Letter reSeArCH
F266), some of which are predicted to be sites of direct DNA contact^10
(Fig. 1a, Extended Data Fig. 1). Another mutational hotspot is at R219,
a DNA-contact site in α-helix 3, which is a highly conserved fold of the
FKHD domain that sits in the major groove of target DNA (Extended
Data Fig. 1). Finally, 20% of FOXA1 mutations encode truncations just
downstream of the FKHD DNA-binding domain, resulting in the loss
of the C-terminal transactivating domain. Annotation of all FOXA1
mutations in the MSK-IMPACT 504 cohort^11 revealed that Wing2-
hotspot mutations—the most common subclass—are found across all
disease stages, but are more prevalent in primary locoregional cases
(Fig. 1c). There are only four cases of R219 mutation in FOXA1 in
this cohort but, notably, two of these had castration-resistant disease.
We therefore expanded the analysis to 1,822 patients by including a
larger cohort from MSK-IMPACT and a published cohort from Weill
Cornell^12 , which is enriched for neuroendocrine prostate cancer
(NEPC), and observed significant enrichment (P < 0.006) of R219
mutations versus other FOXA1 mutations in NEPC (3 out of 4) versus
adenocarcinoma (8 out of 84) (Fig. 1d).
We next investigated whether FOXA1 mutation in patients is associ-
ated with clinical outcome. In the absence of appropriate longitudinalabcEVWT
R219CR219SD226NH247QH247RH247YM253KEV+WT
+R219S+R219C+D226N+H247Q+H247R+H247Y+M253KΔF254/E255ΔM253–N256Y259C
Y259SR261CF266LG275X+Y259C+Y259S+R261C+F266L+G275X01234567Relative response ratio+ΔM253–N256+ΔF254/E255EV+WT
+R219S+R219C+D226N+H247Q+H247R+H247Y+M253K +Y259C+Y259S+R261C+F266L+G275X
+ΔM253–N256+ΔF254/E255010203040Growth relative to EV controlFOXA1 luciferase reporter assayGrowth assay (10 days after seeding)*****
*****
** ** *************** ** ********
** ***NS*** **EV
+FOXA1(WT)
EV (no EGF)
+FOXA1(WT) (no EGF)0510 15050100150200250DayCell growth (normalized to day 1)Growth assay*P < 0.05 (over +WT)
**P < 0.01 (over +WT)*P < 0.05 (compared to WT)
**P < 0.01 (compared to WT)dEV +WT +R219S +ΔF254/E255Bright eldH&E+D226N +R261CFOXA1ARp63e +H247R +F266L +G275X020406080100Organoids with a lumen (%)Lumen formation assay (10 days after seeding)
**
**
**
******
******
******NSNS–2 2+R219S+R261C+D226N+ΔF254/E255+WTLuminal gene setEnriched
in EVEnriched
in +WT or +mut–2 2EMT gene set–2 2shERF up gene setEnriched
in EVEnriched
in EVEnriched
in +WT or +mutEnriched
in +WT or +mutNSNSGSEA normalized enrichment scoresfP = 0.0494P
= 0.0062
P = 0.0023**Fig. 2 | Expression of FOXA1 mutants promotes growth and reveals
distinct morphologies for different classes of alterations. a, FOXA1
luciferase reporter assay with results normalized to level of FOXA1(WT)
activity. Colours indicate the position of the altered amino acid within
the FKHD DNA-binding domain depicted in Fig. 1a. Grey indicates
truncation. b, Overexpression of FOXA1 promotes growth in prostate
organoids in standard medium conditions (solid lines, n = 3 biological
replicates) and in restrictive medium conditions (dashed lines, no
EGF, n = 8 biological replicates). EV, pCW empty vector control.
c, O verexpression of wild-type (+WT) or various FOXA1 mutants
promotes growth ten days after seeding in medium lacking EGF.
d, Quantification of lumen-containing organoids for each line in the
Foxa1 allelic series. All values of P are relative to empty vector control,
calculated using unpaired, two-tailed Student’s t-test. e, Histology and
immunohistochemistry of organoid lines overexpressing variants of
FOXA1 via the doxycycline-inducible pCW vector ten days after seeding.
Images from a single biological experiment. H&E, haematoxylin and
eosin staining. Scale bars: top, 200 μm; other rows, 100 μm. f, Summary
of GSEA comparing Foxa1 wild-type or mutant organoid lines to empty
vector control for a basal low (luminal) gene set, the hallmark EMT gene
set and a gene set of the top 100 genes induced following ERF knockdown
(using short hairpin RNA directed against ERF, shERF) in organoids. Data
are from RNA-sequencing (RNA-seq) analysis of three biological replicates
for each organoid line. Only comparisons with an FDR of <0.25 are shown
with the corresponding normalized enrichment score. Gene sets with a
positive normalized enrichment score are enriched in organoids that have
either Foxa1 wild-type or mutant alleles. Data in a–d are mean ± s.d.
Numbers of biological replicates (indicated as dots) and specific P
values are presented in the source data. *P < 0.05, **P < 0.01, NS, not
significant. All P values are relative to overexpression of wild-type FOXA1
unless otherwise noted; unpaired, two-tailed Student’s t-test.18 JULY 2019 | VOL 571 | NAtUre | 409