SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma

Abstract The suppressor of zest 12 (SUZ12), one of the core polycomb repressive complex 2 (PRC2) components, has increasingly appreciated as a key mediator during human tumorigenesis. However, its expression pattern and oncogenic roles in head and neck squamous cell carcinoma (HNSCC) remain largely unexplored yet. Here, we sought to determine its expression pattern, clinicopathological significance and biological roles in HNSCC. Through data mining and interrogation from multiple publicly available databases, our bioinformatics analyses revealed that SUZ12 mRNA was significantly overexpressed in multiple HNSCC patient cohorts. Moreover, SUZ12 protein was markedly up‐regulated in primary HNSCC samples from our patient cohort as assessed by immunohistochemical staining and its overexpression significantly associated with cervical node metastasis and reduced overall and disease‐free survival. In the 4‐nitroquinoline 1‐oxide (4NQO)‐induced HNSCC mouse model, increased SUZ12 immunostaining was observed along with disease progression from epithelial hyperplasia to squamous cell carcinoma in tongue. Furthermore, shRNA‐mediated SUZ12 knock‐down significantly inhibited cell proliferation, migration and invasion in HNSCC cells, and resulted in compromised tumour growth in vivo. Collectively, our data reveal that SUZ12 might serve as a putative oncogene by promoting cell proliferation, migration and invasion, and also a novel biomarker with diagnostic and prognostic significance for HNSCC.

stage at diagnosis have been recognized as the key factors affecting patient prognosis. 3 Previous intensive efforts were made to unravel the genetic, epigenetic and environmental factors driving HNSCC tumorigenesis. 4 However, in-depth mechanistic understanding about HNSCC initiation and progression still remains far from complete.
Thus, development of effective treatment for HNSCC will largely hinge on the identification of genetic alterations and relevant molecular mechanisms during HNSCC carcinogenesis to find druggable targets of therapeutic values.
Epigenetic abnormality has been increasingly recognized as a hallmark of cancer and significantly contributes to cancer initiation and progression. 5 Until now, dozens of epigenetic modulators have been identified as pivotal mediators driving tumorigenesis and held great promise as therapeutic targets against cancer largely due to their pervasive roles as well as inherent reversible nature of epigenetic alternations. 6 Among these cancer-associated epigenetic modulators, polycomb group (PcG) proteins have stood out as essential participators of malignant transformation, metastatic spreading and promising targets for therapeutic intervention. 7 Mounting evidence has established that these PcG components commonly assemble into protein complexes with additional factors which are recruited to specific chromatin regions to maintain target genes in a transcriptionally repressive state. Briefly, two canonical PcG complexes (PRC1 and PRC2) harbour multiple core members to execute their functions by histone modifications. 8 As expected, our previous studies and others have provided strong evidence that multiple PcG members such as Bmi1 and EZH2 might function as bona fide oncogenes driving tumorigenesis in diverse sites and their elevated abundance associates with cancer aggressiveness and unfavourable prognosis in a myriad of human cancer including HNSCC. 7,[9][10][11][12][13] Recently, another PcG member, suppressor of zest 12 (SUZ12), which is essential for PRC2-mediated gene silencing by generating trimethylation on lysine 27 residue of histone H3 (H3K27me3), has been increasingly appreciated as a putative oncogene or tumour suppressor underlying tumorigenesis. 14-16 SUZ12 has been found to be frequently overexpressed in several solid cancers including colorectal, ovarian and non-small lung cancer. Its aberrant overexpression significantly associated with aggressive clinicopathological features and inferior survival. 14,17,18 Furthermore, SUZ12 knock-down induced impaired tumour growth, invasion and metastasis in bladder, gastric and lung cancer. 17,19,20 However, on contrary, recurrent loss-of-function somatic alterations of SUZ12 have been identified in malignant peripheral nerve sheath tumour and contribute to its initiation and progression. 16 Therefore, these data highlight the complexity of biological roles of SUZ12 underlying tumorigenesis in diverse contexts.
However, to the best of our knowledge, the expression of SUZ12 and its clinicopathological significance in HNSCC have not been established yet.
In this study, we sought to investigate the expression of SUZ12 and its clinicopathological significance in primary human HNSCC samples and chemical-induced animal model. The tumorigenic roles of SUZ12 in HNSCC were also further explored both in vitro and in vivo.

