Functional characterization of ALK5 (TGFBR1) mutations in endometrial cancer

Authors

• M Zamba-Campero
• D Tetreault
• E Petroni
• D Soliman
• J Silberman
• R Fishman
• P Adams

Abstract

ALK5 (TGFBR1) is a transmembrane receptor serine/threonine kinase that transduces TGF-β(Transforming Growth Factor β) signaling to activate SMAD2/3-dependent and -independent pathways. The purpose of this study is to determine the functional effects of ALK5 mutations in endometrial cancer (EC). Using nine in silico algorithms, we found that 79% (23 of 39) of ALK5 kinase domain mutations in EC are predicted to impact protein function. To test these predictions experimentally, constructs expressing wildtype-, constitutively active-, kinase-dead­, or mutant-ALK5, including ALK5-A230V, were transfected into NIH3T3 cells, which have low endogenous ALK5 levels. Following TGF-β stimulation, we observed that transient exogenous expression of ALK5-A230V, located in the ATP-binding pocket, delayed SMAD2/3 signal transduction and altered SMAD-independent signaling. We further showed that the ALK5-A230V mutant has reduced protein stability via a ubiquitin-dependent protein degradation mechanism. Our structural modeling predicts that SB431542, a small molecule ATP-competitive inhibitor of ALK5 will bind to the ALK5-A230V mutant with less affinity than to wildtype ALK5. We, therefore, examined the inhibitory effect of SB431542 on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay in combination with TGF-β stimulation. SBE luciferase activity in ALK5-A230V-transfected cells was inhibited less by SB431542 than in wildtype-ALK5 transfected cells indicating that ALK5-A230V is less sensitive to SB431542 than wildtype ALK5, potentially due to the changes in affinity. Our findings are novel and show that the ALK5-A230V mutant is a partial loss-of-function mutant that attenuates TGF-β signal transduction and has reduced sensitivity to an ALK5 small molecule inhibitor.

Scientific Focus Area: Cancer Biology

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