Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion

<div><p>Objectives</p><p>To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines.</p><p>Materials and methods</p><p>An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA</p><p>Results</p><p>Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches.</p><p>Conclusion</p><p>The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.</p></div

Autophagy is the main mechanism of cell death associated to Sorafenib.

<p>a) Caspase 3/7 activity was evaluated in ACHN cells treated with Sorafenib for 24 hours. b) Viability of ACHN cells was evaluated under the same conditions by MTT assay. c) ACHN cells were exposed to 10 μM Sorafenib for 16 hours. Protein extracts were blotted with the indicated antibodies. d) Caspase 3/7 activity was evaluated in 786-O cells treated with Sorafenib for 24 hours. e) Viability of 786-O cells was evaluated by MTT assay as in B). f) 786-O cells were exposed to 10 μM Sorafenib for 16 hours. Protein extracts were blotted with the indicated antibodies. Tubulin was used as a loading control.</p

Sorafenib toxicity is not related to MAPK.

<p>a) ACHN and 786-O cells were treated for 48 h at the indicated concentrations of Sorafenib and viability was assessed by MTT assay. Black bars indicate ACHN and white bars indicate 786-O. b) ACHN and c) 786-O cells were exposed to 10 μM Sorafenib for 16 hours. Protein extracts were blotted against with the indicated antibodies. Tubulin was used as a loading control. d) Cells were treated for 48 h with 10 μM U0126, 10 μM PD98059 or 10 μM SB203580. Viability was measured by MTT assay. Black bars indicate ACHN and white bars indicate 786-O. e) ACHN and 786-O cells were treated with Sorafenib (10 μM) in combination with the indicated inhibitors (10 μM each) for 48 h. Viability was measured by MTT assay. Black bars indicate ACHN and white bars indicate 786-O. Densitometric quantification of the signals on the Western blots is shown for each picture (fold active/total) below each group.</p

Autophagy mediates cells death associated to Akt inhibition.

<p>a) ACHN cells were treated with MK-2206 at the indicated concentrations (μM) for 24 hours and caspase 3/7 activity was evaluated (Left panel). Cell viability was evaluated under the same conditions by MTT assay (Right panel). b) Caspase 3/7 activity (Left panel) and cell viability (right panel) were evaluated in 786-O cells as indicated in A). c) Cells were exposed to 10 μM MK-2206 for 16 hours. Protein extracts were blotted with the indicated antibodies. Tubulin was used as a loading control.</p

Blockage of autophagy promotes resistance to Sorafenib in ACHN cells.

<p>a)ACHN cells were treated with Sorafenib at the indicated concentrations in the presence or absence of 2.5 mM 3-M.A. Viability was assessed by MTT assay after 48 hours. b) ACHN cells were treated with Sorafenib at 10 μM in the presence or absence of 2.5 mM 3-M.A for 16 hours. The protein extracts were collected and blotted with the indicated antibodies. Tubulin was used as a loading control. c) ACHN cells were infected with lentivirus carrying empty vector or a specific shRNA against ATG5. Selected pools were lysed and protein extracts were blotted with the indicated antibodies. Tubulin was used as a loading control. d) ACHN cells that were infected with lentivirus carrying empty vector (black bars) or a specific shRNA against ATG5 (grey bars) were treated with Sorafenib for 48 hours at the indicated concentrations and viability was assessed by MTT assay.</p