Oncogene FOXK1 Enhances Invasion Of Colorectal Carcinoma By Inducing Epithelial-Mesenchymal Transition

PART1 The Corelation Between The Expression Of FOXK1 And Prognosis In Human Colon CancerBACKBROUND1. The Identification And Characterization Of FOXK1Human Forkhead-box (FOX) gene family consists of at least 43 members, including FOXA1, FOXA2, FOXA3, FOXB1, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXD5 (FOXD4L1), FOXD6 (FOXD4L3), FOXE1, FOXE2, FOXE3, FOXF1, FOXF2, FOXG1 (FOXG1B), FOXH1, FOXI1, FOXJ1, FOXJ2, FOXJ3, FOXKl, FOXK2, FOXL1, FOXL2, F0XM1, FOXN1, FOXN2 (HTLF), FOXN3 (CHES1), FOXN4, FOXN5 (FOXR1), FOXN6 (FOXR2), FOXO1 (FOXO1A), FOXO2 (FOXO6), FOXO3 (FOXO3A), FOXO4 (MLLT7), FOXP1, FOXP2, FOXP3, FOXP4, and FOXQ1. FOXE3-FOXD2 (Ip33), FOXQ1-FOXF2-FOXC1 (6p25.3), and FOXF1-FOXC2-FOXL1 (16q24.1) loci are FOX gene clusters within the human genome. Members of FOX subfamilies A-G, I-L and Q were grouped into class 1 FOX proteins, while members of FOX subfamilies H and M-P were grouped into class 2 FOX proteins. C-terminal basic region within the FOX domain was the common feature of class 1 FOX proteins. FOXH1 and FOXO1 mRNAs are expressed in human embryonic stem (ES) cells. FOXC1, FOXC2, FOXE1, FOXE3, FOXL2, FOXN1, FOXP2 and FOXP3 genes are mutated in human congenital disorders.We describe here the identification and characterization of a human forkhead box gene, FOXK1, that encodes predicted proteins most homologous to the mouse myocyte nuclear factor (MNF)/forkhead box K1 (FoxK1). Human FOXK1 is located at the chromosomal position 7p22. Two transcript variants, FOXK1 a and FOXK1b, containing the divergent 5’-ends are identified. Sequence comparison indicated that the human FOXK1 proteins share 66-74% amino acid identity (99% identity in the forkhead domain) with their mouse ortholog.2. Relationship Between Cancer And FOXK1The forkhead box (Fox) gene family is a group of highly conserved transcription factors that are expressed in diverse species, including yeast and humans. Many FOX protein members have been documented to play critical roles in embryonic development as well as organogenesis and are also involved in the regulation of a variety of physiological processes, such as metabolic processes, cell signaling, and the cell cycle. Consequently, dysregulation of the functions, subcellular localization and expression of FOX transcription factors leads to the development and progression of diseases, in particular, cancer.FOXA1 gene is amplified and over-expressed in esophageal and lung cancer. FOXM1 gene is up-regulated in pancreatic cancer and basal cell carcinoma due to the transcriptional regulation by Sonic Hedgehog (SHH) pathway. FOXO1 gene is fused to PAX3 or PAX7 genes in rhabdomyosarcoma. FOXO3 and FOXO4 genes are fused to MLL gene in hematological malignancies. Deregulation of FOX family genes leads to congenital disorders, diabetes mellitus, or carcinogenesis. Expression profiles, genetic alterations and epigenetic changes of FOX family genes as well as binding proteins and target genes of FOX family transcription factors should be comprehensively investigated to develop novel therapeutics and preventives for human diseases.The human forkhead box k1 gene encodes predicted proteins that are most homologous to the mouse myocyte nuclear factor MNF/Forkhead box K1 (Foxkl). It has been shown in silico that FOXK1 is expressed in immature tissues of the brain, eye, heart, lung and thymus. In adults, FOXK1 is predominantly expressed in many malignant tissues, brain, colon and lymph nodes. This gene has been implicated in normal and neoplastic developmental processes. The data indicate a role for human FOXK1 in regulating the developmental process as well as the potential involvement of FOXK1 in tumorigenesis. In human breast cancer,FOXK1 recruits tetl to act as a tumou suppressor. In a human rhabdomyosarcoma cell line it has been shown that FOXK1 interacts with the MADS-box transcription factor SRF and