Exogenous S100A6 Upregulates Its Own Expression In Colorectal Carcinoma Cells And Its Mechanism

Backgrounds and objectivesColorectal cancer(CRC) is one of the most common malignancies. An approximated population of one million is diagnosed with this fatal disease each year. Although much progress has been made, early diagnosis remains a problem to be solved. Therefore, it is crucial to elucidate the molecular mechanism of CRC development and to find more effective methods of diagnosis and treatment.S100A6(Calcyclin), a member of the S100 family, plays an important role in regulating cell proliferation, cell differentiation, and protein phosphorylation. It was observed that when exogenous S100A6 protein was used to treat tumor cells, the level of S100A6 in the cells also elevated. This suggests that S100A6 in the microenvironment may upregulate the level of S100A6 in tumor cells. Previous studies have identified a connection between S100A6 and β-catenin. These two proteins are often co-expressed in tumor cells due to the regulatory role of β-catenin. Interestingly, our recent studies have proved that this regulatory path goes both ways for the two proteins. Based on the evidence, we speculated that S100A6 in the microenvironment could possibly upregulate S100A6 expression via Wnt/β-catenin signal pathway in cancer cells.In this study, we verified our hypothesis by using CRC cell lines HCT116 and SW480 as models, and provided substantial evidence for illustrating the mechanism of CRC development and also provide potential means of CRC diagnosis and treatment.Methods1. The preparation and validation of recombinant hS100A6 protein(rh S100A6): the plasmids of pGST-ClvHRV3C-hS100A6 and pGST-Moluc-HRV3 C were transformed into E. coli BL21 bacteria by calcium chloride transformation, then cultivated in LB medium. Isopropyl-β-thiogalactoside(IPTG) was used to induce expression of the recombinant protein(GST-HRV3C-hS100A6) and then the bacteria were sonicated on ice. GST-HRV3C-hS100A6 were puried by glutathione-Sepharose 4B(GS4B) beads from bacteria lysate and digested by GST-HRV3 C overnight in 4℃. Then the GST tag and GST-HRV3 C were removed by GS4 B beads. Finally, rh S100A6 was harvested and identified by SDS-PAGE and Western blot, filtered via 0.22 μm membrane, quantified by spectrophotometry and stored at-80 ?C.2. The preparation and validation of recombinant GST-S100A6(GST-hS100A6) and GST protein: the plasmids of pGST- Moluc and p GST- Moluc- hS100A6 were transformed into E. coli BL21 bacteria by calcium chloride transformation and then cultivated in LB medium. Recombinant protein were induced by IPTG and bacteria were sonicated on ice, then recombinant protein were puried by GS4 B beads. Finally, protein was identified by SDS-PAGE and Western blot, filtered via 0.22 μm membrane, quantified by spectrophotometry and stored at-80 ?C.3. Amplification and identification of recombinant adenoviruses: Adβ-cat and Adsiβ-cat(they carry human β-catenin gene and β-catenin-siRNA gene, respectively), and their control adenoviruses(AdGFP and AdRFP), were amplified in human embryonic kidney cell 293(HEK293 cell). To identify the adenoviruses infection efficiency, CRC cell lines HCT116 and SW480 were infected with Adβ-cat and Adsiβ-cat, and then β-catenin expression in these cells was detected by RT-PCR and Western blot.4. Effect of rhS100A6 on S100A6 expression in human CRC cells and its mechanism:4-1 Effect of rhS100A6 on S100A6 expression in human CRC cells: HCT116, SW480 and FHC(normal colon mucosal epithelial cell line) were treated with rhS100A6(10 μg/ml) and then S100A6 mRNA level in these cells was detected by RT-PCR. Subsequently, CRC cells were treated with rhS100A6(10 μg/ml) or GST-hS100A6(100 μg/ml) and their S100A6 protein level was detected by Western blot.4-2 Effect of rhS100A6 on β-catenin expression in human CRC cells: After HCT116 and SW480 cells were treated with rhS100A6(10μg/ml), β-catenin mRNA level was detected by RT-PCR. Total β-catenin protein level, cytoplastic β-catenin protein and nuclear β-catenin protein level were detected by Western blot. The expression and distribution of β-catenin was detected by immunocytochemistry(ICC) staining and immunofluorescence staining.4-3 Effect of Wnt/β-catenin pathway on the expression of S100A6 mRNA by S100A6 in HCT116 and SW480 cells:(1) Verify that the Wnt/β-catenin pathway could modulate S100A6 expression: RT-PCR and Western blot were used to detect S100A6 expression level in HCT116 and SW480 cells infected with Adβ-cat or Adsiβ-cat.