Scribble modulates the MAPK/Fra1 pathway to disrupt luminal and ductal integrity and suppress tumour formation in the mammary gland

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression

Modelling lyssavirus infections in human stem cell-derived neural cultures

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods

VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.

The VANGL family of planar cell polarity proteins is implicated in breast cancer however its function in mammary gland biology is unknown. Here, we utilized a panel of Vang1 and Vangl2 mouse alleles to examine the requirement of VANGL family members in the murine mammary gland. We show that Vang1CKOΔ/Δ glands display normal branching while Vangl2flox/flox and Vangl2Lp/Lp tissue exhibit several phenotypes. In MMTV-Cre;Vangl2flox/flox glands, cell turnover is reduced and lumens are narrowed. A Vangl2 missense mutation in the Vangl2Lp/Lp tissue leads to mammary anlage sprouting defects and deficient outgrowth with transplantation of anlage or secondary tissue fragments. In successful Vangl2Lp/Lp outgrowths, three morphological phenotypes are observed: distended ducts, supernumerary end buds, and ectopic acini. Layer specific defects are observed with loss of Vangl2 selectively in either basal or luminal layers of mammary cysts. Loss in the basal compartment inhibits cyst formation, but has the opposite effect in the luminal compartment. Candidate gene analysis on MMTV-Cre;Vangl2flox/flox and Vangl2Lp/Lp tissue reveals a significant reduction in Bmi1 expression, with overexpression of Bmi1 rescuing defects in Vangl2 knockdown cysts. Our results demonstrate that VANGL2 is necessary for normal mammary gland development and indicate differential functional requirements in basal versus luminal mammary compartments

Isolation and characterization of orthochromatic erythroblasts.

<p><b>(A)</b> Orthochromatic erythroblasts were isolated (gate highlighted in red) from the spleen by FACS (Aria II) based on their Ter119 versus CD44 expression. Hoechst negative cells were excluded from the sort. <b>(B)</b> Cell cycle analysis was performed on the sorted orthochromatic erythroblasts based on <i>in vitro</i> BrdU incorporation and 7-AAD staining and compared to the less mature (“late”) erythroblasts (gate highlighted in blue in Fig 1A). <b>(C)</b> Enucleation in the sorted population was quantified hourly for 6 hours using. Data are means (+/− SD) of 3 independent experiments. Enucleation rate was also compared between 5 and after 10 hours after the FACS sort. Data are means (+/− SD) of 4 independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test). <b>(D)</b> The extent of enucleation was assessed by FACS LSR II shown here for negative and positive plate controls (DMSO and Cytochalasin D (Cyt.D) respectively). Propidium iodide (PI) was added to exclude dead cells from the analysis and to reveal potentially cytotoxic effects of the compounds.</p

Characterization of a role of CDK activity in erythroid enucleation.

<p>Orthochromatic erythroblasts were isolated from the spleen using FACS, and incubated in the presence of the indicated compounds for 5h. <b>(A)</b> Graphs showing percentages of enucleation in the presence of the indicated compounds at the indicated concentrations. Data are means (+/− SD) of 3–4 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). <b>(B)</b> Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated inhibitors for 5h. Data are means (+/− SD) of 3 independent experiments (364–904 cells per experiment treated with Flavopiridol (1μM); 499–730 cells per experiment treated with SNS-032 (1μM); 510–634 cells treated with Dinaciclib (25nM)). Scale bar = 10μm <b>(C)</b> Graph showing the percentages of enucleation after 5h and 10h in the presence of the indicated compounds (1μM). Data are means (+/− SD) of 3 independent experiments analyzed using FACS LSR II. <b>(D)</b> Compounds were washed out and replaced by media containing the vehicle control DMSO. Enucleation was assessed by FACS LSR II 0.5h, 1h, 3h and 5h after the washout of the indicated compounds (1μM). Data are means (+/− SD) of 3 independent experiments analyzed using FACS LSR II.</p

Characterization of a role of the proteasome in erythroid enucleation.

