HBXIP: a potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelial-mesenchymal transition

Journal Pre-proof HBXIP: A potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelialmesenchymal transition

Xuanyu Wang, Qi Feng, Hongnv Yu, Xingzhi Zhou, Changliang Shan, Qinggao Zhang, Shuangping Liu PII:

S0024-3205(20)30101-6

DOI:

https://doi.org/10.1016/j.lfs.2020.117354

Reference:

LFS 117354

To appear in:

Life Sciences

Received date:

9 December 2019

Revised date:

17 January 2020

Accepted date:

22 January 2020

Please cite this article as: X. Wang, Q. Feng, H. Yu, et al., HBXIP: A potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelialmesenchymal transition, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117354

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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HBXIP: a potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelial-mesenchymal transition

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Xuanyu Wang1#, Qi Feng2#, Hongnv Yu3, Xingzhi Zhou4, Changliang Shan2,5, Qinggao Zhang1*, Shuangping Liu1* 1 Chronic Disease Research Center, Medical College, Dalian University, Dalian 116622, Liaoning, China. 2 Biomedical Translational Research Institute, Jinan University, Guangdong 510632, Guangzhou, China. 3 Department of Central laboratory, Xinhua Hospital Affiliated of Dalian University, Dalian 11602, Liaoning, China. 4 Genesino （ Dalian ） Biological S&T Development Co., Ltd, Dalian 116622, Liaoning, China. 5 State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300350, China.

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#Xuanyu Wang and Qi Feng contributed equally to this study.

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*Corresponding to: Dr. Shuangping Liu, Chronic Disease Research Center, Medical College, Dalian University, Dalian 116622, Liaoning, China. Phone: 86-15898113268; E-mail: [email protected] or Dr. Qinggao Zhang, Chronic Disease Research Center, Medical College, Dalian University, Dalian 116622, Liaoning, China. Phone: 86-13322289111; E-mail: [email protected]. Word count：4348 Number of tables:3

Number of figures:5

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Abstract

1 Introduction

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Hepatitis B X-interacting protein (HBXIP) is highly expressed in many cancers, but the correlation between the expression of HBXIP and the clinical significance and underlying molecular mechanisms in colorectal cancer (CRC) is still unclear. We selected 186 specimens from CRC patients for analyzing the relationship between the expression of HBXIP and the clinical-pathological features by immunohistochemistry. Migration and invasion experiments were performed to examine the effect of HBXIP on CRC cell metastasis. Besides, we also explored the possible molecular mechanism of HBXIP regulation of CRC cell metastasis by Western blot. Our data indicated that the HBXIP was overexpressed in CRC tissues. High HBXIP expression was correlated with metastasis and shorter survival times in patients with CRC and served as an independent factor for poor prognosis. Moreover, HBXIP promotes CRC metastasis by enhancing the epithelial-mesenchymal transition (EMT) process. Our findings provide the first evidence that HBXIP induces EMT to promote metastasis and predicts the poor prognosis of CRC. Therefore, HBXIP may become a new target for CRC treatment. Keywords: HBXIP, CRC, prognostic biomarker, EMT

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Colorectal cancer is one of the major malignant tumors in the world [1]. About 1.2 million patients worldwide are diagnosed with colorectal cancer each year, and more than 600,000 patients die directly or indirectly from colorectal cancer, and the incidence of colorectal cancer in men is higher than in women [2]. In China, with the increase of the aging population, changing of the living environment and eating habits, especially the increasing intake of animal protein, the incidence and mortality of CRC are also increasing year by year, which has become the top one in digestive tract cancer [3]. What's more, it's reported that about 15%-25% of patients have occurred liver metastases at the time of diagnosis [4]. Therefore, early diagnosis and treatment are essential for preventing and reducing the cumulative mortality of CRC. The main research topic of our team in recent years is to explore the expression and the clinicopathological significance of HBXIP in tumor tissues. The HBXIP protein is a conserved cellular constituent protein consisting of 173 amino acids with a molecular weight of approximately 18 kDa, which is located on chromosome 1p13.3 [5]. HBXIP protein was first discovered in human hepatoma cell line HepG2 and it can be bind to the C-terminus of hepatitis B virus-encoded protein (HBX), altering the replication cycle of hepatitis B virus [6]. HBXIP is associated with a variety of carcinogenic signals. For example, HBXIP promotes cell proliferation through NF-kB signaling pathway [7]; HBXIP activates mTORC1 signaling pathway through amino acids to promote cancer cell proliferation and migration [8]; HBXIP promotes human breast cancer growth through pAKT / MDM2 pathway [9]. Many studies show that 2

