In vitro effects of FBXW7 mutation in serous endometrial cancer: Increased levels of potentially druggable proteins and sensitivity to SI‐2 and dinaciclib

Abstract

Serous endometrial cancers (ECs) are clinically aggressive tumors that frequently harbor somatic mutations in FBXW7 (F-box and WD repeat domain-containing 7). The FBXW7 tumor suppressor is part of a SCF (complex of SKP1, Cullin 1, F-box protein) ubiquitin ligase complex which controls the degradation of numerous substrates that, if not properly regulated, can contribute to the initiation or progression of tumorigenesis. Despite reports that up to 30% of serous ECs include somatic mutations in FBXW7, the molecular effects of mutated FBXW7 in ECs have not been determined. Here, we used transient transfection and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing in serous EC cell lines to interrogate the molecular effects of six recurrent FBXW7 mutations. We show that FBXW7 mutations lead to increased Cyclin E1, steroid receptor coactivator 3 (SRC-3), c-MYC, Rictor, glycogen synthase kinase 3 (GSK3), P70S6 kinase, and protein kinase B (AKT) phosphorylated protein levels in serous EC cells. Furthermore, we demonstrate that CRISPR-edited FBXW7-mutant ARK1 serous EC cells exhibit increased sensitivity to SI-2 (a SRC inhibitor) and dinaciclib (a cyclin dependent kinase (CDK) inhibitor) compared to parental ARK1 cells. Collectively, our findings reveal biochemical effects of FBXW7 mutations in the context of EC and provide in vitro evidence of sensitivity to targeted inhibitors.

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

“…We CRISPR‐edited the ARK4 serous EC cell line to generate three derivative cell lines, each harboring one recurrent FBXW7 mutation (p.R465C, p.R479Q, or p.R505C) (Figure ). To identify proteomic changes associated with recurrent FBXW7 mutations in serous ECs, we performed LC‐MS/MS proteomic profiling for ARK1 7 and ARK4 parental and CRISPR‐edited FBXW7 ‐mutant cell lines in triplicate and compared phosphorylated and total protein expression of FBXW7 ‐mutated cell lines to that of the matched FBXW7 nonmutated parental lines.…”

“…ARK4 cells were CRISPR‐edited by GEIC to incorporate FBXW7 c.C1393T (p.R465C), c.G1436A (p.R479Q), and c.C1513T (p.R505C). ARK4 was edited following published methods 7 with one exception: RNP complexes were assembled by combining 200 pmol of Alt‐R gRNA (Integrated DNA Technologies) with 80 pmol of Cas9 protein (California Institute for Quantitative Biosciences) at room temperature for 10 minutes. JHUEM‐1 cells were CRISPR‐edited by GEIC to remove the endogenous FBXW7 c.C1513T (p.R505C) mutation following the methods used for ARK4.…”

“…FBXW7 primer sequences are available upon request. All coding exons of FBXW7 were sequenced in ARK4 and JHUEM‐1 parental and derivative lines to verify CRISPR edits (Figure ) using PCR conditions, purification, and Sanger sequencing exactly as previously reported 7 …”

“…Protein isolation, quantification, gel electrophoresis, and transfer were performed following published methods 7 1 day after plating 5.5 × 10 5 cells per 60‐mm dish. Protein was extracted from six primary serous endometrial tumors using a protocol modified from Peña‐Llopis and Brugarolas 18 .…”

“…An ideal model system to examine the effects of FBXW7 mutation has been developed through CRISPR editing of ARK1 serous EC cells to insert recurrent FBXW7 somatic mutations 7 . Research comparing the levels of a small number of proteins in parental and CRISPR‐edited ARK1 cell lines provided the first insights into the direct biochemical effects of FBXW7 mutations in the context of serous EC: increased phosphorylation of seven cancer‐related proteins detected by Western blot 7 . Equivalent protein changes also occurred in ARK1 and ARK2 cells transiently expressing mutant FBXW7 7 …”