Abstract
Background/Aim: Salivary adenoid cystic carcinoma (SACC) is the most common malignancy of the salivary gland with a poor prognosis and survival. The present study aimed to investigate the role of histone methyltransferase WHSC1 in SACC. Materials and Methods: Human SACC specimens were evaluated for WHSC1 expression by RT-PCR and immunohistochemistry. The effects of WHSC1 knockdown on SACC cells proliferation, cell cycle, clone and tumorsphere formation, and apoptosis as well as on the expression of related genes were examined. A xenograft mouse model of SACC was used to evaluate the in vivo effects of WHSC1 knockdown on SACC tumorigenesis. Results: WHSC1 expression was up-regulated in human SACC tissues (p<0.01). WHSC1 knockdown in SACC cells significantly inhibited cell proliferation, clone and tumorsphere formation (p<0.05). Cell distribution at the S and G2/M phases was significantly reduced by WHSC1 knockdown (p<0.05). WHSC1 knockdown significantly increased apoptosis of SACC cells (p<0.05). c-Myc, survivin, Bcl-2 and cyclin B1 genes were significantly down-regulated by WHSC1 knockdown cells (p<0.05). WHSC1 knockdown significantly reduced H3K36me2 modification of the MYC gene promoter in SACC cells and tumorigenesis of SACC cells in vivo (p<0.05). Conclusion: Knockdown of WHSC1 inhibited cell proliferation, induced apoptosis and affected tumorigenesis in SACC.
Salivary adenoid cystic carcinoma (SACC) arises from the secretory epithelial cells of the salivary glands (1) and is the most common malignancy of the salivary glands, accounting for about 25-50% of malignant tumors in the major or the minor salivary glands, respectively (2-4). SACC demonstrates unique characteristics that lead to poor prognosis and low overall survival rates (5). Recent molecular pathology findings suggest that genetic translocation and/or overexpression of oncoproteins is important in salivary gland tumorigenesis and diagnosis. Studies have found that the recurrent MYB-NFIB gene fusion is the main genomic hallmark of SACC (6, 7), However, further molecular genetic studies are needed to identify biomarkers as well as therapeutic targets or prognostic factors of ACC.
Histone methylation modification is an important process involved in the regulation of oncogenic gene expression and is controlled by histone methyltransferases and demethylases. WHSC1 (also known as NSD2) is a histone methyltransferase that mediates di-methylation of histone 3 lysine 36 (H3K36me2). H3K36 methylation has been found to be associated with transcriptional activation in many cancers 8). Increased expression of WHSC1 was found in many solid tumors, such as multiple myeloma, prostate cancer, breast cancer and head and neck cancer (8-10). In vitro studies indicated that WHSC1 modulates Ras-related C3 botulinum toxin substrate 1 (Rac1) transcription, twist family transcription factor 1 (TWIST1), and nuclear factor κB (NF-κB) to promote tumorigenesis and metastasis (11, 12). However, the role of WHSC1 in SACC remains unclear.
In the present study, WHSC1 was found to be up-regulated in human SACC. The effects of WHSC1 knockdown in SACC cells on cell proliferation, apoptosis and tumorigenesis were investigated.
Materials and Methods
Patients and specimens. A total of 23 salivary adenoid cystic carcinomas and 23 matched paracarcinoma normal salivary gland specimens were obtained from patients who underwent surgery between 2002 and 2014 at the Affiliated Shandong Provincial Hospital of Shandong University. Diagnosis was confirmed histologically by experienced pathologists. The study was approved by Research Ethics Committee of Affiliated Shandong Provincial Hospital of Shandong University (Approval number: 2018-230) and written informed consent was obtained from all patients.
