Intraductal Injections into the Mouse Mammary Gland.

The mammary intraductal xenografting technique has been established to inject cells or other substances directly into the mammary ducts of female mice. Using this refined xenografting method provides the possibility of mimicking the normal microenvironment of preinvasive breast lesions including, ductal carcinoma in situ (DCIS), to study of the progression of DCIS to invasive breast cancer in a more relevant manner than with other mammary xenografting methods. Xenografting into the mammary fat pad delivers cells directly into the stroma and bypasses the occurrence of invasive transition, during which cells invade through the basement membrane. Either breast cancer cell lines or patient-derived breast cancer cells can be injected into the mammary duct using this protocol to model breast cancer progression. This protocol will cover the procedures required to perform this technique.

Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1-deficient mouse mammary tumour cells.

Little is known about the role of Sox11 in the regulation of mammary progenitor cells. Sox11 is expressed by mammary bud epithelial cells during embryonic mammary gland development and is not detected in mammary epithelial cells after birth. As Sox11 is an oncofetal gene, we investigated the effects of reducing Sox11 levels in embryonic mammary progenitor cells and found that Sox11 regulates proliferative state, stem cell activity and lineage marker expression. We also investigated the effect of reducing Sox11 levels in two transplantable Brca1-deficient oestrogen receptor-negative mouse mammary tumour cell lines, to assess whether Sox11 regulates similar functions in tumour progenitor cells. When Sox11 levels were reduced in one Brca1-deficient mammary tumour cell line that expressed both epithelial and mesenchymal markers, similar effects on proliferation, stem cell activity and expression of lineage markers to those seen in the embryonic mammary progenitor cells were observed. Orthotopic grafting of mammary tumour cells with reduced Sox11 levels led to alterations in tumour-initiating capacity, latency, expression of lineage markers and metastatic burden. Our results support a model in which tumours expressing higher levels of Sox11 have more stem and tumour-initiating cells, and are less proliferative, whereas tumours expressing lower levels of Sox11 become more proliferative and capable of morphogenetic/metastatic growth, similar to what occurs during embryonic mammary developmental progression.

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.

Regulatory roles for SOX11 in development, stem cells and cancer.

The transcription factor SOX11 (SRY-related high-mobility-group (HMG) box 11), a member of the SOXC group, is expressed during embryogenesis but largely absent in most adult differentiated tissues. SOX11 regulates progenitor and stem cell behavior, and often acts together with the other two SOXC group members, SOX4 and SOX12, in regulating developmental processes, including neurogenesis and skeletogenesis. Dysregulation of SOX11 has been implicated in a number of diseases including, neurodevelopmental disorders and osteoarthritis, and a wide variety of cancers. Functions of SOX11 during both development and disease could be attributed to its context-dependent post-transcriptional modifications or interaction with other co-factors. We review the molecular and functional roles of SOX11 during development where similar processes appear to be deregulated in cancers.

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.

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.

SOX11 promotes invasive growth and ductal carcinoma in situ progression.

Here, we show that SOX11, an embryonic mammary marker that is normally silent in postnatal breast cells, is expressed in many oestrogen receptor-negative preinvasive ductal carcinoma in situ (DCIS) lesions. Mature mammary epithelial cells engineered to express SOX11 showed alterations in progenitor cell populations, including an expanded basal-like population with increased aldehyde dehydrogenase (ALDH) activity, and increased mammosphere-forming capacity. DCIS.com cells engineered to express SOX11 showed increased ALDH activity, which is a feature of cancer stem cells. The CD44+/CD24-/ALDH+ cell population was increased in DCIS.com cells that expressed SOX11. Upregulating SOX11 expression in DCIS.com cells led to increased invasive growth both in vitro and when they were injected intraductally in a mouse model of DCIS that recapitulates human disease. Invasive lesions formed sooner and tumour growth was augmented in vivo, suggesting that SOX11 contributes to the progression of DCIS to invasive breast cancer. We identified potential downstream effectors of SOX11 during both microinvasive and invasive tumour growth stages, including several with established links to regulation of progenitor cell function and prenatal developmental growth. Our findings suggest that SOX11 is a potential biomarker for DCIS lesions containing cells harbouring distinct biological features that are likely to progress to invasive breast cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers.

INTRODUCTION: Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. METHODS: We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. RESULTS: Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell proliferation and cell survival. CONCLUSIONS: Specific subsets of embryonic mammary genes, rather than the entire embryonic development transcriptomic program, are activated in tumorigenesis. Genes involved in embryonic mammary development are consistently upregulated in some breast cancers and warrant further investigation, potentially in drug-discovery research endeavors.

Phosphorylated tubulin adaptor protein CRMP-2 as prognostic marker and candidate therapeutic target for NSCLC.

Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.

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.