(A) Schematic representation of the WT Scrib allele, targeting construct, and mutated Scrib allele. Excision of the Frt-flanked PGK-Neo cassette in the LoxP-flanked targeting vector was accomplished by crossing Scrib^fl-Neo mice to Actin-FLPe mice, producing the Scrib floxed (Scrib^fl) allele. The insertion of EcoRV and KpnI restriction sites permitted screening for recombinant ES cell clones (see Supplemental Figure 1, A and B). Scrib-KO mice were derived by crossing Scrib^+/fl mice with a germline Cre-deleter mouse. Exons are indicated as boxes and LoxP/FLPe sites as triangles. (B) Photographs of WT, Scrib^+/–, and KO E14.5 embryos. Scale bar: 10 mm. (C) Western blot analysis of embryonic brain lysates (E16.5) confirmed Scrib loss in Scrib^+/– and KO compared with WT embryos.

(A) PCR analysis of genomic DNA to detect WT (290 bp) and KO (550 bp) Scrib alleles in Scrib^+/– and WT prostate. (B) qRT-PCR for Scrib mRNA confirmed a significant, 55% reduction in Scrib^+/– compared with WT prostates (P = 0.0065, unpaired t test). Error bars indicate SD. (C) Scrib IF staining of WT and Scrib^+/– hyperplastic prostate. (D) Representative H&E images of normal WT and hyperplastic Scrib^+/– prostate (400 days). (E) Phenotype incidence in WT and Scrib^+/– mice at 100, 200, and 400 days (n ≥ 10). Scale bars: 50 μm (larger panels) and 10 μm (insets).

(A) PCNA IHC shows a significant increase in PCNA-positive cells in Scrib^+/– lesions (2.26% ± 0.62%, mean ± SD) compared with WT (0.47% ± 0.09%, P < 0.0001, unpaired t test, 400 days). (B) IHC in Scrib^+/– hyperplastic foci revealed aberrant E-cadherin and p-ERM compared with WT tissue. Arrows indicate aberrant expression. Right column: IF to detect Par3 (red) and pan-Dlg (green) shows that these polarity regulators are mislocalized in Scrib^+/– prostate hyperplasia compared with WT prostate (400 days). N, normal Scrib^+/– tissue; Hyp, hyperplastic Scrib^+/– tissue. Arrowheads indicate Par3 accumulation at tight junctions. (C) IHC revealed a significant elevation in p-ERK–positive nuclei in Scrib^+/– lesions compared with WT (unpaired t test, 400 days). (D) IHC revealed a significant elevation in p-ELK1–positive nuclei in Scrib^+/– lesions compared with WT (unpaired t test, 400 days). (E) Representative H&E images of Scrib^+/– prostates administered vehicle or PD0325901 (20 mg/kg, 5 days on, 2 days off for 3 weeks at 230–260 days of age), and p-ERK staining showing efficient MEK inhibition. (F) Scrib^+/– prostate weight is significantly decreased upon MEK inhibition (0.30 ± 0.04 g, mean ± SD) compared with administration of vehicle (0.45 g ± 0.08, P = 0.0003, Mann-Whitney U non-parametric test), similar to the level in WT prostate (P = 0.4606, Mann-Whitney U non-parametric t test). Scale bar: 50 μm (larger panels) and 10 μm (insets). Error bars indicate SD.

