Senescence is a cellular process that arrests the cell cycle while maintaining active metabolism and transcription, thereby recruiting immune cells and upregulating ligands for sensitizing senescent cancer cells to clearance by natural killer (NK) cells. However, there are some barriers that prevent senescence induction or change senescence function by inducing inflammation, that in turn promote tumor growth and progression. Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in inflammation and tumorigenesis of the colon. Nonetheless, role of ILK in senescence induction in colorectal cancer (CRC) is still unclear. The aim of present study is to investigate the role of ILK in CRC cell lines growth and senescence induction.
A doxycycline-inducible CRISPR/Cas9 specific for ILK gene was utilized here to generate ILK-deleted CRC lines RKO and HT29 (ILK KO). Cell growth was examined in 2D culture by counting viable cells using trypan blue stain as well as in soft agar 3D culture by measuring colonies areas. Senescence was induced by treating cells with doxorubicin and senescence markers such as p21 expression and senescence associated-β-galactosidase (SA-β-gal) staining were investigated. Protein expression was examined by western blot. Also, the HT29 KO cells have been transfected with ILK WT and ILK mutant (L207W) plasmid DNA to investigate the effect on p21 expression.
The results overall showed that ILK regulates cell growth and senescence in a cell specific manner. The inducible CRISPR/Cas9-mediated ILK KO inhibited ILK protein expression significantly in the CRC cell lines. Consequently, this inhibition in HT29 cells showed a slight reduction on the growth in 2D and 3D culture whereas ILK KO in RKO cells displayed a significant reduction in 3D culture. On the other hand, ILK KO in HT29 cells increased senescence at the basal level and by doxorubicin induction as indicated by increasing SA-β-gal positive cells and p21 expression. Then, the ILK WT transfection into HT29 KO cells rescued the phenotype of p21 expression.
Our results uncover a potential role for ILK in senescence indicating that targeting ILK in CRC could be effective to slow down tumor growth and promote senescence induction enabling recruitment of anti-tumor immune cells into the tumor microenvironment.