Chidamide and Radiotherapy Synergistically Induce Cell Apoptosis and Suppress Tumor Growth and Cancer Stemness by Regulating the MiR-375-EIF4G3 Axis in Lung Squamous Cell Carcinomas

As a selective histone deacetylase (HDAC) inhibitor developed in China, chidamide has been applied for the treatment of refractory peripheral T-cell lymphoma (PTCL) and multiple solid tumors, including lung cancer. However, the underlying mechanisms are not well elucidated. In our present study, we found that chidamide and radiation acted synergistically to suppress cell and xenograft growth of lung squamous cell carcinoma cells by inducing cell apoptosis. Moreover, chidamide alone or a combination of chidamide and radiation treatment inhibited cancer cell stemness. miRNA microarray analysis demonstrated that miR-375 was the highest upregulated microRNA (miRNA) in NCI-2170 and NCI-H226 cells treated with chidamide alone or treated with chidamide plus radiation, compared with normal control. Inhibition of miR-375 attenuated the promoting effect of chidamide alone and chidamide plus radiation-induced NCI-2170 and NCI-H226 cell apoptosis and reverted the suppression of cancer stemness caused by chidamide alone or chidamide plus radiation treatment. Moreover, EIF4G3, a scaffold protein in the translation initiation complex, was found to be a direct target of miR-375 based on the luciferase reporter assay and western blot analysis. Interestingly, both chidamide alone and chidamide plus radiation treatments suppressed the mRNA and protein expression of EIF4G3. Silence of EIF4G3 also induced cell apoptosis and suppressed tumor growth in NCI-2170 and NCI-H226 cells. These data suggest that chidamide shows a synergistic effect with radiation therapy on lung squamous cell carcinomas by modulating the miR-375-EIF4G3 axis, which may afford an effective strategy to overcome the drug resistance of chidamide in clinical cancer therapy.


Introduction
Lung cancer is one of the leading causes of cancer-associated deaths all over the world, with a high metastatic potential [1]. Lung cancer can be divided into two main groups: small-cell lung cancers (SCLCs) and non-small-cell lung cancers (NSCLCs) [2]. NSCLCs account for 85% of lung cancer and can be further classified into four subtypes according to their histological and molecular features: lung large-cell carcinomas (LCLCs), lung neuroendocrine tumors (LungNETs), lung adenocarcinomas (LUADs), and lung squamous cell carcinomas (LSCCs) [3]. LSCCs are the main type of NSCLC showing strong malignancy. Although advanced treatment strategies and technologies such as surgical treatment, radiotherapy, and chemotherapy have been developed rapidly, the five-year survival rate among patients with LSCC remains very poor with increased risk of recurrence [4]. erefore, it is urgently needed to furtherly understand the molecular mechanisms underlying LSCC initiation and progression and to seek effective methods for early detection and treatment.
Chidamide, a selective inhibitor of HDAC1, 2, 3, and 10 developed wholly in China, has been entered into clinical trials both in the United States and China. In December 2014, chidamide has been approved by the China Food and Drug Administration (CFDA) as a treatment strategy for peripheral T-cell lymphoma (PTCL) [5]. Interestingly, accumulating studies have demonstrated that chidamide shows an effective antitumor activity in multiple solid tumors, including liver cancer, colon carcinoma, and lung cancer [6][7][8][9][10][11]. In lung cancer, Hu et al. have performed a phase I trial of chidamide combined with paclitaxel and carboplatin in patients with advanced NSCLC and found that a combination treatment of chidamide and paclitaxel or carboplatin was tolerated without unanticipated toxicities or pharmacokinetic interactions [6]. Chidamide has also been reported to enhance the suppressive effect of platinum on NSCLCs [11]. However, the underlying mechanisms through which chidamide suppresses lung cancer are unclear.
MicroRNAs (miRNAs) represent a class of noncoding short RNAs with 19-24 nucleotides in length, which was highly conserved in eukaryotes. miRNAs play an important role in regulating multiple physiological and pathological processes by binding to the 3′-untranslated regions (3′-UTRs) of target genes [12,13]. Dysregulation of miRNA has been implicated in the initiation and progression of a wide range of cancers, including liver, gastric, breast, lung, and colorectal cancers. For instance, Tian et al. found that silence of miR-203 promotes tumor cell growth and invasion by upregulating the SNAI2 in prostate cancer [14]. miR-22 inhibits breast cancer cell proliferation and increases paclitaxel sensitivity by suppressing N-RAS [15]. Aberrant expression of miRNAs has also been observed in lung cancer. For example, downregulation of miR-98-5p in NSCLC suppresses NSCLC proliferation and metastasis by targeting TGFBR1 [16]. miR-5195-3p inhibits cell proliferation, migration, and invasion in human NSCLC by targeting MYO6 [17].
Among these cancer-related miRNAs, miR-375 was initially identified as a critical regulator of insulin secretion and a novel therapeutic target for diabetes treatment [18]. Further studies have demonstrated that miR-375 participates in various cancer types by targeting several critical target genes including ATG7, AEG-1, YAP1, SP1, IGF1R, JAK2, and PDK1 [19]. e deregulation of miR-375 in tumors can be caused by a variety of mechanisms such as aberrant promoter methylation [20][21][22]. Deregulation of miR-375 can also be used as a biomarker for cancer prediction and diagnosis [23,24]. In lung cancer, Jin et al. have reported that miR-375 expression was obviously increased in lung adenocarcinoma and SCLCs but reduced in LSCCs [25]. However, the exact role of miR-375 in lung cancer, especially in LSCCs, is not fully understood.
In the present study, we found that a combination of chidamide and radiation treatment promoted synergistic cytotoxicity and suppressed tumor stemness in LSCCs. Importantly, miR-375 was upregulated in NCI-2170 and NCI-H226 cells treated with chidamide alone or with chidamide plus radiation, compared with normal control. In addition, suppression of miR-375 attenuated chidamide alone and chidamide plus radiation-induced NCI-2170 and NCI-H226 cell apoptosis and suppressed tumor growth and stemness. Moreover, EIF4G3 was identified as a direct target of miR-375. Interestingly, both chidamide alone and chidamide plus radiation treatments suppressed the mRNA and protein expression of EIF4G3. Silence of EIF4G3 also induced cell apoptosis and suppressed tumor growth in NCI-2170 and NCI-H226 cells. ese data suggest that chidamide shows a synergistic effect with radiation therapy on lung squamous cell carcinomas by modulating the miR-375-EIF4G3 axis.

