Effects And Mechanisms Of EMT-associated Proteins In Invasion And Metastasis In Colon Cancer

Background and ObjectionXIAP associated factor1(XAF1) is a tumor suppressor. It can suppress the anti-caspase activity of XIAP and then inhibit the proliferation of cancer cells. FHL2(four and a half LIM domain2) is an oncogene in gastrointestinal (GI) cancers, and involved in the network of signal pathways. β-catenin is a key molecule in Wnt signaling pathway, mediating the maintain of cell-cell connection through interacting with E-cadherin on cell membrane. Both Snail1and ZEB1(zinc finger E-box-binding homeobox1) are transcription repressors of E-cadherin and transcription factors of epithelial-mesenchymal transition (EMT). Hypoxia induciable factor1alpha (HIF-la) is the central molecule in cell adaption to hypoxia. HIF-1α and (3-catenin synergistically promote the intrahepatic situ invasion and distant metastasis. HIF-la also promotes EMT through directly binding to Twist. Specificity protein1(Spl) is an important component of transcriptional mechanisms of eukaryotic cells. It increases FHL2expression in GI cancers through transcription regulation. In this study, we focused on the roles of onco-proteins in colon cancer and their mechanisms. We also explored the association among the above eight proteins, which provided theoretically strong support for prognosis of colon cancer and the value of interference therapy strategy.Methods and materials1. Interaction between XAF1and FHL21.1Screening and identification of XAFl-interacting proteinsYeast two-hybrid systems, co-immunoprecipitation (co-IP) and co-localization analysis were performed to screen and identify XAF1-interacting proteins. Co-IP was also performed to identify the binding domains in XAF1-FHL2interaction.1.2Effect of XAF1on FHL2expression and transactivityWhole cell lysate, subcellular cell fractions including nuclear, cytosolic and mitochondrial extracts were prepared and applied to detect the expressions and co-localizations of XAF1and FHL2. Dual luciferase assay was used to evaluate the effect of XAF1on FHL2-associated transcription factors, such as β-catenin and AP-1. Effect of XAF1on IL-8and c-jun was also detected by western blot.2.FHL2promotes EMT and metastasis in colon cancer2.1FHL2expression in primary and metastatic colon cancer tissuesImmunohistochemistry (IHC) staining was performed to detect FHL2expression in17pairs of primary and metastatic cancer tissues.2.2Effect of FHL2on colon cancer cell adhesion and invasion capacitiesTissue culture dishes were coated respectively with collagen IV (50μg/ml) or fibronectin (FN,25μg/ml). The cells with weak or strong adhesion capacity were collected followed by qPCR to detect FHL2expression.2.3Effect of FHL2on EMTCells were incubated with rTGF-β1(recombinant human transforming growth factor beta1) and anti-TGF-β1antibody to detect the effect of FHL2on rTGF-β1-induced EMT.SiRNA knockdown, gene over-expression and truncated-mutation system were performed to alter FHL2expression, and then to check the changes of EMT markers.2.4Mechanism of FHL2promoting EMT in colon cancerWestern blot, IHC and dual luciferase assay were applied to detect the associateion between FHL2and E-cadherin, FHL2and β-catenin, respectively. Immunofluorescence was performed to observe the effect of FHL2on the formation of cell membrane-associated E-cadherin/p-catenin complex.3.FHL2inhibited E-cadherin through interacting with Snaill3.1Expressions of FHL2and Snaill in colon cancerWestern blot, immunofluorescence and IHC were performed to detect the expressions of FHL2and Snaill in colon cancer cell lines and clinical tissues.3.2Identification of the interaction between FHL2and SnaillCo-IP was performed to identify the interaction between FHL2and Snaill and the functional binding domain of FHL2.3.3Effect of FHL2on Snaill expression and metabolismWhole cell lysates and subcellular cell fraction were extracted to detect the expressions of FHL2. Snaill and phospho-Snaill by western blot.3.4FHL2regulates E-cadherin transactivity through interacting SnaillWildtype or mutant constructs of E-cadherin promoter including Snaill-binding domain were first generated. Dual luciferase assay was used