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HBXIP is closely related to the occurrence and development of many malignant tumors, including breast cancer [10], liver cancer [11], lung cancer [12], etc, which may be involved as an oncoprotein in the proliferation, apoptosis, and migration of various tumor cells. However, the expression and the clinicopathological significance of HBXIP in CRC have not been reported. Here we performed immunohistochemical staining of HBXIP protein in 186 colorectal tissues and found that HBXIP was increased more frequently in CRC compared with the adjacent non-tumor colorectal tissues and the overexpression of HBXIP was closely related to the survival of CRC patients. Clinical stage and Lymph node status were related to HBXIP overexpression in CRC. We also described the role of HBXIP in CRC metastasis, providing a deeper insight into the role of HBXIP in the development of CRC and its related molecular mechanisms.

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2 Materials and methods 2.1 Ethics statement

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This research complied with the Helsinki Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of Dalian University Medical College. Patients were informed that the resected specimens were potentially used for scientific research and that their privacy would be maintained.

2.2 Clinical samples

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A total of 186 clinical samples, including 93 CRC tissue samples, and 93 adjacent non-tumor colorectal tissues, were collected from Shanghai Outdo Biotech Co., Ltd. Pathological parameters of all 93 CRC specimens included gender, age, tumor size, histological grade, clinical stage, lymph node status, Ki67, MSH2, MSH6, P53, and survival data. All CRC patients did not receive any chemotherapy or radiotherapy before surgery. The staging criteria were followed by the American Joint Committee on Cancer (AJCC) (8th Edition). All patient’s followed-up dates ended on June 6, 2016, or at the time of death. The entire experimental design and process were followed by the Dalian University Review Committee.

2.3 Immunohistochemistry Immunohistochemistry used a SPlink Detection Kit (Biotin-Streptavidin HRP Detection Systems) (Zhongshanjinqiao Biological Technology Ltd., Beijing, China). The tissue sections were deparaffinized with xylene, and then the sections were sequentially placed in 100%, 95%, and 85% ethanol to hydrate. The antigen repair buffer solution (pH 6.0) was boiled and placed at about 80 °C, and the sections were placed for antigen retrieval for 1 h. After adding the appropriate amount of endogenous peroxidase blocker and normal goat serum working solution, the tissue sections were incubated with primary antibody HBXIP (1:400, #14492-1-AP, Proteintech, USA) overnight at 4 °C in a humidified chamber. After incubating the 3

Journal Pre-proof tissue sections with biotin-labeled goat anti-mouse/rabbit IgG polymer and horseradish-labeled streptavidin working solution for a certain time, an appropriate amount of freshly prepared DAB solution (Zhongshanjinqiao Biological Technology Ltd., Beijing, China) is added and then stained with hematoxylin staining solution. Finally, the slices are hydrated and sealed with a neutral resin.

2.4 Interpretation of immunohistochemistry staining

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All tissue sections were independently evaluated by two investigators (Liu S and Zhang Q) who were not known for all clinical data. The semi-quantitative scoring criteria for the positive degree of tumor cells are as follows: the number of negative cells or positive cells is not more than 5%, the score is "-", and the number of positive cells is between 5-25%, which is rated as "+", '++ 'Represents 26-50% of positive cells, '+++' means positive cells account for more than 50%. The expression of HBXIP in cytoplasmic was considered a positive staining. In addition, tissue sections rated "++" and "+++" represent strongly positive expression. For survival data analysis, the expression levels of HBXIP include high expression (++ and +++) and low expression (-&+).

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2.5 Cell culture and siRNA transfection

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Human colonic cancer cell line HCT116 and HCT8 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. These two cell lines were used to investigate the relationship of EMT and HBXIP in vivo [13]. The sequence of HBXIP siRNA was 5′-GTTGCACCGTGATCAATTT-3′ (RiboBio, Guangzhou, China). The cells were plated at 3*105 cells/well in a six-well plate. After 12 h, 100 nM of HBXIP siRNA or negative control (NC) siRNA and roboFECTTM CP Transfection Kit (RiboBio, Guangzhou, China) were added to roboFECTTM CP Buffer and added to the cells. After incubation for 24h-48h, subsequent experiments were performed.

2.6 Cell proliferation assay

For the cell counting experiment, the transfected HCT116 and HCT8 cells were plated in a twenty-four-well plate at 10,000 cells/well for four consecutive days. For the colony formation experiment, the transfected HCT116 and HCT8 were plated in a six-well plate at 500 cells/well and incubation continued for 10-14 days, stained with 0.1% crystal violet stain solution (Solarbio, Beijing, China) and counted.

