(A)
P63 mRNA levels (fold-change ± SD, n=4) in Pt-T PDXs
(carbo) vs. controls (Ctrl). (B)
P63 mRNA levels (fold-change ± SD, n=3–6) in
FZD7(+) versus FZD7(−)cells sorted from SKOV3, OVCAR5, and COV362 cell
lines, and cell suspensions from human tumors. (C)
P63 mRNA expression levels (fold-change ± SD, n = 4) in
OVCAR5 cells transduced with shRNAs targeting FZD7 (shFZD7) vs.
control shRNA (shctrl). (D) Western blot for β-catenin, P63,
and GAPDH in SKOV3 and OVCAR5 cells transduced with shRNAs targeting
FZD7 (shFZD7) or control shRNA (shctrl). Quantification
shows fold change of β-catenin and P63 expression across 3 experiments.
(E, F)
P63 mRNA levels (fold-change ± SD, n=3) in OVCAR5
(E) and SKOV3 (F) cells transfected with FZD7 vs.
control vector (Ctrl). (G) Western blot for β-catenin, P63,
and GAPDH in OVCAR5 cells transfected with control (ctrl) or FZD7-pcDNA3.1
(FZD7). Quantification shows fold change of β-catenin and P63 expression
across 3 experiments. (H) Western blot for FZD7, β-catenin,
P63, GPX4 and GAPDH in OVCAR5 cells transfected with shctrl, shFZD7
(J) treated with WNT3a (150ng/ul) and/or IWR-1-endo (1
μM) for 24 hours (n = 2). (I-K)
FZD7(I), P63
(J), and GPX4
(K) mRNA expression levels (fold-change ± SD, n=3–4)
in OVCAR5 cells transduced with control shRNAs (Ctrl_shctrl, Ctrl_shP63,
FZD7_shctrl) or transfected with FZD7 expression vector and subsequently
transduced with shRNA targeting P63 (FZD7_shP63). For all
comparisons: *P<0.05, **P<0.01, ***P<0.001.
(L) Scatter plot shows the correlation between
P63 and FZD7 mRNA expression levels in
HGSOC tumors (n=419) profiled in the TCGA database. Pearson correlation
coefficients and P-values are shown. (M) Kaplan-Meier survival
curves for HGSOC patients profiled in the TCGA having high (n=61) or low (n=318)
P63 (TP63-012) mRNA expression levels.
High or low levels were defined based on statistically determined cutoff point
that maximizes absolute value of the standardized two-sample linear rank
statistic. (N) Model demonstrates the proposed mechanism by which
FZD7 engages the anti-oxidant pathway governed by GPX4.