (CDS) of MAPK4 was amplified using a human genomic DNA extracted from SiHa cells and then inserted into the PCI plasmid (Promega, Madison, WI, USA). Primers sequences for cloning MAPK4 CDS into the expressing vector are listed in Table S
4. CA-AKT plasmid was purchased from Addgene (catalog no. plasmid #14751, Watertown, MA).
Synergistic effect of MAPK4 deletion with PARP1 inhibitors
After cells were incubated with different concentrations of PARP1 inhibitors, olaparib or veliparib, cell viablity was determined by a cell counting kit-8 (CCK-8, Dojindo, Tokyo, Japan). A microplate spectrometer (Thermo Fisher Scientific) was used to determine the absorbance (OD = 450 nm). Next, the 50% inhibitory concentration (IC50) was quantified using the sigmoidal dose-response function of GraphPad Prism. In addition, after knocking out MAPK4, SiHa or caSki cells were over-expressed with MAPK4 or CA-AKT. Accordingly, the IC50 was quantified.
Xenograft mouse model
Animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health [
21] and approved by the Ethics Committee of Henan Cancer Hospital. Thymic BALB/c nude mice (female, four-six weeks, 16-18 g) were housed in a pathogen-free facility and used for xenograft studies. SiHa or caSki cells with MAPK4 deletion (1 × 10
6) and the control cells were subcutaneously injected into the right flanks of mice. When the tumors reached a diameter of 4–5 mm, mice were randomly assigned to different groups (6 mice/group). Tumors were monitored and treated with 5 Gy irradiation every 2 days after the injection. The length (L) and width (W) of tumors were determined every week. Then, the volume of each tumor was calculated using the formula V = 1/2LW
2. At 4 weeks post-implantation, tumors were dissected from the mice. Protein expression of p-AKT, AKT, p-DNA-PK, MAPK and γH2AX in tumor tissues was analyzed in each group established by caSki cells. To study the synergistic effect of MAPK4 deletion with PARP1 inhibitors in vivo, olaparib (100 mg/kg) was administered by oral gavage once daily, in addition to irradiation treatment. Tumor volume (mm
3) of each group median was graphed over time to monitor tumor growth. 4 weeks later, tumors were harvested and photographed.
Statistical analysis
The statistical data were obtained from three independent experiments and fit the normal distribution. Data are presented as the mean ± standard error of the mean (SEM). The investigator was blinded to the group allocation during the experiment. Statistical differences were analyzed using the Student t test for two groups and ANOVA for multiple groups. Variation within each group of data was estimated, and the variance between groups was statistically compared. P < 0.05 was considered as significant.
Discussion
In this study, we have not only demonstrated the suppressive role of MAPK4 knockout on AKT phosphorylation, but also identified the relationship between MAPK4 knockout and enhanced sensitivity of cervical cancer to radiation treatment and PARP1 inhibitors in cells and mouse models. Although intensity-modulated radiotherapy (IMRT) has been adopted to reduce gastrointestinal toxicity and increase the radiotherapy dose, treatment tolerance and severe side effects are dose-limiting factors. Thus, novel developments aim to improve radiotherapy for cervical cancer [
22]. It was demonstrated that concomitant radiochemotherapy could improve disease-free and overall survival compared to radiotherapy alone in early cervical cancer [
23]. For example, methyl jasmonate (MJ), a newly identified cytotoxic agent, when effectively incorporated with X-ray irradiation, can significantly reduce the radiation doses required to inhibit cell survival of cervical cancer cells [
24]. Additionally, the combination of Aloe-emodin (AE), an
Aloe vera leaf exudate and radiation induce apoptosis and further improve Alkaline phosphatase (ALP) activity compared with treatment with AE or radiation alone [
25]. Our data in this study demonstrated that MAPK4 knockout could enhance the sensitivity of cervical cancer cells to radiation treatment both in vitro and in vivo, suggesting that targeting MAPK4 may be a promising radiosensitizer.