| Cell migration and invasion assay
Cell migration and invasion assays in vitro were performed with wound-healing and transwell chambers (8-lm pore size, Corning) with Matrigel (BD Pharmingen) pre-coating, respectively. For woundhealing assay, cells were grown into monolayer and scratched using a sterile 200-lL pipette. Cell migration was observed at various time-points later by microscopy. Images of 10 scratches per cells under each experimental condition were captured at the same locations and then compared with ImageJ software (NIH). For cell invasion assay, Boyden chambers were pre-coated with 100% Matrigel, and then, cells were seeded in the upper chambers with serum-free medium. Complete growth medium containing 10% FBS in the lower chambers served as chemoattractant. The non-invaded cells were gently removed with a cotton swab and those invaded cell located on the lower side were fixed and stained with crystal violet, and counted.

| Immunofluorescence assay
For cell immunofluorescent staining, cells were pre-seeded and grown on glass coverslips 24 h prior to experiment and then fixed with 4% paraformaldehyde and permeabilized in Triton X-100 (0.1% in PBS) and sequentially blocked with 3% bovine serum albumin (BSA) for 30 minutes. Following the overnight incubation with primary antibody against SUZ12 (1:200 dilution), these cells were further incubated with secondary antibody and cytoskeleton actin/ nuclear staining. Immunofluorescence was visualized under a Zeiss fluorescence microscope or confocal microscope, and then image captured using similar parameters.

| HNSCC xenograft model
All experiments involving animal subjects were in accordance with the institutional animal welfare guideline and approved by Institutional Animal Care and Use Committee of Nanjing Medical University. Six-week-old female nu/nu mice were obtained and maintained in a specific pathologic-free environment. Stable SUZ12 knock-down cancer cells (2 9 10 6 ) suspended in total 100 lL PBS and Matrigel (1:1) were inoculated subcutaneously on the right flanks (6 animals per experimental group). Tumour growth was monitored after injection, and tumour diameters were measured by calipers every 3 days when tumour masses were identified. Tumour volume is calculated as follows: volume = a 9 b 2 /2, where a and b are defined as the longest diameter and shortest diameter, respectively. Final tumour weights were also measured upon animals were killed. The tumour samples were further processed for H&E staining, immunohistochemical staining, etc.

| HNSCC patients and tissue samples
Accordingly, the immunoreactivity of each slide was categorized into three subgroups based on the final score: 0, negative; 1-4, low expression; 4-12, high expression, similar as our previous reports. 9,25,26

| Statistical analysis
All quantitative data reported here were shown as mean AE SD from two or three independent experiments as indicated and compared with Student's t test or ANOVA with Bonferroni post hoc test unless otherwise specified. The potential associations between SUZ12 expression and various clinicopathological parameters were evaluated by chi-square or Fisher's exact test. The survival rates of patients were estimated using Kaplan-Meier method and compared with log-rank test. The prognostic analyses were performed by univariate and multivariate Cox regression models to determine the individual clinicopathological variables with patient overall survival. P values <.05 (two-sided) were considered statistically significant. All statistical analyses were performed with GraphPad Prism 6 (Graph-Pad) or SPSS 21.0 software (IBM).

| SUZ12 mRNA is frequently overexpressed in HNSCC as determined via bioinformatics analyses
Accumulating evidence has indicated that SUZ12 is usually up-regulated in multiple cancers and associates with unfavourable prognosis. 14,18,25 Initially, to explore the mutational landscape and mRNA expression of SUZ12 in HNSCC, we utilized three publicly available databases including cBioPortal, Oncomine and TCGA and analysed the relevant data. As shown in Figure 1A-F, data mining and interrogation from Oncomine database indicated significant higher expression of SUZ12 mRNA in HNSCC samples from several independent patient cohorts as compared to normal counterparts, respectively, except the Cromer's cohort. [27][28][29][30][31][32][33] Data integration and analyses from TCGA-HNSCC cohort (502 cases) using cBioPortal platform indicated that total frequency of SUZ12 genetic alterations in HNSCC samples was less than 2.5%. Moreover, SUZ12 mRNA is significantly overexpressed in TCGA-HNSCC samples as compared to non-tumour samples (44 cases) ( Figure. 1H). To uncover potential associations between SUZ12 mRNA expression status and the clinicopathological parameters, we compared its abundance among diverse groups based on pathological grade and clinical stage, respectively. However, as shown in Figure S1A, B, the abundance of SUZ12 mRNA was comparable without significant difference among patient groups stratified by pathological grade and clinical stage. In addition, there was no significant association between SUZ12 mRNA expression and patient overall survival (Kaplan-Meier analysis, logrank test, P = .773), when the median value of SUZ12 mRNA was employed as cut-off to stratify patients into low or high SUZ12expressing groups ( Figure S2).