miRNA Mimic, Inhibitor, and siRNA Transfection.
Cells were cultured to about 75% confluence before transfection. Control mimic, miR-375 mimic, control siRNA, and EIF4G3 siRNA were transfected into cells using Lipofectamine RNAiMAX Reagent ( ermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's manual. Forty-eight hours after transfection, the cells were applied for the following experiment. e siRNA oligos were synthesized by Santa Cruz (CA, USA).

Cell Proliferation.
Cell Counting Kit-8 was applied to determine the proliferation rate of NCI-H2170 and NCI-H226 cells with indicated treatment as previously described. Briefly, two thousand treated cells were plated into 96-well plates. en, CCK-8 solution was added at the harvest time and incubated for an additional 30 min. e absorbance was determined at 450 nm on the microplate reader (Molecular Devices, Walpole, MA, USA).

Apoptosis
Assay. Apoptosis assay was performed by Annexin V/PI double staining using the Annexin V-FITC apoptosis detection kit (BD Biosciences, Pharmingen, CA, USA) following the standard manual. Briefly, treated cells were washed with cold 1x PBS and resuspended in 1x binding buffer. Five μl of FITC-labeled Annexin V and 5 μl of propidium iodide (PI) were added to the suspended cells and gently mixed, following by the incubation at room temperature for 15 min in dark. At last, the samples were detected on the flow cytometer (BD Biosciences).

Luciferase Reporter Assay.
e luciferase reporter assay was determined using the psi-CHECK2 dual-luciferase system (Promega, Madison, WI, USA) following the standard manual.

ALDEFLUOR Assay and Flow Cytometry.
e ALDE-FLUOR Kit (Stemcell Technologies) was used for ALDH + cell analyses according to the manufacturer's manual. For each sample, one-half of cells was treated with 50 mM of diethylaminobenzaldehyde (DEAB) to define negative gates.

Sphere Formation Assay.
Sphere formation assay was determined as previously described [29].

