- Email: [email protected]
Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation
Accepted Manuscript Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation Yu Qian, Suhua Xia, Zhenyu Feng PII:
S0006-291X(16)32178-7
DOI:
10.1016/j.bbrc.2016.12.119
Reference:
YBBRC 36993
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 13 December 2016 Accepted Date: 18 December 2016
Please cite this article as: Y. Qian, S. Xia, Z. Feng, Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2016.12.119. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation Yu Qian1*, Suhua Xia2, Zhenyu Feng3
RI PT
1 Translational Medicine Research Center, Shanxi Medical University, Taiyuan, Shanxi 030001, China 2 Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, China 3 Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu, China
SC
Correspondence to: Dr. Yu Qian, Translational Medicine Research Center, Shanxi Medical University, No.56 South Xinjian Street, Taiyuan, Shanxi 030001, China. Tel: +86-0351-4135676; E-mail: [email protected]
EP
TE D
M AN U
Abstract FOXK2, which belongs to the fork head DNA binding protein family, has been shown to play a critical role in tumorigenesis. Here, we detected FOXK2 expression and its clinical significance in colorectal cancer, which has not been fully investigated before. Results from public database and our cohort indicated that FOXK2 was transcriptionally activated in colorectal cancer tissues compared to non-cancer tissues. High expression of FOXK2 was significantly correlated with poor survival. In vitro cell experiments suggested that FOXK2 promoted cell proliferation. Furthermore, we found that oncogene SOX9 was responsible for the up-regulation of FOXK2 by directly binding on its promoter. Depletion of FOXK2 attenuated SOX9 induced cell growth. In addition, we observed that the expression of FOXK2 was significantly associated with the expression of SOX9 both in the public database and our colorectal cancer tissues. The patients with SOX9+FOXK2+ had a poor overall survival (p=0.0084). In conclusion, our data suggested that SOX9 transcriptionally activated FOXK2 was involved in the pathogenesis of colorectal cancer and might be a novel target for colorectal cancer therapy.
AC C
Keywords: FOXK2, SOX9, colorectal cancer 1. Introduction
Colorectal cancer is one of the most common malignancies worldwide with highly mortality [1]. Surgery combined with chemotherapy is the main care for colorectal cancer [2, 3]. However, due to the frequent metastasis and chemo resistance, the prognosis of advanced colorectal cancer is still unfavorable [4]. The mechanisms underlying the pathogenesis and development of colorectal cancer remain poorly unknown. Genome alterations, including copy-number alterations (CNA), mutants and gene fusion, are identified as the hallmarks of cancer. Previous studies have demonstrated that oncogenes and tumor suppressor genes deregulation played critical roles in colorectal cancer progression, such as KRAS mutant, TP53 mutant and TERT amplification [5-7]. FOXK2, as known as ILF (interleukin enhancer binding factor), belongs to the Forkhead box (FOX) transcription factors which have been demonstrated to control a wide rage of cellular
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
RI PT
processes, such as DNA repair, cell cycle and survival [8, 9]. FOXK2 could be phosphorylated by CDKs during cell division and colocalized with AP-1, promoting AP-1 mediated transcriptional activation [10, 11]. Previous study indicated that FOXK2 interacted with deubiquitinase BAP1 and recruited BAP1 to DNA which promoted local histone deubiquitation and caused changes in target genes [12, 13]. The functions of FOXK2 in cancers remain to be further investigated. FOXK2 binds to BRCA1/BARD1 and enhances the degradation of ERα in breast cancer, leading to cellular suppression [14]. It also promotes cell survival and activates Wnt signal by promoting DVL nuclear translocation in colorectal cancer [15, 16]. However, the clinical significance and prognostic value of FOXK2 in colorectal cancer are still largely unknown. Sex-determining region Y box 9 (SOX9) is determined as oncogene, which are activated in stem cells and critical for cancer progression. Recent studies have reported that uncontrolled expression of SOX9 in several types of cancers, such as colorectal cancer, pancreatic cancer and glioma and lung cancer [17, 18]. More importantly, the up-regulation of SOX9 indicates poor prognosis in these cancers. SOX9 has been shown to induce self-renewal of cancer stem cells and inhibit Notch activity in hepatocellular carcinoma and to regulate metastasis by activating Wnt/β-catenin signal [19, 20]. Also, SOX9 could be regulated by several critical factors such as epigenetically activated by NF-κB signals, stabilized by SLUG and suppressed by ZBTB7A [21-23]. However, the mechanisms underlying SOX9 mediated tumorigenesis remain elusive. In the present study, we showed that FOXK2 expression was significantly elevated in colorectal cancer tissues and correlated with poor prognosis. Cellular experiments indicated that FOXK2 promoted cell growth and could be transcriptionally activated by SOX9. The coincident up-regulation of FOXK2 and SOX9 in colorectal cancer tissue indicated the pathological association of SOX9-FOXK2 axis in the progression of colorectal cancer. 2. Materials and methods
2.1 Ethics statements and tissue samples
AC C
EP
The study was approved by the Ethics Committee of the first Affiliated Hospital of Soochow University. A total of 145 colorectal cancer samples were collected from the Department of Pathology, the first and second Affiliated Hospital of Soochow University from February 2004 to January 2009. Written informed consent was obtained form all patients. None of the patients had received chemotherapy, radiotherapy or related therapies before surgery and their complete clinical data including follow-up information was available and reviewed. 2.2 Cell culture
Human colorectal cancer cell lines HCT116, SW480, SW620, DLD-1, LOVO, COLO205 and HEK293T cell line were obtained from American Type Culture Collection (ATCC) and Cell Bank at the Chinese Academy of Sciences. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS, 100 U/ml penicillin and 100mg/ml streptomycin. All cells were cultured at 37 with humidified atmosphere containing 5% CO2. 2.3 Lentivirus infection
ACCEPTED MANUSCRIPT The lentiviral for ectopic expression and knockdown of FOXK2 were obtained form Hanbio Co., Ltd (Hanbio, Shanghai, China). For establishing stable overexpression or knockdown cells, lentivirus was added to infect cells, followed by the positive selection using puromycin (Sigma, 5µg/ml).
RI PT
2.4 Oncomine and cBioPortal databases analysis
SC
The mRNA expression of FOXK2 in colorectal cancer was identified from Oncomine database as circumscribed in the criteria of p value less than 10-4 and fold change more than 2. The top 10% ranking lists were shown (https://www.oncomine.org/resource/login.html). The FOXK2 mRNA expression was identified from Kaiser’s colon dataset. The correlation between DNA copy number and mRNA expression of FOXK2 in colorectal cancer was identified by cBioPortal dababase (http://www.cbioportal.org/)
M AN U
2.5 MTT assay
The cell proliferation assay was performed by MTT assay. Indicated cells were seeded into the 96-well plates at the density of 2000/well. 20µl of MTT reagent (5mg/ml) was added and the plates were incubated for 3 hours and cell viability was determined at the absorption of 590nm. 2.6 Colony formation assay
TE D
Colony formation was performed by seeding cells at the concentration of 2000 per well in the 6-wells plates. After culturing for 15 days, the colonies were fixed and stained with 0.1% crystal violet and counted.
EP
2.7 Immunohistochemical staining (IHC)
AC C
The immunohistochemical staining was performed as described previously [24]. Antibodies used in the study were as follows: anti-FOXK2 (ab50946, Abcam, Cambridge, MA, USA) and anti-SOX9 (#82630, Cell Signaling Technology, Cambridge, MA, USA). To quantify to level of FOXK2 and SOX9 expression, all sections were photographed by microscope (Carl Zeiss, Jena, Germany) and scored by two independent pathologists. The semi-quantification was performed according to the staining intensity (0, 0-15%, 1, 15-50%, 2, 50-70%, 3, above 70%) and the ratio of positive staining cells (0, low staining, 1, moderate staining, 2, high staining, 3, extremely high staining). The overall score was calculated by multiplying intensity score and percentage score. A cut-off of score ≥4 was defined as high expression. 2.8 Quantitative real-time PCR Total RNA was isolated form cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The first strand cDNA was synthesized using PrimeScript RT Master kit (Takara, Dalian, China). Real-time PCR was performed by SYBR Green methods on 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as described
ACCEPTED MANUSCRIPT previously. The primers used in the detection were as follows: FOXK2, forward, 5’-GGAGGCGTCTGAGTCTCCA-3’, reverse, 5’-CCCACCTTGTACCCTGAAGA-3’; SOX9, forward, 5’-CGGAGGAAGTCGGTGAAG-3’, reverse, 5’-CTGGGATTGCCCCGAGTGCT-3’; GAPDH, forward, 5’-CATGAGAAGTATGACAACAGCCT -3’, reverse, 5’-AGTCCTTCCACGATACCAAAGT -3’. The quantification of gene expression was normalized to GAPDH by 2-△△Ct method.