| SUZ12 overexpression positively associates with cervical node metastasis and overall survival
The detailed correlations between SUZ12 expression and the clinicopathological parameters in HNSCC are analysed and shown in F I G U R E 1 Overexpression of SUZ12 mRNA in multiple HNSCC cohorts. The mRNA levels of SUZ12 (log2-transformed) were compared between HNSCC samples and normal counterparts in multiple patient cohorts. (A-H) The original data were retrieved from Oncomine database and TCGA and then plotted using GraphPad Prism 6.0 software. Y-axis represents the median intensity, 25th and 75th percentile data. *P < .05; **P < .01; ns, not significant; Student's t test or Mann-Whitney U test as appropriate To probe prognostic value of SUZ12 expression for patients with HNSCC, we next aimed to determine possible relationship

| SUZ12 is involved in chemical-induced HNSCC tumorigenesis
Having revealed aberrantly high expression of SUZ121 in human HNSCC samples, we next developed a well-established chemical-induced animal model to characterize the expression pattern of SUZ12 during HNSCC initiation and progression ( Figure 4A). Pathological lesions were primarily identified in tongue following 4NQO treatment and displayed typical changes from epithelial hyperplasia, dysplasia, carcinoma in situ and invasive SCC, thus largely recapitulating the multiple-staged tumorigenic process in human HNSCC.
Furthermore, as shown in Figure 4B, immunohistochemical staining of SUZ12 in these samples indicated negative or low staining in normal tongue mucosa and epithelial with hyperplasia, while prominent strong nuclear staining in carcinoma in situ and invasive carcinoma.
Data from immunohistochemical staining revealed that significant SUZ12 overexpression was observed in carcinoma (62.5%, 5/8), whereas much less were detected in healthy mucosa (16.7%, 1/6), samples with hyperplasia (16.7%, 1/6) or dysplasia/carcinoma in situ (50.0%, 3/6). Together, our findings from chemical-induced animal model provide support to the notion that SUZ12 might be critically involved in HNSCC development probably by serving as a putative oncogene.

| SUZ12 knock-down inhibits proliferation, migration and invasion in HNSCC cells
Having revealed the overexpression of SUZ12 and its clinical significance in HNSCC, we next sought to dissect its oncogenic roles driving HNSCC initiation and progression by shRNA-mediated lossof-function approach. We designed and synthesized two independent shRNA sequences targeting human SUZ12 mRNA which were cloned into lentiviral vectors, sequenced and packaged. We  Figure 5C,D). However, the ratios of cells undergoing apoptosis upon SUZ12 knock-down were comparable to control cells without significant difference (data not shown). Moreover, the migratory and invasive potentials of cell following SUZ12 knock-down were also measured using wound-healing and transwell assays, respectively. As shown in Figure 5E,F, SUZ12 knock-down significantly reduced both migratory and invasive properties of cells in vitro. In agreement with these observed phenotypical changes following SUZ12 depletion, the expression of cell proliferation marker cyclin D1 was markedly reduced in SUZ12 knock-down cells, while the EMT/metastasis-associated The number in bold indicates statistical significance with P-values <.05.
marker Vimentin was down-regulated concomitant with E-cadherin up-regulation ( Figure 5G).
To complement the in vitro loss-of-function assay in exploring pro-tumorigenic functions of SUZ12, we further performed bioinformatics analyses using TCGA-HNSCC data to identify the candidate genes whose expression was potentially correlated with SUZ12 which were subjected to gene ontology (GO) and pathway analyses.  Figure 6B). In addition, as shown in Figure 6C, analyses through cBioPortal platform revealed interactive network containing 51 nodes including SUZ12 Consistent with its primary roles as a chromatin modifier, most altered neighbours of SUZ12 were histone-related genes and PRC components. Notably, some well-established oncogenes such as DNMT1, 3 as well as JARID2 were also identified. Taken together, our in vitro cellular assay and bioinformatics assay both strongly favour the notion that SUZ12 is a novel putative oncogene in HNSCC.
F I G U R E 5 SUZ12 knock-down resulted in impaired cell proliferation, migration and invasion, while enhanced chemosensitivity to 5-FU. A, The knock-down efficiency of two shRNA lentiviral vectors targeting human SUZ12 was measured by Western blot. The shSUZ12-1 with higher knock-down potency was selected for the following experiments. Representative images of Western blot were shown from three independent experiments. B, Cell proliferation was determined after Cal27 and FaDu cells were infected with shSUZ12-1 lentivirus by MTT assay. C, D, Cell viability was probed in Cal27 (C) and FaDu (D) cells treated with shSUZ12-1 lentivirus and 5-FU alone or in combination for 48 h by MTT assay. E, Cell migration was determined in cells with stable SUZ12 knock-down by wound-healing assay. F, Cell invasion was determined in cells with stable SUZ12 knock-down by transwell invasion assay. Scale bar: 100 lm. G, The abundance of proliferative marker cyclin D1 and migration/invasion-relevant marker E-cadherin and Vimentin was compared in cells infected shSUZ12 lentivirus or control virus. Representative images of Western blot were shown from three independent experiments. # P > .05, *P < .05, **P < .01, Student's t test  Figure 7E,F). Moreover, the number of Ki67-positive cells was significantly reduced in tumour samples from SUZ12 knock-down cells as compared to samples formed from control cells ( Figure 7E,F).
Together, these findings revealed that SUZ12 knock-down impaired tumour growth of HNSCC in vivo, suggesting that SUZ12 might be required for HNSCC growth.

