Functional characterization of mutant ALK5 (TGFBR1) in endometrial cancer

Authors

• E YU
• DW BELL

Abstract

ALK5 (TGFBR1) is a druggable receptor serine/threonine kinase that transduces TGF-β (Transforming Growth Factor β) signaling to activate SMAD2/3-dependent and -independent pathways. ALK5 is mutated in a subset of endometrial cancers (ECs). We aim to functionally characterize a somatic mutation that occurs at a highly conserved kinase domain residue of ALK5 in EC. Our in silico analyses using nine algorithms predicted that this mutation will impact protein function. To test this prediction experimentally, constructs expressing wildtype-, constitutively active-, kinase-dead­, or EC-mutant-ALK5 were transfected into NIH/3T3 cells, which have low endogenous ALK5 levels. In response to TGF-β1 stimulation, transient exogenous expression of the EC-mutant-ALK5 showed an increase in canonical SMAD2/3-dependent kinase activity in a luciferase assay and resulted in increased cell survival. We used CRISPR-Cas9 editing to knock in the EC-ALK5 mutation into an ALK5-wildtype EC cell line, and showed that the mutation knockin cells displayed increased migration, in a transwell assay. Interestingly, the mutation knockin cells exhibited neomorphic ligand responsiveness to Activin A compared to the mock CRISPR-edited cell line, increased phosphorylation levels of SMAD3 as well as of non-canonical SMAD1/5/9. To further investigate molecular signaling altered by the mutant protein, we treated the isogenic EC cell lines with either Activin A or vehicle and assessed the phosphorylation level of proteins associated with the TGF-β signaling pathway on a protein microarray and showed alterations in mitogen-activated protein kinases (MAPKs) in the mutant cells. In conclusion, our findings provide novel perspectives on mutation of the ALK5 kinase domain in EC.

Scientific Focus Area: Cancer Biology

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