RI PT
2.9 Western blot
2.10 Dual luciferase reporter assay
M AN U
SC
Primary antibodies against FOXK2, SOX9 and GAPDH (Santa Cruz Biotechnology, sc-365062, Santa Cruz, CA, USA) were used in western blotting. Cell lysates were harvested in RIPA buffer (Thermo Scientific) containing the protease and phosphatase inhibitors cocktail. Total protein concentration was measured using BCA method. An equal amounts of protein samples (about 60µg) were loaded and separated by SDS-PAGE, transferred on to PVDF membranes, blocked with non-fat milk and incubated with primary antibodies at 4 overnight. After washing, the blots were incubated with secondary antibody and visualized by ECL methods.
TE D
The promoter sequences of FOXK2 were cloned and inserted into pGL3-basic vector (Promega, WI, USA). For luciferase assay, HEK293T cells were transiently transfected with indicated luciferase reporter plasmid, SOX9 or control plasmids. Renilla luciferase plasmid was co-transfected and used as normalized control. After 48 hours incubation, the firefly and Renilla luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instruction. 2.11 Chromatin immunoprecipitation (ChIP) analysis
AC C
EP
Chromatin was immunoprecipitationed with ChIP assay kit (Millopore, Billerica, MA, USA) according to the manufacturer’s recommendation. The antibody used in ChIP experiment was anti-SOX9 (ab3697, Abcam, Cambridge, MA, USA). The immunoprecipitated DNA was measured by PCR. Primer sequences used were as follows: forward, 5’-CTTGATTTAGATGGGATCTTGT-3’ and reverse, 5’-GAGGCAGGAGAATTGCTTAAAC-3’. 2.12 Statistical analysis
Data were analyzed using SPSS 21.0 software (IBM, Chicago, IL, USA) and presented by GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Correlation between FOXK2 expression and clinicopathological features was measured by Chi-squared test. Overall survival was assessed by Kaplan-Meier method with log-rank test. The correlation between protein expression levels was analyzed using Spearman correlation test. Data were presented as mean±SEM from at least three independent in vitro experiments. Student’s t test and ANOVA test were used to compare the results between groups. A p value less than 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1 FOXK2 is highly expressed in colorectal cancer tissues
3.2 FOXK2 promotes cell proliferation
M AN U
SC
RI PT
To elucidate the expression pattern of FOXK2 in colorectal cancer, we first explored the relative expression value of FOXK2 using Oncomine database (http://www.oncomine.com/). Oncomine database analysis of colorectal cancer tissues and normal tissues indicated that FOXK2 was significantly up-regulated in cancer tissues (Fig. 1A). The gene copy number variant and transcription deregulation are critical for tumor pathogenesis and progression. We therefore detected whether the alternation of gene copy number was involved in the up-regulation of FOXK2 in colorectal cancer. The results from cBioPortal database (http://www.cbioportal.org/) indicated that the increase of FOXK2 mRNA was mainly caused by the gain and amplification, indicating that DNA copy number was associated with the mRNA expression (Fig. 1B). Furthermore, we measured the FOXK2 expression using IHC in 145 cases of colorectal cancer. The results indicated that expression of FOXK2 was significantly higher in cancer tissues than that in adjacent non-cancer tissues (Supplementary Fig. 1). Of note was the fact that high level of FOXK2 was correlated with poor survival rate, indicting the prognostic value of FOXK2 (Fig. 1C).
EP
TE D
To further explore the biological function of FOXK2 in colorectal cancer cells, we detected the expression of FOXK2 in 6 cell lines (Supplementary Fig. 2). Based on the relative expression level of FOXK2, we performed gain and lost function analysis in HCT116, SW480 and SW620 cell lines (Fig. 2A). By MTT and colony formation assay, we found that knocking down of FOXK2 reduced the cell growth rate, while ectopic expression of FOXK2 promoted the cell proliferation (Fig. 2B-D). Also, ectopic expression of FOXK2 in SW620-sh-FOXK2 cells restored the cell viability. Taken together, these data suggested that FOXK2 promoted cell proliferation. 3.3 FOXK2 could be transcriptionally activated by SOX9 oncogene
AC C
Since we observed that FOXK2 was transcriptionally activated in colorectal cancer tissues, we analyzed the promoter of FOXK2 and found that oncogene SOX9 might be involved in the up-regulation of FOXK2. To further evaluate our hypothesis, we utilized luciferase reporter assay. As shown in Fig. 3A and B, the overexpression of SOX9 significantly increased that activity of the pGL3-P1 and -P2 promoter rather than -P3 or -P4 in 293T cells. The results indicated that SOX9 regulating site was ranged form -1500 bp to -1000 bp ahead of transcriptional start site (TSS). To determine the SOX9 binding sites location, we further evaluated the sequence of the promoter of FOXK2 and found that there were repeated SOX9 binding motif which could contribute to the transcriptional activation (Fig. 3C). Indeed, a ChIP assay was performed using SOX9 antibody to immunoprecipitate the endogenous SOX9 from HCT116 and SW620 cells. As shown in Fig. 3D, a clear enrichment of the binding sequences was detected. We ectopic expressed SOX9 in SW480 and DLD-1 cells and FOXK2 was subsequently elevated (Fig. 3E and F). In addition, knocking down of FOXK2 attenuated SOX9 induced cell growth (Fig. 3G and H). Taken together, these
ACCEPTED MANUSCRIPT data suggested that SOX9 could directly activate FOXK2 transcription. Depletion of FOXK2 profoundly suppressed SOX9 induced cancer cell proliferation. 3.4 FOXK2 and SOX9 expression are correlated in colorectal cancer tissues
M AN U
SC
RI PT
To investigate the correlation between FOXK2 and SOX9 expression in human colorectal cancer tissues, IHC staining of FOXK2 and SOX9 was analyzed in our cohort. We observed that both FOXK2 and SOX9 expression were coincidently enhanced in cancer tissues compared to non-cancer tissues (Fig. 4A). According to the IHC scores, SOX9 positive rate was approximately 63.4% (45/71) in FOXK2 positive group whereas 45.9% (34/74) in FOXK2 negative group (Spearmen correlation, p=0.035). Furthermore, we examined the GSE9348 as a validation cohort containing 12 normal tissues and 70 cancer tissues. As shown in Fig. 4C and D, FOXK2 and SOX9 were both significantly elevated in cancer tissues. Also, FOXK2 and SOX9 expression were significantly correlated (p=0.003). We then found that SOX9+FOXK2+ patients had a poorer survival rate than other patient (p=0.0084, Supplementary Fig. 3). Taken together, these data suggested that FOXK2 and SOX9 expression were correlated and indicated poor survival of colorectal cancer patients. 4. Discussion
AC C
EP
TE D
In the present study, by IHC staining, we observed FOXK2 expression was significantly enhanced in colorectal cancer tissues compared to that in normal tissues, which was coincident with previously reports [16]. Through exploring the GEO and TCGA database, we detected FOXK2 mRNA and genome expression pattern in colorectal cancer. FOXK2 was up-regulated in both mRNA level and protein level. Moreover, we analyzed the association of the expression of FOXK2 and prognosis information, which was studied for the first time. The data indicated that patients with high expression of FOXK2 had shorter survival, indicating the proto-oncogenic functions of FOXK2 in colorectal cancer. By in vitro experiments, we found that FOXK2 promotes cell growth, which was consistent with previous reports. Van der Heide et al demonstrated that FOXK2 was required for cellular growth and survival, partially by up-regulating Bcl-2 and inhibiting Puma, Noxa [15]. Also, depletion of FOXK2 could be restored by mTOR activation. These results indicated the potential mechanisms underlying FOXK2 induced cell growth. Moreover, FOXK2 activated Wnt signal by translocating DVL, an activator for stabilizing β-catenin, and conditional expression of FOXK2 promoted intestinal proliferation in mouse model [16]. However, the precise role of FOXK2 remains elusive and sometimes even controversial. For instance, FOXK2 could directly binds to the promoter of FOXO3a and interact with transcription corepressor complexes such as NCoR/SMRT, SIN3A, NuRD, and REST/CoREST to repress hypoxia response and sensitize cells to drug resistance in breast cancer [25, 26]. These data seems contrary with our observation and we thought the tumor heterogeneity and regulatory pattern might be the main reasons. Further investigation would focus on the FOXK2 regulating downstream signals in colorectal cancer. SOX9 was involved in regulating tumor chemoresistance by several signals [27-29]. We previously suggested that SOX9 participated with DNMT1 to predict chemoresistance of colorectal cancer [30]. Here, we explored the sequence of FOXK2 promoter and predicted the
ACCEPTED MANUSCRIPT
SC
Conflict of interest No potential conflicts of interest were disclosed.