Xenograft Mouse Model.
Treated cells were subcutaneously injected into both sides of flank areas of 6-8-weekold BALB/c nude mice for 42 days. Tumor volumes were measured using the following equation: 0.5 × length × width 2 each other day after palpable tumors appeared. e study protocol was approved by the Animal Care and Use committee of Harbin Medical University.
2.13. Statistical Analysis. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using the software GraphPad Prism 5. Student's t-test was used to determine the statistical differences, in which p < 0.05 was considered to be significant.

Both Chidamide Alone and Chidamide Plus Radiation Combinational Treatment Synergistically Promote Cell Apoptosis and Suppressed Cancer Cell Stemness in NCI-H2170 and NCI-226 Cells.
Initially, to determine the effect of chidamide on cellular proliferation in LSCC cells, we treated NCI-H2170 and NCI-226 cells with 300 nM of chidamide for 24 h or/and 6 mV X-ray radiation using a linear accelerator at single doses of 0, 1, 2, and 4 Gy. e results indicated that chidamide, radiotherapy, and their combinational treatment inhibited cell proliferation in NCI-H2170 and NCI-H226 cells (Figures 1(a)-1(c)). Next, to explore the possible mechanism underlying chidamideregulating proliferation of LSCC cells, we intended to testify whether cellular apoptosis could be contributed to the synergistic anticancer effect of chidamide and radiation on LSCC. After treating the NCI-H2170 and NCI-226 cells with 300 nM of chidamide for 24 h and/or with 2 Gy radiation, the effect of chidamide, radiotherapy, and their combinational treatment on cell apoptosis was determined by flow cytometry. Both the early and late apoptosis rates were significantly increased in cells treated with chidamide, radiotherapy, and their combination ( e results demonstrate that chidamide and radiation synergistically inhibit LSCC cell proliferation potentially via inducing cellular apoptosis. It has been known that cancer stem cells (also named cancer-initiating cells or cancer stem-like cells) play a central role in tumor progression, metastasis, recurrence, and chemotherapy resistance. Herein, we also detect the effect of chidamide on lung cancer stemness. Results of sphere formation assay demonstrated that the sizes and number of spheres were suppressed by chidamide alone or a combination treatment of chidamide and radiation (Figures 2(a)-2(j)). To determine the population of cancer stem cells, ALDEFLUOR assay was performed.
e results demonstrated that chidamide alone or a combination treatment of chidamide and radiation reduced the population of ALDH + cells (Figures 2(k)-2(m)).   Journal of Oncology

Chidamide Alone or a Combinational Treatment with Chidamide and Radiation Upregulates miR-375 Expression.
Next, we sought to elucidate the molecular mechanism underlying antitumor activity of chidamide. As miRNAs play a critical role in tumorigenesis, we hence focused on the expression profile alterations of miRNAs. e Affymetrix miRNA 2.0 Array was applied to identify differentially expressed miRNAs in NCI-H2170 and NCI-H226 cells in response to chidamide, radiotherapy, and their combinational treatment. Among the upregulated miRNAs, miR-375 showed the most remarkable fold (Figure 3(a)). Moreover, real-time PCR results also validated that both chidamide and chidamide plus radiation combinational treatment elevated the expression of miR-375 in NCI-H2170 and NCI-H226 cells (Figure 3(b)). Interestingly, radiation alone could not upregulate miR-375 expression, which indicates a crucial role of miR-375 in mediating the antitumor activity of chidamide against LSCC.

Suppression of miR-375 Reverses the Promoting Effect of Chidamide on LSCC Cell Apoptosis and Attenuates Reduction of Cancer Stem Cells Caused by Chidamide.
To delineate the role of miR-375 in the chidamide-induced LSCC cell apoptosis, we transfected NCI-H2170 and NCI-H226 cells with control inhibitor and miR-375 inhibitor following chidamide, radiation, and their combinational treatment, respectively. CCK8 assay was performed to measure the proliferation rate in these treated cells. As shown in       Journal of Oncology candidate target gene for miR-375 for further analysis, as its 3′-UTR contains two conserved binding regions of miR-375. e binding sites between miRNA-375 and EIF4G3 are shown in Figure 7(a). To clarify whether EIF4G3 is a direct target of miR-375, a dual-luciferase reporter assay was applied to determine the luciferase activities of EIF4G3 3′-UTR. As shown in Figure 7(b), transfection of miR-375 mimic significantly reduced the luciferase activity of the wild-type 3´-UTR of EIF4G3 compared with the control mimic-transfected cells (Figure 7(b)). However, less significant differences were found between cells transfected with control mimic and the miR-375 mimic when cotransfected with the mutated 3′-UTR of EIF4G3 (Figure 7(b)). Additionally, results of western blotting revealed that miRNA-375 significantly downregulated EIF4G3 (Figure 7(c)). (a-j) Sphere formation assay to determine the self-renewal capacities of NCI-H2170 and NCI-H226 cells treated with chidamide alone or a combination of chidamide and radiation. e sphere sizes and number were calculated in Figures 2(c)-2(j). (k-m) Treated cells with chidamide alone (300 nM) or combined with radiation treatment were subjected to ALDEFLUOR assay and the population of ALDH + cells were counted by flow cytometry. Statistical significance was determined by Student's t-test and indicated by * p < 0.05; * * p < 0.01; and * * * p < 0.001 (vs. control); ## p < 0.01; ### p < 0.001 (vs. Gy).

