Mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer

Abstract

Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway.

Figure 1. Genomic changes in SCRIB associated with human breast cancer

(A) The cBioPortal was used to detect genomic alterations and gene expression changes (mutations, copy number alterations, mRNA expression) in the SCRIB gene among 748 human breast tumors. (B) SCRIB gene expression in breast cancer molecular subtypes. Normalized and median-centered SCRIB gene expression levels were compared in breast cancer molecular subtypes as defined in a publically available dataset of breast tumors. SCRIB expression is expressed as log2 values. Mean SCRIB expression between tumors within each subtype is represented by a black line. Lines above the graph represent statistical significance of variance between subtypes as determined by Kruskall-Wallace non-parametric method. Asterisks represent p values; *** = p<0.001; ** = p<0.01; * = p<0.05; n.s. = p>0.05. (C) Analysis of SCRIB mRNA across TNBC subtypes. Gene expression (GE) profiles were obtained from 21 publicly available data sets that contained 3,247 primary human breast cancers. Gene expression for SCRIB (212556_at) was extracted and used for all comparisons. The lower panel displays p-values representing the difference between SCRIB expression in each subgroup (D) SCRIB expression stratifies overall survival in breast cancer. Kaplan-Meier estimates of overall survival were calculated in a publically available integrated multi-study breast cancer transcriptomic dataset through the KMplotter tool. The upper quartile of SCRIB expression used to dichotomise data into high versus low expression groups. Number of total patients are shown, significance of differences in survival curves are determined by log-rank p-values and hazard ratios are calculated by cox regression analysis. (E) (i) Human breast tumor with predominantly membrane SCRIB localization. (ii) Human breast tumor with membrane and cytosolic SCRIB localization. (iii) Human breast tumor with predominantly cytosolic SCRIB localization.

Figure 2. Generation and characterization of MMTV-hSCRIBP305L mice

(A) Schematic of MMTV-hSCRIBP305L targeting construct. (B) Cell lysates from mammary glands isolated from FVB and hSCIBP305L mice were analyzed for expression of SCRIB and actin (loading control) by Western blot. (C) Expression of SCRIB, CK14 and E-cad was analyzed by IHC in mammary glands isolated from FVB and hSCRIBP305L mice. (D) Expression of SCRIB, CK14 and E-cad was analyzed by IHC in mammary glands isolated from FVB and hSCRIBP305L mice. Scale bars = 10μm.

Figure 3. Mislocalization of SCRIB in the mouse mammary gland disrupts morphogenesis

(A) Whole mount analysis of FVB and hSCRIBP305L mammary glands collected at the indicated time points. (B) Quantitative analysis of the results presented in (A), from three mice per condition. Error bars represent standard error of the mean (SEM). * denotes p<0.05. (C) FACS analysis of luminal progenitor cell (CD49f^low/CD61⁺) content in mammary epithelial cells isolated from FVB and hSCRIBP305L mice. * denotes p<0.05.

Figure 4. Mislocalization of SCRIB induces hyperplastic lesions in the mammary gland

(A) Whole mount analysis of FVB and hSCRIBP305L mammary glands collected at the indicated time point. (B) H&E staining of mammary glands collected from FVB and P305L mice. (C) Analysis of hyperplastic phenotype in two distinct hSCRIBP305L lines. (D) Expression of CK18 and CK14 were analyzed by IHC in mammary glands isolated from FVB and hSCRIBP305L mice. Scale bars = 20μm. (E) Quantitative analysis of the results presented in (D).

Figure 5. Mislocalization of SCRIB generates tumors with basal characteristics

(A) Kaplan-Meier curve of tumor onset in FVB and two distinct hSCRIBP305L lines. (B) H&E staining of mammary tumors collected from hSCRIBP305L mice. (i) Glandular adenocarcinoma, (ii) microacinar tumor, (iii) spindle cell tumor, (iv) adenosquamous carcinoma. (C) Pathological classification of hSCRIBP305L tumors. (D) H&E staining of tumor emboli within lung from hSCRIBP305L tumor bearing mouse. (E) Expression of CK18 and CK14 were analyzed by IHC in mammary tumors isolated from hSCRIBP305L mice. Scale bars = 50μm. (F) Expression of Ki67 and cleaved caspase 3 was analyzed by IHC in mammary tumors isolated from hSCRIBP305L mice. Each image was taken from a different tumor. (G) Quantitative PCR analysis was utilized to examine expression of luminal markers (GATA3, CK18) on cDNA generated from NDL and hSCRIBP305L tumors. (H) Quantitative PCR analysis was utilized to examine expression of basal markers (CK5, CK6, CK14) on cDNA generated from NDL and hSCRIBP305L tumors. (I) Western blot analysis was utilized to examine expression of the luminal marker E-cad, and the basal markers Vimentin and CK14 in NDL and P305L tumors.