RI PT
SOX9 binding site by Jaspar database [31]. We then constructed the different forms of luciferase reporter and found that the repeat ATATAGTTC motif was involved, followed by the assessment of ChIP assay. We indicated that SOX9 could transcriptionally activate FOXK2 and depletion of FOXK2 abrogated SOX9 induced cell growth. Further study would be performed to explore FOXK2 in colorectal cancer chemoresistance model. In conclusion, we showed in this study that FOXK2 was a target and transcriptionally activated by oncogene SOX9, and identified the clinical value of FOXK2 in colorectal cancer. The association of FOXK2 and SOX9 was showed both in our cohort and other published data source and FOXK2, SOX9 double positive patients had shorter survival. Therefore, SOX9-FOXK2 axis could be the potential target for colorectal cancer and further work is desirable, such as more investigation of the downstream of FOXK2 and the regulation mechanisms.
Appendix A. Supplementary data
M AN U
Acknowledgments We thank Dr. Jinlong Liu at Shanghai Institutes for Biological Science, Chinese Academy of Sciences for his generosity and advising in the experiments.
AC C
EP
TE D
References [1] R. Siegel, C. Desantis, A. Jemal, Colorectal cancer statistics, 2014, CA Cancer J Clin, 64 (2014) 104-117. [2] B. Nordlinger, H. Sorbye, B. Glimelius, G.J. Poston, P.M. Schlag, P. Rougier, W.O. Bechstein, J.N. Primrose, E.T. Walpole, M. Finch-Jones, D. Jaeck, D. Mirza, R.W. Parks, M. Mauer, E. Tanis, E. Van Cutsem, W. Scheithauer, T. Gruenberger, E.G.-I.T.C. Group, U.K. Cancer Research, O. Arbeitsgruppe Lebermetastasen und-tumoren in der Chirurgischen Arbeitsgemeinschaft, G. Australasian Gastro-Intestinal Trials, D. Federation Francophone de Cancerologie, Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial, The Lancet. Oncology, 14 (2013) 1208-1215. [3] D. Schrag, M.R. Weiser, K.A. Goodman, M. Gonen, E. Hollywood, A. Cercek, D.L. Reidy-Lagunes, M.J. Gollub, J. Shia, J.G. Guillem, L.K. Temple, P.B. Paty, L.B. Saltz, Neoadjuvant chemotherapy without routine use of radiation therapy for patients with locally advanced rectal cancer: a pilot trial, J Clin Oncol, 32 (2014) 513-518. [4] Y. Toiyama, K. Hur, K. Tanaka, Y. Inoue, M. Kusunoki, C.R. Boland, A. Goel, Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer, Ann Surg, 259 (2014) 735-743. [5] S. Misale, R. Yaeger, S. Hobor, E. Scala, M. Janakiraman, D. Liska, E. Valtorta, R. Schiavo, M. Buscarino, G. Siravegna, K. Bencardino, A. Cercek, C.T. Chen, S. Veronese, C. Zanon, A. Sartore-Bianchi, M. Gambacorta, M. Gallicchio, E. Vakiani, V. Boscaro, E. Medico, M. Weiser, S. Siena, F. Di Nicolantonio, D. Solit, A. Bardelli, Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer, Nature, 486 (2012) 532-536.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[6] T. Cooks, I.S. Pateras, O. Tarcic, H. Solomon, A.J. Schetter, S. Wilder, G. Lozano, E. Pikarsky, T. Forshew, N. Rosenfeld, N. Harpaz, S. Itzkowitz, C.C. Harris, V. Rotter, V.G. Gorgoulis, M. Oren, Mutant p53 prolongs NF-kappaB activation and promotes chronic inflammation and inflammation-associated colorectal cancer, Cancer Cell, 23 (2013) 634-646. [7] T. Fernandez-Marcelo, A. Sanchez-Pernaute, I. Pascua, C. De Juan, J. Head, A.J. Torres-Garcia, P. Iniesta, Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer, PLoS One, 11 (2016) e0149626. [8] J.R. Friedman, K.H. Kaestner, The Foxa family of transcription factors in development and metabolism, Cellular and molecular life sciences : CMLS, 63 (2006) 2317-2328. [9] S. Nakagawa, S.S. Gisselbrecht, J.M. Rogers, D.L. Hartl, M.L. Bulyk, DNA-binding specificity changes in the evolution of forkhead transcription factors, Proc Natl Acad Sci U S A, 110 (2013) 12349-12354. [10] A. Marais, Z. Ji, E.S. Child, E. Krause, D.J. Mann, A.D. Sharrocks, Cell cycle-dependent regulation of the forkhead transcription factor FOXK2 by CDK.cyclin complexes, The Journal of biological chemistry, 285 (2010) 35728-35739. [11] Z. Ji, I.J. Donaldson, J. Liu, A. Hayes, L.A. Zeef, A.D. Sharrocks, The forkhead transcription factor FOXK2 promotes AP-1-mediated transcriptional regulation, Mol Cell Biol, 32 (2012) 385-398. [12] Y. Okino, Y. Machida, S. Frankland-Searby, Y.J. Machida, BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes the ubiquitin-mediated activation of FoxK2 target genes, The Journal of biological chemistry, 290 (2015) 1580-1591. [13] Z. Ji, H. Mohammed, A. Webber, J. Ridsdale, N. Han, J.S. Carroll, A.D. Sharrocks, The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex, Nucleic Acids Res, 42 (2014) 6232-6242. [14] Y. Liu, X. Ao, Z. Jia, X.Y. Bai, Z. Xu, G. Hu, X. Jiang, M. Chen, H. Wu, FOXK2 transcription factor suppresses ERalpha-positive breast cancer cell growth through down-regulating the stability of ERalpha via mechanism involving BRCA1/BARD1, Scientific reports, 5 (2015) 8796. [15] L.P. van der Heide, P.J. Wijchers, L. von Oerthel, J.P. Burbach, M.F. Hoekman, M.P. Smidt, FoxK2 is required for cellular proliferation and survival, J Cell Physiol, 230 (2015) 1013-1023. [16] W. Wang, X. Li, M. Lee, S. Jun, K.E. Aziz, L. Feng, M.K. Tran, N. Li, P.D. McCrea, J.I. Park, J. Chen, FOXKs promote Wnt/beta-catenin signaling by translocating DVL into the nucleus, Dev Cell, 32 (2015) 707-718. [17] N. Liu, L. Zhang, Z. Wang, Y. Cheng, P. Zhang, X. Wang, W. Wen, H. Yang, H. Liu, W. Jin, Y. Zhang, Y. Tu, MicroRNA-101 inhibits proliferation, migration and invasion of human glioblastoma by targeting SOX9, Oncotarget, (2016). [18] C.H. Zhou, L.P. Ye, S.X. Ye, Y. Li, X.Y. Zhang, X.Y. Xu, L.Y. Gong, Clinical significance of SOX9 in human non-small cell lung cancer progression and overall patient survival, Journal of experimental & clinical cancer research : CR, 31 (2012) 18. [19] C. Liu, L. Liu, X. Chen, J. Cheng, H. Zhang, J. Shen, J. Shan, Y. Xu, Z. Yang, M. Lai, C. Qian, Sox9 regulates self-renewal and tumorigenicity by promoting symmetrical cell division of cancer stem cells in hepatocellular carcinoma, Hepatology, 64 (2016) 117-129. [20] H. Liu, Z. Liu, B. Jiang, R. Peng, Z. Ma, J. Lu, SOX9 Overexpression Promotes Glioma Metastasis via Wnt/beta-Catenin Signaling, Cell Biochem Biophys, 73 (2015) 205-212.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[21] L. Sun, L.A. Mathews, S.M. Cabarcas, X. Zhang, A. Yang, Y. Zhang, M.R. Young, K.D. Klarmann, J.R. Keller, W.L. Farrar, Epigenetic regulation of SOX9 by the NF-kappaB signaling pathway in pancreatic cancer stem cells, Stem Cells, 31 (2013) 1454-1466. [22] S. Luanpitpong, J. Li, A. Manke, K. Brundage, E. Ellis, S.L. McLaughlin, P. Angsutararux, N. Chanthra, M. Voronkova, Y.C. Chen, L. Wang, P. Chanvorachote, M. Pei, S. Issaragrisil, Y. Rojanasakul, SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma, Oncogene, 35 (2016) 2824-2833. [23] G. Wang, A. Lunardi, J. Zhang, Z. Chen, U. Ala, K.A. Webster, Y. Tay, E. Gonzalez-Billalabeitia, A. Egia, D.R. Shaffer, B. Carver, X.S. Liu, R. Taulli, W.P. Kuo, C. Nardella, S. Signoretti, C. Cordon-Cardo, W.L. Gerald, P.P. Pandolfi, Zbtb7a suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion, Nat Genet, 45 (2013) 739-746. [24] J. Jiang, C. Yu, M. Chen, S. Tian, C. Sun, Over-expression of TRIM37 promotes cell migration and metastasis in hepatocellular carcinoma by activating Wnt/beta-catenin signaling, Biochem Biophys Res Commun, 464 (2015) 1120-1127. [25] G. Nestal de Moraes, P. Khongkow, C. Gong, S. Yao, A.R. Gomes, Z. Ji, N. Kandola, D. Delbue, E.P. Man, U.S. Khoo, A.D. Sharrocks, E.W. Lam, Forkhead box K2 modulates epirubicin and paclitaxel sensitivity through FOXO3a in breast cancer, Oncogenesis, 4 (2015) e167. [26] L. Shan, X. Zhou, X. Liu, Y. Wang, D. Su, Y. Hou, N. Yu, C. Yang, B. Liu, J. Gao, Y. Duan, J. Yang, W. Li, J. Liang, L. Sun, K. Chen, C. Xuan, L. Shi, Y. Wang, Y. Shang, FOXK2 Elicits Massive Transcription Repression and Suppresses the Hypoxic Response and Breast Cancer Carcinogenesis, Cancer Cell, 30 (2016) 708-722. [27] P. Camaj, C. Jackel, S. Krebs, E.N. De Toni, H. Blum, K.W. Jauch, P.J. Nelson, C.J. Bruns, Hypoxia-independent gene expression mediated by SOX9 promotes aggressive pancreatic tumor biology, Molecular cancer research : MCR, 12 (2014) 421-432. [28] L. Garros-Regulez, P. Aldaz, O. Arrizabalaga, V. Moncho-Amor, E. Carrasco-Garcia, L. Manterola, L. Moreno-Cugnon, C. Barrena, J. Villanua, I. Ruiz, S. Pollard, R. Lovell-Badge, N. Sampron, I. Garcia, A. Matheu, mTOR inhibition decreases SOX2-SOX9 mediated glioma stem cell activity and temozolomide resistance, Expert Opin Ther Targets, 20 (2016) 393-405. [29] Y. Xie, W. Lu, S. Liu, Q. Yang, B.S. Carver, E. Li, Y. Wang, L. Fazli, M. Gleave, Z. Chen, Crosstalk between nuclear MET and SOX9/beta-catenin correlates with castration-resistant prostate cancer, Molecular endocrinology (Baltimore, Md.), 28 (2014) 1629-1639. [30] Z.Y. Suhua Xia, Xiaowei Qi, Yong Pu, Yankui Liu, Boshi Wang, Yun Liu, Li Zhang, Yu Qian, Aihui Ma, Guiqin Xu, Hong Tu, Yongzhong Liu, Overexpression of SOX9 and DNMT1 predicts poor prognosis and chemoresistance of colorectal cancer, Int J Clin Exp Pathol, 9 (2016) 589-600. [31] A. Mathelier, O. Fornes, D.J. Arenillas, C.Y. Chen, G. Denay, J. Lee, W. Shi, C. Shyr, G. Tan, R. Worsley-Hunt, A.W. Zhang, F. Parcy, B. Lenhard, A. Sandelin, W.W. Wasserman, JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles, Nucleic Acids Res, 44 (2016) D110-115. Legends Fig. 1. FOXK2 is overexpressed in colorectal cancer. A. FOXK2 mRNA expression in colorectal cancer tissues was explored in the Oncomine database. The box plot represents the FOXK2
ACCEPTED MANUSCRIPT expression in colorectal cancer tissues and normal tissues. B. The correlation between gene copy number and mRNA expression of FOXK2 in colorectal cancer was identified from cBioPortal database. C. The overall survival from our cohort containing 145 colorectal cancer samples was examined by Kaplan-Meier analysis.
RI PT
Fig. 2. FOXK2 promotes cellular proliferation. A. FOXK2 overexpression, knockdown and reversed expression were examined by Western blot. Cell proliferation was measured by MTT assay (B) and colony formation assay (C) in aforementioned cells. D. Statistical analysis of colony formation assay. *, p<0.05, **, p<0.01.
M AN U
SC
Fig. 3. FOXK2 is transcriptionally activated by SOX9. A. Scheme of the FOXK2 promoter luciferase vector construction. B. Relative luciferase activity assays of luciferase reporters with different length of FOXK2 promoter were performed after co-transfection with SOX9 or control. The Renilla luciferase vector was co-transfected as an internal control. C. Putative SOX9–binding site in FOXK2 promoter were shown. D. HCT116 and SW620 cells were subjected to ChIP assay using SOX9 antibody and isolated DNA was employed to PCR by specific promoter primers. E. Overexpression of SOX9 was examined in SW480 and DLD-1 cells. F. FOXK2 expression was examined after SOX9 overexpression. G. FOXK2 expression was examined after SOX9 overexpression followed by knocking down FOXK2. H. Colony formation assay was performed in cells after SOX9 overexpression followed by knocking down FOXK2. *, p<0.05, **, p<0.01.
AC C
EP
TE D
Fig. 4. FOXK2 expression is associated with SOX9 in colorectal cancer tissues. A. Representative images of FOXK2 and SOX9 expression in colorectal cancer tissues (upper panel, low expression of FOXK2 and SOX9, lower panel, high expression of FOXK2 and SOX9, bar, 100µm). B. The statistical analysis of FOXK2 and SOX9 expression in 145 cases of colorectal cancer tissues (Spearman correlation test). The relative expression of FOXK2 (C) and SOX9 (D) in GSE9348 dataset. E. The correlation test of FOXK2 and SOX9 in GSE9348 dataset.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Highlights FOXK2 is up-regulated in colorectal cancer and associated with poor prognosis. FOXK2 promotes proliferation of CRC cells. FOXK2 is transcriptional activated by SOX9. FOXK2 expression is correlated with SOX9 in CRC tissues.