(2) Role of Wnt/β-catenin pathway in the upregulation of S100A6 mRNA by S100A6 in HCT116 and SW480 cells: There were five experimental groups: Blank group, AdRFP group, Adsiβ-cat group, rhS100A6 group and(Adsiβ-cat+rhS100A6) group. RT-PCR and qRT-PCR were applied to examine S100A6 mRNA level in each group.4-4 Effect of downregulating Wnt/β-catenin pathway on proliferation and migration of CRC cells enhanced by S100A6: the experimental groups were same as in method 4-3(2). After inhibiting the Wnt/β-catenin pathway by Adsiβ-cat, MTT, wound healing assay and transwell(without matrigel) assay were applied to detect the proliferation and migration of CRC cells.5. Exploration of the related mechanisms of β-catenin transportation to the nucleus enhanced by exogenous S100A6: HCT116 and SW480 cells were treated with rhS100A6(10μg/ml), Western blot was applied to detect the level of p-AKT(Ser473), p-AKT(Ser308), t-AKT, p-GSK3β and t-GSK3β.Results1. The molecular weight of recombiant protein rhS100A6, GST-hS100A6 and GST were all correct and rhS100A6, GST-hS100A6 were recognized by anti-S100A6 monoclonal antibody.2. β-catenin in HCT116 and SW480 cells was upregulated and downregulated after Adβ-cat and Adsiβ-cat infection, respectively.2. Exogenous S100A6 upregulated the expression of S100A6 in CRC cells and its mechanism involved activating the Wnt/β-catenin pathway:3-1 S100A6 mRNA level increased in HCT116 and SW480 cells after being treated with rhS100A6(P<0.01); however, there was no change in FHC cells(P>0.05). S100A6 protein level increased in HCT116 and SW480 cells after being treated with rhS100A6 or GST-hS100A6. These results suggested that exogenous S100A6 could upregulate its own expression in CRC cells(positive regulation of S100A6).3-2 β-catenin mRNA in HCT116 and SW480 cells after being treated with rhS100A6 had no obvious change(P>0.05), but both of the total β-catenin and nuclear β-catenin protein level elevated(P<0.05). The ICC and immunofluorescence staining showed that rh S100A6 treatment promoted β-catenin translocation from cytoplasm to nucleus in CRC cells. These results suggested that exogenous S100A6 protein could activate the Wnt/β-catenin pathway.3-3 S100A6 level in HCT116 and SW480 cells increased and decreased after infection with Adβ-cat and Adsiβ-cat, respectively. The results revealed that Wnt/β-catenin pathway takes part in modulating S100A6 expression in CRC cells, namely, S100A6 is one of the target genes of Wnt/β-catenin pathway.3-4 Compared with rhS100A6 treatment group, the level of S100A6 mRNA decreased in co-treatment group by Adsiβ-cat and rhS100A6 protein(P<0.01). The result suggested that the role of rhS100A6 in promoting S100A6 mRNA in HCT116 and SW480 cells can be partly reversed by Adsiβ-cat infection, which can downregulate β-catenin and therefore inhibit the Wnt/β-catenin pathway.3-5 Cell proliferation and migration in the co-treatment group by Adsiβ-cat and rhS100A6 protein were weaker than that of the rhS100A6 treatment group(P<0.01), which indicated that the proliferation and migration of CRC cells were enhanced by S100A6, but this effect was significantly inhibited when the Wnt/β-catenin pathway was inhibited. This result also provides important support for our conclusion, namely, Wnt/β-catenin pathway is involved in the positive regulation of S100A6 in CRC cells. So, downregulation of the pathway can partly inhibit S100A6 positive regulation and therefore inhibit the proliferation and migration of CRC cells enhanced by S100A6 to some extent.4. Protein levels of p-AKT(Ser473) and p-GSK3β all increased in CRC cells treated with rhS100A6(P<0.01). The results suggested that the mechanism that exogenous S100A6 promotes β-catenin transportation to the nucleus may be involved in the activation of AKT signal pathway.Conclusion1. S100A6 in the microenvironment upregulates its own expression in CRC cells( positive regulation of S100A6) and its mechanism was involved in the activation of Wnt/β-catenin pathway.2. The mechanism that S100A6 in the microenvironment promotes β-catenin transportation to the nucleus may be involved in the activation of AKT signal pathway.