Neuregulin-3 Regulates Epithelial Progenitor Cell Positioning and Specifies Mammary Phenotype

Mutation of Neuregulin-3 (Nrg3) results in defective embryonic mammary gland development. Here, we investigate functions of Nrg3 signaling in embryonic mammary morphogenesis. Nrg3 regulates the distribution of epithelial progenitor cells within the presumptive mammary-forming region during early mammary morphogenesis. Basal and suprabasal epithelial cells are significantly smaller within the hypoplastic mammary primordium (MP) that forms in Nrg3 mutants, indicative of failure to acquire mammary epithelial cell (MEC) morphological phenotype. Activation of Erbb4 JM-a CYT-1, an Erbb4 isoform expressed in the developing MP, leads to MEC spreading and migration. Nrg3 promotes the accumulation of epithelial progenitor cells at the MP site in embryo explant cultures. Our results implicate Nrg3 signaling in mediating key events of mammary mesenchyme specification, including mesenchymal condensation, mitosis, and induction of mammary marker expression. Taken together, our results show Nrg3 has a major role in conferring specification of the mammary phenotype to both epithelial and mesenchymal progenitor cells

Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells.

Embryonic mammary cells are a unique population comprised of undifferentiated, highly plastic progenitor cells that create normal mammary tissues. The mammary gland continues to develop after birth from descendants of embryonic mammary cells. Here, we establish cell lines from mouse mammary organs, immediately after they formed during prenatal development, to facilitate studies of primitive mammary cells, which are difficult to isolate in sufficient quantities for use in functional experiments. We show that some lines can be induced to secrete milk, a distinguishing feature of mammary epithelial cells. Targeted deletion of Sox9, from one clone, decreases the ability to respond to lactogenic stimuli, consistent with a previously identified role for Sox9 in regulating luminal progenitor function. Sox9 ablation also leads to alterations in 3D morphology and downregulation of Zeb1, a key epithelial-mesenchymal transition regulator. Prenatal mammary cell lines are an invaluable resource to study regulation of mammary progenitor cell biology and development

A Sox2–Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer

Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

Background Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Methods Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. Results The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. Conclusions For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells

Función de la proteína mediadora de respuesta a colapsinas 2 (CRMP-2) en cáncer de pulmón no microcítico

Los microtúbulos desarrollan una función importante en procesos celulares tan cruciales como la división celular, la adhesión a sustrato, la migración o el transporte de proteínas y orgánulos. Durante la mitosis, los microtúbulos constituyen el huso mitótico. Debido a su intervención en la división celular, los microtúbulos son dianas moleculares de primera magnitud en las terapias frente al cáncer. En los últimos años se ha descubierto que es esencial la colaboración de las Proteínas Asociadas a Microtúbulos (MAPs) para que los microtúbulos puedan realizar su función correctamente. La familia de las proteínas de respuesta a colapsina o CRMPs constituye un ejemplo de este tipo de moléculas. La proteína mediadora de respuesta a colapsina tipo 2 (CRMP-2) es una proteína adaptadora de tubulina que interacciona con los heterodímeros de tubulina para unirlos a los microtúbulos crecientes, de modo dependiente de su estado de fosforilación. A pesar de su interacción con tubulina, CRMP-2 ha sido descrita fundamentalmente en tejido nervioso. Pensamos que debido a su participación en la dinámica de los microtúbulos, CRMP-2 puede presentar una expresión o regulación diferencial en las células transformadas con respecto a células primarias y con ello contribuir a la progresión tumoral. En concreto en este estudio nos propusimos estudiar la función de CRMP-2 en el contexto de carcinoma pulmonar y nos planteamos los siguientes objetivos: 1- Estudiar la expresión de CRMP-2 en células primarias de epitelio bronquial, células inmortalizadas y células transformadas de pulmón y comparar los niveles de expresión y fosforilación de esta proteína en células normales y transformadas, así como su localización subcelular y co-localización con tubulina. 2- Analizar si las variaciones en el estado de fosforilación de CRMP-2 pueden contribuir a la adquisición de propiedades características de tumores agresivos: mayor proliferación y capacidad de migración. 3- Estudiar la participación de CRMP-2 en la mitosis y evaluar si las alteraciones en su grado de fosforilación afectan a la correcta división de las células tumorales

Función de la proteína mediadora de respuesta a colapsinas 2 (CRMP-2) en cáncer de pulmón no microcítico

Los microtúbulos desarrollan una función importante en procesos celulares tan cruciales como la división celular, la adhesión a sustrato, la migración o el transporte de proteínas y orgánulos. Durante la mitosis, los microtúbulos constituyen el huso mitótico. Debido a su intervención en la división celular, los microtúbulos son dianas moleculares de primera magnitud en las terapias frente al cáncer. En los últimos años se ha descubierto que es esencial la colaboración de las Proteínas Asociadas a Microtúbulos (MAPs) para que los microtúbulos puedan realizar su función correctamente. La familia de las proteínas de respuesta a colapsina o CRMPs constituye un ejemplo de este tipo de moléculas. La proteína mediadora de respuesta a colapsina tipo 2 (CRMP-2) es una proteína adaptadora de tubulina que interacciona con los heterodímeros de tubulina para unirlos a los microtúbulos crecientes, de modo dependiente de su estado de fosforilación. A pesar de su interacción con tubulina, CRMP-2 ha sido descrita fundamentalmente en tejido nervioso. Pensamos que debido a su participación en la dinámica de los microtúbulos, CRMP-2 puede presentar una expresión o regulación diferencial en las células transformadas con respecto a células primarias y con ello contribuir a la progresión tumoral. En concreto en este estudio nos propusimos estudiar la función de CRMP-2 en el contexto de carcinoma pulmonar y nos planteamos los siguientes objetivos: 1- Estudiar la expresión de CRMP-2 en células primarias de epitelio bronquial, células inmortalizadas y células transformadas de pulmón y comparar los niveles de expresión y fosforilación de esta proteína en células normales y transformadas, así como su localización subcelular y co-localización con tubulina. 2- Analizar si las variaciones en el estado de fosforilación de CRMP-2 pueden contribuir a la adquisición de propiedades características de tumores agresivos: mayor proliferación y capacidad de migración. 3- Estudiar la participación de CRMP-2 en la mitosis y evaluar si las alteraciones en su grado de fosforilación afectan a la correcta división de las células tumorales

All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application

SOX11 promotes epithelial/mesenchymal hybrid state and alters tropism of invasive breast cancer cells.

SOX11 is an embryonic mammary epithelial marker that is normally silenced prior to birth. High SOX11 levels in breast tumours are significantly associated with distant metastasis and poor outcome in breast cancer patients. Here, we show that SOX11 confers distinct features to ER-negative DCIS.com breast cancer cells, leading to populations enriched with highly plastic hybrid epithelial/mesenchymal cells, which display invasive features and alterations in metastatic tropism when xenografted into mice. We found that SOX11+DCIS tumour cells metastasize to brain and bone at greater frequency and to lungs at lower frequency compared to cells with lower SOX11 levels. High levels of SOX11 leads to the expression of markers associated with mesenchymal state and embryonic cellular phenotypes. Our results suggest that SOX11 may be a potential biomarker for breast tumours with elevated risk of developing metastases and may require more aggressive therapies