<p><b>(A)</b> Orthochromatic erythroblasts were cytospun immediately (0h) after FACS enrichment, Rapid Diff stained and imaged with the Olympus BX-51 microscope (100x/1.40 NA oil objective) using the Spot Advanced software (version 4.7). For quantitative analysis of the cytospins cells were manually examined and assigned a morphological class as per illustration. Data are means (+/− SD) of 4 independent experiments (112–541 cells per experiment were enumerated). <b>(B)</b> Orthochromatic erythroblasts were incubated in media containing DMSO (vehicle control) or Cytochalasin D (1μM) for 5h and subsequently cytospun. For quantitative analysis 355–740 cells per experiment were enumerated for the 5h time point in DMSO; 640–712 cells per experiment for the 5h time point in 1μM Cytochalasin D. Data are means (+/− SD) of 4 independent experiments. <b>(C)</b> Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated proteasome inhibitors (1μM) for 5h. Data are means (+/− SD) of 3 independent experiments (347–585 cells per experiment treated with Bortezomib; 422–428 cells per experiment treated with MLN9708; 351–698 cells per experiment treated with MLN2238). <b>(D)</b> Quantitative analysis of cytospun orthochromatic erythroblasts treated with Bortezomib and Cytochalasin D (1μM each). Data are means (+/− SD) of 3 independent experiments (448–516 cells per experiment treated with Bortezomib in combination with Cyt.D). <b>(E)</b> Graph showing percentages of enucleation after 5h and 10h in the presence of Bortezomib (1μM). Data are means (+/− SD) of 3 independent experiments analyzed using FACS LSR II. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test).</p

<i>Scrib</i> loss in the mammary gland results in duct hyperplasia.

<p><b>A.</b> Confirmation of <i>Scrib</i> deletion in mammary glands of <i>MMTV-Cre;Scrib^flox/−</i> 6 week old virgin mice by immunoblotting. <b>B.</b> Mammary whole mount analysis of 6 and 12 week old <i>MMTV-Cre</i>, <i>MMTV-Cre;Scrib^flox/+</i> and <i>MMTV-Cre;Scrib^flox/−</i> virgin mice. Scale bar = 2 mm. <b>C.</b> Quantitation of increased terminal end bud number during duct morphogenesis of peripubescent <i>MMTV-Cre;Scrib^flox/−</i> 6 wk virgin mice compared to control mice. ± SEM. Mann Whitney t-test, (n = 5–9). <b>D.</b> Quantitation of increased branching in mammary glands of post-pubescent <i>MMTV-Cre;Scrib^flox/−</i> 12 wk virgin mice compared to control mice. ± SEM. Mann Whitney t-test, (n = 3–6). <b>E.</b> Histological analysis by H&E staining show normal duct formation in peri-pubescent 6 wk virgin <i>MMTV-Cre</i> (a) and <i>MMTV-Cre;Scrib^flox/−</i> (b) mice. Longitudinal sections of mature mammary ducts show organized epithelial bi-layer in 12 wk virgin <i>MMTV-Cre</i> control mice (e), whereas mammary ducts from 12 wk <i>MMTV-Cre;Scrib^flox/−</i> mice (f) have a highly disorganized epithelium with luminal occlusion resulting from an abundance of poorly differentiated and multilayered mammary epithelial cells. IHC shows basolateral localization of Scribble in luminal epithelial cells of 6 (c) and 12 week old (g) <i>MMTV-Cre</i> control mice and validates <i>Scrib</i> loss in mammary epithelium of 6 (d) and 12 week old (h) <i>MMTV-Cre;Scrib^flox/−</i> mice. Scale bar = 100 µm. <b>F.</b> Quantitation of multilayering phenotype performed by counting layers of luminal epithelial cells along length of longitudinal duct sections. ± SEM. Mann Whitney t-test, (n = 3–5). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004323#pgen.1004323.s001" target="_blank">Figure S1</a>.</p

Scribble loss enhances mammary tumourigenesis.

