The mutational spectrum of FOXA2 in endometrioid endometrial cancer points to a tumor suppressor role

Abstract

BACKGROUND Forkhead box protein A2 (FOXA2) plays an important in development, cellular metabolism and tumorigenesis. The Cancer Genome Atlas (TCGA) identified a modest frequency of FOXA2 mutations in endometrioid endometrial cancers (EEC). The current study sought to determine the relationship between FOXA2 mutation and clinicopathologic features in EEC and FOXA2 expression. METHODS Polymerase chain reaction (PCR) amplification and sequencing were used to identify mutations in 542 EEC. Western blot, quantitative reverse transcriptase PCR (qRT-PCR) and immunohistochemistry (IHC) were used to assess expression. Methylation analysis was performed using combined bisulfite restriction analysis (COBRA) and sequencing. Chi-squared, Fisher's exact, student's t- and log-rank tests were performed. RESULTS Fifty-one mutations were identified in 49 tumors (9.4% mutation rate). The majority of mutations were novel, loss of function (LOF) (78.4%) mutations, and most disrupted the DNA-binding domain (58.8%). Six recurrent mutations were identified. Only two tumors had two mutations and there was no evidence for FOXA2 allelic loss. Mutation status was associated with tumor grade and not associated with survival outcomes. Methylation of the FOXA2 promoter region was highly variable. Most tumors expressed FOXA2 at both the mRNA and protein level. In those tumors with mutations, the majority of cases expressed both alleles. CONCLUSION FOXA2 is frequently mutated in EEC. The pattern of FOXA2 mutations and expression in tumors suggests complex regulation and a haploinsufficient or dominant-negative tumor suppressor function. In vitro studies may shed light on how mutations in FOXA2 affect FOXA2 pioneer and/or transcription factor functions in EEC.

Functional characterization of recurrent FOXA2 mutations seen in endometrial cancers

“…The majority of FOXA2 mutations are loss-of-function defects (frameshift and nonsense alleles), consistent with FOXA2 ’s role as a tumor suppressor. 26, 27 Primary tumor specimens rarely have a second mutation or other identifiable abnormality. Haploinsufficiency for FOXA2 could be sufficient to drive endometrial tumorigenesis, as has been proposed for a number of tumor suppressors.…”

“…Myc-tagged FOXA2 expression constructs were developed for four recurrent mutations that together are representative of the most frequently seen endometrioid endometrial cancer mutations 27 (Figure 1a). Two frameshifts N-terminal of the forkhead domain (both involving p.Q122) and the recurrent p.S169W missense allele in the forkhead/DNA binding domain were studied.…”

“…FOXA2 mutations in EC occur in the last exon (2 coding exons) and thus transcripts from the stop/frameshift mutations would not be subject to nonsense-mediated decay. 26, 27, 37 Changes in abundance and activity of the truncated/frameshift mutant proteins are expected given loss of C-terminal amino acids and/or addition of novel sequences in the frameshift proteins. Previous studies have shown that FOXA2 stability is determined by both sumoylation and acetylation, and is context dependent.…”

“…The missense mutant we studied (p.S169W within the DNA binding domain) had significantly reduced in vitro transcriptional activity relative to WT FOXA2. Serine 169 is potential phosphorylation site 27 and it is possible that phosphorylation at this site increases transcriptional activity or influences protein stability. Given the fact that the level of expression of the p.S169W mutant protein was much lower than that for WT FOXA2 in transfection assays (Figure 1b), it is most likely that the relative abundance of p.S169W FOXA2 explains the reduced transcriptional activity seen with the CDH1 /luciferase reporter assay.…”

Forkhead box a2 (FOXA2) is essential for uterine function and fertility

“…Thus, Lif gene activation by ligand-activated ESR1 is postulated to involve FOXA2 pioneer transcription activity (48,49). Future studies should focus on the molecular mechanism by which FOXA2 programs the GE transcriptome and influences steroid hormone responses of uterine glands, particularly because FOXA2 is expressed in the glands of the human uterus, is involved in endometrial hyperplasia (19), is frequently mutated and inactivated in endometrioid endometrial cancer (50), and is down-regulated in ectopic endometrium in women with endometriosis (51).…”