2.7 Wound healing assay Transfected HCT116 and HCT8 were evenly spread in a six-well plate at 106 cells/well. After 12 hours, a wound was placed in the middle of each well with a 10 μl pipette tip, and culture was continued with serum-free medium and recorded at 0h, 24h, 48h with a microscope. 4

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2.8 Migration and invasion assay The matrigel was diluted 1:8 with pre-cooled serum-free medium, and then 50 μl of the diluted matrigel was evenly spread in a transwell chamber and placed in 37 ° C incubator for 0.5-1 h. Transfected HCT116 and HCT8 cells were suspended in a serum-free medium and uniformly placed in a chamber at 105 cells/well. The transwell chamber was placed in a 24-well plate containing 10% serum medium and cultured for 24 h. Cells that passed through the chamber were stained with 0.1% crystal violet (Solarbio, Beijing, China) and finally counted with a microscope.

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2.9 Western blot analysis

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The transfected HCT116 and HCT8 cells were centrifuged and collected. Cell lysate (Solarbio, Beijing, China) was added to the cells and lysed at 4 °C for 40 minutes. The cells were centrifuged at 12000 r/min for 20 minutes at 4 °C and the supernatant was collected. The collected proteins were separated by SDS-PAGE gel electrophoresis, and then the proteins on the gel were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% skim milk powder for 1 h at room temperature and combined with HBXIP antibody (1:1000, Proteintech, USA), E-cadherin (1:5000, Proteintech, USA), MMP2 (1:500, Proteintech, USA), Vimentin (1:1000, Proteintech, USA), Snail (1:500, Proteintech, USA) at 4°C overnight. The membrane was then incubated with goat anti-rabbit IgG HRP-linked antibodies (1:2500, Life Science, China) for 1 h at room temperature. The protein signal is then detected using a high-sensitivity chemiluminescence detection kit (ECL Western Blot Kit, CWbiotech, China).

3.0 Statistical analysis

Statistical analysis was performed by SPSS (version 20.0). Kaplan - Meier method (logarithmic rank test) was used for survival curve analysis. Chi-square test and Fisher precise tests were used for establishing the correlation between HBXIP expression and clinicopathological features in CRC. The independence of HBXIP in predicting prognosis was evaluated by multivariate Cox proportional hazards regression model. The analysis of two groups’ comparisons was used by two-tailed student’s t-tests. Each experiment was repeated at least three times, and the P-value was less than 0.05 for a significant difference.

3 Results 3.1 HBXIP is upregulated in CRC We detected the expression of the HBXIP in CRC patients’ tissues. 5

Journal Pre-proof Immunohistochemistry staining demonstrated that the positive rate of HBXIP was 81.7% (76/93) in CRC compared with adjacent non-tumor colorectal tissues (30.1%, 28/93) (p<0.01) (Table 1). The strong positive rate of HBXIP was 61.3% (57/93) (p<0.01) in CRC, which was also significantly higher than adjacent non-tumor colorectal tissues (4.3%, 4/93) (p<0.01) (Table 1). As shown in Fig. 1, HBXIP was detected in the cytoplasm of CRC cells.

3.2 Relationship between HBXIP overexpression level and the clinicopathological features of CRC

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The relationship between HBXIP and clinicopathological features of CRC was summarized in Table 2. HBXIP overexpression was significantly correlated with lymph node metastasis and clinical stage of CRC (p<0.01). The positive rate of HBXIP with metastatic was significantly higher than non-metastatic (p<0.01). For the clinical stage of CRC, we found that tissues with strong positive HBXIP staining were more common in stage Ⅲ~Ⅳ, comparing with stage Ⅰ~Ⅱ (p<0.01). However, HBXIP protein expression level was not correlated with the patients’ gender, age, tumor size, histological grade, Ki67, MSH2, MSH6, P53 in CRC (p0.05).

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3.3 Association between HBXIP expression and prognosis of CRC

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We analyzed 93 cases of CRC by the Kaplan-Meier method to demonstrate the role of HBXIP in CRC prognosis. We found that patients with high HBXIP expression had lower overall survival (OS) than patients with low HBXIP expression (log rank=18.847, p=0.000) (Fig. 2a). Similarly, the patients with high HBXIP expression had decreased OS compared to those with low HBXIP expression in either LN metastasis (+) cases (log rank=5.443, p=0.020) or LN metastasis (-) cases (log rank=5.507, p=0.019) (Fig. 2b, c). Interestingly, the patients with high HBXIP expression had decreased OS compared to those with low HBXIP expression in early-stage cases (log rank=7.291, p=0.007), but there is no difference in late-stage (log rank=1.987, p=0.159) (Fig. 2d, e). It indicates that there is no significant difference in the expression of HBXIP in patients with late-stage, and we guess that HBXIP could be an early prognostic indicator of CRC.

