European Review for Medical and Pharmacological Sciences 2017; 21: 3821-3826 SOX12 upregulation is associated with metastasis of hepatocellular carcinoma and increases CDK4 and IGF2BP1 expression

P. Y UA N1, L. MENG2, N. WANG3

1Department of Interventional Therapy, the People’s Hospital of Jianhu, Jianhu, Jiangsu, China 2Clinical Laboratory, the People’s Hospital of Lanling, Linyi, Shandong, China 3Department of Ultrasound, the First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China

Introduction Abstract. – OBJECTIVE: In this study, we studied the expression profile of SOX12 in different pathological stages of hepatocellular Hepatocellular carcinoma (HCC) is one of carcinoma (HCC) and explored the possible the most common cancers and is also a leading downstream related to its regulation of cause of cancer-related death worldwide1. Local HCC invasion and migration. and distant metastases are indicators of poor MATERIALS AND METHODS: Bioinformatic prognosis and are also the main causes of can- data mining was performed in liver cancer co- cer-related death2,3. Therefore, elucidating the hort in TCGA. HepG2 cells were used as in vitro cell model to assess the effect of SOX12 on molecular mechanisms underlying HCC metas- CDK4 and IGF2BP1 expression. The association tasis is critical for identifying novel therapeutic between the expression of SOX12, CDK4 or IG- targets. F2BP1 and survival time in HCC patients was SYR-related high mobility group (HMG) box also assessed based on the data in TCGA. (SOX) family are a conserved group RESULTS: The regional lymph node metasta- of transcriptional factors widely participate in sis (N1) and distant metastasis (M1) cases had both normal embryonic development and onco- significantly increased SOX12 expression than 4,5 5,6 the lymph node negative (N0) and distant metas- genic processes . Previous studies found that tasis negative (M0) cases respectively. CDK4 the SOX factors have extraordinary regulations and IGF2BP1 were among the top 20 SOX12 on cancer cell proliferation, migration, inva- co-upregulated genes in HCC, which have well sion and tumor metastasis in multiple types of characterized enhancing the effect on HCC cell cancer. SOX12 belongs to the SOXC group of growth, invasion, and metastasis. In HepG2 SOX family, together with SOX4 and SOX117. cells, knockdown of SOX12 reduced IGF2BP1 These three SOX members show overlapping and CDK4 expression, while enforced SOX12 ex- 8 pression significantly enhanced their expres- expression patterns and molecular properties . sion. Low SOX12 or CDK4 expression was asso- SOX12-null mice are viable and do not show ciated with significantly better 3-year survival obvious malformations due to the compensation and better 5-year survival. Low IGF2BP1 expres- of SOX4 and SOX117. The oncogenic effect of sion was associated with significantly better SOXC group in HCC has been reported. Briefly, 3-year survival in HCC patients. CONCLUSIONS: SOX4 contributes to hepato-carcinogenesis via SOX12 upregulation is asso- 9 ciated with regional and distant metastasis of inhibiting -mediated apoptosis . SOX12 in- HCC. SOX12 can increase the expression of duces epithelial-mesenchymal transition (EMT) CDK4 and IGF2BP1, which confer malignant by trans-activating Twist1 expression and also phenotypes to HCC. The expression of SOX12, enhances cancer cell invasion and metastasis via IGF2BP1 or CDK4 might be potential clinical increasing the expression of fibroblast growth prognostic markers for HCC. factor binding 1 (FGFBP1)10. Among the Key Words 16 SOX genes, SOX12 was identified as one of SOX12, CDK4, IGF2BP1, Metastasis, Hepatocellular the most up-regulated genes in HCC compared carcinoma. to adjacent normal tissues10.