to detect the effect of FHL2on the transactivities of the above constructs. Combinational experiments were designed to detect the effect of Snaill inhibition on FHL2-overexpression-suppressed the transactivities of E-cadherin promoter constructs.4. HIF-la promotes EMT and metastasis in colon cancer 4.1Effect of HIF-la on cell morphology and EMT markers’expressionsCells were treated with deferoxamine (DFO) or Ad5-HIF-la-EGFP. After that, cell morphology and E-cadherin localization were observed under microscope. The effect of HIF-la on EMT markers was detected by western blot.4.2Effect of HIF-1α on cell adhesion, invasion and migration in vitroAfter DFO or Ad5-HIF-la-EGFP treatment, adhesion assay was performed to evaluate the adhesion capacity of cells expressing different status of HIF-1α. Invasion assay was perfonned with transwell chamber to detect the effect of HIF-1α on cell invasion. Wound healing was applied to determine the migration capacity.4.3Effect of HIF-la on cancer metastasis in vivoHT29cells were first infected with Ad5-EGFP or Ad5-HIF-la-EGFP. The above single cell suspension (1.2X106) was injected into the splenic capsule of5-6weeks old BALB/c female nude mice. After36days, mice were anatomized to identify whether there were the occurrence of in situ tumors, as well as the existence of liver, lung and renal cancer metastasis. Spleen and liver tissues were collected for diagnosis with H&E staining and IHC.4.4HIF-1α interacts with ZEB1The expressions and co-localization of HIF-1α and ZEB1were explored in colon cancer tissue and adjacent normal colon epithelium. Twenty-two pairs of primary and metastatic lymph nodes were collected to observe the expressions of HIF-1α, ZEB1, E-cadherin and Vimentin. HIF-1α level was altered through chemical treatment or adenovirus infection to detect ZEB1expression. Potential hypoxia response elements (HRE) in ZEB1promoter were analyzed with bioinformatics. Electrophoresis mobility shift Assay (EMSA) was performed to verify the direct interaction between HIF-1α and ZEB1.5.The role of Spl on colon CSCs 5.1The separation and culture of colon CSCsCSCs were isolated by density gradient centrifugation through Ficoll-PaqueTM PLUS (Endotoxin tested, sterile, GE Healthcare). Stem cell medium was prepared in DMEM/F12with N-2Plus media Supplement (Invitrogen), lOng/ml recombinant human fibroblast growth factor-basic (FGF-2, IMGENX),20ng/ml recombinant human epidermal growth factor (EGF, IMGENX),100μg/mL streptomycin,100U/mL penicillin and without serum. CSCs were cultured in ultra-low attachment dish or plate that do not allow adherence to a substratum.5.2Expressions of EMT markers and stem cell surface markers in CSCsCell proliferation was determined by flow cytometry analysis and Ki-67staining. The expressions of Snail, E-cadherin, Vimentin, c-kit, CD133, CD44and CD166were detected in both CSCs and parental cell lines.5.3Spl expression in colon cancer tissues and CSCsSpl expression was observed in45pairs of tumor and neighboring normal tissue by IHC. QPCR and western blot were carried out to assay Spl expression in both CSCs and parental cell lines.5.4Effect of Spl on CSCs growth, colony formation and apoptosisSpl siRNA was transfected or500nM MIA was added into SW480spheres. Cell numbers were counted everyday for3days. Soft agar assay was performed to evaluate the effect of Spl on colony formation. The apoptosis was detected by flow cytometry and analyzed with Winmdi2.9software.5.5Effect of Spl inhibition on CD44and CD166expressions in vivo and in vitroSW480or HCT116(2×106) were subcutaneously injected into the flank of5-6weeks old BALB/c nude mice. After tumors became palpable, mice were randomly assigned to treat with25mg/kg of MIA or dissolvent control (PBS) every3days for3weeks. After the last treatment, tumors were removed and digested into single cell suspension to analysis the expressions of CD44and CD166by flow cytometry.Spl siRNA was transfected or500nM MIA was added into SW480spheres. QPCR and flow cytometry analysis were performed to detect the expressions of CD44and CD166.6. Statistical analysisResults obtained from IHC, qPCR, adhesion, invasion, cell growth, soft agar and dual luciferase assay experiments were expressed as the means±SD. If equal variances assumed, two-tailed Student’s t test or one way ANOVA were performed; if equal variances not assumed, Satterthwaite t test or Welch method was used to analyze the quantities data with significant difference being considered if P values were less than0.05.Results1.XAF1interacts with and attenuates the expression and transactivity of FHL21.1Screening and identification of XAF1-interacting proteinsFull-length XAF1protein was expressed in yeast using the bait plasmid pGBKT7. A human ovary complementary DNA library was screened, and22interacting clones were selected. Gene sequencing showed that two clones interacting specifically with XAF1contained the598bp coding sequence of FHL2gene. GST-pull down, co-IP and immunofluorescence all verified the above findings.1.2Identification of the binding domains between XAF1and FHL2The removal of the first one and half Lim domain abrogated the interaction between FHL2and XAF1, wherever further removal of Lim2restored the binding between the two proteins. This result indicated that LIM2had negative influence on FHL2-XAF1interaction while the last two domain mediated FHL2binding to XAF1protein. Regarding XAF1, we found both the N-terminal Zinc finger domain and the C-terminal non-ZF structure could bind to FHL2with the later segment possessing higher binding capacity.1.3Effect of XAF1on the expression and localization of FHL2Cell fractionation analysis revealed that both proteins were mainly localized to the nucleus with dispersal to mitochondria and to less extent, cytoplasm. Co-IP using cell fractions showed the association of two proteins in all three subcellular compartments, cytoplasm, mitochondria, and nucleus. XAF1negatively regulates FHL2expression and inhibited its level in both cell nuclear and cytoplasm, but increased the level in mitochondria.1.4Effect of XAF1on the transcriptional activity of FHL2XAF1over-expression suppressed the transcription activity of pTOPFLASH. The RLU of pTOPFLASH were476.85±30.65and267.20±0.48, respectively in AGS/vector and AGS/XAF1stable transfectants (P=0.000). On the contrary, XAF1siRNA increased the promoter activity of pTOPFLASH. The RLU were309.67±31.94and752.13±108.97, respectively in cells transfected with the control and XAF1siRNA (P=0.003). Similarly, XAF1over-expression suppressed while XAF1siRNA increased the transcription activity of Luc/AP-1. The RLU of Luc/AP-1were122.21±3.31and71.06±6.92, respectively in AGS/vector and AGS/XAF1stable transfectants (P=0.000); and110.30±30.36and250.07±33.94, respectively in cells transfected with the control and XAF1siRNA (P=0.006). Additionally, XAF1also negatively regulates the expressions of IL-8and c-jun.2.FHL2promotes EMT and metastasis in colon cancer through modulating the formation of E-cadherin/β-catenin complex2.1FHL2expression in primary and metastatic colon cancer tissues.Strong FHL2positive signaling was presented in the cytoplasm and nucleus of cancer cells;16/17of the primary cancer tissues and all of the metastatic tissues take from lymph node, lung or liver highly expressed FHL2; Quantitatively analysis showed near90%of the primary and metastatic cancer cells were FHL2positive; FHL2positive cells were also presented in the compartments of sub-basement memberane in the primary site of cancer.2.2Effect of FHL2on cell adhesion, invasion and migration capacitiesThe attached cells expressed higher level of FHL2than the suspended cells; Knockdown of FHL2inhibited cancer cell adhesion to collagen IV and FN matrix when compared with Ctrl siRNA; FHL2expression positively correlated with cell invasion and migration capacities.2.3FHL2acts as a mediator of EMT-inducerrTGF-β1induced FHL2expression in a dose-dependent pattern; Blockage of TGF-β1with its neutralizing antibody suppressed the endogenous FHL2expression slightly (P>0.05) and rTGF-β1-induced FHL2expression significantly (P<0.05); rTGF-β1could markedly induce the expression of Vimentin and inhibit E-cadherin expression. While the inhibition of Smad2/3or Smad4had no effect on the expressions of E-cadherin and Vimentin.2.4Effect of FHL2on several EMT markers in colon cancer cellsFHL2expression was negatively correlated with E-cadheirn. While a positive relationship was found between FHL2and Vimentin, MMP-9, Snaill and Twist.2.5Effect of FHL2on E-cadherinThe expression of FHL2in cancerous and normal colon cancer tissues were inversely correlated with that of E-cadherin. FHL2-overexpression downregulated, while knockdown of FHL2with siRNA increased E-cadherin expression. Loss of FHL2protein integrity increased E-cadherein expression when compared to full length FHL2protein.2.6Effect of FHL2on β-catenin. FHL2had no effect on the total P-catenin expression level, but significantly inhibited phosphor-β-catenin expression in both cell nuclear and cytoplasm. FHL2overexpression promoted, while FHL2siRNA inhibited the transcriptional activity of β-catenin. The TBE-specific transcription activity (relative luciferase unit ratio of pTOPFLASH/pFOPFLASH) were15.10±1.75and30.10±4.47in DLD1-pcDNA and DLD1-FHL2-pcDNA (P=0.006). FHL2overexpression increased the transitivity of β-catenin. Similarly, FHL2siRNA decreased the pTOPFLASH/pFOPFLASH ratio significantly in comparison with control siRNA (21.82±11.31versus1.47±0.65, P=0.036). FHL2siRNA inhibited survivin and cyclinDl expressions on both mRNA and protein levels.2.7FHL2negatively regulated the formation of E-cadherin/β-catenin complex.The membrane-associated E-cadherin expression was low, while the positive signals of P-catenin disseminated in the whole cell in HCT116ctrl siRNA. After transfection with FHL2siRNA, the levels of both proteins in cell membrane were increased and resulted in formation of the obvious complexes, especially in the intercellular junctions.3. FHL2negatively regulates the transcription of E-cadherin through interaction with Snaill3.1Expressions of FHL2and Snaill in colon cancerWestern blot results showed that FHL2was expressed at a relative high level in SW480, HCT15and SW1116cell lines while at a relative low level in HCT116, DLD1and HT29. Similarly, Snaill was also expressed in SW480, HCT15and SW1116at a relative high level while at a low level or was undetectable in HCT116, DLD1and HT29. The cellular localization of double-staining immunofluorescence analysis indicated that most of the positive cellular signals of FHL2and Snaill showed a predominant nuclear staining. With regard to IHC results, Snail1was expressed by all17primary and16/17of the metastatic cancer tissues, which was similar to FHL2. Nearly90%of cancer cells and few of the adjacent normal epithelial cells expressed Snail1. Semi-quantitatively, scoring of the two proteins showed that the expressions of both proteins in cancerous tissues were significantly higher than that of the adjacent normal colon tissues (P<0.05).3.2The interaction between FHL2and Snail1Co-IP showed that FHL2can physically interact with Snail1. In addition, the removal of the first one and a half, the two and a half or the three and a half LIM domain abrogated the interaction. These results indicate that all of the four and a half of LIM domains are essential during the interaction.3.3The effect of FHL2on the level of total and phosphate Snail1We first confirmed our previous finding about the inverse correlation between FHL2and E-cadherin. Furthermore, a decrease of Snail1phosphorylation at S246was observed along with the increase of Snail1in DLD1-FHL2-pcDNA3.1. Cell fraction experiments showed that FHL2decreased the level of phospho-Snaill but increased the level of Snail1in the nucleus in DLD1-FHL2-pcDNA3.1. These findings suggest that FHL2is able to induce the nuclear accumulation of Snaill.3.4Effect of FHL2on the transactivities of wildtype and mutant E-cadherin promoter constructsTo examine the effect of FHL2inhibition on E-cadherin expression, we suppressed FHL2expression by siRNA transfection and found that FHL2knockdown was accompanied with the decrease of Snail1, while there is a significant increase of E-cadherin. On the molecular mechanism, FHL2siRNA could increase the transcriptional activities of pLuc232-wt and pLuc601-wt in SW480cells. The RLU of pLuc232in DLD1-FHL2-pcDNA3.1was100.40±7.47compared with 176.03±26.15in control cells (P=0.009); similarly, the RLU of pLuc601in DLD1-FHL2-pcDNA3.1cells was68.08±7.93compared with102.65±10.27in control cells (P=0.010). The RLU of