| Background and Objective:Colorectal cancer (CRC) is one of the crucial malignancies in the worldwide and the leading causes of cancer deaths. Clinically, metastasis is still the main cause of death. It’s reported that more than half of the colorectal cancer patients have various degrees of micrometastasis before or undergoing surgery operation. Moreover, micrometastasis is the direct reason of recurrence and metastasis after surgery. An initiating mechanism in the early stage of distant metastasis is related to epithelial-mesenchymal transition (EMT), a complex process that permited a polarized epithelial cell to gain mesenchymal-cell like properties. Cancer cells acquire aggressive phenotypes to detach from the primary tumor mass through EMT, enter the surrounding stroma and migrate to the distant sites. An increasing body of evidence from clinical and experimental studies has supported a role for EMT in CRC’s dissemination. TGF-β is an important induced factor in epithelial-mesenchymal transition process. Tumor metastasis are closely related to EMT, which can help cells acquire the ability of agressiveness and migration while the cells have none of those abilities before, thus making metastasis to other organs and emerging distant metastasis, and finally colonization through mesenchymal-epithelial transition. Therefore, it is of great significance to clarify the molecular mechanism between EMT and CRC metastasis. it is significancant to predict, diagnose and prognosis of the metastasis of colorectal cancer.LIM and SH3 protein 1 (LIM and SH3 protein, LASP-1) was a protein related to CRC metastasis, which was screened and identified by our research team before. LASP-1 cDNA(No.X82456) was initially acquired from cDNA library of metastatic axillary lymph nodes. Its full-length of mRNA contains approximately 4000 nucleotides. Its encoding protein is made up of 261 amino acid. LIM domain (1 Lin-1, Isl-1 and Mec-3) was contained at the end of its amino terminal and Src homology domain 3 (SRC homology 3, SH3) was contained at the end of carboxyl terminal. It’s reported that LASP1 protein is closely related to the aggressiveness and metastasis of tumor. To explore mechanisms of LASP-1 involved in metastastic colorectal cancer, we previously used high-throughput fluorescence of two-dimensional difference gel electrophoresis technology (2-DDIGE) to screening LASP-1 associated protein, and succeeded in screening and identificating the members of the S100 protein family, S100A11 protein.S100 calcium binding protein A11(S100A11) is an important member of the S100 family, also known as S100 calcium binding protein C(S100C), calgizzarin, metastastic lymph node gene 70 protein (MLN70). It was found in chicken gizzard smooth muscle cells for the first time in 1991. S100A11 has been reported in many tumors and it has bidirectional regulation to tumor cells. S100A11 may be localized in the nucleus which is different from other S100 family proteins. It’s suggested that it has the potential function of transcriptional regulation. When the concentration of Ca2+and TGF-β were increased, S100A11 would binding with nuclear protein, then S100A11 translocated to nucleus and involve in the regulation of growth in normal human keratin cells (NHK). In normal human fibroblast cells, S100A11 is located in the cytoplasm. However, when the fibroblast cells mutated, it could be found in the nucleus, which suggesting that TGF-β is involved in the regulation of S100A11. It has been reported that the TGF-β/Smad signaling pathway is a new target for the treatment of colorectal cancer patients. Moreover, there also pointed out that the TGF-β could activate the Smad signaling pathway to induce the classical EMT process by contributing to the expression of LASP1. The paper intended to solve these problems through molecular pathological and molecular biological methods. It intended to combined with study in vitro and in vivo, and then clarified that S100A11 can promote colorectal cancer metastasis clearly. It provided the direct evidence that LASP1 involve in the regulation of S100A11’s biological function. Our diversely research indicated that classical TGFβ/Smad could induce the metastasis of CRC through LASP1-S100A11 axis. It provided adequate scientific evidence that TGFβ-LASP1-S100A11 axis is regarded