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BACKGROUND: A better understanding of ductal carcinoma in situ (DCIS) is urgently needed to identify these preinvasive lesions as distinct clinical entities. Semaphorin 3F (SEMA3F) is a soluble axonal guidance molecule, and its coreceptors Neuropilin 1 (NRP1) and NRP2 are strongly expressed in invasive epithelial BC cells. METHODS: We utilized two cell line models to represent the progression from a healthy state to the mild-aggressive or ductal carcinoma in situ (DCIS) stage and, ultimately, to invasive cell lines. Additionally, we employed in vivo models and conducted analyses on patient databases to ensure the translational relevance of our results. RESULTS: We revealed SEMA3F as a promoter of invasion during the DCIS-to-invasive ductal carcinoma transition in breast cancer (BC) through the action of NRP1 and NRP2. In epithelial cells, SEMA3F activates epithelialmesenchymal transition, whereas it promotes extracellular matrix degradation and basal membrane and myoepithelial cell layer breakdown. CONCLUSIONS: Together with our patient database data, these proof-of-concept results reveal new SEMA3F-mediated mechanisms occurring in the most common preinvasive BC lesion, DCIS, and represent potent and direct activation of its transition to invasion. Moreover, and of clinical and therapeutic relevance, the effects of SEMA3F can be blocked directly through its coreceptors, thus preventing invasion and keeping DCIS lesions in the preinvasive state.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Invasividade Neoplásica , Proteínas do Tecido Nervoso , Neuropilina-1 , Neuropilina-2 , Humanos , Neuropilina-1/metabolismo , Neuropilina-1/genética , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neuropilina-2/metabolismo , Neuropilina-2/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Transição Epitelial-Mesenquimal/genética , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/genética , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
BACKGROUND: Deeper insights into ERBB2-driven cancers are essential to develop new treatment approaches for ERBB2+ breast cancers (BCs). We employed the Collaborative Cross (CC) mouse model to unearth genetic factors underpinning Erbb2-driven mammary tumour development and metastasis. METHODS: 732 F1 hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains were monitored for mammary tumour phenotypes. GWAS pinpointed SNPs that influence various tumour phenotypes. Multivariate analyses and models were used to construct the polygenic score and to develop a mouse tumour susceptibility gene signature (mTSGS), where the corresponding human ortholog was identified and designated as hTSGS. The importance and clinical value of hTSGS in human BC was evaluated using public datasets, encompassing TCGA, METABRIC, GSE96058, and I-SPY2 cohorts. The predictive power of mTSGS for response to chemotherapy was validated in vivo using genetically diverse MMTV-Erbb2 mice. FINDINGS: Distinct variances in tumour onset, multiplicity, and metastatic patterns were observed in F1-hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains. Besides lung metastasis, liver and kidney metastases emerged in specific CC strains. GWAS identified specific SNPs significantly associated with tumour onset, multiplicity, lung metastasis, and liver metastasis. Multivariate analyses flagged SNPs in 20 genes (Stx6, Ramp1, Traf3ip1, Nckap5, Pfkfb2, Trmt1l, Rprd1b, Rer1, Sepsecs, Rhobtb1, Tsen15, Abcc3, Arid5b, Tnr, Dock2, Tti1, Fam81a, Oxr1, Plxna2, and Tbc1d31) independently tied to various tumour characteristics, designated as a mTSGS. hTSGS scores (hTSGSS) based on their transcriptional level showed prognostic values, superseding clinical factors and PAM50 subtype across multiple human BC cohorts, and predicted pathological complete response independent of and superior to MammaPrint score in I-SPY2 study. The power of mTSGS score for predicting chemotherapy response was further validated in an in vivo mouse MMTV-Erbb2 model, showing that, like findings in human patients, mouse tumours with low mTSGS scores were most likely to respond to treatment. INTERPRETATION: Our investigation has unveiled many new genes predisposing individuals to ERBB2-driven cancer. Translational findings indicate that hTSGS holds promise as a biomarker for refining treatment strategies for patients with BC. FUNDING: The U.S. Department of Defense (DoD) Breast Cancer Research Program (BCRP) (BC190820), United States; MCIN/AEI/10.13039/501100011039 (PID2020-118527RB-I00, PDC2021-121735-I00), the "European Union Next Generation EU/PRTR," the Regional Government of Castile and León (CSI144P20), European Union.
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Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.
Assuntos
Exposição à Radiação , Radiometria , Humanos , Camundongos , Animais , Espectroscopia de Infravermelho com Transformada de Fourier , Análise de Fourier , Radiometria/métodos , Proteínas , Radiação Ionizante , Exposição à Radiação/análise , Doses de RadiaçãoRESUMO
Hematologic toxicity is a common side effect of multimodal cancer therapy. Nearly all animal studies investigating the causes of radiotherapy-induced hematologic toxicity use inbred strains with limited genetic diversity and do not reflect the diverse responses observed in humans. We used the population-based Collaborative Cross (CC) mouse resource to investigate the genetic architecture of the acute and persistent immune response after radiation exposure by measuring 22 immune parameters in 1,720 CC mice representing 35 strains. We determined relative acute and persistent radiation resistance scores at the individual strain level considering contributions from all immune parameters. Genome-wide association analysis identified quantitative trait loci associated with baseline and radiation responses. A cross-species radiation resistance score predicted recurrence-free survival in medulloblastoma patients. We present a community resource of immune parameters and genome-wide association analyses before and after radiation exposure for future investigations of the contributions of host genetics on radiosensitivity.