coregulates the expression of SM alpha-actin and PPGB.It is reported that FOXK1 and FOXK2 promote Wnt/β-catenin signaling by translacating DVL into the nucleus and is highly expressed in HT29 and DLD1. In this study, we report on FOXK1, a gene that is over-expressed in various cancers, including CRC, and acts as an important oncogene. WE will focus on the topic of the relationship between the expression of FOXK1 and the clinical characteristics of colon cancer patients. We wish to develop novel therapeutics and preventives for human colon cancerMATERIALSCell cultures and cell lines:FHC, HT-29, SW480, LOVO, SW1116, SW620, Colo205 and DLD1 cells,93 pairs of colon cancer tissue, TMA (Tissue multi-array) 15 types of different nomal and carcinoma tissue. Reagents:reverse transcription assay kit, Recombinant human TGF-β1, Dual luciferase reporter gene detection kit, Rabbit anti-human FOXK1 moloclonal antibody, Goat anti rabbit polyclonal antibody labeled with horseradish peroxidase Rhodamine and phalloidin, RNA isolation kit, reagents of IHC.METHODS1. FOXK1 Expression In 15 Types Of Normal And Cancer TissuesTMAs were purchased from Alenabio Co., Ltd.,Xian, Shanxi, China (BN 1002a and FDA800). TMAs with 15types (3 spots for each) of normal and cancer tissues contained brain, breast, esophagus, kidney, lung, cervix uteri, ovary, pancreas, prostate, skin, small intestine, stomach, testis, large intestine and rectum. In accordance with the manufacturer’s information, all tissue samples were fixed in formalin, processed in a tissue processor (no more than 24 h of fixation) and finally embedded in paraffin, to analyse the expression of FOXK1 between normal and cancer of multiple organs.2. FOXK1 expression in tissues and cells of colon cancer2.1 expression of FOXK1 in matched colon normal and cancer tissuesWe then measured FOXK1 expression in 9 pairs of matched colon normal (N) and cancerous (T) tissues by Western blotting.2.2 FOXK1 expression in 7 colon cancer cell linesBased on Western blotting, we demonstrated FOXK1 expression in the following seven CRC cell lines compared with the FHC, HT-29, SW480, LoVo, SW1116, S W620, Colo215 and DLD1.3. Influence of FOXK1 to the expression of oncogenes.3.1 To establish a stable FOXKl expression cell lineTo establish stable cell lines, SW480 was transfected with empty pcDNA 3.1 vector and pcDNA3.1-FOXK1. After 48 hours, we cultured cells in RPMI 1640 medium supplemented with1000μg/ml geneticin(G418) for 2 weeks more, then select monoclonal cells which are cultured in RPMI 1640 medium supplemented with 1000μg/ml geneticin for 2 weeks again. The expression of FOXK.1 in stable cell lines was verified by qRT-PCR and Western-blotting.3.2 The influence FOXK1 on mRNA expression of oncogenesWe screened for potential target genes by examining the expression changes of 3 major oncogenes that are known to be involved in proliferation and transformation after ectopic FOXK1 expression in SW480. The mRNA expression of Survivin, Cyclin D1, AP-1 and FOXK1 was detected by qRT-PCR analysis.3.3 The promoter activity of oncogenes regulated by FOXK1 was testified by Dual-luciferase reporter gene detection assay.We cloned the promoter region of human Survivin, Cyclin D1 and AP-1 upstream of a luciferase gene in a reporter plasmid and then transfected in the stable colon cell lines, we testified the promoter activity by Dual-luciferase reporter gene detection assay.4. FOXK1 expression correlated with tumor progression and poor prognosis of CRC patientsTo evaluate the relationship between FOXK1 protein and CRC prognosis, we analyzed the correlation between high FOXK1 expression and clinicopathological features of CRC which containing age, gender, location, tumor size, TNM stage, differentiation and lymph node metastasis. Next, we evaluated the prognostic effect of FOXK1 on overall survival by comparing the overall survival of CRC patients with high and low FOXK1 protein levels.5. Statistical AnalysesStatistical analyses