S0006-291X(16)32178-7
DOI:
10.1016/j.bbrc.2016.12.119
Reference:
YBBRC 36993
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 13 December 2016 Accepted Date: 18 December 2016
Please cite this article as: Y. Qian, S. Xia, Z. Feng, Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2016.12.119. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation Yu Qian1*, Suhua Xia2, Zhenyu Feng3
RI PT
1 Translational Medicine Research Center, Shanxi Medical University, Taiyuan, Shanxi 030001, China 2 Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, China 3 Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu, China
SC
Correspondence to: Dr. Yu Qian, Translational Medicine Research Center, Shanxi Medical University, No.56 South Xinjian Street, Taiyuan, Shanxi 030001, China. Tel: +86-0351-4135676; E-mail: [email protected]
EP
TE D
M AN U
Abstract FOXK2, which belongs to the fork head DNA binding protein family, has been shown to play a critical role in tumorigenesis. Here, we detected FOXK2 expression and its clinical significance in colorectal cancer, which has not been fully investigated before. Results from public database and our cohort indicated that FOXK2 was transcriptionally activated in colorectal cancer tissues compared to non-cancer tissues. High expression of FOXK2 was significantly correlated with poor survival. In vitro cell experiments suggested that FOXK2 promoted cell proliferation. Furthermore, we found that oncogene SOX9 was responsible for the up-regulation of FOXK2 by directly binding on its promoter. Depletion of FOXK2 attenuated SOX9 induced cell growth. In addition, we observed that the expression of FOXK2 was significantly associated with the expression of SOX9 both in the public database and our colorectal cancer tissues. The patients with SOX9+FOXK2+ had a poor overall survival (p=0.0084). In conclusion, our data suggested that SOX9 transcriptionally activated FOXK2 was involved in the pathogenesis of colorectal cancer and might be a novel target for colorectal cancer therapy.
AC C
Keywords: FOXK2, SOX9, colorectal cancer 1. Introduction
Colorectal cancer is one of the most common malignancies worldwide with highly mortality [1]. Surgery combined with chemotherapy is the main care for colorectal cancer [2, 3]. However, due to the frequent metastasis and chemo resistance, the prognosis of advanced colorectal cancer is still unfavorable [4]. The mechanisms underlying the pathogenesis and development of colorectal cancer remain poorly unknown. Genome alterations, including copy-number alterations (CNA), mutants and gene fusion, are identified as the hallmarks of cancer. Previous studies have demonstrated that oncogenes and tumor suppressor genes deregulation played critical roles in colorectal cancer progression, such as KRAS mutant, TP53 mutant and TERT amplification [5-7]. FOXK2, as known as ILF (interleukin enhancer binding factor), belongs to the Forkhead box (FOX) transcription factors which have been demonstrated to control a wide rage of cellular
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
RI PT
processes, such as DNA repair, cell cycle and survival [8, 9]. FOXK2 could be phosphorylated by CDKs during cell division and colocalized with AP-1, promoting AP-1 mediated transcriptional activation [10, 11]. Previous study indicated that FOXK2 interacted with deubiquitinase BAP1 and recruited BAP1 to DNA which promoted local histone deubiquitation and caused changes in target genes [12, 13]. The functions of FOXK2 in cancers remain to be further investigated. FOXK2 binds to BRCA1/BARD1 and enhances the degradation of ERα in breast cancer, leading to cellular suppression [14]. It also promotes cell survival and activates Wnt signal by promoting DVL nuclear translocation in colorectal cancer [15, 16]. However, the clinical significance and prognostic value of FOXK2 in colorectal cancer are still largely unknown. Sex-determining region Y box 9 (SOX9) is determined as oncogene, which are activated in stem cells and critical for cancer progression. Recent studies have reported that uncontrolled expression of SOX9 in several types of cancers, such as colorectal cancer, pancreatic cancer and glioma and lung cancer [17, 18]. More importantly, the up-regulation of SOX9 indicates poor prognosis in these cancers. SOX9 has been shown to induce self-renewal of cancer stem cells and inhibit Notch activity in hepatocellular carcinoma and to regulate metastasis by activating Wnt/β-catenin signal [19, 20]. Also, SOX9 could be regulated by several critical factors such as epigenetically activated by NF-κB signals, stabilized by SLUG and suppressed by ZBTB7A [21-23]. However, the mechanisms underlying SOX9 mediated tumorigenesis remain elusive. In the present study, we showed that FOXK2 expression was significantly elevated in colorectal cancer tissues and correlated with poor prognosis. Cellular experiments indicated that FOXK2 promoted cell growth and could be transcriptionally activated by SOX9. The coincident up-regulation of FOXK2 and SOX9 in colorectal cancer tissue indicated the pathological association of SOX9-FOXK2 axis in the progression of colorectal cancer. 2. Materials and methods
2.1 Ethics statements and tissue samples
AC C
EP
The study was approved by the Ethics Committee of the first Affiliated Hospital of Soochow University. A total of 145 colorectal cancer samples were collected from the Department of Pathology, the first and second Affiliated Hospital of Soochow University from February 2004 to January 2009. Written informed consent was obtained form all patients. None of the patients had received chemotherapy, radiotherapy or related therapies before surgery and their complete clinical data including follow-up information was available and reviewed. 2.2 Cell culture
Human colorectal cancer cell lines HCT116, SW480, SW620, DLD-1, LOVO, COLO205 and HEK293T cell line were obtained from American Type Culture Collection (ATCC) and Cell Bank at the Chinese Academy of Sciences. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS, 100 U/ml penicillin and 100mg/ml streptomycin. All cells were cultured at 37 with humidified atmosphere containing 5% CO2. 2.3 Lentivirus infection
ACCEPTED MANUSCRIPT The lentiviral for ectopic expression and knockdown of FOXK2 were obtained form Hanbio Co., Ltd (Hanbio, Shanghai, China). For establishing stable overexpression or knockdown cells, lentivirus was added to infect cells, followed by the positive selection using puromycin (Sigma, 5µg/ml).
RI PT
2.4 Oncomine and cBioPortal databases analysis
SC
The mRNA expression of FOXK2 in colorectal cancer was identified from Oncomine database as circumscribed in the criteria of p value less than 10-4 and fold change more than 2. The top 10% ranking lists were shown (https://www.oncomine.org/resource/login.html). The FOXK2 mRNA expression was identified from Kaiser’s colon dataset. The correlation between DNA copy number and mRNA expression of FOXK2 in colorectal cancer was identified by cBioPortal dababase (http://www.cbioportal.org/)
M AN U
2.5 MTT assay
The cell proliferation assay was performed by MTT assay. Indicated cells were seeded into the 96-well plates at the density of 2000/well. 20µl of MTT reagent (5mg/ml) was added and the plates were incubated for 3 hours and cell viability was determined at the absorption of 590nm. 2.6 Colony formation assay
TE D
Colony formation was performed by seeding cells at the concentration of 2000 per well in the 6-wells plates. After culturing for 15 days, the colonies were fixed and stained with 0.1% crystal violet and counted.
EP
2.7 Immunohistochemical staining (IHC)
AC C
The immunohistochemical staining was performed as described previously [24]. Antibodies used in the study were as follows: anti-FOXK2 (ab50946, Abcam, Cambridge, MA, USA) and anti-SOX9 (#82630, Cell Signaling Technology, Cambridge, MA, USA). To quantify to level of FOXK2 and SOX9 expression, all sections were photographed by microscope (Carl Zeiss, Jena, Germany) and scored by two independent pathologists. The semi-quantification was performed according to the staining intensity (0, 0-15%, 1, 15-50%, 2, 50-70%, 3, above 70%) and the ratio of positive staining cells (0, low staining, 1, moderate staining, 2, high staining, 3, extremely high staining). The overall score was calculated by multiplying intensity score and percentage score. A cut-off of score ≥4 was defined as high expression. 2.8 Quantitative real-time PCR Total RNA was isolated form cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The first strand cDNA was synthesized using PrimeScript RT Master kit (Takara, Dalian, China). Real-time PCR was performed by SYBR Green methods on 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as described
ACCEPTED MANUSCRIPT previously. The primers used in the detection were as follows: FOXK2, forward, 5’-GGAGGCGTCTGAGTCTCCA-3’, reverse, 5’-CCCACCTTGTACCCTGAAGA-3’; SOX9, forward, 5’-CGGAGGAAGTCGGTGAAG-3’, reverse, 5’-CTGGGATTGCCCCGAGTGCT-3’; GAPDH, forward, 5’-CATGAGAAGTATGACAACAGCCT -3’, reverse, 5’-AGTCCTTCCACGATACCAAAGT -3’. The quantification of gene expression was normalized to GAPDH by 2-△△Ct method.
RI PT
2.9 Western blot
2.10 Dual luciferase reporter assay
M AN U
SC
Primary antibodies against FOXK2, SOX9 and GAPDH (Santa Cruz Biotechnology, sc-365062, Santa Cruz, CA, USA) were used in western blotting. Cell lysates were harvested in RIPA buffer (Thermo Scientific) containing the protease and phosphatase inhibitors cocktail. Total protein concentration was measured using BCA method. An equal amounts of protein samples (about 60µg) were loaded and separated by SDS-PAGE, transferred on to PVDF membranes, blocked with non-fat milk and incubated with primary antibodies at 4 overnight. After washing, the blots were incubated with secondary antibody and visualized by ECL methods.
TE D
The promoter sequences of FOXK2 were cloned and inserted into pGL3-basic vector (Promega, WI, USA). For luciferase assay, HEK293T cells were transiently transfected with indicated luciferase reporter plasmid, SOX9 or control plasmids. Renilla luciferase plasmid was co-transfected and used as normalized control. After 48 hours incubation, the firefly and Renilla luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instruction. 2.11 Chromatin immunoprecipitation (ChIP) analysis
AC C
EP
Chromatin was immunoprecipitationed with ChIP assay kit (Millopore, Billerica, MA, USA) according to the manufacturer’s recommendation. The antibody used in ChIP experiment was anti-SOX9 (ab3697, Abcam, Cambridge, MA, USA). The immunoprecipitated DNA was measured by PCR. Primer sequences used were as follows: forward, 5’-CTTGATTTAGATGGGATCTTGT-3’ and reverse, 5’-GAGGCAGGAGAATTGCTTAAAC-3’. 2.12 Statistical analysis
Data were analyzed using SPSS 21.0 software (IBM, Chicago, IL, USA) and presented by GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Correlation between FOXK2 expression and clinicopathological features was measured by Chi-squared test. Overall survival was assessed by Kaplan-Meier method with log-rank test. The correlation between protein expression levels was analyzed using Spearman correlation test. Data were presented as mean±SEM from at least three independent in vitro experiments. Student’s t test and ANOVA test were used to compare the results between groups. A p value less than 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1 FOXK2 is highly expressed in colorectal cancer tissues
3.2 FOXK2 promotes cell proliferation
M AN U
SC
RI PT
To elucidate the expression pattern of FOXK2 in colorectal cancer, we first explored the relative expression value of FOXK2 using Oncomine database (http://www.oncomine.com/). Oncomine database analysis of colorectal cancer tissues and normal tissues indicated that FOXK2 was significantly up-regulated in cancer tissues (Fig. 1A). The gene copy number variant and transcription deregulation are critical for tumor pathogenesis and progression. We therefore detected whether the alternation of gene copy number was involved in the up-regulation of FOXK2 in colorectal cancer. The results from cBioPortal database (http://www.cbioportal.org/) indicated that the increase of FOXK2 mRNA was mainly caused by the gain and amplification, indicating that DNA copy number was associated with the mRNA expression (Fig. 1B). Furthermore, we measured the FOXK2 expression using IHC in 145 cases of colorectal cancer. The results indicated that expression of FOXK2 was significantly higher in cancer tissues than that in adjacent non-cancer tissues (Supplementary Fig. 1). Of note was the fact that high level of FOXK2 was correlated with poor survival rate, indicting the prognostic value of FOXK2 (Fig. 1C).
EP
TE D
To further explore the biological function of FOXK2 in colorectal cancer cells, we detected the expression of FOXK2 in 6 cell lines (Supplementary Fig. 2). Based on the relative expression level of FOXK2, we performed gain and lost function analysis in HCT116, SW480 and SW620 cell lines (Fig. 2A). By MTT and colony formation assay, we found that knocking down of FOXK2 reduced the cell growth rate, while ectopic expression of FOXK2 promoted the cell proliferation (Fig. 2B-D). Also, ectopic expression of FOXK2 in SW620-sh-FOXK2 cells restored the cell viability. Taken together, these data suggested that FOXK2 promoted cell proliferation. 3.3 FOXK2 could be transcriptionally activated by SOX9 oncogene
AC C
Since we observed that FOXK2 was transcriptionally activated in colorectal cancer tissues, we analyzed the promoter of FOXK2 and found that oncogene SOX9 might be involved in the up-regulation of FOXK2. To further evaluate our hypothesis, we utilized luciferase reporter assay. As shown in Fig. 3A and B, the overexpression of SOX9 significantly increased that activity of the pGL3-P1 and -P2 promoter rather than -P3 or -P4 in 293T cells. The results indicated that SOX9 regulating site was ranged form -1500 bp to -1000 bp ahead of transcriptional start site (TSS). To determine the SOX9 binding sites location, we further evaluated the sequence of the promoter of FOXK2 and found that there were repeated SOX9 binding motif which could contribute to the transcriptional activation (Fig. 3C). Indeed, a ChIP assay was performed using SOX9 antibody to immunoprecipitate the endogenous SOX9 from HCT116 and SW620 cells. As shown in Fig. 3D, a clear enrichment of the binding sequences was detected. We ectopic expressed SOX9 in SW480 and DLD-1 cells and FOXK2 was subsequently elevated (Fig. 3E and F). In addition, knocking down of FOXK2 attenuated SOX9 induced cell growth (Fig. 3G and H). Taken together, these
ACCEPTED MANUSCRIPT data suggested that SOX9 could directly activate FOXK2 transcription. Depletion of FOXK2 profoundly suppressed SOX9 induced cancer cell proliferation. 3.4 FOXK2 and SOX9 expression are correlated in colorectal cancer tissues
M AN U
SC
RI PT
To investigate the correlation between FOXK2 and SOX9 expression in human colorectal cancer tissues, IHC staining of FOXK2 and SOX9 was analyzed in our cohort. We observed that both FOXK2 and SOX9 expression were coincidently enhanced in cancer tissues compared to non-cancer tissues (Fig. 4A). According to the IHC scores, SOX9 positive rate was approximately 63.4% (45/71) in FOXK2 positive group whereas 45.9% (34/74) in FOXK2 negative group (Spearmen correlation, p=0.035). Furthermore, we examined the GSE9348 as a validation cohort containing 12 normal tissues and 70 cancer tissues. As shown in Fig. 4C and D, FOXK2 and SOX9 were both significantly elevated in cancer tissues. Also, FOXK2 and SOX9 expression were significantly correlated (p=0.003). We then found that SOX9+FOXK2+ patients had a poorer survival rate than other patient (p=0.0084, Supplementary Fig. 3). Taken together, these data suggested that FOXK2 and SOX9 expression were correlated and indicated poor survival of colorectal cancer patients. 4. Discussion
AC C
EP
TE D
In the present study, by IHC staining, we observed FOXK2 expression was significantly enhanced in colorectal cancer tissues compared to that in normal tissues, which was coincident with previously reports [16]. Through exploring the GEO and TCGA database, we detected FOXK2 mRNA and genome expression pattern in colorectal cancer. FOXK2 was up-regulated in both mRNA level and protein level. Moreover, we analyzed the association of the expression of FOXK2 and prognosis information, which was studied for the first time. The data indicated that patients with high expression of FOXK2 had shorter survival, indicating the proto-oncogenic functions of FOXK2 in colorectal cancer. By in vitro experiments, we found that FOXK2 promotes cell growth, which was consistent with previous reports. Van der Heide et al demonstrated that FOXK2 was required for cellular growth and survival, partially by up-regulating Bcl-2 and inhibiting Puma, Noxa [15]. Also, depletion of FOXK2 could be restored by mTOR activation. These results indicated the potential mechanisms underlying FOXK2 induced cell growth. Moreover, FOXK2 activated Wnt signal by translocating DVL, an activator for stabilizing β-catenin, and conditional expression of FOXK2 promoted intestinal proliferation in mouse model [16]. However, the precise role of FOXK2 remains elusive and sometimes even controversial. For instance, FOXK2 could directly binds to the promoter of FOXO3a and interact with transcription corepressor complexes such as NCoR/SMRT, SIN3A, NuRD, and REST/CoREST to repress hypoxia response and sensitize cells to drug resistance in breast cancer [25, 26]. These data seems contrary with our observation and we thought the tumor heterogeneity and regulatory pattern might be the main reasons. Further investigation would focus on the FOXK2 regulating downstream signals in colorectal cancer. SOX9 was involved in regulating tumor chemoresistance by several signals [27-29]. We previously suggested that SOX9 participated with DNMT1 to predict chemoresistance of colorectal cancer [30]. Here, we explored the sequence of FOXK2 promoter and predicted the
ACCEPTED MANUSCRIPT
SC
Conflict of interest No potential conflicts of interest were disclosed.
RI PT
SOX9 binding site by Jaspar database [31]. We then constructed the different forms of luciferase reporter and found that the repeat ATATAGTTC motif was involved, followed by the assessment of ChIP assay. We indicated that SOX9 could transcriptionally activate FOXK2 and depletion of FOXK2 abrogated SOX9 induced cell growth. Further study would be performed to explore FOXK2 in colorectal cancer chemoresistance model. In conclusion, we showed in this study that FOXK2 was a target and transcriptionally activated by oncogene SOX9, and identified the clinical value of FOXK2 in colorectal cancer. The association of FOXK2 and SOX9 was showed both in our cohort and other published data source and FOXK2, SOX9 double positive patients had shorter survival. Therefore, SOX9-FOXK2 axis could be the potential target for colorectal cancer and further work is desirable, such as more investigation of the downstream of FOXK2 and the regulation mechanisms.
Appendix A. Supplementary data
M AN U
Acknowledgments We thank Dr. Jinlong Liu at Shanghai Institutes for Biological Science, Chinese Academy of Sciences for his generosity and advising in the experiments.
AC C
EP
TE D
References [1] R. Siegel, C. Desantis, A. Jemal, Colorectal cancer statistics, 2014, CA Cancer J Clin, 64 (2014) 104-117. [2] B. Nordlinger, H. Sorbye, B. Glimelius, G.J. Poston, P.M. Schlag, P. Rougier, W.O. Bechstein, J.N. Primrose, E.T. Walpole, M. Finch-Jones, D. Jaeck, D. Mirza, R.W. Parks, M. Mauer, E. Tanis, E. Van Cutsem, W. Scheithauer, T. Gruenberger, E.G.-I.T.C. Group, U.K. Cancer Research, O. Arbeitsgruppe Lebermetastasen und-tumoren in der Chirurgischen Arbeitsgemeinschaft, G. Australasian Gastro-Intestinal Trials, D. Federation Francophone de Cancerologie, Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial, The Lancet. Oncology, 14 (2013) 1208-1215. [3] D. Schrag, M.R. Weiser, K.A. Goodman, M. Gonen, E. Hollywood, A. Cercek, D.L. Reidy-Lagunes, M.J. Gollub, J. Shia, J.G. Guillem, L.K. Temple, P.B. Paty, L.B. Saltz, Neoadjuvant chemotherapy without routine use of radiation therapy for patients with locally advanced rectal cancer: a pilot trial, J Clin Oncol, 32 (2014) 513-518. [4] Y. Toiyama, K. Hur, K. Tanaka, Y. Inoue, M. Kusunoki, C.R. Boland, A. Goel, Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer, Ann Surg, 259 (2014) 735-743. [5] S. Misale, R. Yaeger, S. Hobor, E. Scala, M. Janakiraman, D. Liska, E. Valtorta, R. Schiavo, M. Buscarino, G. Siravegna, K. Bencardino, A. Cercek, C.T. Chen, S. Veronese, C. Zanon, A. Sartore-Bianchi, M. Gambacorta, M. Gallicchio, E. Vakiani, V. Boscaro, E. Medico, M. Weiser, S. Siena, F. Di Nicolantonio, D. Solit, A. Bardelli, Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer, Nature, 486 (2012) 532-536.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[6] T. Cooks, I.S. Pateras, O. Tarcic, H. Solomon, A.J. Schetter, S. Wilder, G. Lozano, E. Pikarsky, T. Forshew, N. Rosenfeld, N. Harpaz, S. Itzkowitz, C.C. Harris, V. Rotter, V.G. Gorgoulis, M. Oren, Mutant p53 prolongs NF-kappaB activation and promotes chronic inflammation and inflammation-associated colorectal cancer, Cancer Cell, 23 (2013) 634-646. [7] T. Fernandez-Marcelo, A. Sanchez-Pernaute, I. Pascua, C. De Juan, J. Head, A.J. Torres-Garcia, P. Iniesta, Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer, PLoS One, 11 (2016) e0149626. [8] J.R. Friedman, K.H. Kaestner, The Foxa family of transcription factors in development and metabolism, Cellular and molecular life sciences : CMLS, 63 (2006) 2317-2328. [9] S. Nakagawa, S.S. Gisselbrecht, J.M. Rogers, D.L. Hartl, M.L. Bulyk, DNA-binding specificity changes in the evolution of forkhead transcription factors, Proc Natl Acad Sci U S A, 110 (2013) 12349-12354. [10] A. Marais, Z. Ji, E.S. Child, E. Krause, D.J. Mann, A.D. Sharrocks, Cell cycle-dependent regulation of the forkhead transcription factor FOXK2 by CDK.cyclin complexes, The Journal of biological chemistry, 285 (2010) 35728-35739. [11] Z. Ji, I.J. Donaldson, J. Liu, A. Hayes, L.A. Zeef, A.D. Sharrocks, The forkhead transcription factor FOXK2 promotes AP-1-mediated transcriptional regulation, Mol Cell Biol, 32 (2012) 385-398. [12] Y. Okino, Y. Machida, S. Frankland-Searby, Y.J. Machida, BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes the ubiquitin-mediated activation of FoxK2 target genes, The Journal of biological chemistry, 290 (2015) 1580-1591. [13] Z. Ji, H. Mohammed, A. Webber, J. Ridsdale, N. Han, J.S. Carroll, A.D. Sharrocks, The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex, Nucleic Acids Res, 42 (2014) 6232-6242. [14] Y. Liu, X. Ao, Z. Jia, X.Y. Bai, Z. Xu, G. Hu, X. Jiang, M. Chen, H. Wu, FOXK2 transcription factor suppresses ERalpha-positive breast cancer cell growth through down-regulating the stability of ERalpha via mechanism involving BRCA1/BARD1, Scientific reports, 5 (2015) 8796. [15] L.P. van der Heide, P.J. Wijchers, L. von Oerthel, J.P. Burbach, M.F. Hoekman, M.P. Smidt, FoxK2 is required for cellular proliferation and survival, J Cell Physiol, 230 (2015) 1013-1023. [16] W. Wang, X. Li, M. Lee, S. Jun, K.E. Aziz, L. Feng, M.K. Tran, N. Li, P.D. McCrea, J.I. Park, J. Chen, FOXKs promote Wnt/beta-catenin signaling by translocating DVL into the nucleus, Dev Cell, 32 (2015) 707-718. [17] N. Liu, L. Zhang, Z. Wang, Y. Cheng, P. Zhang, X. Wang, W. Wen, H. Yang, H. Liu, W. Jin, Y. Zhang, Y. Tu, MicroRNA-101 inhibits proliferation, migration and invasion of human glioblastoma by targeting SOX9, Oncotarget, (2016). [18] C.H. Zhou, L.P. Ye, S.X. Ye, Y. Li, X.Y. Zhang, X.Y. Xu, L.Y. Gong, Clinical significance of SOX9 in human non-small cell lung cancer progression and overall patient survival, Journal of experimental & clinical cancer research : CR, 31 (2012) 18. [19] C. Liu, L. Liu, X. Chen, J. Cheng, H. Zhang, J. Shen, J. Shan, Y. Xu, Z. Yang, M. Lai, C. Qian, Sox9 regulates self-renewal and tumorigenicity by promoting symmetrical cell division of cancer stem cells in hepatocellular carcinoma, Hepatology, 64 (2016) 117-129. [20] H. Liu, Z. Liu, B. Jiang, R. Peng, Z. Ma, J. Lu, SOX9 Overexpression Promotes Glioma Metastasis via Wnt/beta-Catenin Signaling, Cell Biochem Biophys, 73 (2015) 205-212.