Fig 1.
Increased expression of AXL, but not MERTK or TYRO3, in late state colorectal cancer.
(A) Colorectal cancers stratified based on total expression of TAM RTKs from TCGA Illumina Hi-Seq RNA-seq data. Abundance of TYRO3, AXL and MERTK in each tumor (an individual bar) is represented by red, green and blue segments as indicated. (B) Abundance of AXL, MERTK and TYRO3 in colon and rectal adenocarcinomas at the indicated stages based on RNA-seq data from TCGA data sets. Cohen’s d for change in AXL expression between Stage I versus Stage III colorectal cancer is 0.52 (moderate effect size) and between Stage I and Stage IV is 0.54 (moderate effect size). (C) Expression of AXL, MERTK and TYRO3 in colorectal adenocarcinomas at the indicated stages, as detected by RT-qPCR. Data are presented as individual samples, mean ± SEM per tumor stage. * p<0.05; n.s., non significant.
Fig 2.
Expression of AXL correlates with the expression of cell migration-associated genes.
Correlation between the expression of selected cell migration-associated genes, Integrins alpha 1, 5, 7 and 11, Integrins beta 1, 2 and 3, E-selectin, VCAM1, ICAM1, neuropilin 1 and 2 and ZEB1 and 2, and AXL, MERTK and TYRO3 in TCGA. Strong Pearson correlation values > 0.7 are indicated in bold, while moderate Pearson correlation values (>0.5—<0.69) are underlined and in italics.
Fig 3.
Silencing or inhibition of AXL kinase activity inhibits migration and invasion of human colon cancer cells.
(A) Representative images and (B) relative cell migration in a Boyden chamber assay of RKO-AS45-1 and HCT116 cells transfected with scrambled or AXL siRNAs. (C) Representative images and (D) relative cell invasion in a Matrigel-coated Boyden chamber assay of RKO-AS45-1 and HCT116 cells transfected with scrambled or AXL siRNAs. (E) Representative images and (F) relative cell migration in a scratch wound healing assay of RKO-AS45-1 treated with the indicated concentrations of the small molecule AXL inhibitor, R428 after 24 or 48h. (G) Representative images and (H) relative cell migration in a scratch wound healing assay of HCT116 cultured in the presence of the indicated concentrations of the small molecule AXL inhibitor, R428 after 24 or 48h. Data is presented as representative samples or as mean ± SEM of 3 independent experiments, * p<0.05; ** p<0.01; *** p<0.001; n.s., non significant.
Fig 4.
Silencing AXL in human colon cancer cells does not decrease proliferation.
(A) Percentage of EdU incorporation normalized to scrambled siRNA control, in the indicated cell types transfected with scrambled or AXL siRNAs. (B) Representative FACS cell cycle analysis and (C) proportion of control siRNA or AXL siRNA-treated RKO-AS45-1 cells in G1, S and G2/M. (D) Representative FACS cell cycle analysis and (E) percentage of HCT116 cells at the indicated stages of the cell cycle after scrambled or AXL siRNAs transfection. (F) Percentage of viable RKO-AS45-1 and HCT116 after scrambled or AXL siRNAs transfection, as measured by Alamar Blue cell viability assay. (G) Representative FACS plots and (H) fold change in Annexin V+/Propidium Iodide (PI)- and Annexin V+/(PI)+ RKO-AS45-1 cells after scrambled or AXL siRNAs transfection. (I) Representative FACS plots and (J) fold change in Annexin V+/Propidium Iodide (PI)- and Annexin V+/(PI)+ HCT116 cells after scrambled or AXL siRNAs transfection. Data is presented as representative samples or as mean ± SEM of 3 independent experiments, * p<0.05; n.s., non significant.