as therapeutic targets in patients with advanced colorectal cancer.Statistical analysis:Data was analyzed by SPSS 19.0 statistical software (SPSS, Chicago, USA). Quantitative data was collected by three independent experiments, which represented as mean±standard deviation. Independent sample T test was applied when comparing to data between two groups. However, multiple groups of data was compared by using One-way ANOVA. The results of CCK8 were assessed by factorial design analysis of variance. Relationship between S00A11 and clinical pathology parameters was analyzed by Pearson’s chi-square test (χ2 test). Kaplan Meier survival curve was used to analyze the related patients’prognosis correlation by regarding S100A11 as a single variant factor, Cox proportional hazards regression was uesd as Multi-factor variable analysis. P< 0.05 was considered to be significant.Method:1. LASP1 positively regulates the expression of S100A11 through protein-protein interaction in a protein level1.1 LASP1 positively regulates the expression of S100A11To further validate the results of proteomic screening before, we transferred LASP1cDNA to SW480 and HCT116 cell lines and used siRNA interference to interfere the expression of LASP1. S100A11 mRNA or protein expression were measured by RT-PCR, Western Bloting and immunoflurescence. The results of Q-PCR indicated that S100A11 mRNA expression were not influenced by LASP1 (P>0.05)and Western Bloting revealed that LASP1 regulated the expression of S100A11. Immunoflurescence found that LASP1 and S100A11 were related and colocalized in protein level. Furthermore, we used IHC to detect the expression of LASP1 and S100A11 in 50 formalin-fixed, paraffin-embedded tissues and analyzed their correlation using statistical methods (P<0.05).1.2 The interaction between LASP1 and S100A11 were analyzedWe used COIP assays to re-examine the interaction of LASP1 and S100A11 in SW620 and HCT116 cell lines and the results confirmed LASP1 was binded with S100A11.2. LASP1 mediated the invasive phenotype and EMT in colorectal carcinoma through S100A112.1 LASP1 mediated the invasive phenotype of colorectal carcinoma through S100A11In order to detect the changes of invasive phenotype resulted by S100A11’s protein levels in cells mediated by LASP1, we performed rescue assays. Firstly, we established several groups:BLANK, NC, LASP1 and LASP1+si-S100A11. We employed transwell to explore the influence of S100A11 on cell migration capacity mediated by LASP1 in BLANK, NC, si-LASP1, si-LASP1+S100A11 groups. We found that overexpression of LASP1 and S100A11 knockdown meanwhile could significantly weaken the migration capacity mediated by LASP1 (P<0.05),whereas LASP1 inhibition and S100A11 overexpression meanwhile rescued migration capacity (P<0.05).2.2 LASP1 mediated EMT in colorectal carcinoma through S100A11We used gain-in and loss assays to detect the changes of EMT resulted by S100A11’s protein levels in cells mediated by LASP1. Firstly, we established several groups:BLANK, NC, LASP1 and LASP1+si-S100A11. We employed Western Bloting to validate the markers of EMT. We found that co-transfer of LASP1cDNA3.0 and si-S100A11 in SW480 and HCT116 cell lines could markedly reversed EMT mediated by LASP1 (i.e. EMT markers FN and E-cadherin were decreased and increased respectively). On the contrary, co-transfer of si-LASPl and S100A11 plasmids could significantly inhibit EMT (i.e. FN increased while E-cadherin decreased)2.3 S100Allwas involved in EMT mediated by TGFβIn order to illuminate the relationship between S100A11 and TGFβ, we stimulated SW480 and HCT116 cells using TGFβ for Oh,24h and 48h. We observed S100A11 could promote EMT of colorectal carcinoma cell lines, which was inhibited by adding TGFβ receptor inhibitor SB431542.3. The expression of subcellular S100A11 in colorectal carcinoma and its relevance with clinical pathology development3.1 S100A11 expression in colorectal carcinoma tissuesIHC indicated that S100A11 was located in both the cytoplasma and nucleus and its expression was much stronger in carcinoma tissues compared with normal colorectal tissues. The results was also corroborated by Western Bloting (P<0.05). Immunoflurescence showed that the expression of S100A11 was low in normal tissues while high in carcinoma tissues. It expressed both in the