| DISCUSSION
The epigenetic modifying complexes PRC1 and PRC2 regulate downstream targets by regulating chromatin structure and have been critically involved in multiple physiological and pathological processes including stem cell self-renewal and differentiation, cell apoptosis as well as tumorigenesis. 7,34 Previous studies have suggested that SUZ12 might be a putative oncogene involving tumorigenesis and F I G U R E 6 Gene ontology and KEGG pathway analyses of SUZ12-related genes in HNSCC. A, GO biological process analyses of SUZ12 positively and negatively related genes identified from TCGA-HNSCC database. B, KEGG pathway analyses of SUZ12 positively and negatively related genes identified from TCGA-HNSCC database. C, Network formed by SUZ12 and its most frequently altered neighbour genes (50 of a total of 69) produced by cBioPortal platform serve as a potential diagnostic biomarker as well as anticancer therapeutic target. 14,15,17,35 Herein, we revealed the expression pattern of SUZ12 in HNSCC, determined its clinicopathological and prognostic significance and also uncovered its pro-tumorigenic roles by loss-offunction approach. Our findings together indicate that SUZ12 serves as a novel putative oncogene to promote HNSCC tumorigenesis and also a new biomarker with translational potentials.
HNSCC initiation and progression are characterized by multiple stages from normal epithelial to SCC driven by genetic predisposition, activation of oncogenes and inactivation of tumour suppressor genes. 4 Particularly, epigenetic modifications underlying oncogene or tumour suppressor dysregulations have been found to contribute to almost all stages during HNSCC tumorigenesis. 36 Among these cancer epigenetic modulators, PRC1/2 has been increasingly recognized as key mediators to facilitate cancer initiation, unchecked growth and metastatic dissemination, and also has been demonstrated as therapeutic targets with considerable promise. 7,34 Here, we found that SUZ12 is aberrantly up-regulated in a large fraction of HNSCC samples as evidenced by significantly elevated SUZ12 mRNA in multiple cancer clinical data sets as well as overexpression of SUZ12 protein in our patient cohort. This is consistent with previous findings which revealed elevated SUZ12 in gastric, bladder, lung and colorectal cancer. 14 Accumulating evidence has indicated that SUZ12 is critically involved in tumorigenesis by promoting cell proliferation, migration and suppressing apoptosis. 14,15,17,19 In line with this, our findings from in vitro loss-of-function assay reveal that loss of SUZ12 resulted in reduced proliferation, migration and invasion in HNSCC cells. These findings were also further substantiated by the facts such as reduced xenograft tumour growth upon SUZ12 depletion and positive association between SUZ expression and cervical node metastasis in patient cohort. Consistently, previous studies have revealed that loss of miR-200b in breast cancer increased SUZ12 expression and H3K27me3 abundance, in turn, resulted in repression of E-cadherin and impaired invasiveness and metastasis. 15 Complementary, Herranz N and his colleagues revealed that the EMT master factor Snail interacted with SUZ12 and recruited PRC2 to the E-cadherin promoter, which in turn repressed E-cadherin expression and enhanced cancer metastasis. 37 In bladder cancer, SUZ12 was critically involved in TGF-b1-induced up-regulation of malat1, EMT and metastasis. 38 Of note, pharmacological or genetic depletion of SUZ12 inhibited cell proliferation, impaired the migratory and invasive properties and disrupted the maintenance of cancer stem cell, ultimately induced tumour regression and reduced tumour metastasis. 15,17,35,39 Together, our findings together with others strongly suggest that SUZ12 probably functions as a putative pro-tumorigenic gene via enhancing cancer cell proliferation and invasion. Selective targeting of SUZ12 by genetic or pharmacological approach might hold translational promise against HNSCC.
In conclusion, our findings reveal aberrantly overexpressed SUZ12 in a significant subset of HNSCC and unravel its oncogenic roles to promote initiation and progression of HNSCC. Large knowledge gap still remains between the functional roles of SUZ12 during HNSCC tumorigenesis and efficient therapeutic approaches to targeting SUZ12.

ACKNOWLEDG EMENTS
This work is financially supported, in whole or in part, by National

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.