Silence of EIF4G3 Promotes Cell Apoptosis and Suppresses Xenograft Growth in NCI-H2170 and NCI-H226 Cells.
To determine the role of EIF4G3 in the biological behaviors of LSCC cells, we first detected the expression of EIF4G3 in NCI-H2170 and NCI-H226 cells treated with chidamide or chidamide plus radiation combinational treatment. e results demonstrated that both chidamide and chidamide plus radiation combinational treatment suppressed EIF4G3 expression both at the mRNA and protein levels (Figures 7(d)-7(f)). Next, we silenced the expression of EIF4G3 in NCI-H2170 and NCI-H226 cells and found that silence of EIF4G3 significantly induced cell apoptosis (Figures 7(g)-7(i)). Moreover, silence of EIF4G3 in NCI-H2170 cells obviously suppressed xenograft growth (Figures 7(j)-7(l)).
Taken together, our study systematically demonstrates that chidamide and radiation synergistically promote LSCC cell apoptosis and suppressed tumor growth and stemness by modulating the miR-375-EIF4G3 axis.

Discussion
e HDAC inhibitors can be used for various diseases, some of which have entered clinical trials. In cancer, HDAC inhibitors are becoming promising novel tumor therapeutic drugs exerting anticancer function across a wide range of cancers, especially in leukemia. Chidamide, an orally active novel HDAC inhibitor of the benzamide class, selectively inhibits HDAC1, HDAC2, HDAC3, as well as HDAC10. In pancreatic cancers, chidamide augments gemcitabine-induced cell growth arrest and apoptosis by downregulating the antiapoptotic gene MCL-1 [31]. Chidamide has been tested extensively for its tumor inhibitory activity. In colon cancer cells, chidamide suppresses cell proliferation and induces cell cycle arrest by inhibiting the PI3K/AKT and RAS/MAPK signaling pathways. In non-small-cell lung cancer cell lines, chidamide and carboplatin synergistically induce cell growth arrest [11]. In the present study, we demonstrated that chidamide-induced cellular growth inhibition of NCI-H2170 and NCI-H226 LSCC cells and it synergistically augmented radiation-induced cell apoptosis. Previous reported studies have demonstrated that HDAC inhibitors show significant single-agent anticancer activity in T-cell lymphomas. Consistent with these results, our data also show that chidamide alone induces cell apoptosis and suppresses tumor growth in LSCC.
In the study of NCI-H2170 and NCI-H226 cell lines, it has been found that chidamide, radiation, and their combinational treatment could exert an anti-LSCC effect. However, unlike chidamide and chidamide plus radiation combinational treatment, radiation alone could not upregulate the expression of miR-375 and downregulate the expression of EIF4G3. ese results suggest the miR-375/EIF4G3 axis may not be involved in the regulation of radiotherapy alone-induced cell apoptosis. On the other hand, acquired drug resistance frequently occurred to destroy effective therapy with chemotherapeutic agents, leading to an unsatisfactory clinical outcome. erefore, HDAC inhibitor combined with radiation at different dose might have multiple targets and pathways to induce cell apoptosis. and 5(i), respectively. All the data were statistically analyzed by Student's t-test. * p < 0.05; * * p < 0.01; and * * * p < 0.001.
In conclusion, our results systematically explored the role of miR-375/EIF4G3 axis in chidamide-induced LSCC apoptosis and tumor growth arrest, which may afford an effective strategy to overcome the drug resistance of chidamide in clinical cancer therapy.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All the authors declare that they have no conflicts of interest regarding the publication of this paper.