cytoplasma and nucleus.3.2 S100A11 expression in colorectal carcinoma cell linesWestern Bloting indicated that S100A11 was expressed in human colorectal carcinoma cell lines and its expression level was gradually increased in SW480,SW620, SW480/M5 which were derived from a common origin but possessed increasingly higher metastatic potention, indicating the possible relationship between S100A11 and metastasis of colorectal carcinomas. Our immunoflurescence results showed that S100A11 expression was mainly found in the cytoplasm and its expression in SW480, SW620, SW480/M5 and RKO was located in the cytoplasm and nucleus.3.3 S100A11 expression level was correlated with patient prognosisKaplan-Meier analysis indicated that S100A11 overexpression in the cytoplasm and nucleus of colorectal carcinoma cells presented poorer prognosis, which was statistically significant (P<0.05, P<0.05)3.4 Analysis of single variant and muti-variant that impacted prognosis of patients with CRCUnvariant analysis indicated that overexpression of S100A11 in the cytoplasm and nucleus was correlated with clinical prognosis in patients with CRC, while multi-variant anlysis did not support the overexpression of S100A11 in the cytoplasm and nucleus as an independent factor for poorer prognosis (P<0.05).4. The overexpression of S100A11 in the cytoplasm and nucleus promoted invasive phenotypes and EMT of colorectal carcinoma cells.4.1 The impact of overexpressed S100A11 transiently on the capability of proliferation, migration of wound-healing in CRC cells﹙alidation of overexpressed S100All transientlyThe HA vector and HA-S100A11, HA-NLS-S100A11, HA-NES-S100A11 recombinant expression vector were transfected into SW480 cells. The results of Western Bloting showed that overexpressed subcelular localized S100A11 was successfully constructed, which were named as SW480/HA, SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11; The results of immunofluorescence showed that exogenous S100A11 of SW480/HA-S100A11 localized in the cytoplasm and the nucleus, SW480/HA-NLS-S100A11 of exogenous S100A11 was localized in the nucleus, SW480/HA-NES-S100A11 exogenous S100A11 located in the cytoplasm, the immunofluorescence assays showed that overexpression of S100A11 cell line was constructed successfully, The NLS nuclear localization signal of S100A11 can be localized in the nucleus, NES nucleus transport signal can make S100A11 located in the cytoplasm of the cell.②The effect of overexpressed S100A11 transiently on the capability of proliferation in CRC cancer cellsCCK8 results showed that S100A11 overexpressed transiently in SW480 and HCT116 cells (HA-NLS-S100A11-, HA-NES-S100A11-, HA-S100A11-) resulted in statistically significantly increasing tumor’s growth compared with control (HA) (SW480, P<0.05; HCT116, P<0.05). The results was in line with the notion that S100A11 could promote the proliferative capacity of CRC cells.③ The effect of overexpressed S100A11 transient on the capability of migration in CRC cancer cellsThe results of Transwell showed that S100A11 overexpressed transiently in SW480 and HCT116 cells (HA-NLS-S100A11-, HA-NES-S100A11-, HA-S100A11-) resulted in statistically significantly increasing number of migrated cells compared with control(HA) (P<0.05)④The effect of transient S100A11 overexpression on the healing capacity of scratch test of CRC cancer cellsWound-healing assays indicated that S100A11 overexpressed transiently in HA-NLS-S100A11-, HA-NES-S100A11- and HA-S100A11- cell lines resulted in statistically significantly increasing migrated rate compared with control (HA) (P<0.05).These results showed that S100A11 overexpressed in vitro could promote the capacity of proliferation, migration and wouding healing in colorectal carcinoma cells.4.2 The impact of silencing S100A11 transiently on the capacity of proliferation, invasion and woud-healing in CRC cells.①validation the efficiency of S100A11 which was transiently silencingThe negative control terminal with green fluorescence was used as transfection efficiency evaluation. Results showed that siRNAoligo transfection efficiency can reach 60-70% and it was easy to achieve better effects through gene silencing; siRNA-192 and siRNA were transfected in over-expressed cells, such as HCT116 cells and M5 cells, The results of Western Boting showed:that transfection siRNA-92 interference fragment in the cell, The levels of S100A11 were significantly decreased, indicating that siRNA-92 fragments could knock down S100A11 successfully.