FLOT1 promotes gastric cancer progression and metastasis through BCAR1/ERK signaling

Figures

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  Figure 1. FLOT1 promotes proliferation, migration and invasion in gastric cancer cells. A. Western blotting for FLOT1 expression level in GES-1 and cultured GC cell lines. B. Western blotting for FLOT1 expression level in FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines. C. Cell proliferation assay for FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines. D. Colony formation assay and quantification data for FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines. E. Transwell invasion assay and quantification data for for FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines. F. Wound-healing assay and quantification data for FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines at 0 and 24h after wound scratch.

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  Figure 2. FLOT1 promotes tumorigenesis in mouse model. A-J. FLOT1-OE HGC-27 cells or FLOT1-KD AGS cells and the control cell lines were inoculated subcutaneously on the back of male NOD-SCID mice. (C,E) Tumors were measured and tumor volume were calculated on the indicated days. 36 or 24 days after inoculation, mice were sacrificed and tumors were collected (A,B) and weighted (D,F). The expression of FLOT1 in tumors was examined by western blotting (G,H). Ki-67 in mouse xenograft tumors were examined by Immunohistochemical staining (I,J).

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  Figure 3. FLOT1 promotes metastasis in mouse model. A-F. FLOT1-OE HGC-27 cells or FLOT1-KD AGS cells and the control cell lines were injected into NOD-SCID mice through tail vein. 30(FLOT1-OE and control) or 60(FLOT1-KD and control) days later before sacrificed, mice were scanned by 18F-FDG PET-CT to check lung metastatic nodules (A,B) and the uptake rates of 18F-FDG (tumor/muscle ratio) were collected and compared (C-D). (E-F) Representative images of lungs and hematoxylin and eosin staining for paraffin-embedded lung sections. Arrows indicate lung metastatic nodules.

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  Figure 4. FLOT1 interacts with BCAR1 and regulates BCAR1 phosphorylation and translocation. A. FLOT1 co-immunoprecipitation with BCAR1(left) or BCAR1 co-immunoprecipitation with FLOT1(right) in AGS and HGC-27 cells. B. Western blotting for the expression of indicated proteins in FLOT1-OE HGC-27 cell lines or FLOT1-KD AGS cell lines and the control cell lines C. Confocal microscopy imaging analysis for the colocalization and translocation of exogenous pDsRed2-N1-FLOT1 and p-EGFP-C1-BCAR1 in 293T cell.

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  Figure 5. BCAR1 plays oncogenic role in gastric cancer. A. Western blotting for BCAR1 expression level in BCAR1-OE or KD HGC-27 cell lines and the control cell lines. B. Cell proliferation assay for the BCAR1-OE and control HGC cells (left) or BCAR1-KD and control HGC-27 cells(right). C. Colony formation assay and quantification data for BCAR1-OE or KD HGC-27 cell lines and the control cell lines. D. Wound-healing assay for BCAR1-OE or KD HGC-27 cell lines and the control cell lines at 0 and 48h after wound scratch. E. Transwell invasion assay for BCAR1-OE or KD HGC-27 cell lines and the control cell lines. F-K. BCAR1-OE or KD HGC-27 cell lines and the control cell lines were inoculated subcutaneously into NOD-SCID mice. (G,J) Tumors were measured and tumor volumes were calculated on the indicated days. 32 or 28 days after inoculation, mice were sacrificed and tumors were collected (F,I) and weighted (H,K).

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  Figure 6. BCAR1 mediates FLOT1 caused gastric cancer cell proliferation, migration and invasion through ERK signaling. A. Colony formation assay and quantification data for BCAR1- KD and control HGC-27 cells transfected with FLOT1 overexpression or control plasmid. B. Wound-healing assay for BCAR1- KD and control HGC-27 cells transfected with FLOT1 overexpression or control plasmids at 0 and 48h after scratching. C. Trans-well invasion assay for BCAR1- KD and control HGC-27 cells transfected with FLOT1 overexpression or control plasmids. D. Western blotting for the expression of indicated proteins in BCAR1-OE or KD and the control cell lines in AGS (left) and HGC-27 (right) cell. E. Western blotting for the expression of indicated proteins in BCAR1- KD and control HGC-27 cells transfected with FLOT1 overexpression or control plasmid.

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  Figure 7. Phosphorylation of BCAR1 on Tyr410 is essential for FLOT1 induced GC cell migration and invasion. A. Colony formation assay and quantification data for FLOT1- KD AGS cell transfected with BCAR1-WT, BCAR1-MT or control plasmids and the control AGS cells. B. Wound-healing assay and quantification data for FLOT1- KD AGS cell transfected with BCAR1-WT, BCAR1-MT or control plasmids and the control AGS cells at 0 and 48h after scratching. C. Trans-well invasion assay for and quantification data for FLOT1- KD AGS cell transfected with BCAR1-WT, BCAR1-MT or control plasmids and the control AGS cells.

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  Figure 8. FLOT1/BCAR1/ERK signaling is closely correlated with human gastric cancer. A. Representative images of the IHC staining for FLOT1 and BCAR1 expression level in human gastric cancer tissues and paired adjacent normal gastric mucosa. B. The correlation between FLOT1 and BCAR1 expression in human gastric cancer tissues. C. The correlation between FLOT1 and Ki67 expression in human gastric cancer tissues. D. The correlation between BCAR1 and Ki67 expression in human gastric cancer tissues. E. The correlation between FLOT1 and p-ERK1/2 expression in human gastric cancer tissues. F. The correlation between BCAR1 and p-ERK1/2 expression in human gastric cancer tissues. Kaplan-Meier survival analysis showed the correlation between overall survival rate and FLOT1 expression. G. IHC staining score of FLOT1(left panel) and BCAR1(right panel) in gastric cancer tissues and adjacent normal mucosa (P < 0.0001). H. Kaplan-Meier survival analysis for the correlation of overall survival rate and FLOT1 expression. I. Kaplan-Meier survival analysis for the correlation of overall survival rate and BCAR1 expression. J. Comparison of FLOT1 mRNA expression in gastric cancer tissues (n = 414) and normal…