Proteomic profiling of FBXW7‐mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target

Abstract

Background:Despite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7). Methods: To identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)-edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry-based proteomic profiles of CRISPR-edited ARK1 and ARK4 serous EC cells to matched parental cells. Results: Among distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7-mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR-edited FBXW7-mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM-1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors. Conclusion: Our findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors.

High‐risk endometrial cancer proteomic profiling reveals that FBXW7 mutation alters L1CAM and TGM2 protein levels

“…4A). 18 On the basis of MSS, POLE-ED, and TP53 status 25,26 (Supporting Table 10), we determined that HEC-50B cells aligned best to the p53-abnormal molecular subgroup, whereas ARK1 and ARK4 were TP53-mutant with an unknown MSS status (Supporting Table 10). Of the unique total and phosphorylated proteins exhibiting significantly different levels in FBXW7-mutated HEC-50B cells, 372 also exhibited significantly different levels in ARK1 and/ or ARK4 FBXW7-mutant cell lines in comparison with the matched parental line (Fig.…”

“…1A) were verified with FBXW7 primers (Supporting Table 1), polymerase chain reaction conditions, purification, and Sanger sequencing exactly as previously reported for ARK1, ARK4, and JHUEM-1. 18,20 Likewise, the FBXW7-mutant status of parental HEC-1-B cells and the wild-type status of all other parental coding exons as well as all coding exons of the matched CRISPRedited cell line were verified with these exact methods (Supporting Fig. 1B).…”

“…16 Herein we molecularly classified HEC-50B cells as p53 abnormal because they are MSS, 17 POLE wild-type, and TP53 mutant. We compared our findings with analogous results from TP53-mutant ARK1 and ARK4 SEC cells 18 and identified 372 nonredundant proteins that exhibited altered levels in FBXW7-mutated derivative lines from multiple high-risk ECs, including 2 druggable proteins: L1 cell adhesion molecule (L1CAM) and transglutaminase 2 (TGM2). We further validate these findings by demonstrating that the reversion of endogenous FBXW7 mutations to a wild-type genotype in 2 grade 2 endometrioid endometrial cancer (G2EEC) cell lines (JHUEM-1 and HEC-1-B), molecularly classified herein as part of the mismatch repair deficiency (MMRd)/MSI subgroup, results in decreased L1CAM and TGM2 protein levels.…”

“…1) via methods previously described for the generation of CRISPR-edited ARK1, ARK4, and JHUEM-1 cell lines. 18 DNA Extraction, Polymerase Chain Reaction Amplification, and Sanger Sequencing DNA was extracted with the Gentra Puregene Kit (Qiagen, Germantown, Maryland) according to the manufacturer's instructions. The wild-type status of all coding exons of FBXW7 in HEC-50B parental cells and all exons except those harboring CRISPR-edited mutations in derivative lines (Supporting Fig.…”

“…Proteins detected at significantly different levels in CRISPR-edited FBXW7mutation knock-in ARK1 and ARK4 cells in comparison with matched parental cells were previously published. 18 Unsupervised hierarchical clustering was performed with the Partek Genomics Suite.…”