were performed using the SPSS 13.0. Correlation between FOXK1 expression and clinicopathological characteristics were evaluated by Chi-square test. By drawing Kaplan-Meier survival curves to show the expression of FOXK1 influence on overall survival. Survival analysis method using log-rank test The χ2, Fisher exact probability, and Student’s t tests were used for comparison between groups. P<0.05 was considered significant.RESULT1. FOXK1 Expressed Highly in all Cancer Tissues And is Negative In Normal TissueNormal tissues of skin, testis and kidney showed weakly positive expression, other 12 tissues were negative, including the stomach, small intestine, large intestine, rectum, and gastrointestinal tissues. However, all cancerous tissues showed strongly positive staining.2. FOXK1 Expressed Highly In Tissues And Cells Of Colon Cancer2.1 Increased expression detectived in colon cancer tissuesExpression in 9 pairs of matched colon normal (N) and cancerous (T) tissues are tested by Western blotting. In the 9 cancerous tissues,8 tissues expressed higher levels of FOXK1 than the normal ones.2.2 FOXK1 expression in 7 colon cancer cell linesStrongly expression is tested in the cells of SW1116 and Colo205, Lovo and DLD1 show the moderate result, while low expression occurs in HT29, SW480, SW620 by western-blotting.3. Effects of FOXK1 on the expression of oncogenes.3.1 To establish stable transfectants of FOXK1Overexpressed FOXK1 in the stable cell lines of SW480 which was confirmed by Western blotting and qRT-PCR analysis. qRT-PCR analysis was repeat for 2 times and the average was kept to analyse with Independent Samples T-Test. The result of qRT-PCR in two groups are 4.621±0.43and 1.005±0.147, The difference was statistically significant (t=11.256, p=0.008)3.2 FOXK1 promotes the mRNA expression of oncogenesThe mRNA expression of Survivin, Cyclin D1, AP-1 and FOXK1 was up-regulated in stable FOXK1 transfectants. The result of the three oncogenes in the two groups described as follows:the relative ratio of Survivin-mRNA:2.245±0.153 and 1.0017±0.083, Cyclin D1-mRNA:3.638±0.338 and 1.0017±0.098, while the AP-1-mRNA:2.298±0.090 and 1.0024±0.083, The difference was statistically significant (t= 10.044、10.700、13.759, p= 0.01、0.009、0.005)3.3、 The promoter activity of oncogenes are up-regulated by FOXK1 was testif-ied by Dual-luciferase reporter gene detection assay.The RLU of Survivin, Cyclin D1 和 AP-1 in the group of FOXK1-pcDNA 3.1-SW480 are respectively 1475.41±205.36,1090.57±206.12,1220.98±246.62. While the control group are 411.33±23.63,240.35±45.36,402.58±30.42, The difference was statistically significant (t=8.916,6.978,4.435, p=0.001,0.02,0.021)4. FOXK1 Correlated With the Poor Prognosis Of CRC PatientsNo significant association was observed between FOXK1 expression and age (P =0.534), gender (P=0.606), location (P=0.264). However, FOXK1 expression significantly correlated with tumor size (P=0.019), TNM stage (P= 0.002), differentiation (P=0.000), AJCC Ⅰ, Ⅱ stage (P=0.031),Ⅲ, Ⅳ stage (P=0.04) and lymph node metastasis (P= 0.013). Of the 93 surgical CRC specimens,63 cases exhibited a high expression of FOXK1, whereas low expression was found in the other 30 cases. Among the participants, patients with higher FOXK1 expression were associated with a significantly lower 7-year survival rate than those with a lower expression, according to Kaplan-Meier curve assessment (P=0.000, log-rank test;). Such a relationship observed in patients with early-stage CRC (i.e., American Joint Committee on Cancer (AJCC) stage Ⅰ and Ⅱ; P=0.000,) was more obvious than that in late-stage CRC (i.e., AJCC stage Ⅲand IV; P=0.099,). Unfortunately, the prognostic value of FOXK1 expression in selective patient subgroups stratified according to AJCC stage was not evident.DISCUSSION1. FOXK1 facts as an oncogene in the brain, breast, esophagus, kidney, lung, cervix uteri, ovary, pancreas, prostate, skin, small intestine, stomach, testis, large intestine and rectum2. Over-expressed FOXK1 promotes the carcinogenesis3. FOXK1 expression positively correlated with tumor size、TNM stage、 differentiation and lymph node metastasis,4. High FOXK1 expression is a poor prognosis factorPART 2:FOXK1 Enhances Invasion Of Colorectal Carcinoma By Inducing Epithelial-Mesenchymal TransitionBACKGROUNDColorectal cancer is one of the clinical common malignant tumor of digestive tract, the incidence continues to rise, and colorectal cancer incidence rate rise velocity year after year, its incidence has been ranked the third, a quarter of patients with colorectal cancer has associated with lymph node or distant metastasis,90% of cancer patients die of metastasis. the median survival time was only 5-6 months in patients without any treatment. The metastasis of colorectal cancer has become the focus and difficulty of cancer treatment and scientific research.Epithelial-mesenchymal transition (EMT), which plays an essential role in tumor invasion and metastasis, is an essential phenotypic event during embryonic development, tissue remodeling and wound healing. EMT is also a reversible process that often occurs at the invasive front of many metastatic cancers. EMT is physiologically initiated by certain autocrine factors, with rTGF-β being the strongest inducer that functions in the majority of epithelial cell types tested in vivo. Others have found that the Fox gene family and EMT play important roles in cancer metastasis. It is reported that FOXK1 and FOXK2 promote Wnt/β-catenin signaling by translacating DVL into the nucleus and is highly expressed in HT29 and DLD1. However, the role of FOXK1 played in EMT and the fuction of invasion and metastasis in colon cancer is still unknown. What the study mainly centre on is to uncover the influence to EMT with the knowdown or overexpression of FOXK1. What’s more, in vitro and in vivo, to indicate the importance of FOXK1 in the invasion and metastasis. At last, in the hope of FOXK1 being used in tumor biological target therapy.MATERIALSCell cultures and cell lines:SW480, SW1116 cells,93 pairs of colon cancer tissue, lymph node metastatic cancer tissue, metastatic liver cancer tissueReagents:reverse transcription assay kit, Recombinant human TGF-β1, Dual luciferase reporter gene detection kit, Rabbit anti-human E-cadherin moloclonal antibody. Rabbit anti-humanβ-catenin monoclonal antibody, Rabbit anti-human Vimentin moloclonal antibody, Rabbit anti-human FOXK1 moloclonal antibody, goat anti-human Snail moloclonal antibody, Rhodamine and phalloidin, RNA isolation kit, qRT-PCR assay kits, reagents of IHC, Transwell chamber.,Relevant animal surgical instrumentsMETHODS:1. FOXK1 Expression In Primary Colon Cancer And Lymph Node CancerWe examined FOXK1 expression in primary colon cancer tissues by IHC as described in part 1 and we describe the position FOXK1 expressed in cells. Next, we detected the expression of FOXK1 in regional lymph node metastatic cancer tissue.2. Effect of low expression of FOXK1 on migration and invasion2.1 Confirm the low expression of FOXK1 in the cell lines of SW1116 and SW480Scrambled-siRNA and FOXK1-siRNA were transfected to SW1116 and SW480. After 48 hours, Protein was extracted for Western blotting to detect the expression of FOXK1.2.2 Wound healing verify the influence of FOXK1 on migration of SW1116 and SW480SW1116 and SW480 were transfected with Scrambled-siRNA or FOXK1-siRNA plated in 6-well plates with 100% confluence. A linear wound was made by scraping a 1000μl-pipette tip across the confluent cell layer.24hs after treatment by serum free medium. Cells were washed twice to remove detached cells, size of the wounds were observed and measured at Ohr、12hr、24hr、36hr、48hr、 60hr.2.3 Transwell verify the influence of FOXK1 on invasion of SW1116 and SW480SW1116 and SW480 were transfected with Scrambled-siRNA or FOXK1-siRNA plated in transwell chamber coated with Matrigel. Cells were seeded onto the upper chamber (2×105 cells/chamber) and