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[21] L. Sun, L.A. Mathews, S.M. Cabarcas, X. Zhang, A. Yang, Y. Zhang, M.R. Young, K.D. Klarmann, J.R. Keller, W.L. Farrar, Epigenetic regulation of SOX9 by the NF-kappaB signaling pathway in pancreatic cancer stem cells, Stem Cells, 31 (2013) 1454-1466. [22] S. Luanpitpong, J. Li, A. Manke, K. Brundage, E. Ellis, S.L. McLaughlin, P. Angsutararux, N. Chanthra, M. Voronkova, Y.C. Chen, L. Wang, P. Chanvorachote, M. Pei, S. Issaragrisil, Y. Rojanasakul, SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma, Oncogene, 35 (2016) 2824-2833. [23] G. Wang, A. Lunardi, J. Zhang, Z. Chen, U. Ala, K.A. Webster, Y. Tay, E. Gonzalez-Billalabeitia, A. Egia, D.R. Shaffer, B. Carver, X.S. Liu, R. Taulli, W.P. Kuo, C. Nardella, S. Signoretti, C. Cordon-Cardo, W.L. Gerald, P.P. Pandolfi, Zbtb7a suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion, Nat Genet, 45 (2013) 739-746. [24] J. Jiang, C. Yu, M. Chen, S. Tian, C. Sun, Over-expression of TRIM37 promotes cell migration and metastasis in hepatocellular carcinoma by activating Wnt/beta-catenin signaling, Biochem Biophys Res Commun, 464 (2015) 1120-1127. [25] G. Nestal de Moraes, P. Khongkow, C. Gong, S. Yao, A.R. Gomes, Z. Ji, N. Kandola, D. Delbue, E.P. Man, U.S. Khoo, A.D. Sharrocks, E.W. Lam, Forkhead box K2 modulates epirubicin and paclitaxel sensitivity through FOXO3a in breast cancer, Oncogenesis, 4 (2015) e167. [26] L. Shan, X. Zhou, X. Liu, Y. Wang, D. Su, Y. Hou, N. Yu, C. Yang, B. Liu, J. Gao, Y. Duan, J. Yang, W. Li, J. Liang, L. Sun, K. Chen, C. Xuan, L. Shi, Y. Wang, Y. Shang, FOXK2 Elicits Massive Transcription Repression and Suppresses the Hypoxic Response and Breast Cancer Carcinogenesis, Cancer Cell, 30 (2016) 708-722. [27] P. Camaj, C. Jackel, S. Krebs, E.N. De Toni, H. Blum, K.W. Jauch, P.J. Nelson, C.J. Bruns, Hypoxia-independent gene expression mediated by SOX9 promotes aggressive pancreatic tumor biology, Molecular cancer research : MCR, 12 (2014) 421-432. [28] L. Garros-Regulez, P. Aldaz, O. Arrizabalaga, V. Moncho-Amor, E. Carrasco-Garcia, L. Manterola, L. Moreno-Cugnon, C. Barrena, J. Villanua, I. Ruiz, S. Pollard, R. Lovell-Badge, N. Sampron, I. Garcia, A. Matheu, mTOR inhibition decreases SOX2-SOX9 mediated glioma stem cell activity and temozolomide resistance, Expert Opin Ther Targets, 20 (2016) 393-405. [29] Y. Xie, W. Lu, S. Liu, Q. Yang, B.S. Carver, E. Li, Y. Wang, L. Fazli, M. Gleave, Z. Chen, Crosstalk between nuclear MET and SOX9/beta-catenin correlates with castration-resistant prostate cancer, Molecular endocrinology (Baltimore, Md.), 28 (2014) 1629-1639. [30] Z.Y. Suhua Xia, Xiaowei Qi, Yong Pu, Yankui Liu, Boshi Wang, Yun Liu, Li Zhang, Yu Qian, Aihui Ma, Guiqin Xu, Hong Tu, Yongzhong Liu, Overexpression of SOX9 and DNMT1 predicts poor prognosis and chemoresistance of colorectal cancer, Int J Clin Exp Pathol, 9 (2016) 589-600. [31] A. Mathelier, O. Fornes, D.J. Arenillas, C.Y. Chen, G. Denay, J. Lee, W. Shi, C. Shyr, G. Tan, R. Worsley-Hunt, A.W. Zhang, F. Parcy, B. Lenhard, A. Sandelin, W.W. Wasserman, JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles, Nucleic Acids Res, 44 (2016) D110-115. Legends Fig. 1. FOXK2 is overexpressed in colorectal cancer. A. FOXK2 mRNA expression in colorectal cancer tissues was explored in the Oncomine database. The box plot represents the FOXK2
ACCEPTED MANUSCRIPT expression in colorectal cancer tissues and normal tissues. B. The correlation between gene copy number and mRNA expression of FOXK2 in colorectal cancer was identified from cBioPortal database. C. The overall survival from our cohort containing 145 colorectal cancer samples was examined by Kaplan-Meier analysis.
RI PT
Fig. 2. FOXK2 promotes cellular proliferation. A. FOXK2 overexpression, knockdown and reversed expression were examined by Western blot. Cell proliferation was measured by MTT assay (B) and colony formation assay (C) in aforementioned cells. D. Statistical analysis of colony formation assay. *, p<0.05, **, p<0.01.
M AN U
SC
Fig. 3. FOXK2 is transcriptionally activated by SOX9. A. Scheme of the FOXK2 promoter luciferase vector construction. B. Relative luciferase activity assays of luciferase reporters with different length of FOXK2 promoter were performed after co-transfection with SOX9 or control. The Renilla luciferase vector was co-transfected as an internal control. C. Putative SOX9–binding site in FOXK2 promoter were shown. D. HCT116 and SW620 cells were subjected to ChIP assay using SOX9 antibody and isolated DNA was employed to PCR by specific promoter primers. E. Overexpression of SOX9 was examined in SW480 and DLD-1 cells. F. FOXK2 expression was examined after SOX9 overexpression. G. FOXK2 expression was examined after SOX9 overexpression followed by knocking down FOXK2. H. Colony formation assay was performed in cells after SOX9 overexpression followed by knocking down FOXK2. *, p<0.05, **, p<0.01.
AC C
EP
TE D
Fig. 4. FOXK2 expression is associated with SOX9 in colorectal cancer tissues. A. Representative images of FOXK2 and SOX9 expression in colorectal cancer tissues (upper panel, low expression of FOXK2 and SOX9, lower panel, high expression of FOXK2 and SOX9, bar, 100µm). B. The statistical analysis of FOXK2 and SOX9 expression in 145 cases of colorectal cancer tissues (Spearman correlation test). The relative expression of FOXK2 (C) and SOX9 (D) in GSE9348 dataset. E. The correlation test of FOXK2 and SOX9 in GSE9348 dataset.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Highlights FOXK2 is up-regulated in colorectal cancer and associated with poor prognosis. FOXK2 promotes proliferation of CRC cells. FOXK2 is transcriptional activated by SOX9. FOXK2 expression is correlated with SOX9 in CRC tissues.