② The effect of silencing S100A11 transiently on the capability of proliferation in CRC cancer cellsThe results of CCK.8 showed that interferencing S100A11 transiently in HCT116 and M5 cells resulted in statistically significantly decreasing the ability of growth compared with control (HA) (P<0.05). This results were in line with the notion that S100A11 silencing in vitro could inhibit the capacity of proliferation in CRC cells.③The effect of silencing S100A11 transient on the capability of migration in CRC cancer cellsThe results of Transwell showed that S100A11 interference(siRNA-192) transiently in M5 and HCT116 cells resulted in statistically significantly decreasing the number of migrated cells compared with control(HA)(P<0.05). This result indicated that S100A11 silencing in vitro could inhibit the migration capability of CRC cells.④The effect of transient S100A11 silencing on the healing capacity of scratch test of CRC cancer cellsScratch test indicated that S100A11 interference(siRNA-192) transiently in HCT116 and M5 cell lines resulted in statistically significantly decreasing healing capacity compared with control (HA) (P<0.05).These results showed that S100A11 silencing in vitro could inhibit the capacity of proliferation, migration and wound-healing in colorectal carcinoma cells.4.3 Molecular mechanism for the regulation of colorectal carcinoma metastasis by S100A11To explore alterations of EMT marker in CRC cells after S100A11 was overexpressed, we transfected the plasmid of HA, HA-S100A11-, HA-NLS-S100A11-, HA-NES-S100A11-to SW480 and HCT116 cells and examine changes of EMT markers through Western Bloting. We found that the expression of epithelial marker (E-cadherin) was lower in cells transfected with HA-S100A11, HA-NLS-S100A11 and HA-NES-S100A11 compared with cells treated with HA. Furthermore, we noticed that the expression of mesenchymal markers (Vimentin, Fibronectin) and transcription factor Slug was significantly higher in cells transfected with HA-S100A11-, HA-NLS-S100A11-and HA-NES-S100A11-compared with cells transfected with HA. Expressions of p-smad2 and p-smad3 were also markedly increased in the treatment groups. These observations pointed that S100A11 both in the cytoplasm and nucleus could activate the classical TGFβ/Smad signaling pathway, which could be reversed by the knockdown of S100A11.5. The overexpression of S100A11 both in the cytoplasm and nucleus in vivo could promote tumor-forming capacity and the lung homing potential in CRC cells5.1 Construction and verification of SW480 stable cell lines with overexpressed S00A11We transfected SW480 cells with HA, HA-S100A11-, HA-NLS-S100A11-and HA-NES-S100A11-respectively and selected single colonies to culture enlargely by G418. Immunofluorescence examination revealed exogenous S100A11 in SW480/HA-S100A11 was located in the cytoplasm and nucleus, while exogenous S100A11 in SW480/HA-NLS-S100A11 was located in the nucleus and exogenous S100A11 in SW480/HA-NES-S100A11 was located in the cytoplasm. Western Bloting indicated we have successfully constructed stable cell line with overexpressed S100A11. We named these cells as SW480/HA, SW480/HA-S100A11, SW480/HA-NLS-S1OOA11 and SW480/HA-NES-S100A11. Immunoflurescence and immunoblot reveled that cells with stable S100A11 overexpression was successfully constructed by showing that when constructed nucleus-locating NLS signal, S100A11 could locate in the nucleus, when constructed with NES signal, S100A11 could located in the cytoplasm.5.2 The capability of overexpressed S100A11 in vitroThe results of CCK8 showed that cells with stable S100A11 overexpression (SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11) resulted in statistically significantly increasing the ability of growth compared with control (SW480/HA) (P<0.005). Stable subcelluar overexpression of S100A11 could promote the ability of proliferation in CRC cells. Transwell study showed that cells with stable S100A11 overexpression (SW480/HA-S100A11, S W480/H A-NLS-S100A11,S W480/HA-NES-S100A11) manifested significantly increased migration capability. Scratch test revealed that cells with stable