pLuc232-wt in cells treated with control or FHL2siRNA were76.36±18.32and147.61±16.63(P=0.033) while that of pLuc601-wt were61.80±12.99and84.30＋8.90(P=0.018), respectively. Furthermore, significant difference was found between pLuc232-wt and pLuc232-mt (76.36±18.32versus128.84±6.92, P=0.033). Combinational analysis with FHL2over-expression and Snail1knockdown was carried out. Snail1siRNA was transfected into DLD1-FHL2-pcDNA and DLDl-pcDNA, and then dual luciferase assay was performed24h later. We found that the magnifying effect of FHL2on E-box mutation was minimised by the transfection with Snail1siRNA.4.HIF-la promotes EMT and cancer metastasis in colon cancer through interacting with ZEB14.1Effect of HIF-la on cell morphology and EMT markersThe expression of HIF-la was induced by DFO-treatment and Ad5-HIF-la-EGFP-infection in mRNA or protein level. HIF-la overexpresion was able to induce the cells morphology transition from round, oval or other epithelial shapes to fibroblast-like shape. Besides, the cell-cell connection and cell polarity were integrated and good in control cells, while the gap from cell to cell increased in HIF-la-overexpressing cells. Hypoxia or HIF-la overexpression induced the expressions of Vimentin and N-cadherin, while decreaed E-cadherin and Plakoglobin expressions. After DFO treatment, the cell membrane-associated E-cadheirn was decreased, while the level in nuclear was increased.4.2Effect of hypoxia or HIF-1α on the capacities of invasion and migration in vitroThe expression of HIF-la was significantly increased in cell population with higher adhesion capacity (P<0.05). The capacities of adhesion, invasion and migration in cells with hypoxia or HIF-1α overexpression were significantly increased compared with the control cells (P<0.05).4.3Effect of hypoxia or HIF-1α on the capacities of invasion and migration in vivoTo confirm the activation of HIF-la increases metastasis in vivo, HT29cells infected with Ad5-EGFP or Ad5-HIF-1α-EGFP were injected into the splenic capsule of5-6weeks old BALB/c female nude mice. After injection, the general status of nude mice had no significant difference compared with that before cells injection. Thirty-six days latere, mice were killed and anatomized. We found that no spleen in situ tumor presented in any mice, and no lung or renal cancer metastasis occured. In control group, one mouse presented liver metastasis (1/8); two mice presented puncate ischemia and necrosis (2/8). In HT29-Ad5-HIF-1α-EGFP group, three presented puncate ischemia and necrosis in most of the liver or enven the entire liver (3/12); eight mice presented liver metastasis (8/12); and the number of liver metastasis was much more than the control mouse.4.4HIF-1α and ZEB1expressions in colon cancer and the adjacent normal tissuesBoth HIF-la and ZEBl were overexpressed in colon cancer tissues compared paired adjacent normal tissues. Furthermore, both of them expressed in cytoplasm and nuclear, especially in the nuclear. The same results were found by qPCR.4.5The regulation of HIF-la on ZEB1expressionZEB1expression was increased accompany with HIF-1α upregualtion.4.6HIF-1α interacted with ZEB1through binding HRE3Bioinformatics analysis suggested that there were four potential HRE upstream of the ATG translation starting codon (<3500nt) in ZEB1promoter. They located at-517～-521bp(HREl),-525～-529bp (HRE2),-630～-634bp (HRE3) and-1342～-1347bp (HRE4), respectively. The EMS A results showed that HIF-la-binding was increased after incubation of nuclear extracts from HIF-1α-overexpressing cells with the HRE3-containing oligonucleotide from the ZEB1promoter.4.7Expressions of HIF-1α, ZEB1, E-cadherin and Vimentin in primary and metastatic colon cancer tissuesThe levels of HIF-1α and ZEB1were high in both primary colon cancer tissues and metastatic lymph nodes. Furthermore, both of them mainly expressed in cytoplasm and nuclear of cancer cells, especially in the nuclear. Vimentin mainly located at the mesenchymal tissue in primary and metastatic colon cancer tissues. E-cadherin expressed in cell membrane, cytoplasm and nuclear, especially in the cell membrane, while little or no expression in lymph nodes. The expressions of HIF-la, ZEB1and Vimentin