COLO-678

ALS Modulates CRC Cell Proliferation and the Active Form of RAS

To determine the effects of Aurora kinases A (AURKA) inhibition on RAS signal output, the effects of alisertib (ALS) on cell viability were examined against a panel of colorectal cancer (CRC) cell lines bearing Kirsten rat sarcoma virus (KRAS) WT, G12D, G12V, G13D, A146T, and BRAF V600E mutations. The KRAS G13D-expressing cell line, HCT116, was the most susceptible to ALS treatment over 48 and 96 h (Fig. 1A and B). Colo-678_{KRAS G12D}, SK-CO-1_{KRAS G12V}, and CCCL-18_{KRAS A146T} were also susceptible but to a lesser extent. KRAS WT and BRAF V600E-expressing cell lines exhibited a moderate response to ALS treatment (Fig. 1A and B). Subsequently, the RAS-GTP level was evaluated following the treatment of cells with ALS. RAF1-RBD was used to pull down the active form of RAS from Caco-2_{KRAS WT}, Colo-678_{KRAS G12D}, SK-CO-1_{KRAS G12V}, HCT116_{KRAS G13D}, CCCL-18_{KRAS A146T} and HT29_{BRAF V600E} cells. In KRAS WT-expressing Caco-2 cells, ALS increased the level of RAS-GTP in a concentration-dependent manner (Fig. 2). In KRAS mutant-expressing cells, ALS increased RAS-GTP level in SK-CO-1_{KRAS G12V}, HCT116_{KRAS G13D} and CCCL-18_{KRAS A146T} cells, whereas ALS decreased the RAS-GTP level in Colo-678_{KRAS G12D} cells. Notably, in HT29 cells expressing BRAF V600E, a constitutively active BRAF mutant commonly found in CRC that does not rely on RAS activation to activate the RAS-RAF-MEK-ERK signal, ALS decreased the level of the active form of RAS. In combination, these results suggested that the inhibition of AURKA exerts different inhibitory effects on cell proliferation and regulates the active form of RAS in a KRAS allele-specific manner.

Fig. 1 ALS differentially inhibits the proliferation of CRC cells. (Ren B, et al., 2023)

Fig. 2 ALS modulates RAS-GTP level in an RAS allele-specific manner in colorectal cancer cell lines. (Ren B, et al., 2023)

CM from CAFs Exposed to TNF-α Increase Migration of Colo-678 Cells

IL-8 has been previously described to promote the migration of colon cancer cells and endothelial cells in vitro. The increased IL-8 protein levels in cell culture supernatants of cancer-associated stromal fibroblasts (CAFs) exposed to 10 ng/ml TNF-α would increase the migration of these cells in comparison with conditioned medium (CM) of nonstimulated CAFs. As shown in Figure 3, Colo-678 cells showed increased migration toward CM from CAFs incubated with TNF-α in comparison with CM of CAFs that were incubated in 0.5% FBS for the same period. Blockage of IL-8 by neutralizing antibody showed a mild, but not significant, inhibitory effect (Fig. 3C). This lack of evidence was followed for the role of IL-8 as a chemoattractant of the tested cell lines by investigating their migration toward increasing concentrations of IL-8 in the lower chamber. Among the tested cell lines, these experiments revealed only slight increases in migratory activity in Colo-678 cells and HUVECs (data not shown). For the concentration-dependent increases, the lack of responsiveness to IL-8 in our Boyden system strongly indicates that CAFs release other as yet unidentified chemoattractants.

Fig. 3 Effect of TNF-α on chemotactic properties of CAFs. The migration of HuH7 hepatoma cells (A), DLD1 colon carcinoma cells (B), Colo-678 colon carcinoma cells (C), and HUVECs (D) toward the conditioned medium of CAFs that were incubated for 24 hours in 10 ng/ml TNF-α [CAF-CM (TNF)] and toward CM of nonstimulated CAFs (CAF-CM) were compared. (Mueller L, et al., 2007)