GAS6 is highly expressed in the thyroid carcinoma cell lines.
GAS6 was differentially expressed in the thyroid carcinoma cell lines, and GAS6 was highly expressed in cancer tissues. In comparison to other tumors, GAS6 expression was significantly higher in papillary thyroid Carcinoma (Figure.1A and 1B). In a recent study, the survival curve drawn based on the TCGA data predicted that the up-regulation of GAS6 was closely associated with the unproductive diagnosis in the thyroid tumor (19). Herein, the survival rate was predicted as lower in cancer cells with the lower expression of GAS6 according to the TCGA data (Figure.1C). RT-qPCR and western blot results confirmed the differential expression of GAS6 in the four thyroid cancer cell lines including CAL62, 850-5C, NIM, and OCUT-1 at mRNA and protein levels, respectively. GAS6 exhibited the highest expression in CAL62 cell lines, up to two folds higher than that of 850-5C, which exhibited the lowest expression (Figure.1D). These TCGA database results confirmed that GAS6 could play potential roles in thyroid carcinoma evolution.
Overexpression of GAS6 enhanced proliferation of CAL62 cells.
First of all, 850-C5 (which initially displayed the lowest expression GAS6) and CAL62 (which initially had the highest expression GAS6) thyroid were transfected with a GAS6 over-expressive vector (pcDNA3.1-GAS6) and GAS6 inhibitor (siGAS6), respectively. Then, the expression characterization of GAS6 in the thyroid cancer cells was carried out using both qPCR and western blotting. The results showed that compared to normal cell lines Nthy-ori3-1 (control), the expression of GAS6 was almost four folds higher in cells under pcDNA3.1-GAS6 treatment and less than three folds downregulated in siGAS6 cells (Figure.2A). Moreover, the MTT assay revealed that overexpression of GAS6 lightly promoted thyroid cancer cell viabilities while its inhibition suddenly dropped down the cell viability rate at more than two folds smaller than that of the control groups (Figure.2B).
GAS6 expression regulates PI3K and p-Akt protein expression in 850-05 and CAL62 cancer cells.
The PI3K/Akt pathway is well known for its dominant role in thyroid carcinomas (20). Given the evidence for GAS6 implication in cell growth and development, we evaluated the effect of GAS6 on the PI3K/Akt pathway using RT-qPCR and western blot analysis in different groups. We found that GAS6 mRNA expression significantly diminished in all siGAS6 groups when compared to control cells while treatment with GAS6 overexpression vectors improved the protein mRNA of GAS6 850-C5 cells. Moreover, western blot assessments showed that the protein expression of GAS6 was suddenly lowered in CAL62 cells under siGAS6 treatment but upregulated in 850-C5-GAS6 overexpression cells. On the other hand, the expression levels of PI3K/Akt pathway biomarkers (PI3K AKT and p-Akt), were well improved in 850-C5-GAS6 overexpression cells when compared to normal cells, but greatly reduced when GAS6 was inhibited (Figure 3A and 3B). This section of findings demonstrated that modification of GAS6 expressions directly affects the PI3K/Akt signaling pathway in thyroid cancer cells.
XL184 inhibits the protein expressions of PI3K and p-Akt in 850-05 and CAL62 cancer cells.
Previously described as a potent inhibitor of VEGFR2 (IC50=0.035 nmol/L), MET (IC50=1.3 nmol/L), and RET (IC50=5.2 nmol/L), XL184 can also inhibit both GAS6-AXL and HGF-MET pathways in cancer cells (21). Given the evidence for the co-expression of GAS6 and AXL in thyroid cancer cells, we performed a second Western bolt assay to characterize the effects of XL184 treatments on the PI3K/AKT signaling pathway. We found that the protein expressions of PI3K, AKT, and p-Akt in CAL62 cancer cells were also downregulated (Figure 4A). Moreover, the MTT assay showed that the viability rate of Cal62 cells was markedly also reduced in cells treated with XL184 (Figure 4B). These results were almost similar to those gotten from the siGAS6 groups, suggesting that GAS6 might play essential roles in key processes of thyroid cancer growth. Thus, to confirm the interaction between GAS6 with AXL in thyroid cancer cells, cells were co-transfected FLAG-tagged AXL with or without HA-tagged GAS6. Co-IP assay with HA flag antibodies showed that GAS6-HA could interact with AXL protein (Figure 4C)
GAS6 mediated the apoptosis of CAL62 cells
flow cytometry and TUNEL assays were monitored to examine apoptotic events in transfected cells. The flow cytometry assay was carried out in order to evaluate the differential levels of transfected cells apoptosis. According to the flow cytometry results, compared with human papillary thyroid cancer cells transfected with empty vector, CAL62 cells under siGAS6 and XL184 treatments had stronger apoptotic abilities, while GAS6 overexpression suppressed apoptosis of CAL62 cells when compared with the control groups (Figure.5A). In addition, we TUNEL assay was used to visualize apoptotic cells and the results also confirmed that the apoptotic rates of GAS6 overexpression cells were strongly reduced in comparison with those of the control groups and significantly enhanced in siGAS6 and XL184 groups (Figure. 5B). According to these results, GAS6 inhibition promotes the apoptosis of thyroid cancer cells by activating the PI3K/Akt pathway.
GAS6 mediates the proliferation and migration of CAL62 cells through the PI3K/Akt pathway
Both Transwell and wound healing experiments were used to determine the cell migration abilities of CAL62 cells treated with a GAS6 over-expressive vector (pcDNA3.1-GAS6), GAS6 inhibitor (siGAS6), and XL184, respectively. The Transwell assay results showed that compared with CAL62 cells transfected with empty vector, GAS6 overexpression (pcDNA3.1-GAS6) promotes the cells migration, while inhibition of GAS6 expression (si-GAS6 group) and XL184 treatment slows down the migration of human papillary thyroid cancer cells (Figure.6A). In addition, the wound healing results showed that compared with the control group, the migration abilities of both siGAS6 and XL184 groups were significantly reduced. Then, the protein level of PI3K, AKT, and the phosphorylation of AKT (p-AKT) was greatly decreased in the CAL62 cells treated with siGAS6. On contrary, GAS6 overexpression cells (pcDNA3.1-GAS6 groups) exhibited accelerated rates of migration when compared with the control group (Figure.6B).