3.4 Univariate and multivariate analyses of prognostic variables in CRC We performed univariate and multivariate analyses to assess whether HBXIP expression is an independent risk factor for CRC prognosis. Univariate analysis data indicated that histological grade, clinical stage, lymph node status, and HBXIP 6

Journal Pre-proof expression were all demonstrated to be prognostic variables for OS in CRC patients. Besides, in the multivariate analysis, only histological grade (p<0.05) and HBXIP expression (p<0.01) were found to be independent prognostic variables of OS (Table 3), suggesting that HBXIP overexpression is an important independent worse prognostic factors of CRC.

3.5 HBXIP significantly promoted the proliferation and colony formation of CRC cells

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The effect of HBXIP on proliferation and migration of HCT116 and HCT8 cells was investigated using NC siRNA and HBXIP siRNA. Western blot analysis showed that HBXIP expression was significantly downregulated by HBXIP siRNA compared to the NC siRNA in HCT116 cells (Fig. 5a, b) and HCT8 cells (Fig. 5c, d). As shown in Fig. 3, whether in cell counting (Fig. 3a, b) or colony formation assay (Fig. 3c, d), proliferation of HCT116 and HCT8 cells transfected with HBXIP siRNA was significantly inhibited.

3.6 HBXIP promotes the migration, invasion, and EMT of CRC cells

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Migration and invasion experiments were performed to examine the effects of HBXIP on CRC cell motility. As shown in Fig. 4, the numbers of migrated (Fig. 4a, b, c) and invaded cells (Fig. 4d, e) with low HBXIP levels were significantly decreased compared to the control group. To further investigate its metastatic mechanism, we tested whether the level of HBXIP induces EMT in CRC cells. Our results showed that low expression of HBXIP up-regulates the expression levels of epithelial marker (E-cadherin), while down-regulated the expression levels of mesenchymal markers (Vimentin, Snail) and MMP2 (Fig. 5), suggesting that HBXIP could promote the EMT of CRC HCT116 cells (Fig. 5a, b) and HCT8 cells (Fig. 5c, d). Collectively, our observations suggested that HBXIP may promote CRC cell migration and invasion via the induction of EMT.

4 Discussion HBXIP has been identified as a kind of oncoprotein, which is highly expressed in different tumors. Our previous results showed that the HBXIP exhibited oncogene characteristics in ovarian cancer [14], pancreatic ductal adenocarcinoma [15], non-small cell lung cancer [16], and cervical cancer [17]. Other research groups have found that HBXIP is associated with poor prognosis in breast cancer [18], and hepatocellular carcinoma cells [19]. What’s more, HBXIP can play different roles through different molecular mechanisms, such as it can promote the proliferation of tumor cells by inhibiting apoptosis [20], promoting centrosome replication [21], inhibiting the expression of tumor suppressor gene [22], and promoting lipid 7

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metabolism [23]. In recent years, research on HBXIP promoting tumor cell migration and invasion has become a hot topic. Our results showed that the expression of HBXIP is associated with lymphatic metastasis in CRC patients, and HBXIP can promote the migration and invasion of CRC cells. There are many studies on the mechanism of tumor cell metastasis promoted by HBXIP. For example, HBXIP promotes breast cancer cell metastasis by activating KEAP1 and NRF2 [24]. HBXIP activates MMP15 expression and promotes metastasis of hepatic carcinoma [19]. EMT refers to the phenomenon that epithelial cells differentiate into mesenchymal cells under specific physiological and pathological conditions., which enhance the metastasis and invasiveness of cancer cells [25]. It has been reported that low expression of e-cadherin contributes to metastasis and recurrence of invasive lobular breast cancer and mesenchymal-associated transcription factors such as Snail, Slug can reduce E-cadherin expression [26-27]. Vimentin is a type of intermediate filament present in various non-epithelial cells, especially interstitial cells, which has great significance for stabilizing cytoplasmic structures [25]. Our results indicated that the down-regulation of HBXIP inhibits EMT progression, resulting in increased expression of the epithelial marker (E-cadherin) and decreased expression of the mesenchymal cell marker (Vimentin, Snail). MMPs digest and degrade extracellular matrix components and participate in tumor metastasis [24]. Cancer cells can destruct the barrier of the basement membrane by adjusting the activity of MMPs, allowing cancer cells to directly invade the interstitial compartment [24]. Studies have shown that MMP2 is associated with tumor cell metastasis [28]. We found that the expression of MMP2 was decreased in HBXIP knockdown CRC cells. These results support that HBXIP is an important regulator of EMT in CRC. Mismatch repair (MMR) plays an important role in cell replication, maintenance of genetic information stability, regulation of gene mutations, and repair of DNA base mismatches. Once a mutation occurs, it will cause defects in the repair function [29]. MSH2 and MSH6 are important members of the MMR family. The deletion of MMR protein may affect the occurrence, invasion, and metastasis of early CRC [30]. Our results showed that the expression of HBXIP does not appear to be related to the MMR proteins MSH2 and MSH6, suggesting that HBXIP is a prognostic indicator independent of MMR disorders. As a nuclear protein, Ki-67 participates in cell proliferation and may be obligatory for it. Besides, P53 works as a contributing factor in a multicellular organism, which can prevent the formation of cancer and thus inhibit cancer [31]. We showed that the expression of HBXIP in CRC does not seem to be related to Ki-67 or P53. We suspected that the above consequence may result from that the cases are not enough. If the number of cases is enlarged, we may get more meaningful data. Interestingly, Our previous results indicated that in both early and late stages, patients with high HBXIP expression had lower OS than those with low HBXIP expression ones in NSCLC [16]. but it seems to be different in CRC patients. In this experiment, we found that the patients with high HBXIP expression had lower OS than those with low HBXIP expression ones, but there is no difference in the late 8

