EURURO Vol. 72 No. 4

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to human orthologs using the homoloGene database
).
Pathway enrichment was assessed using gene set enrichment
analysis (GSEA) to query pathways collected from the C2 database,
in which normalized enrichment scores (NES) and p values were
estimated using 1000 gene permutations. Master regulator (MR)
analysis was performed using the Master Regulator Inference algorithm
(MARINa) by interrogating a human PCa interactome using
‘‘humanized’’ mouse signatures and human signatures. The statistical
significance of each MR was estimated using 1000 gene permutations,
and p < 0.05 was considered significant. To estimate clinical relevance,
we used a Cox proportional hazards model for univariate analysis and
Kaplan-Meier survival analysis for multivariate analysis, performed with
the surv and coxph functions from the survcomp package (Bioconductor;
). Before the Kaplan-Meier analysis, k-means
clustering was used to segregate patients into two groups corresponding
to low or high MR activity. The p values were estimated using a Wald test
for the Cox proportional hazards model, and a log-rank test for Kaplan-
Meier survival analysis. Significance for statistical tests was assumed at
p < 0.05. Statistical analysis was performed using GraphPad Prism
software (Version 6.0), R-studio (0.99.902, R v3.3.0), and Matlab (2012a).
2.4. Accession numbers
Raw and normalized expression profiling data are publicly available
through the Gene Expression Omnibus database via accession number
GSE92999.
3. Results
3.1. Finasteride abrogates PIN in a GEMM of early-stage PCa
Nkx3.1 mutant mice develop PIN as a consequence of aging,
and share conserved molecular features with early-stage
human PCa . We reasoned that this GEMM could
provide information on the efficacy of finasteride in delaying
progression of early-stage PCa and on the molecular
programs associated with such[7_TD$DIFF] response. Cohorts of litter-
mate wild-type (Nkx3.1+/+
[15_TD$DIFF]) and homozygous mutant (Nkx3.1-
/ ) mice were randomly assigned to the vehicle or
finasteride arms at age 4 mo A), which is before the
onset of PIN in these mice Treatment (10 mg/kg/d, five
times weekly was continued for up to 12 mo, by which
point untreated Nkx3.1 / mice display PIN . At the
conclusion of the study, mice were euthanized to assess DHT
levels ), histopathological phenotype C,D), and
expression of markers of prostate differentiation, cancer
initiation, and androgen response and 2A).
Finasteride was highly effective in inhibiting 5a-reduc-
tase, as evidenced by the nearly complete inhibition of
serum DHT levels in both Nkx3.1 +/+ and Nkx3.1 / mice
(n = 5 per group; p = 0.024 and p 0.0001, respectively;
B). In addition, several AR-regulated genes, including
Tmprss2 and Foxa1, were downregulated to a similar extent
in Nkx3.1+/+ and Nkx3.1 / prostates (n = 5 per group;
p < 0.001; E), whereas expression levels of AR itself
and a non-AR regulated gene, Hoxb13, were not affected
(n = 5 per group; ). Notably, expression of Nkx3.1 , a
known target of AR, was not affected in finasteride-treated
Nkx3.1+/+ mice, reflecting the complexity of the relationship
between AR and NKX3.1 expression .
However, finasteride had a profound effect on the
histological phenotype of Nkx3.1 / but not Nkx3.1 +/+
prostates C,D). Specifically, the PIN phenotype of
Nkx3.1 / prostates was significantly abrogated following
treatment, while the normal prostate histology of Nkx3.1 +/+
mice was virtually unaffected (n = 25–30 per group,
p < 0.001; C,D). Notably, abrogation of the PIN
phenotype was not observed in finasteride-treated Pten
heterozygous mutant mice (Pten+/ ), which develop PIN but
are wild type for Nkx3.1 (Supplementary Fig. 1). The striking
reversal of the PIN phenotype in finasteride-treated
Nkx3.1 / mice was accompanied by increased expression
of markers of prostate differentiation, including E-cadherin
and probasin (n = 5 per group; p < 0.0001; ,E).
RNA sequencing comparing prostate tissues from vehi-
cle- and finasteride-treated Nkx3.1+/+ or Nkx3.1 / mice
further indicated the similar inhibition of 5a-reductase in
Nkx3.1+/+ and Nkx3.1 / prostates, as evidenced from their
comparable reduction in expression of AR-regulated genes
(p < 0.001; A) . However, GSEA comparing a
differential gene expression signature between finasteride-
and vehicle-treated Nkx3.1 / prostates with a comparable
signature from Nkx3.1+/+ prostates revealed a strong
difference after treatment. Specifically, genes downregu-
lated in finasteride-treated Nkx3.1 / prostates were
Table 1 – Characteristics of the patients from the 5-ARI cohorts
WCM cohort (training) FHCRC (validation)
Patients (n)
5-ARI–treated 9 10 [9_TD$DIFF]
Untreated 18 5
Sample collection period 2012–2015 2003–2005
Age (yr) 64 (59.5–68.5) 61 (46–73)
PSA range (ng/ml) 2.39–14.5 2.5–10
Pathological Gleason score 6 (6–9)
Treated patients 3 + 3 (n = 4), 3 + 4 (n = 4), 4 + 3 (n = 1)
Untreated patients 3 + 4 (n = 7), 4 + 3 (n = 8), 4 + 4 (n = 3)
5-ARI = 5 a-reductase inhibitor; WCM = Weill Cornell Medicine; FHCRC = Fred Hutchinson Cancer Research Center; PSA = prostate-specific antigen.
a Of the ten patients, five received a low (0.5 mg) and five a high ARI dose (3.5 mg).
b Median (interquartile range).
c Mean (range).
d Median (range).
E U R O P E A N U R O L O G Y 7 2 ( 2 0 1 7 ) 4 9 9 – 5 0 6 501