Figure 6. Mislocalization of SCRIB enhances EGF-induced AKT activation

(A) Expression of SCRIB was analyzed in MCF-10A stably expressing SCRIBWT or hSCRIBP305L. (B) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were analyzed by IF for SCRIB localization. (C) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were grown in the absence of EGF overnight, then allowed to recover in EGF-containing medium for the indicated lengths of time. Protein lysates from each condition were analyzed for phosphorylation of AKT at Thr308 and Ser473, and ERK1/2 at Thr202/Tyr204. (D) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were grown in the absence of EGF overnight, then allowed to recover in EGF-containing medium for the indicated lengths of time. Protein lysates from each condition were analyzed for phosphorylation of TSC2 at Thr1462 and PRAS40 at Thr246. (E) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were grown in the absence of EGF overnight, then allowed to recover in EGF-containing medium for the indicated lengths of time. Protein lysates from each condition were analyzed for phosphorylation of p70S6K at Thr389 and S6 at Ser235/236. (F) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were grown in the absence of EGF overnight, pretreated with rapamycin for 30 minutes, then allowed to recover in EGF-containing medium for 45 minutes. Protein lysates from each condition were analyzed for phosphorylation of p70S6K at Thr389 and S6 at Ser235/236. (G) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were grown in the absence of EGF overnight, pretreated with rapamycin for 30 minutes, then allowed to recover in EGF-containing medium for 30 minutes. Protein lysates from each condition were analyzed for phosphorylation of Akt at Ser473.

Figure 7. SCRIB is found in a protein complex with PTEN

(A) Control (IgG) and SCRIB immunoprecipitations were performed on Eph4 protein lysates. (B) HEK293 cells were transfected with an empty vector, T7-tagged SCRIBWT (WT), T7-tagged hSCRIBP305L (PL), and GFP-WT-PTEN. Immunoprecipitations were carried out from total cell lysates utilizing the T7 antibody or control IgG. (C) HEK293 cells were transfected with an empty vector, T7-tagged SCRIB, GFP-WT-PTEN or GFP-PTEN399. Immunoprecipitations were carried out from total cell lysates utilizing the T7 antibody. (D) HEK293 cells were transfected with an empty vector, T7-tagged SCRIB or T7-tagged SCRIB PDZ mutants, and GFP-WT-PTEN. Immunoprecipitations were carried out from total cell lysates utilizing the T7 antibody. (E) Quantification of results presented in (D), from three independent experiments. (F) MCF-10A cells, either control or overexpressing SCRIBWT or hSCRIBP305L, were fractionated to separate the membrane and cytosol fractions. Protein lysates from each condition were analyzed for PTEN, caspase 3 (cytosolic marker) and c-Met (membrane marker). (G) MCF-10A cells were transfected with an empty vector, WT-PTEN (WT) or PTEN-399 (399), grown in the absence of EGF overnight, then allowed to recover in EGF-containing medium for 10 minutes. Protein lysates from each condition were analyzed for phosphorylation of AKT at Thr308. (H) Phosphorylation of Akt and S6 was analyzed by WB in mammary glands isolated from FVB and hSCRIBP305L mice. (I) Phosphorylation of S6 was analyzed by WB in mammary glands isolated from FVB and hSCRIBP305L mice. (J) Western blot analysis was utilized to examine phosphorylation of S6 in NDL and P305L tumors. (K) Phosphorylation of Akt was analyzed by IHC in NDL and P305L tumors. (L) Analysis of phosphorylation of S6 at Ser235 in human tumor samples with amplification of SCRIB. (M) Mutual exclusivity calculations of alterations in selected genes in human breast cancer. p>0.05 for pairs outlined in red. Orange=strong tendency towards co-occurrence (odds ratio>10); Yellow=tendency towards co-occurrence (2<odds ratio<10); Blue=strong tendency towards mutual exclusivity (0<odds ratio<0.1); White=no association (0.5<odds ratio<2).