Immunohistochemistry (IHC). IHC was performed on 5 μm-thick formalin fixed paraffin embedded slides. After deparafinization, rehydration, antigen retrieval, and endogenous peroxidase blockage. Samples were then incubated with the following antibodies: anti-WHSC1 (ab75359, Abcam, Cambridge, UK), anti-cleaved caspase-3 (9661, Cell Signaling Technology, Boston, USA), anti-Ki67 (GB13030-2, Servicebio, Wuhan, P.R. China) at 4°C for 8-10 h. The sections were then incubated with secondary antibody (7074, Cell Signaling Technology, Boston, MA, USA) for 30 min at room temperature. Color development was performed with 3’-diaminobenzidine (DAB) (K5007, DAKO, Copenhagen, DK). Nuclei were lightly counter stained with hematoxylin. The slides were viewed using a microscope (DM 4000B, Leica, Wetzlar, Germany) and positive cells were recognized by the appearance of brown staining. Expression levels were assessed by staining indexes using a 10-point quantification method (13). Scores of 0-5 indicate low expression, while scores of 6-10 indicate high expression.
Cell culture. Human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM were purchased from American Type Culture Collection. Cells were grown in DMEM (Hyclone, UT, USA) with 10% FBS (Gibco, CA, USA) and antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml) at 37°C in a humidified atmosphere with 5% CO2.
shRNAs transfection. Two WHSC1-specific short hairpin RNAs (WHSC1-KD1 and WHSC1-KD2 shRNA) were designed and cloned into the pLVX-shRNA1 plasmid. A scramble nonsense sequence was used as a negative control. The plasmids were packaged into lentivirus using a three-plasmid system, pLVX-shRNA1 with targeted sequence, psPAX2 and pMD2G. SACC-83 and SACC-LM cells were transfected and screened with 2 mg/ml puromycin for 2 weeks. The WHSC1-KD1 shRNA sequence was TGCCAATAACACGTCCACT, and the WHSC1-KD2 shRNA sequence was CCCTTCGCAGTGTTTGTCT.
Cell proliferation assay. Cell proliferation was measured using the CCK8 assay. The cells (5×103) were transferred to a 96-well plate in RPMI 1640 containing 10% FBS. After 24, 48 and 72 h of incubation, CCK8 reagent (1:10 dilution) was added to each well for 2 h. The optical density (OD) at a wavelength of 450 nm was determined by a microplate reader (Bio-Rad, CA, USA). Proliferation curves were plotted for each of the cell groups.
Cloning efficiency assay. 1000 cells were added in each well of 6-well plate, and the resulting colonies were fixed, and stained with 0.1% crystal violet at day 7 and colony number was counted.
Tumorsphere formation. Single-cell suspensions were added to ultralow-attachment plates in defined medium at a density of 1000 cells/well. The defined serum-free medium contained DMEM/F-12 supplemented with B27 (1:50) supplement, EGF (20 ng/ml), and FGF (10 ng/ml). Seven days after continuous cultivation, the number of spheres was recorded under a microscope. One-half volume of the medium was added every second day. The images were collected on day 7th with an inverted microscope, only spheres larger than 100 μm were quantitated.
Apoptosis assay. The percentage of cells undergoing apoptosis was evaluated by flow cytometry (Beckman Gallios, Brea, CA, USA). After incubating with cisplatin for 48 h, cells were harvested, washed and resuspended in 100 μl staining buffer added with 1 μl 7-AAD (559925, BD Biosciences, NJ, USA) and 5 μl Annexin-V-APC (556421, BD Biosciences).
Cell cycle analysis. 5-ethynyl-2 deoxyuridine (EdU) assay was performed according to the manufacturer's instructions (C0071L, Beyotime, Shanghai, P.R. China), SACC-83 and SACC-LM cells were incubated with EdU for 2 h.
RNA extraction and real-time quantitative PCR. Total RNA was isolated from cells using TRIzol Reagent (15596-018, Invitrogen, Carlsbad, CA, USA). 1 μg total RNA was reversely transcribed using the TaKaRa RT-PCR kit (Takara, Shiga, Japan) according to the manufacturer's instructions. followed by PCR amplification with specific primers (Table I). Data were analyzed by the relative standard curve method and normalized to GAPDH expression. Relative RNA expression in the cells was calculated using the 2−ΔΔCt method.