were increased in metastatic lymph node compared with primary colon cancer tissue, with P<0.05for HIF-1α and ZEB1, while P=0.203for Vimentin. The expression of E-cadherin was significantly decreased in metastatic lymph nodes compared with primary colon cancer tissues (P=0.000).5.The role of Spl in colon cancer stem cells (CSCs)5.1Isolation and identification of colon CSCsColon CSCs with typical stem cell properties were successfully isolated by Ficoll-Paque. They were suspended, round or irregular with clear bound. The percentage of cells in GO state was34.88%in CSCs, while7.11%in parental cells. The percentage of Ki-67-positive cells was12.29%in SW480-CSCs compared with87.06%in SW480(P=0.000), and9.9%in HCT116-CSCs compared with94%in HCT116(P=0.000).5.2Cells in spheroid acquired EMT featureQPCR and western blot analysis showed that E-cadherin expression was decreased in CSCs compared with parental cells. Concomitant upregulation of Snail, Vimentin and c-kit were detected.5.3The expressions of stem cell surface markers CD44was expressed in SW480, DLD1, HCT116, HT-29, HCT15, LoVo and SW1116cells; CD166was expressed in the above same cell lines except LoVo; whereas, CD133expression was very low, even undetectable in all applied cell lines. QRT-PCR showed that all of CD133, CD44and CD166were significantly upregulated in CSCs. More importantly, CD44and CD166expressed at a level of10-2-10-3, while CD133was10-5in SW480or HCT116. The percentage of CD44+CD166+cells was25.89%in SW480-CSCs compared with6.55%in SW480. Similarly,18.63%in HCT116-CSCs compared with4.56%in HCT116.5.4Spl expression in colon cancer tissue and CSCsSpl was mainly expressed in the nuclear of colon cancer cell, while little or undetectable in adjacent normal colon epithelial tissue.57.8%of cells were Spl-negative and only5.9%were strong Spl-positive in normal colon tissues, while25.2%of cancer cells were Spl-negative and74.8%were Spl-positive (10.4%:+;34.1%:++;30.3%:+++) in colon cancer tissues. Both the mRNA and protein levels of Spl were much higher in SW480or HCT116spheres than SW480or HCT116, respectively. Spl expressed at a higher level in human colon cancer specimens than adjacent normal colon epithelium, and the highest level in human colon CSCs.5.5Spl suppression inhibited cell growth and colony formation, and induced cell apoptosisSp1siRNA and MIA abolished the sphere cell growth (27%vs79.95%and14.41%vs78.42%). The inhibition of Spl sharply reduced the colony formation from100%to less than10%(100%vs9.3%in siRNA group;100%vs4.65%in MIA-treated cells; P<0.05). The percentage of apoptotic CSCs increased from8.75%to52.69%between control and Spl siRNA transfection groups, or4.89%to55.59%between control and MIA-treated CSCs cells.5.6Inhibition of Spl inhibited the expressions of CD44and CD166in vivo The percentage of CD44+CD166+cells significantly decreased from18.02%to2.14%after MIA treatment in SW480-injected group. In consistent, the percentage decreased from23.6%to2.9%in HCT116-injected group. Furthermore, the percentage of CD44+cells were7.53%in control group, while1.11%after MIA treatment. As for CD166+, the cells degraded from18.03%to16.18%, which suggested that CD44was more sensitive than CD166to the change of microenvironment.5.7Inhibition of Spl downregulated the expressions of CD44and CD166in vitroSpl suppression by MIA treatment also degraded the expressions of CD44and CD166, which was in a concentration-depend manner. FCM showed that Sp1siRNA treatment resulted in6.77%of CD44+CD166+from32.05%in Ctrl siRNA group, and MIA treatment decreased from20.08%to0.93%.Conclusion1.FHL2is a XAF1-binding protein; XAF1negatively regulates FHL2expression and transacti vity.2.FHL2promotes EMT and cancer metastasis in colon cancer through regulating the composition of E-cadherin/β-catenin complex.3.FHL2inhibits the transcription activity and expression of E-cadherin through interacting with Snail1.4.HIF-1α promotes colon cancer metastasis and EMT through interacting with ZEB1.5. Spl inhibition attenuates the characterizations of colon CSCs.6.XAF1and E-cadherin are tumor suppressors; while FHL2, β-catenin, Snaill, HIF-1α and ZEB1are oncogenes, which are all important in colon cancer metastasis and EMT.