Journal Pre-proof stage. It indicates that HBXIP seems to be an early prognostic indicator of CRC.

5 Conclusion In short, our consequences suggested that HBXIP stands an important place in the development of CRC. Our data also indicated that the expression of HBXIP is improved in CRC samples and is significantly associated with lymph node status and clinical staging. It brings about short survival and plays a role as an independent factor for poor prognosis. What’s more, HBXIP promoted CRC cell migration and invasion via the induction of EMT. The high proportion and prognostic value of HBXIP expression suggested that it may be a significant biomarker and a potential therapeutic target for patients with CRC.

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Conflict of interest No conflict of interest was declared.

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Funding This work was supported by grants from the National Nature Science Foundation of China (61671098, 31560312). This study was also supported by the Science and Technology Program of Guangzhou (Grant 201807010003). The funding source didn’t involve the design of the study, the collection, and analysis of data, and the publication of the manuscript.

References

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Acknowledgment This research complied with the Helsinki Declaration. It has been approved by the Human Ethics Committee and the Research Ethics Committee of Dalian University.

[1] R.L. Siegel, K.D. Miller, A. Jemal. Cancer statistics, 2016. CA Cancer J Clin. 66271-289. https://doi.org/10.3322/caac.21254 [2] R. Siegel, D. Naishadham, A. Jemal. Cancer statistics, 2013, CA Cancer J Clin. 63 (2013) 11–30. https://doi.org/10.3322/caac.21166 [3] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He. Cancer statistics in China, 2015, CA Cancer J Clin. 66 (2016) 115-132. https://doi.org/10.3322/caac.21338 [4] A. Sadanandam, C.A. Lyssiotis, K. Homicsko, E.A. Collisson, W.J. Gibb, S. Wullschleger. A colorectal cancer classification system that associates cellular phenotype and responses to therapy, Nat Med. 19 (2013) 619-625. https://doi.org/10.1038/nm.3175 [5] M. Melegari, P.P. Scaglioni, J.R. Wands. Cloning and characterization of a novel 9

Journal Pre-proof hepatitis B virus x binding protein that inhibits viral replication, J Virol. 72 (1998) 1737-1743. https://doi.org/0000-0002-6036-5138 [6] R. Fujii, C. Zhu, Y. Wen, H. Marusawa, B. Bailly-Maitre, S. Matsuzawa, H. Zhang, Y. Kim, C.F. Bennett, W. Jiang, J.C. Reed. HBXIP, cellular target of hepatitis B virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis, Cancer Res. 66 (2006) 9099–9107. https://doi.org/10.1158/0008-5472.CAN-06-1886 [7] Q. Liu, W. Lu, C. Yang, Y. Wang, W. Li, Y. Chu, J. Deng, Y. Hou, J. Jin. HBXIP activates the PPAR δ /NF- κ B feedback loop resulting in cell proliferation, Oncotarget. 9 (2017) 404-417. https://doi.org/10.18632/oncotarget.23057

ro

of

[8] L. Bar-Peled, L. Schweitzer, R. Zoncu, D. Sabatini. Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1, Cell. 150 (2012) 1196–1208. https://doi.org/10.1016/j.cell.2012.07.032.

lP

re

-p

[9] H. Li, Z. Wang, M. Jiang, R. Fang, H. Shi, Y. Shen, X. Cai, Q. Liu, K. Ye, SJ. Fan, W. Zhang. The oncoprotein HBXIP promotes human breast cancer growth through down-regulating p53 via miR-18b/MDM2 and pAKT/MDM2 pathways, Acta Pharmacol Sin. 39 (2018) 1787-1796. https://doi.org/10.1038/s41401-018-0034-6.