Western blotting. Protein cell extracts were obtained with RIPA buffer (Roche Diagnostics, Indianapolis, IN, USA). 30 μg of protein lysate was separated by 10% SDS–PAGE gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were incubated with 10% nonfat milk solution for blocking non-specific binding and then with anti-c-Myc (9402, Cell Signaling Technology, Boston, MA, USA), anti-WHSC1 (ab75359, Abcam, Cambridge, UK), anti-Bcl-2 (4223S, Cell Signaling Technology), anti-cyclin B1 (4138, Cell Signaling Technology), anti-Survivin (2808, Cell Signaling Technology) or anti-GAPDH antibodies (5174S, Cell Signaling Technology). After washing twice with Tris buffered saline, the membranes were incubated with appropriate secondary antibody conjugated with HRP for 2 h at room temperature. Analysis of electrochemiluminescence was performed according to the manufacturer's instructions (WBKLS0050, Millipore).
In vivo study. BALB/C male nude mice, aged 4-6 weeks and weighing 20-25 g, were purchased from SHANGHAI SLAC (Shanghai, P.R. China). All mice were maintained in a HEPA-filtered environment at 24-25°C and humidity was maintained at 50-60%. All animal experiments were approved by the Institutional Animal Care and Use Committee and performed in compliance with the NIH Guide.
Xenograft mouse models of salivary adenoid cystic carcinoma were established by subcutaneously injecting 1×106 transfected SACC cells in the flank of nude mice. Tumor growth was measured weekly with calipers. Tumor volume was calculated using the formula (L × W2) × ½, where W and L represent the perpendicular minor and major dimension, respectively. All animals were sacrificed 21 days after tumor cell implantation. At autopsy the tumor was removed and weighed.
ChIP-qPCR analysis. 2×107 SACC-83 and WHSC1-knockdown SACC-83 cells were crosslinked, lysed and sheared to about 200-700 DNA base pairs in length using UCD-300 (Bioruptor, BE). ChIP was performed using Magnetic ChIP kit according to manufacturer's instructions (17-371, Millipore). Quantification of ChIP-enriched DNA was then performed by qPCR. The ChIP antibodies used were anti-H3K36me2 (61019, Active Motif, CA, USA) and normal rabbit IgG. Primers used are listed in Table II.
Statistical analysis. All experiments were repeated at least three times as indicated. Data are expressed as mean±standard deviation. All statistical analyses were performed using SPSS16.0 software (SPSS inc., Chicago, IL, USA). Data comparisons between two groups were performed using the Student's t-test. A value of p<0.05 was regarded as statistically significant.
Results
WHSC1 expression is upregulated in human SACC. To assess WHSC1 expression in human SACC specimens, GSE59701 datasets were obtained from the GEO website and a list of expression of 20,201 genes from 12 SACC samples with matched normal tissues was recorded. WHSC1 mRNA expression was significantly higher in SACC tissues compared with paired normal controls (p<0.01) (Figure 1A). WHSC1 expression was further investigated in SACC specimens. RT-qPCR analysis showed that WHSC1 mRNA expression was also significantly higher in 23 SACC patients' biopsies compared with their paracarcinoma normal salivary gland specimens (p<0.01) (Figure 1B). Immunohistochemical analyses further revealed significantly higher WHSC1 expression in 23 SACC patients' biopsies compared with their paracarcinoma normal salivary gland specimens (p<0.05) (Figure 1C and D).
WHSC1 knockdown inhibits SACC cell growth and cancer stem cells. Knockdown of WHSC1 in SACC-83 and SACC-LM cells was performed by transfecting them with WHSC1-specific short hairpin RNA. The effect of WHSC1 knockdown on cell growth was assessed by examining cell proliferation as well as clone and tumorsphere formation. The SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 showed significantly reduced cell proliferation compared to SACC-83 and SACC-LM cell controls (p<0.05) (Figure 2A and B). Clone formation was significantly inhibited in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to SACC-83 and SACC-LM cell control (p<0.05) (Figure 2C and 2D).
Cancer stem cells (CSCs) are closely correlated with tumorigenesis in many solid tumors (14, 15). Intriguingly, WHSC1 knockdown markedly reduce the ability of SACC cells to form tumor spheres (Figure 2E and F), suggesting that WHSC1 may affect cancer stem cell properties in SACC.
Effect of WHSC1 knockdown on the cell cycle. The effect of WHSC1-knockdown on the cell cycle was assessed by the Edu assay. As shown in Figure 3A and B, cell distribution at phase S and G2/M was significantly reduced in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to SACC-83 and SACC-LM cell control (p<0.05).