Corresponding Author: Na Wang, MD; e-mail: [email protected] 3821 P. Yuan, L. Meng, N. Wang

As a , the downstream Quantitative Real-Time-PCR (QRT-PCR) regulation of SOX12 in HCC is still not fully 24 h after infections, Total RNA was extracted understood. In this paper, we further studied its from cell samples by using the High Pure RNA expression profile in different pathological stages Isolation Kit (Bio-Rad, Hercules, CA, USA) and of HCC and explored the possible downstream then was reversely transcribed into cDNA using genes related to its regulation of HCC invasion the iScript cDNA Synthesis Kit (Bio-Rad, Hercu- and migration. les, CA, USA). Then, QRT-PCR was performed to detect SOX12 expression using TaqMan Master Mix (Applied Biosystems, Foster City, CA, USA) Materials and Methods and the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The Data Mining in Liver Cancer Cohort relative mRNA expression was normalized to in TCGA GAPDH. The primers for SOX12 were: forward, in liver cancer cohort in TC- 5’-CGCGATGGTGCAGCAGCG-3’ and reverse, GA was analyzed using the UCSC Xena browser 5’-GCCACTGGTCCATGATCTTC-3’. Relative (http://xena.ucsc.edu/). The genes co-expressed mRNA expression levels (fold changes) between with SOX12 in liver cancer cohort in TCGA was groups were calculated using the 2-ΔΔCT method. analyzed using the cBioPortal for Cancer Ge- nomics11,12 and further verified using the UCSC Western Blotting Xena. The associations between the expression Total protein were extracted from HepG2 cells of SOX12, CDK4 or IGF2BP1 and survival of 48 h after infection by using a lysis buffer. Then HCC patients were also assessed using the UCSC the samples were subjected a conventional western Xena. blotting as described previously15. The primary antibodies used include anti-SOX12 (SAB1411973, Protein-Protein Interaction (PPI) Sigma-Aldrich, Carlsbad, CA, USA), anti-CDK4 Network (ab137675, Abcam, Cambridge, MA, USA), an- The genes co-upregulated with SOX12 in liv- ti-IGF2BP1 (ab82968, Abcam), and anti-β-actin er cancer cohort in TCGA were loaded into (ab8226, Abcam). After incubation and washing, the Search Tool for the Retrieval of Interacting the membranes were further incubated with HRP Genes (STRING) (http://string-db.org/) database conjugate secondary antibodies. The blot signals for analysis of PPI network. The evidence level were visualized using the ECL Western blotting was set to > 0.7, which indicates experimentally substrate (Promega, Madison, WI, USA). validated interactions. Statistical Analysis Cellular SOX12 Expression Identification Statistical analysis was performed using Graph- Immunostaining of the distribution and ex- Pad Prism 6.0. The difference between groups was pression of SOX12 in HCC cell line HepG2 cells evaluated by unpaired, two-tailed Student’s t-test. were reviewed in Human Protein Atlas, an online The difference between Kaplan-Meier curves were database offering the combination of transcrip- assessed using the log-rank test. The test statistics tomics and antibody-based proteomics for map- (χ2) and p-value (χ2 distribution) were given. p < ping the human proteome down to the single cell 0.05 indicates statistical significance. level13,14.

Cell Culture and Transfection Results Human HCC HepG2 cells were purchased from the American Type Culture Collection and SOX12 Upregulation is Associated with were cultured in Dulbecco’s Modified Eagle Me- Regional and Distant Metastasis of HCC dium (DMEM) at 37°C in a 5% CO2 incubator. One recent study reported that SOX12 can pro- The medium was supplemented with 10% FBS, mote metastasis of HCC10. In this study, we further 100 μg/ml penicillin, and 100 μg/ml strepto- characterized the expression profile of SOX12 in mycin. SOX12 lentiviral shRNA and lentiviral different stages of HCC. By using the data from expression particles were obtained from Geneco- liver cancer cohort in TCGA, we compared the poeia. HepG2 were infected with the lentiviral expression profiles of SOX12 in different patho- particles in the presence of Polybrene. logical stages (Figure 1A). The results showed

3822 SOX12 upregulation is associated with metastasis of hepatocellular carcinoma

Figure 1. SOX12 upregulation is associated with regional and distant metastasis of HCC. A-D, Heat map (left) and bar chart (right) of SOX12 mRNA expression in different pathological stages (A), different T stages (B), different N stages (C) and different M stag- es (D) of HCC. The number of patients in different stages were listed below the bar chart. Analysis was performed using the UCSC Xena based on liver cancer cohort in TCGA database. that SOX12 expression gradually increased with 2A, red arrows), which have well characterized en- the development of pathological stages (Figure hancing effect on HCC cell growth, invasion and 1A). Then, we compared SOX12 expression in metastasis16-18. Therefore, we decided to further different T stages. The results showed similar investigate whether there is any regulative effect SOX12 expression from T1 to T4 stages (Figure of SOX12 on CDK4 and IGF2BP1. By reviewing 1B). However, by comparing SOX12 expression the heat map and performing regression analy- between the cases with or without regional lymph sis using the UCSC Xena, we further confirmed node metastasis and between the cases with or moderate level of co-upregulation between SOX12 without distant metastasis, we observed that the and CDK4 (Figure 3A-B) and between SOX12 regional lymph node metastasis (N1) and distant and IGF2BP1 (Figure 3A and C). SOX12 immu- metastasis (M1) cases had significantly increased nostaining in Human Protein Atlas showed that SOX12 expression (Figure 1C-D). SOX12 has nucleoplasm expression in HepG2 cells (Figure 3D), which is consistent with its regulation SOX12 is Co-Upregulated at the transcriptional level. Then, HepG2 cells with CDK4 and IGF2BP1 in HCC were infected with lentiviral SOX12 shRNA or SOX12 can upregulate Twist1 and FGFBP1 in lentiviral SOX12 expression particles respectively HCC, thereby promoting metastasis10. As a tran- (Figure 3E and G). Following Western blotting scription factor, SOX12 might regulate expression data showed that knockdown of SOX12 reduced of some other genes. Via data mining in liver can- IGF2BP1 and CDK4 expression (Figure 3F), while cer cohort in TCGA using cBioPortal for Cancer enforced SOX12 expression significantly enhanced Genomics, we identified the most co-upregulated their expression (Figure 3H). genes with SOX12 (Pearson’s r ≥ 0.5) (Figure 2A). By performing PPI network analysis, we failed to Low SOX12, CDK4 or IGF2BP1 identify any known interactions between SOX12 Expression is Associated with Better and the co-upregulated genes (Figure 2B). Interest- Survival Among HCC Patients ingly, we found CDK4 and IGF2BP1 were among Since metastasis is an indicator of poor sur- the top 20 co-upregulated genes in HCC (Figure vival, we further assessed whether the expression