As an atypical member of the mitogen-activated protein (MAP) kinase family, MAPK4 knockout mice are viable and fertile and exhibit no gross morphological or physiological anomalies. However, MAPK4-deficient mice manifest depression-like behavior in forced-swimming tests, indicating that the MAPK4 has acquired specialized functions through evolutionary diversification [
26]. So far, little is known about the physiological function of MAPK4 and its involvement in diseases, including cancer. Although gene expression profiling data provided by The Cancer Genome Atlas (TCGA) show that MAPK4 expression is correlated with the survival rates in patients with lung cancer, bladder cancer and glioma, its functions and mechanism of actions in lung cancer and colon cancer were recently identified [
13]. Wang et al. demonstrated that over-expression of MAPK4 leads to oncogenic effects, and MAPK4 inhibition suppresses cell proliferation and xenograft tumor growth. Mechanistically, MAPK4 activates the phosphorylation of AKT at threonine 308 and serine 473 [
14]. Our data in this study demonstrated that cervical cancer patients with high MAPK4 expression had lower survival probability and MAPK4 deletion blocked AKT phosphorylation in cervical cancer cells. AKT phosphorylation has previously been described to cooperate with DNA-PKcs and was involved in DNA damage repair. AKT1 is a regulatory component in the homologous recombination repair of DNA-DSB in a Rad51-dependent manner in non-small cell lung cancer cells [
27]. Single knockdown of Akt1 and Akt2 leads to a decrease in Rad51 foci formation and significantly reduces Rad51 protein level in colon cancer cells [
28]. Moreover, Akt1-T308A/S473A-expressing cells are characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays [
29]. Dual targeting of mTORC1 and AKT1 inhibits DNA-DSB repair, leading to radiosensitization of solid tumor cells [
30]. We found that MAPK4-knockout cervical cancer cells showed lower AKT phosphorylation level, and had heightened sensitivity to radiation treatment and PARP1 inhibitors.
In regard to the upstream regulation of MAPK4, two miRNAs have been reported to specifically target MAPK4. Over-expression of miR-767-5p functions as a tumor drive through targeting MAPK4 in multiple myeloma [
31]. miR-127 was found to target both MAPK4 and HOXC6, and promotes cell proliferation and decreases differentiation in porcine [
32]. These indicate that the expression and functions of MAPK4 may vary depending on the cellular context. To date, the regulatory mechanism of MAPK4 in cervical cancer remains unclear, and whether or not miR-767-5p and miR-127 could target MAPK4 and other potential transcriptional regulatory factors will require further investigation.
Because radiotherapy alone or concurrent chemoradiation fail to control advanced cervical cancer, surgery, chemotherapy or targeted therapy have been used in combination to improve the radiotherapy and minimize the side effects. Studies are currently being carried out to investigate potential suppressors of survival pathways and promoters of apoptotic pathways as novel chemotherapy approaches for the treatment of cervical cancer [
33]. An association between poly ADP-ribose polymerase 1 (PARP1) Val762Ala polymorphism (rs1136410) and cancer therapy response has been identified, and PARP1 genotypes have been proposed to be an independent prognostic factor in cervical cancer [
34]. In addition, it has been shown that PARP-1 inhibition may augment cisplatin cytotoxicity in cervical cancer cells by diminishing DNA repair and suppressing β-catenin signaling pathway [
35]. miR-7-5p was demonstrated to negatively regulate PARP-1 protein and gene expression in cisplatin-resistant cervical cancer cells, and facilitates DNA repair and maintain cell survival [
36]. However, little is known about the therapeutic efficacy of PARP inhibitors in the treatment of cervical cancer, either as a single agent or in combination with MAPK4 knockout. PARP1 inhibition along with superoxide dismutase 1 (SOD1) inhibition could promote the synthetic lethal killing of RAD54B-deficient colorectal cancer cells [
37]. Previous study has also shown that simultaneous treatment with PARP and RAD52 inhibitors exerts dual synthetic lethality in BRCA-deficient tumors [
38]. Currently, PARP inhibitors are under clinical trials for BRCA1/BRCA2-deficient breast cancer and ovarian cancer by the approach of synthetic lethal [
39,
40]. Our results demonstrate for the first time that addition of PARP inhibitor may improve therapeutic outcome of MAPK4-deficient cervical cancer treated with radiation.
Conclusions
Altogether, our data revealed that cervical cancer patients with high MAPK4 mRNA expression have lower survival rate. After radiation treatment, the colony number of MAPK4 knockout cells was markedly reduced, and the markers for DNA double-chain breakage were significantly up-regulated. In addition, MAPK4 knockout reduced the phosphorylation of AKT, whereas its over-expression exerted opposite effects. In MAPK4 KO cells with irradiation treatment, inhibition of AKT phosphorylation promoted DNA double-chain breakage, and constitutively activated AKT (CA-AKT) increased the levels of p-AKT and DNA repair related proteins, p-DNA-PK and RAD51. MAPK4 is further found to affect the sensitivity of cervical cancer cells to PARP1 inhibitors by activating AKT phosphorylation. Moreover, MAPK4 knockout enhanced the sensitivity of cervical cancer to radiation and PARP1 inhibitors in mouse xenograft models. Collectively, our data suggest that combined application of MAPK4 knockout and radiation treatment or PARP1 inhibition can be used as therapeutic strategy for advanced cervical carcinoma.
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