WHSC1 regulates cell apoptosis- and stemness-related genes. The effects of WHSC1-knockdown on cell apoptosis were assessed by the Annexin V-7AAD assay. WHSC1 knockdown in SACC-83 and SACC-LM cells significantly increased cell apoptosis compared to SACC-83 and SACC-LM cell control (p<0.05) (Figure 3C and D).
c-Myc, a tumor stem cell differentiation-related factor, was markedly down-regulated in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to the control (p<0.05) (Figure 4A and B). Survivin and Bcl-2, two anti-apoptotic factors (16, 17), were significantly inhibited in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to the control (p<0.05) (Figure 4B). Furthermore, cyclin B1, which is related to G2/M checkpoint (18), was also reduced in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to the control (p<0.05) (Figure 4B). These results indicate that WHSC1 knockdown downregulates the expression of apoptotic and stemness-related genes in SACC cells.
WHSC1 mediates H3K36me2 modification in MYC loci. WHSC1 is an H3K36me2 histone methyltransferase. H3K36 methylation promotes gene transcription. ChIP-qPCR analysis demonstrated that WHSC1 knockdown reduced H3K36me2 modification in the MYC gene promotor (Figure 4C). These finding indicated that the depletion of WHSC1 may lead to more condensed chromatin in the MYC gene locus and thereby inhibit the transcription of c-Myc in SACC cells. Therefore, WHSC1 may directly regulate c-Myc expression by mediating H3K36me2 modification.
WHSC1 knockdown inhibits SACC tumorigenesis in vivo. The effects of WHSC1-knockdown on tumorigenesis were evaluated in a mouse model of SACC. The animals, with the tumor derived from the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2, showed significantly reduced tumor size and weight as compared to the control (p<0.05) (Figure 5A-C). Cleaved caspase 3 positive cells were significantly increased in the SACC-83 and SACC-LM cells transfected with WHSC1-KD1 or WHSC1-KD2 compared to the control (p<0.05). However, Ki67 expression was similar in all groups (Figure 5D). These results demonstrate that WHSC1 knockdown suppresses tumorigenesis and enhances apoptosis in vivo.
Discussion
In the present study, WHSC1 was found to be up-regulated in human SACC, and WHSC1 upregulation inhibited SACC cell line apoptosis both in vitro and in vivo. WHSC1 regulated transcription of key intracellular signaling molecules including BCL-2 and SOX2, by mediating their H3K36me2 modification.
It is shown, in this report, that high levels of WHSC1 in SACC cells promoted proliferation and inhibited apoptosis through epigenetically reprograming a subset of genes, indicating the MYC gene. WHSC1 knockdown significantly reduced H3K36me2 modifications in the MYC promoter region, likely leading to more condensed chromatin and reduced MYC transcription.
Our results suggest a close relationship between the epigenetic regulator, WHSC1, and key intracellular transcription factors and molecules. MYC is a key stemness-related gene and an oncogenic driver that promotes malignant transformation of many tumors (19, 20). WHSC1 knockdown significantly decreased the H3K36me2 modification level in MYC gene locus, leading to inhibition of transcription of this key oncogene.
In conclusion, WHSC1 expression was up-regulated in human SACC patients. Knockdown of WHSC1 in SACC cell lines was associated with decreased cell proliferation, increased apoptosis and decreased tumorigenesis. WHSC1 may be a potential target for effective therapy of SACC.
Acknowledgements
This study is financially supported by the grants of National Natural Science Foundation of China (Nos. 81771087 and 81470755), and Shandong Provincial Natural Science Foundation, China (No. ZR2017BH005).
Footnotes
↵* These Authors contributed equally to this study.
Authors' Contributions
CHAO LIU and JIA-WEI ZHENG designed the study, analyzed the data, prepared the figures, and wrote draft manuscript; CHAO LIU and ZE-LIANG ZHAO performed in vitro study; HAI-WEI WU and LING ZHANG performed in vivo study; ZHIJIAN YANG and ROBERT M. HOFFMAN oversaw the study and revised the manuscript.
This article is freely accessible online.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received January 19, 2019.
- Revision received February 4, 2019.
- Accepted February 5, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved