AXL and transforming growth factor β (TGF-β) are correlated with epithelial to mesenchymal transition, invasiveness, angiogenesis and immune modulation in colorectal cancer (CRC). We have previously demonstrated that targeting AXL caused a significant blockade of cancer cell proliferation and migration. Here we have evaluated the role of TGF-β signalling and the potential interaction between TGF- β and AXL in human CRC cell lines.
We assessed the expression and activation of TGF-β and AXL in a panel of human CRC cell lines (HCT116, SW480, LOVO, LIM 1215 and SW48) by Western Blot (WB) and Real time PCR. We tested the sensitivity of Galunisertib (LY21209761), a TGF-β R1 inhibitor, in HCT116 and LOVO cells the treatment by using MTT, soft-agar colony forming, cell invasion and wound healing assays. To study the correlation between these two pathways, we generated stable LOVO short hairpin RNA (shRNA)-sh-AXL cells clones, in which AXL expression was decreased, and stimulated both parental and shAXL LOVO cells with TGF-β1.
TGF-β receptors 1 and 2 were expressed in all cell lines, whereas AXL was expressed only in HCT116, SW480, LOVO cells. Treatment with Galunisertib had a modest effect on cancer cell growth, whereas it significantly decreased TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). The stimulation of HCT116 and LOVO cells with TGF-β1 resulted in increased levels of phosphorylated (p) AXL, pAKT, and p38 MAPK proteins. However, in contrast to parental LOVO cells, no increase in p38 MAPK was found in LOVO shAXL clones, upon TGF-β stimulation.
In HCT116 and LOVO cells, TGF-β mediated cell migration, invasion and soft agar colony growth formation were significantly inhibited by Galunisertib treatment. Furthermore, a functional potential cross talk between TGF-β induced signalling and AXL could converge on p38 MAPK activation. In this respect, combined treatments with Galunisertib and AXL inhibitors are ongoing to evaluate their antitumor effects in CRC cells.
Clinical trial identification
Legal entity responsible for the study
Dipartimento di Medicina di Precisione, Università degli Studi della Campania Luigi Vanvitelli.
AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG-2015-ID:7778.
All authors have declared no conflicts of interest.