maintained in serum-free medium. After 48hs, Cells dyeing with crystal violet that had invaded the Matrigel and had reached the lower surface of filter were counted under a light microscope at a magnification of× 400. We chose five visual fields and counted the number of migrated cells on the lower surface of the filter.3. Influence Of FOXK1 To EMT Of Colon Cancer3.1 Changes of Cell morphology after over-expressed FOXK1The morphology of FOXK1 stable cell lines transfected with SW480-pcDNA3.1 and SW480-pcDNA3.1-FOXK1 was observed in white light microscope. Cells were dyed by Rhodamine and phalloidin and F-actin were observed in fluorescence microscope3.2 EMT-associated proteins regulated by FOXK1After the knockdown of FOXK1, we assessed the expression of EMT markers. E-cadherin and Vimentin in immunofluorescence analysis. Mesenchymal markers (Vimentin and Snail) and the epithelial markers (E-cadherin and y-catenin) were observed by Western blotting after FOXK1-siRNA transfection.4. Explore The Role Of FOXK1 In rTGF-β1-Induced EMT And Cell Invasion4.1 rTGF-β1 induce the expreesion of FOXK1 and EMT-associated proteinsTo evaluated SW480 response to the most potent EMT inducer rTGF-β1. rTGF-β1 (10μg/ml) were added into cells for Ohr,24h、48h, then three groups of total cell protein were extracted, Western blot was performed to detect FOXK、 E-cadherin and Vimentin expression.4.2 Down-expressed FOXK1 neutralized the influence of TGF-β1 on EMTThe SW480 and SW1116 cells were seeded in a 6-well plate. Each kind of cells inoculated with three holes, when the cell confluence is up to 30-40%, Scramble-siRNA were transfected in to the group 1 and 2. The third was transfecred with FOXK1-siRNA, while the second and third group were added with rTGF-β1(2μg/mL), respectively nameed Scr-siRNA group, Scr-siRNA+　rTGF-β1 group, FOXK1-siRNA+　rTGF-β1group. Together with the cell morphology、 Transwell assay and Western blotting, to analysis whether down-expressing FOXK1 could neutralize the influence of rTGF-β1 on EMT.5. Knockdown of FOXK1 influenced the invasion and matastasis in vivo5.1 Construct the down-expressed FOXK1 stable cell line of SW480An antisense lentiviral (LV) RNAi targeting the FOXK1 gene with short hairpin (sh) RNA (FOXK1-shRNA-LV) was designed, synthesized and stably transfected into SW480 cells, The transfection efficiency was observed by eGFP fluorescence intensity, and validated by Western blotting.5.2 In Vivo Metastasis AssaysFour to six-week-old BALB/C-nu/nu nude mice were obtained from the Laboratory Animal Unit, Southern Medical University, China. To further test the association of FOXK1 with metastasis, the role of FOXK1 in metastases was tested by injection of SW480/pEGFP-FOXK1 and SW480/pEGFP-N1 (Vector), or SW480/pEGFP-FOXK1 shRNA and SW480/pEGFP-src shRNA expressing with Green Fluorescent Protein (GFP) into nude mice.At 30 days post-injection, the mice were sacrificed, the individual organs were removed, and the metastatic tissues were analyzed with H&E staining. The expression of E-cadherin is proved by qRT-PCR and IHC.RESULT1. FOXK1 Highly Expressed In Primary Colon Cancer And Lymph Node Metastatic Cancer TissueWe found that strong FOXK1-positive signals were present in the nucleus of cancer cells (primary cancer tissue) and tumor-associated stroma cells. Notably, no positive signal was found in the epithelial cells of normal colon glands. In the 32 patient with matastasis,27 of 32 primary colon cancer specimens showed high expression of FOXK1,25 of 32 lymph node metastasis showed high expression of FOXK1, the expression of FOXK1 was no significant difference between the two positons, P>0.05.2. Knock-down of FOXK1 reduce the invasion and matatsis of colon cancer cell2.1 Confirm the low expression of FOXK1 in the cell lines of SW1116 and SW480Western-blotting confirmed that the expression of FOXK1 after FOXK1-siRNA transfected SW480 cells and SW1116 was significantly lower than the control group.2.2 FOXK1-siRNA