Jo ur

na

[10] F. Liu, X. You, Y. Wang, Q. Liu, Y. Liu, S. Zhang, L. Chen, X. Zhang, L. Ye. The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF, Carcinogenesis. 35 (2014) 1144-1153. https://doi.org/10.1093/carcin/bgu021 [11] Y. Wang, R. Fang, M. Cui, W. Zhang, X. Bai, H. Wang, B. Liu, X. Zhang, L. Ye. The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Lett. 385 (2017) 234-242. https://doi.org/10.1016/j.canlet.2016.10.018. [12] E. Krepela, P. Dankova, E. Moravcikova, A. Krepelova, J. Prochazka, J. Cermak, J. Schützner, P. Zatloukal, K. Benkova. Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma, Int J Oncol. 35 (2009) 1449-1462. https://doi.org/10.3892/ijo_00000464 [13] K Zeng, X Chen, X Hu, X Liu, T Xu, H Sun, Y Pan, B He, S Wang. LACTB, a novel epigenetic silenced tumor suppressor, inhibits colorectal cancer progression by attenuating MDM2-mediated p53 ubiquitination and degradation, Oncogene. 37(2018) 5534-5551. https://doi.org/10.1038/s41388-018-0352-7 [14] Y. Wang, J. Sun, N. Li, S. Che, T. Jin, S. Liu, Z. Lin. HBXIP overexpression is correlated with the clinical features and survival outcome of ovarian cancer, J 10

Journal Pre-proof Ovarian Res. 10 (2017) 26. https://doi.org/10.1186/s13048-017-0322-7 [15] Zhou X, Wang X, Duan J, Sun W, Chen Z, Li Q, Z. Ou, G. Jiang, X. Ren, S. Liu. HBXIP protein overexpression predicts the poor prognosis of pancreatic ductal adenocarcinomas, Pathol Res Pract. 215 (2018) 343-346. https://doi.org/10.1016/j.prp.2018.12.016 [16] Y. Wang, N. Li, S. Che, T. Jin, J. Piao, S. Liu, Z. Lin. HBXIP suppression reduces cell proliferation and migration and its overexpression predicts poor prognosis in non-small-cell lung cancer, Tumour Biol. 39 (2017) 1010428317709675. https://doi.org/10.1177/1010428317709675

ro

of

[17] N. Li, Y. Wang, S. Che, Y. Yang, J. Piao, S. Liu, Z. Lin. HBXIP overexpression as an independent biomarker for cervical cancer, Exp Mol Pathol. 102 (2017) 133-137. https://doi.org/10.1016/j.yexmp.2017.01.009

re

-p

[18] D. Cheng, B. Liang, Y. Li. HBXIP expression predicts patient prognosis in breast cancer, Med. Oncol. 310 (2014) 210. https://doi.org/ 10.1007/s12032-014-0210-6

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[19] S. Zheng, H. Wu, F. Wang, J. Lv, J. Lu, Q. Fang, F. Wang, Y. Lu, S. Zhang, Y. Xu, Q. Bao, C. Xie, Z. Yin. The oncoprotein HBXIP facilitates metastasis of hepatocellular carcinoma cells by activation of MMP15 expression, Cancer Manag Res. 16 (2019) 4529-4540. https://doi.org/10.2147/CMAR.S198783

Jo ur

na

[20] H. Marusawa, S. Matsuzawa, K. Welsh, H. Zou, R. Armstrong, I. Tamm, J.C. Reed. HBXIP functions as a cofactor of survivin in apoptosis suppression, EMBO J. 22 (2003) 2729-2740. https://doi.org/10.1093/emboj/cdg263 [21] Y. Wen, V.S. Golubkov, A.Y. Strongin, W. Jiang, J.C. Reed. Interaction of hepatitis B viral oncoprotein with cellular target HBXIP dysregulates centrosome dynamics and mitotic spindle formation, J Biol Chem. 283 (2018) 2793-2803. https://doi.org/10.1074/jbc.M708419200 [22] Y. Zhao, H. Li, Y. Zhang, L. Li, R. Fang, Y. Li, Q. Liu, W. Zhang, L. Qiu, F. Liu, X. Zhang, L. Ye. Oncoprotein HBXIP modulates abnormal lipid metabolism and growth of breast cancer cells by activating the LXRs/SREBP-1c/FAS signaling cascade, Cancer Res. 76 (2016) 4696-4707. https://doi.org/10.1158/0008-5472.CAN-15-1734 [23] M. Minczuk, S. Mroczek, S.D. Pawlak, P.P. Stepien. Human ATP-dependent RNA/DNA helicase hSuv3p interacts with the cofactor of survivin HBXIP, FEBS J. 272 (2005) 5008-5019. https://doi.org/10.1111/j.1742-4658.2005.04910.x [24] X. Zhou, C. Zhu, Z. Wu, X. Guo, W. Zou. The oncoprotein HBXIP competitively 11