S100A11 overexpression (SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11) significantly increased migrated capability.5.3 S100A11 overexpression in vivoSubcutaneous tumor formation test showed that cells with stable S100A11 overexpression (SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11) grew faster and has a larger volume (P<0.05) and exhibited higher Ki-67 index compared with control group(SW480/HA) (P<0.05, P<0.05, P<0.05). Tail vein injection in nude mice showed that cells with stable S100A11 overexpression (SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11) located in the lungs with higher capability and exhibited more lung tumor nodules compared with control group (SW480/HA) (P<0.05, P<0.05, P<0.05). IHC examination of subcutaneous tumors showed the expression of Fibronectin was increased and the expression of E-cadherin was decreased in cells with stable S100A11 overexpressed (SW480/HA-S100A11, SW480/HA-NLS-S100A11, SW480/HA-NES-S100A11) compared with control group (SW480/HA). HE observation revealed tumor necrosis significantly and blurred boundary with normal tissues.6. Preliminary investigation of the molecular mechanism of regulation in CRC metastasis thorough S100A116.1 Screening for molecules that interacted with S100A11HA antibody was used to combine the total protein of HA and the total protein was separated by SDS-PAGE. The experimental group was SW480/HA-NES-S100A11, SW480/HA-NLS-S100A11, SW480/HA-S100A11, and the control group was SW480/HA. SDS-PAGE electrophoresis and mass spectrometry combined with silver staining clearly were used to detect protein bands. Analysis showed mutiple protein bands. We preliminarily screened and identified Flotillin-1 and Histonehlt. S100A11 could pull-down Histone hit as a bait protein in HCT116 cells, SW620 cells and HA-NES-S100A11 SW480 cells. Reversely, Flotillin-1 could pull-down S100A11 as a bait protein in HCT116 cells, SW620 cells and HA-NES-S100A11 SW480 cells. Histone hit could pull-down S100A11 as a bait protein in HCT116 cells, SW620 cells and HA-NES-S100A11 SW480 cells. These results were re-confirmed by IF. Co-localization of S100A11 and Flotillin-1, Histone hit were observed in HCT116 and SW620. Immunoprecipitation and immunofluorescence validated the interaction between S100A11 and Flotillin-1, Histone hlt.6.2 Biological function of Flotillin-1 and Histone hltWe synthesised specific siRNA and constructed overexpressed vector of Flotillin-1 and Histone hit. Transwell experiments show that when Flotillin-1 and Histone hit were overexpressed in HCT116 cells, the capability of migration was significantly enhanced (P<0.05, P<0.05), and the classical TGFβ/Smad signaling pathway was activated. On the contrary, when interferenced the expression of Flotillin-1 and Histone hit in HCT116 cells, the migrated abilities were decreased significantly (P<0.05, P<0.05), and the classical TGFβ/Smad signaling pathway was suppressed.6.3 Flotillin-1 and Histone h1t were participated into inducing EMT in colorectal cancer by TGFβ/LASP1/S100A11In order to explicit the molecular mechanisms of S100A11 intercting with Flotillin-1 in cellular and Histone hit in nucleus respectively, we conducted the following recovery test. Groups of Control, S100A11, S100A11+si-Histone hit, and groups of Control, S100A11, S100A11+si-Flotillin-1, TGFβ+si-Flotillin-1, LASP1+si-Flotillin-1 were setted up respectively. Both Transwell and Western Bloting confirmd that weaken of Flotillin-1 and Histone hit could lead to the counteract effects of migration ability (P<0.05) and EMT in colorectal cells, which was mediated by TGF β/LASP1/S100A11.Conclusion:1. Colorectal cancer metastasis related protein(LASP1) positive regulates the expression of S100A11 by protein-protein interaction.2. S100A11 is essential for LASP1-mediated EMT and cell aggressive phenotype3. Both cytoplasmic and nuclear S100A11 is associated with poor prognostic phenotype of CRCs4. Exogenous S100A11 localized to cytoplasm and nucleus contributes aggressive phenotypes and induces EMT in vitro.5. Subcellular localization of endogenous S100A11 overexpression promotes CRC growth and progression in vivo6. FLOT1 and histone H1 are respectively downstream factors for cytoplasmic and nuclear pathway of S100A11... |
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