Fig. 1

FSCN1 expression is linked to β-catenin activation in ACC. (A) The β-catenin inhibitor PNU-74654 inhibits expression of FSCN1, AXIN2 and TCF7 in H295R cells. Cells were treated with drug (100 μM) or vehicle for 24 h, then gene expression was measured by RT-qPCR. Results are shown as percentage of expression compared with vehicle-treated cells. n (independent experiments) = 5–6. Mean ± SD is shown. ***P < .001; ****P < .0001, one-way ANOVA with Šidák's multiple comparisons test. (B) The FSCN1 promoter is repressed by inhibition of β-catenin transcriptional activity in H295R cells. Cells were transfected with FSCN1 promoter-luciferase reporter (pFSCN1) or the control β-catenin-responsive TOPFLASH reporter. To inhibit β-catenin, cells were either treated with PNU-74654 (100 μM) or cotransfected with a dominant-negative TCF4 (ΔN-TCF4) expression plasmid. Results are shown as percentage of luciferase activity compared with vehicle-treated cells (PNU-74654) or empty expression vector (ΔN-TCF4). n (independent experiments) = 2-4. Mean ± SD is shown. **P < .01; ***P < .001; ****P < .0001, one-way ANOVA with Šidák's multiple comparisons test. (C) Higher FSCN1 mRNA levels in CTNNB1-mutated ACC than in tumors without CTNNB1 mutations in the Florence cohort; n (patients number) = 11. Mean ± SD is shown. ***P < .001, t-test. (D) Higher FSCN1 mRNA levels in CTNNB1-mutated and (E) beta-catenin pathway (APC, ZNRF3, CTNNB1)-mutated ACC compared with other tumors in the TCGA cohort; n (patients number) = 78. Mean ± SD is shown. ****P < .0001, t test. Data were retrieved and analyzed using Xena (https://xenabrowser.net). (F) Positive correlation of expression between FSCN1-CTNNB1 and β-catenin target genes AXIN2, LEF1, AFF3, FAM194A, ISM1, PXYLP1 and NKD1 in ACC from the TCGA cohort. Data were retrieved and analyzed using GEPIA (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation).