Daphne W. Bell

Proteomic Changes Associated With Endogenous FBXW7 Mutations in Moderately Differentiated Endometrial Cancer Cells Include Increased TROP2 and Galectin‐3 Levels

ABSTRACTBackgroundEndometrial cancer (EC) is the fourth most commonly diagnosed cancer among women in the US and the fifth leading cause of cancer death in this population. The FBXW7 tumor suppressor gene is frequently mutated in all molecular subtypes of EC. The encoded protein is part of a ubiquitin ligase complex that targets substrate proteins for ubiquitination and, in most instances, proteasome‐mediated degradation.AimsThe purpose of this investigation was to identify the proteomic changes associated with endogenous FBXW7 mutations in EC.Materials & MethodsQuantitative LC–MS/MS was used to identify significant (p < 0.05) differences in the proteomes and phosphoproteomes of two FBXW7‐mutated EC cell lines, HEC‐1‐BFBXW7−R367X and JHUEM‐1FBXW7−R505C, as compared to isogenic mutation‐corrected cell lines. Western blotting was performed to orthogonally validate a subset of protein changes.ResultsAnalysis of LC–MS/MS results identified 397 total proteins and/or phosphoproteins with significantly different levels in both HEC‐1‐BFBXW7−R367X and JHUEM‐1FBXW7−R505C, as compared to isogenic mutation‐corrected cell lines. This protein set included increased levels of TROP2, galectin‐3, ASS1, and PLCG2 in both HEC‐1‐BFBXW7−R367X and JHUEM‐1FBXW7−R505C cells; these perturbations orthogonally validated by western blotting.ConclusionThis study provides novel insights into the proteomic and phosphoproteomic effects of the endogenous FBXW7−R367X and FBXW7−R505C mutations in EC cells, including increased levels of galectin‐3, a potentially druggable target, and of TROP2, which is a druggable target in EC.

The endometrial cancer A230V-ALK5 (TGFBR1) mutant attenuates TGF-β signaling and exhibits reduced in vitro sensitivity to ALK5 inhibitors

The ALK5 (TGFBR1) receptor serine/threonine kinase transduces TGF-β (Transforming Growth Factor beta) signaling to activate SMAD2/3-dependent and -independent pathways. Here, we aimed to determine the functional consequences of ALK5 mutations in human endometrial cancer (EC). Somatic mutation data were retrieved from publicly available databases. Using seven in silico algorithms, 78.5% (11 of 14) of ALK5 kinase domain mutations in EC, including A230V-ALK5, were predicted to impact protein function. For in vitro studies, we focused on A230V-ALK5 because it was the only mutated residue located within the ATP-binding pocket, which is an important region for both ATP-binding and binding of ATP-competitive inhibitors. Constructs expressing wildtype-, constitutively-active-, kinase-dead-, or mutant A230V-ALK5, were transfected into NIH/3T3 cells. Following TGF-β1 stimulation, transient exogenous expression of A230V-ALK5 resulted in attenuated SMAD2/3 signal transduction and reduced AKT activation. We further showed that the A230V-ALK5 mutant had reduced stability resulting from increased ubiquitin-dependent protein degradation. Our structural modeling predicted that SB-431542, a small molecule ATP-competitive inhibitor of ALK5, binds to the A230V-ALK5 mutant with reduced affinity compared to wildtype-ALK5. We therefore examined the inhibitory effect of SB-431542 and galunisertib on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay combined with TGF-β1 stimulation, in NIH/3T3 cells and HEC-265 EC cells. SBE luciferase activity in A230V-ALK5 transfected cells was inhibited less by SB-431542 and galunisertib than in wildtype-ALK5 transfected cells indicating that A230V-ALK5 is less sensitive to inhibition by these agents than wildtype-ALK5, potentially due to changes in SB-431542/A230V-ALK5 binding affinity. Our findings are novel and show that A230V-ALK5 is a partial loss-of-function mutant that attenuates TGF-β1 signal transduction and has reduced sensitivity to ALK5 small molecule inhibitors.

Somatic mutation profiles of clear cell endometrial tumors revealed by whole exome and targeted gene sequencing.

The molecular pathogenesis of clear cell endometrial cancer (CCEC), a tumor type with a relatively unfavorable prognosis, is not well defined. We searched exome-wide for novel somatically mutated genes in CCEC and assessed the mutational spectrum of known and candidate driver genes in a large cohort of cases. We conducted whole exome sequencing of paired tumor-normal DNAs from 16 cases of CCEC (12 CCECs and the CCEC components of 4 mixed histology tumors). Twenty-two genes-of-interest were Sanger-sequenced from another 47 cases of CCEC. Microsatellite instability (MSI) and microsatellite stability (MSS) were determined by genotyping 5 mononucleotide repeats. Two tumor exomes had relatively high mutational loads and MSI. The other 14 tumor exomes were MSS and had 236 validated nonsynonymous or splice junction somatic mutations among 222 protein-encoding genes. Among the 63 cases of CCEC in this study, we identified frequent somatic mutations in TP53 (39.7%), PIK3CA (23.8%), PIK3R1 (15.9%), ARID1A (15.9%), PPP2R1A (15.9%), SPOP (14.3%), and TAF1 (9.5%), as well as MSI (11.3%). Five of 8 mutations in TAF1, a gene with no known role in CCEC, localized to the putative histone acetyltransferase domain and included 2 recurrently mutated residues. Based on patterns of MSI and mutations in 7 genes, CCEC subsets molecularly resembled serous endometrial cancer (SEC) or endometrioid endometrial cancer (EEC). Our findings demonstrate molecular similarities between CCEC and SEC and EEC and implicate TAF1 as a novel candidate CCEC driver gene. Cancer 2017;123:3261-8. © 2017 American Cancer Society.