<p><b>A.</b> Histological analysis of ductal architecture in 75 week mice by H&E staining show hyperplastic alveolar nodules (HAN) in <i>MMTV-Cre;Scrib^flox/−</i> mice (c, d) compared to control (a, b) as indicated by appearance of circular lobules and lipid droplets (d, black arrow). <b>B.</b> Quantitation of rare (<20%, +), frequent (20–80%, ++), or extensive (>80%, +++) observable alveolar or lipid droplets within mammary epithelium. <b>C.</b> IHC of Scribble (a, b), pSTAT5 (c, d) and Ki67 (e, f) in mammary ducts (du) or HAN of 75 week <i>MMTV-Cre</i> control or <i>MMTV-Cre;Scrib^flox/−</i> mice. Scale bar = 100 µm. <b>D.</b> Tumour-free plot for <i>MMTV-Cre</i> (n = 19), <i>MMTV-Cre;Scrib^flox/+</i> (n = 20) and <i>MMTV-Cre;Scrib^flox/−</i> (n = 19) virgin mice palpated weekly for mammary tumours. Scribble loss detected by IHC in tumours from <i>MMTV-Cre;Scrib^flox/−</i> aged mice. Scale bar = 100 µm. <b>E.</b> Incidence of mammary tumours amongst cohorts of MMTV-Cre, MMTV-Cre;Scribflox/+ and MMTV-Cre;Scribflox/− 525 day old virgin mice. <b>F.</b> Tumours from <i>MMTV-Cre;Scrib^flox/−</i> aged mice show increased cell turnover rates as quantified by PCNA and Cleaved caspase 3 IHC. Scale bar = 100 µm. <b>G.</b> Comparative tumour pathologies show tumours from <i>MMTV-Cre;Scrib^flox/−</i> aged mice (n = 11) are more progressed and lack structural acinar/glandular characteristics compared to tumours from <i>MMTV-Cre</i> mice (n = 11). Representative H&E staining of tumour classifications. Scale bar = 100 µm. see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004323#pgen.1004323.s006" target="_blank">Figure S6</a>.</p

<i>Scrib</i> loss impacts on basal progenitors activity but not mammary stem cell turnover <i>in vivo</i>.

<p><b>A.</b> Limiting dilution analysis of the repopulating frequency of CD29^hiCD24⁺ cells from <i>MMTV-Cre;Scrib^flox/−</i> and Control mice. Number of positive outgrowths is shown as number of outgrowths per number of mammary fat pads injected. <b>B.</b> Colony formation assay measuring increased clonogenic potential of FACS purified lin⁻/CD24⁺/CD29^hi basal cell populations from <i>MMTV-Cre;Scrib^flox/−</i> mice grown on irradiated 3T3s. n = 3.</p

Hyperplasia is preceded by defects in cell polarity, apoptosis and spindle orientation during the remodelling and maturation of <i>Scrib</i>-deficient mammary ducts.

<p><b>A.</b> Confocal immunofluorescence microscopy of apical membrane marker pERM (green) and lateral membrane marker E-cadherin (red) show polarised membrane segregation in mammary ducts of 6 week <i>MMTV-Cre</i> control mice (a), and partial to complete disruption of polarity in ducts of <i>MMTV-Cre;Scrib^flox/−</i> mice (b, c, d). Scale bar = 50 µm. <b>B.</b> IHC and quantitation of proliferation (BrDU) and <b>C.</b> apoptosis (Cleaved Caspase 3) in terminal end buds and immature ducts of 6 week old <i>MMTV-Cre</i>, <i>MMTV-Cre;Scrib^flox/+</i> and <i>MMTV-Cre;Scrib^flox/−</i> virgin mice. ± SEM. Mann Whitney t-test, (n = 3–5, 5–9 TEB/mouse). Scale bar 100 µm. <b>D.</b> Spindle orientation of dividing luminal cells in 6 wk virgin mice. Mitotic cells were identified by PHH3 (light blue) immunofluorescence and spindle angle determined by centrosome positioning (pericentrin, red) compared to the lower layer of myoepithelial cells (CK5, green) (a). Representative images of a division within a <i>Z</i>-stack (b). Rosette plots showing percentage of spindle orientations in ducts of <i>MMTV-Cre</i> and <i>MMTV-Cre;Scrib^flox/−</i> mice (c).</p