Journal Pre-proof binds KEAP1 to activate NRF2 and enhance breast cancer cell growth and metastasis, Oncogene. 38 (2019) 4028-4046. https://doi.org/10.1038/s41388-019-0698-5 [25] A. Bhatt, M. Gupta, Hoang. V, S. Chakrabarty, T.D. Wright, S. Elliot, IK. Chopra, D. Monlish, K. Anna, M.E. Burow, J.E. Cavanaugh, P.T. Flaherty. Novel Diphenylamine Analogs Induce Mesenchymal to Epithelial Transition in Triple Negative Breast Cancer, Front Oncol. 9 (2019) 672. https://doi.org/10.3389/fonc.2019.00672

of

[26] R. Moll, M. Mitze, U. Frixen, W. Birchmeier. Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas, Am J Pathol. 143 (1993) 1731–1742. https://doi.org/10.1097/00000433-199312000-00015

-p

ro

[27] Z. Wei, Z. Shan, Z.A. Shaikh. Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail, Toxicol Appl Pharmacol. 344 (2018) 46-55. https://doi.org/10.1016/j.taap.2018.02.022

lP

re

[28] D. Huang, X. Du, R. Yuan, L. Chen, T. Liu, C. Wen, M. Huang, M. Li, L. Hao, J. Shao. Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2 ubiquitination and degradation, Biochem Biophys Res Commun. 453 (2014) 49-56. https://doi.org/10.1016/j.bbrc.2014.09.061

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na

[29] A.B. Buermeyer, S.M. Deschênes, S.M. Baker, R.M. Liskay. Mammalian DNA mismatch repair, Allnu Rev Genet. 33 (1993) 533-564. https://doi.org/10.1146/annurev.genet.33.1.533 [30] C.M. Zhang, J.F. Lv, L. Gong, L.Y. Yu, X.P. Chen, H.H. Zhou, L. Fan. Role of Deficient Mismatch Repair in the Personalized Management of Colorectal Cancer, Int J Environ Res Public Health. 13 (2016) 892. https://doi.org/10.3390/ijerph13090892 [31] S. Surget, M.P. Khoury, J.C. Bourdon. Uncovering the role of p53 splice variants in human malignancy: a clinical perspective, OncoTargets and Ther. 19 (2013) 57-68. https://doi.org/10.2147/OTT.S53876

Figure legends Fig. 1 IHC staining for HBXIP protein in CRC and adjacent tissues HBXIP protein was not expressed in non-tumor tissues (a). HBXIP was positively expressed in cancerous tissue, but negative expressed in adjacent non-tumor colorectal tissues (b). The expression of HBXIP was negative in CRC tissue (c). Weakly positive (d), moderately positive (e-f) and strongly positive (g-i) HBXIP protein expressions were detected in CRC. 12

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Fig. 2 Kaplan–Meier analyses of survival rates in 93 CRC patients concerning HBXIP protein overexpression. a shows OS rates of patients with high and low HBXIP expression. b and c show the comparison of OS, respectively, with HBXIP low-expression and high-expression in those patients with LN metastatic and LN non-metastatic. (d, e) shows the comparison of OS, respectively, with HBXIP low-expression and high-expression in those patients with early-stage and late-stage.