depressed the migration of colon cancer cellThe Migration index of FOXK1-knockdown cells was decreased by 52.02±0.087%　and 58.9±0.03%　respectively at 60hs in SW480 and at 48 hs in SW1116. While the control groups were both totally confluenced. The difference was statistically significant (t=7.04,7.888, p=0.002,0.01). So we could infer that FOXK1-siRNA depressed the migration of colon cancer cell2.3 Knocking-down FOXK1 inhibit the invasion of SW1116 and SW480The number of cells with FOXK1 knockingdown are 48.5±4.93 compared with the control group 72.5±6.14. The same result could be observed in SW1116. FOXK1-siRNA-SW1116 with the number of 61.25±6.65 and control with 118.5±15.76. The difference was statistically significant (t= 6.096,6.694, p= 0.001, 0.01).3. FOXK1 Correlate With The EMT-Associated Protein3.1 FOXK1-transfectants exhibited a spindle-like, fibroblastic morphologyThe stable transfectants of vector-transfected cells displayed a round or flat morphology with short cytoplasmic processes. However, pcDNA3.1-FOXK1-transfectants exhibited a spindle-like, fibroblastic morphology, one of the main characteristics of EMT. Long or dendritic-like cytoplasmic processes were visible under a phase-contrast microscope Compared with the vector-expressing cells, FOXK1-overexpressing cells showed F-actin staining throughout the cytoplasm and at the rim zone of the protrusion. Moreover, filopodia and lamellipodia were identified as dynamic cellular features on the cell membrane surfaces that require actin polymerization and are involved in the invasion and metastasis of cancer cells.3.2 EMT-associated proteins up-regulated by FOXK1E-cadherin was up-regulated and Vimentine was down-regulated in immunofluorescence analysis after the knockdown of FOXK1. The down-regulation of mesenchymal markers (Vimentin and Snail) and the up-regulation of epithelial markers (E-cadherin and y-catenin) were observed by Western blot after FOXK1-siRNA transfection.4. FOXK1-siRNA Reverse EMT And Cell Invasion Induced By rTGF-β14.1 rTGF-β1 induce the expreesion of FOXK1 and EMT-associated proteinsrTGF-β1 induced FOXK1 expression in a time-dependent way. This induction also increased the expression of the mesenchymal marker, Vimentin, whereas it decreased the expression of E-cadherin, an epithelial marker.4.2 Down-expressed FOXK1 neutralized the influence of rTGF-β1 on EMTA higher expression of epithelial markers and a reduced expression of mesenchymal markers were evident after FOXK1-siRNA transfected in the group treated with rTGF-β1 for 48 h. In addition, the results indicated that FOXKl-siRNA neutralized the influence of rTGF-β1 on cell phenotype. Coupled with the morphologic changes of EMT, the knockdown of FOXK1 decreased the invasive ability of tumor cells in transwell assay.5. Knockdown Of FOXK1 Depressed The Invasion And Matastasis In Vivo5.1 Vertify the down-expressed FOXK1 stable cell line of SW480Fluorescent microscopy revealed a transfection rate of 95%.The expression of FOXK1 is tested by Western-blotting.5.2 FOXK1 induced EMT and metastasis in CRC in vivo.Thirty days after injection, the mice with FOXK1 overexpressing SW480 cells, but not vector SW480 cells, formed a variety of large metastatic nodules in livers, whereas the mice with FOXK1 knockdown were in liver small nodules compared with those in the src shRNA. The presence of liver metastases from CRC was confirmed by histological analysis.To further demonstrate whether FOXK1 is required for EMT, the explanted liver tissue showed that, compared with SW480/Vector the orthotopic implantation of SW480/FOXK1 expressing cells resulted in decreased E-cadherin expression in an IHC assay and qRT-PCR.DISCUSSION1. FOXK1 is highly expressed in the primary and metastatic colon cancer.2. Knockingdown of FOXK1 suppresses migration and invasion of colon cancer cell by inhibiting EMT.3. Knockdown of FOXK1 inhibits rTGF-β1-induced EMT and invasion.4. FOXK1-shRNA inhibits EMT and metastasis in CRC in vivo.