(A) Scrib mRNA is decreased in PBCre⁺;Scrib^+/fl (45.2%) and PBCre⁺;Scrib^fl/fl (14.5%) compared with PBCre⁺ prostates at 400 days. (B) Scrib IF shows reduced Scrib expression in PBCre⁺;Scrib^fl/fl prostate epithelium (400 days). (C) Representative H&E images of PBCre⁺, PBCre⁺;Scrib^+/fl, and PBCre⁺;Scrib^fl/fl prostates (400 days). (D) Prostate phenotype incidence at 100, 200, and 400 days (n ≥ 10). (E) PCNA IHC shows a significant increase in the number of PCNA-positive cells in PBCre⁺;Scrib^fl/fl mice (4.0% ± 0.24%) compared with PBCre⁺;Scrib^+/fl (2.1% ± 0.82%) and PBCre⁺ mice (0.6% ± 0.38%) at 400 days. (F) p-ERK staining revealed a significant increase in MAPK signaling in PBCre⁺;Scrib^+/fl (7.1% ± 1.64%) and PBCre⁺;Scrib^fl/fl mice (13.1% ± 4.18%) compared with PBCre⁺ controls (1.8% ± 0.58%) at 400 days. (G) p-ELK1 staining shows a significant increase in p-ELK1 expression in PBCre⁺;Scrib^+/fl (1.8% ± 0.25%) and PBCre⁺;Scrib^fl/fl (4.2% ± 1.67%) compared with WT tissue (0.4% ± 0.13%) at 400 days. Arrows indicate positive nuclei. Scale bars: 50 μm (larger panels) and 10 μm (insets). Data are mean ± SD; n = 3; P values represent unpaired t test. Error bars indicate SD.

(A) Kaplan-Meier survival plot shows a significant reduction in PBCre⁺;Scrib^fl/fl;LSL-K-ras^G12D/+ average survival (372 days) compared with PBCre⁺;LSL-K-ras^G12D/+ mice (χ² = 4.20, P = 0.0449, log-rank test, n ≥ 10). (B) Representative H&E images of PBCre⁺;LSL-K-ras^G12D/+, PBCre⁺;Scrib^+/fl;LSL-K-ras^G12D/+, and PBCre⁺;Scrib^fl/fl;LSL-K-ras^G12D/+ prostates (400 days). (C) Prostate phenotype incidence (400 days, n ≥ 10). (D) IHC to detect p-ERK reveals that the number of p-ERK–positive nuclei in PBCre⁺;LSL-K-ras^G12D/+ (21% ± 11.24%), PBCre⁺;Scrib^+/fl;LSL-K-ras^G12D/+ (20% ± 11.41%), and PBCre⁺;Scrib^fl/fl;LSL-K-ras^G12D/+ (17% ± 6.35%) prostate tumors was significantly elevated compared with control prostate (*P ≤ 0.0496), yet no statistical difference was determined between the double mutants and K-ras activation alone (P > 0.6168) at 400 days. (E) IHC to detect p-ELK1 revealed a significant increase in p-ELK1 expression in PBCre⁺;Scrib^+/fl;LSL-K-ras^G12D/+ (36.6% ± 6.54%), and PBCre⁺;Scrib^fl/fl;LSL-K-ras^G12D/+ (50.5% ± 0.43%) prostate lesions compared with PBCre⁺;LSL-K-ras^G12D/+ (17.7% ± 0.55%, P ≤ 0.0448) and PBCre⁺ (0.4% ±0.07, P ≤ 0.0052) prostate epithelium at 400 days. Scale bars: 50 μm (larger panels) and 10 μm (insets). In D and E, data are mean ± SD; n = 3; P values represent unpaired t test. Error bars indicate SD.

Representative images of TMA samples stained by IHC to detect SCRIB indicating the scoring strategy employed to grade SCRIB intensity (0 negative, +1 weak, +2 moderate, and +3 strong) (A) and mislocalization (B). Scale bars: 50 μm (larger panels) and 10 μm (insets). Samples displaying uniform basolateral membrane expression were designated as normal. Mislocalized expression was classified as clustered accumulation of SCRIB (group A: at cell-cell junctions; group B: along the membrane circumference; group A/B displays features of groups A and B). Scale bars: 50 μm (larger panels) and 10 μm (insets). (C) Kaplan-Meier plot indicates no significant difference between overall SCRIB intensity and PSA recurrence–free survival (P = 0.0687, log-rank test). Moreover, negative samples showed no significant difference in PSA recurrence–free survival compared with positive samples (P > 0.2196, log-rank test). (D) Kaplan-Meier plot comparing normal and mislocalized SCRIB expression revealed a significant reduction in PSA recurrence–free survival for all modes of mislocalization (P = 0.0001, log-rank test).