3823 P. Yuan, L. Meng, N. Wang

Figure 2. PPI analysis of SOX12 and its co-upregulated genes in HCC. A, The most co-upregulated genes with SOX12 (Pearson’s r ≥0.5) in liver cancer cohort in TCGA. Data was obtained by using cBioPortal for Cancer Genomics. B, PPI network among SOX12 and its co-upregulated genes in HCC. of SOX12, CDK4 or IGF2BP1 is related to surviv- pression in breast cancer cells, thereby increasing al among the patients. In a two-group analysis by their migration and invasion capability20. In HCC, setting median gene expression as the cutoff, Ka- SOX12 also functions as a metastasis enhancer plan-Meier curves and the log-rank tests showed via upregulating the expression of Twist1 and FG- that low SOX12 expression was associated with FBP110. These findings suggest that the functional significantly better 3-year survival (p=0.008) role of SOX12 might depend on the specific type (Figure 4A) and better 5-year survival (p=0.008) of cancer. In this work, we further examined the (Figure 4B). Low CDK4 expression was also as- expression profile of SOX12 in different patho- sociated with significantly better 3-year survival logical stages of HCC, based on the liver cancer (p=0.005) (Figure 4C) and better 5-year survival cohort in TCGA. Our comparisons showed that (p=0.007) (Figure 4D). Low IGF2BP1 expression SOX12 upregulation was associated with regional was associated with significantly better 3-year lymph node metastasis and distant metastasis. survival (p=0.018) (Figure 4E). However, this Although Twist1 and FGFBP1 were identified association was not observed in 5-year survival as the downstream effectors of SOX12 in HCC, (p=0.073) (Figure 4F). whether other genes are regulated by SOX12 is not clear. Our further data mining in TCGA da- tabase showed that CDK4 and IGF2BP1 were sig- Discussion nificantly co-expressed with SOX12, which have well characterized effect on HCC cell growth and The regulative effect of SOX12 on cancer cell invasion. CDK4 upregulation is associated with behaviors were mainly observed in colon cancer, enhanced HCC cell proliferation and growth of breast cancer and HCC. In colon cancer, SOX12 xenografts16. Its upregulation might predict more acts as metastasis suppressor via modulating the malignant characteristics in human HCC tissues21 WNT-TCF signaling pathway19. However, SOX12 and also a poor survival rate in the patients22. can significantly upregulate MMP9 and Twist ex- Palbociclib, a well-tolerated and selective CDK4/6

3824 SOX12 upregulation is associated with metastasis of hepatocellular carcinoma

Figure 3. SOX12 increases CDK4 and IGF2BP1 expression in HCC cells. A-C, Heat map of SOX12, CDK4 and IGF2BP1 expres- sion (A) and regression analysis of the correlation between SOX12 and CDK4 (B) and between SOX12 and IGF2BP1 (C). D, Immu- nostaining of SOX12 in HepG2 cells. Blue: DAPI; Green: SOX12. Images were obtained from Human Protein Atlas. E and G, QRT- PCR analysis of SOX12 mRNA expression 24 h after transfection of SOX12 shRNA (E) or SOX12 expression particles (G). F and H, Immunoblotting image of SOX12, IGF2BP1 and CDK4 expression 48 h after transfection of SOX12 shRNA (F) or SOX12 ex- pression particles (H). **, p < 0.01. inhibitor, can suppress cell proliferation in human their prognostic values were still not quite clear. liver cancer cell lines by promoting a reversible By data mining in liver cancer cohort in TC- cell cycle arrest17. As to IGF2BP1, it also plays an GA based on survival data of 365 patients, we important role in malignant behaviors of HCC. observed that low SOX12 or CDK4 expression Mechanistically, IGF2BP1 can bind and stabilize was associated with significantly better 3-year the c- and MKI67 mRNAs and increase survival and also better 5-year survival, while c-Myc and Ki-67 protein expression, two potent low IGF2BP1 expression was associated with regulators of cell proliferation and apoptosis18. significantly better 3-year survival. Therefore, the Knockdown of endogenous IGF2BP1 in HCC cells expression of SOX12, IGF2BP1 or CDK4 might can significantly suppress cell growth, migration be potential clinical prognostic markers for HCC. and invasiveness both in vitro and in vivo23-25. In HepG2 cells, we demonstrated that knockdown of SOX12 reduced IGF2BP1 and CDK4 expression, Conclusions while enforced SOX12 expression significantly enhanced IGF2BP1 and CDK4 expression. In com- SOX12 upregulation is associated with region- bination with previously research findings, we al and distant metastasis of HCC. SOX12 can infer that IGF2BP1 and CDK4 are two important increase the expression of CDK4 and IGF2BP1, downstream effectors of SOX12 in HCC. which confer malignant phenotypes to HCC. The Although the oncogenic properties of IG- expression of SOX12, IGF2BP1 or CDK4 might F2BP1 and CDK4 in HCC were widely reported, be potential clinical prognostic markers for HCC.

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