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Transforming growth factor beta receptor (TGF&bgr;R) pathway is involved in ligand independent transactivation of AXL receptor in colorectal cancer (CRC) cell lines

Date

10 Oct 2016

Session

Poster display

Presenters

Elisena Franzese

Citation

Annals of Oncology (2016) 27 (6): 1-14. 10.1093/annonc/mdw362

Authors

E. Franzese, G. Martini, C. Cardone, T. Troiani, V. Sforza, M.L. Ferrara, V. Belli, P.P. Vitiello, S. Napolitano, N. Zanaletti, P. Vitale, F. De Vita, M. Orditura, F. Morgillo, F. Ciardiello, E. Martinelli

Author affiliations

  • Dipartimento Medico-chirurgico Di Internistica Clinica E Sperimentale, AOU Seconda Università degli Studi di Napoli (AOU-SUN), 80131 - Napoli/IT
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Background

It has been described that AXL,a tyrosine kinase receptor, can be activated through different mechanisms: GAS6-ligand dependent dimerization and ligand-independent activation. As we previously demonstrated the absence of GAS6 in a panel of CRC cell lines, we tried to investigate the possible mechanisms of AXL activation in our in vitro CRC model.

Methods

We used a panel of CRC cell lines (HCT116, SW480, SW620 and LOVO). Expression and activation of AXL and its ligand GAS6 were analyzed by Western Blot (WB) and real time-PCR (RTPCR). We generated, in our laboratory, stable (shRNA)-sh-AXL LOVO cells clones (shAXL clone 1, shAXL clone3 and shAXL clone 5) to evaluate the effect of AXL specific knockdown. GAS6, TGF-ß1 levels were measured by ELISA assay.

Results

TGF-ß1 was detected at variable levels in AXL expressing CRC cell lines (HCT116, SW480, SW620 and LOVO). We stimulated CRC cells with TGF-ß1 finding increased levels of pAXL, pAKT and pMAPK by WB. To reveal how TGF-ß1 actives AXL we measured the mRNA levels of AXL or GAS6 by RT-PCR after TGF-ß1 24h stimuli. No fold mRNA increased was observed, suggesting a different interaction between AXL and TGFß pathway. To further correlate TGFßR pathway with AXL transactivation, we stimulated LOVO cell line, shAXL clone1, 3 and 5 with TGF-ß1 for 24 hrs. An increased activation of p38MAPK in LOVO cells was demonstrated, accompanied by no change in shAXL clones, suggesting that, at least in our model, the downstream protein p38 MAPK is strictly correlated with AXL transactivation stimulated by TGF-ß1. Further experiments with p38MAPK inhibitor are currently ongoing.

Conclusions

Our findings suggested a relationship between TGF-b1, p38 MAPK and AXL as a possible mechanism of AXL independent ligand activation. Further investigations are ongoing.

Clinical trial identification

Legal entity responsible for the study

Second University of Naples

Funding

Second University of Naples

Disclosure

All authors have declared no conflicts of interest.

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