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Fig. 3 HBXIP regulates CRC cell growth. Cell proliferation was examined by cell counts assay (a, b) and colony formation assay (c, d) in HCT116 and HCT8 cells. Quantitative data were represented as mean ± SD for three independent experiments. (*P<0.05; ** P < 0.01)

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Fig. 4 HBXIP promotes metastasis and invasion in CRC cells. Wound-healing analysis of HCT116 and HCT8 cells transfected with HBXIP siRNA. Images were obtained at 0, 24 and 48 hours (a, b, c). The invasion abilities of HBXIP were explored by Transwell assays of HCT116 and HCT8 cells. The number of cells that crossed the matrix membrane was counted (d, e). Quantitative data were represented as mean ± SD for three independent experiments. (*P<0.05; ** P < 0.01)

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Fig. 5 HBXIP promotes EMT in CRC cells. The expression of HBXIP and EMT markers in HCT116 (a, b) and HCT8 (c, d) cells transfected with control siRNA and HBXIP siRNA were analyzed by Western blotting, β-actin was used as a loading control. Data represent the mean±SD of three independent experiments. (*P<0.05; ** P < 0.01)

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Table 1. Expression of HBXIP protein in colorectal cancer and adjacent tissue HBXIP Diagnosis

No.of case

-

+

++

+++

Positive cases

strong positive

rate (%)

cases rate(%)

Colorectal cancer

93

17

19

37

20

81.7%**

62.3%**

Adjacent tissue

93

65

24

3

1

30.1%

4.3%

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Positive rate: percentage of positive cases with +, ++, and +++ staining score.

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Strongly positive rate (high-level expression): percentage of positive cases with ++ and +++

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** p<0.01 compared with adjacent tissue.

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staining score.

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Table 2. Relationship between HBXIP protein overexpression and the clinicopathological features of colorectal cancer HBXIP No. of case (n)

Variables

χ2

P value

strong positive rate (%) 52 41

0.061

0.805

0.264

0.607

5.761

0.056

16.631

0.000**

26(53.1) 21(47.7)

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49 44

5(50.0) 38(56.7) 14(87.5)

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10 67 16

50 43

0.956

27(60.0) 30(62.5)

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45 48

53 40

0.003 32(61.5) 25(61.0)

40 53

23(39.6) 34(85.0) 16.961

0.000**

0.407

0.523

0.207

0.649

0.818

0.366

0.023

0.880

21(42.0) 36(83.7) 26(65.0) 31(58.5)

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Gender Male Female Age (years) >67 ≤67 Tumor size ≤5cm ＞5cm Histological grade Grade-1 Grade-2 Grade-3 TNM stage Ⅰ~Ⅱ Ⅲ~IV Lymph node status N0 N＋ Ki67 0-1 2-3 MSH2 0-1 2-3 MSH6 0-1 2-3 P53 0-1 2-3

16 77

9(56.3) 48(62.3)

39 54

26(66.7) 31(57.4)

43 50

26(60.5) 31(62.0)

** p<0.01

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Journal Pre-proof Table 3. Cox regression model analysis of the clinicopathological features in 93 patients with clone cancer Characteristics

B

SE

Wald

HR

95%Cl

p value

Lower

Upper

0.213

2.679

0.706

0.465

1.071

0.102

Age

0.132

0.209

0.399

1.141

0.758

1.719

0.528

Tumor size

0.362

0.212

2.932

1.437

0.949

2.176

0.087

Histological grade

0.524

0.208

6.340

1.688

1.123

2.538

0.012*

TNM stage

0.780

0.215

13.110

2.182

1.430

3.329

0.000**

Lymph node status

0.715

0.214

11.137

2.043

1.343

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3.109

0.001**

Ki67

-0.240

0.212

1.284

0.786

0.519

1.192

0.257

MSH2

-0.521

0.282

3.418

0.594

0.342

1.032

0.064

MSH6

-0.333

0.212

2.458

0.717

0.473

1.097

0.117

P53

0.156

0.210

0.551

1.169

0.774

1.765

0.458

HBXIP

0.820

0.223

13.494

2.271

1.466

3.517

0.000*

6.051

1.677

1.111

2.532

0.014*

TNM stage

0.517

lP

0.210

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Histological grade

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-0.349

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Gender

-p

Univariate

0.337

0.539

0.391

1.401

0.487

4.033

0.532

Lymph node status

0.081

0.528

0.024

1.085

0.385

3.055

0.878

HBXIP

0.686

0.255

7.257

1.986

1.206

3.273

0.007*

B:coefficient; SE:standard error; Wald:Wald statistic; HR:hazard ratio; Cl:confidence interval; * p<0.05;** p<0.01

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Highlights

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Hepatitis B X-interacting protein (HBXIP) has been identified as a kind of oncoprotein, which is highly expressed in different tumors. 1. We found HBXIP was overexpressed in colorectal cancer (CRC) tissues. 2. High HBXIP expression was correlated with metastasis 3. High HBXIP expression was correlated with shorter survival times in patients with CRC and served as an independent factor for poor prognosis. 4. HBXIP promotes CRC metastasis by enhancing the epithelial-mesenchymal transition (EMT) process. Therefore, HBXIP may become a new target for CRC treatment.

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Figure 3

Figure 4

Figure 5