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High dose rate radiation has gained considerable interest recently as a possible avenue for increasing the therapeutic window in cancer radiation treatment. The sparing of healthy tissue at high dose rates relative to conventional dose rates, while maintaining tumor control, has been termed the FLASH effect. Although the effect has been validated in animal models using multiple radiation sources, it is not yet well understood. Here, we demonstrate a new experimental platform for quantifying oxidative damage to protein sidechains in solution as a function of radiation dose rate and oxygen availability using liquid chromatography mass spectrometry. Using this reductionist approach, we show that for both X-ray and electron sources, isolated peptides in solution are oxidatively modified to different extents as a function of both dose rate and oxygen availability. Our method provides an experimental platform for exploring the parameter space of the dose rate effect on oxidative changes to proteins in solution.
Assuntos
Neoplasias , Animais , Estresse Oxidativo , Peptídeos , Oxigênio , Dosagem RadioterapêuticaRESUMO
Microbacterium sp. BDGP8 is a species of facultative anaerobic gram-positive bacterium of the family Microbacteriaceae. The complete genome consists of a single circular chromosome of 3,293,567 bp with a G + C content of 69.84% and two plasmids of 49,365 bp and 32,884 bp.
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Increasing evidence has shown that thirdhand smoke (THS) exposure is likely to induce adverse health effects. An important knowledge gap remains in our understanding of THS exposure related to cancer risk in the human population. Population-based animal models are useful and powerful in investigating the interplay between host genetics and THS exposure on cancer risk. Here, we used the Collaborative Cross (CC) mouse population-based model system, which recapitulates the genetic and phenotypic diversity observed in the human population, to assess cancer risk after a short period of exposure, between 4 and 9 weeks of age. Eight CC strains (CC001, CC019, CC026, CC036, CC037, CC041, CC042 and CC051) were included in our study. We quantified pan-tumor incidence, tumor burden per mouse, organ tumor spectrum and tumor-free survival until 18 months of age. At the population level, we observed a significantly increased pan-tumor incidence and tumor burden per mouse in THS-treated mice as compared to the control (p = 3.04E-06). Lung and liver tissues exhibited the largest risk of undergoing tumorigenesis after THS exposure. Tumor-free survival was significantly reduced in THS-treated mice compared to control (p = 0.044). At the individual strain level, we observed a large variation in tumor incidence across the 8 CC strains. CC036 and CC041 exhibited a significant increase in pan-tumor incidence (p = 0.0084 and p = 0.000066, respectively) after THS exposure compared to control. We conclude that early-life THS exposure increases tumor development in CC mice and that host genetic background plays an important role in individual susceptibility to THS-induced tumorigenesis. Genetic background is an important factor that should be taken into account when determining human cancer risk of THS exposure.
Assuntos
Neoplasias , Poluição por Fumaça de Tabaco , Humanos , Animais , Camundongos , Poluição por Fumaça de Tabaco/efeitos adversos , Camundongos de Cruzamento Colaborativo , Fatores de Risco , Neoplasias/etiologia , Neoplasias/genética , Carcinogênese/genética , Carcinogênese/induzido quimicamente , Transformação Celular NeoplásicaRESUMO
Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.
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Laminina/metabolismo , Transdução de Sinais , Matriz Extracelular/metabolismo , Fractais , Laminina/química , PolimerizaçãoRESUMO
The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.
Assuntos
Técnicas de Cultura de Células/métodos , Estruturas do Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imageamento Tridimensional , Actinas/metabolismo , Biomimética , Mama/citologia , Adesão Celular , Comunicação Celular , Pontos de Checagem do Ciclo Celular , Estruturas do Núcleo Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Células Epiteliais/ultraestrutura , Espaço Extracelular/metabolismo , Feminino , Humanos , Queratinas/metabolismo , Microscopia de Fluorescência , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestruturaRESUMO
Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout of Mmp14 (KO) in mice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organ model for understanding why KO mice fail to thrive. A global analysis of the mammary glands' proteome in the wild type (WT) and KO mice provided insight into an unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO rudiments were reduced, and the circulating levels of triglycerides and glucose were lower. Analysis of the ultrastructure of mammary glands imaged by electron microscopy revealed a significant increase in autophagy signatures in KO mice. Finally, Mmp14 silenced mammary epithelial cells displayed enhanced autophagy. Applied to a systemic level, these findings indicate that MMP14 is a crucial regulator of tissue homeostasis. If operative on a systemic level, these findings could explain how Mmp14KO litter fail to thrive due to disorder in metabolism.
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The development of the mammary gland is unique: the final stages of development occur postnatally at puberty under the influence of hormonal cues. Furthermore, during the life of the female, the mammary gland can undergo many rounds of expansion and proliferation. The mammary gland thus provides an excellent model for studying the 'stem/progenitor' cells that allow this repeated expansion and renewal. In this Review, we provide an overview of the different cell types that constitute the mammary gland, and discuss how these cell types arise and differentiate. As cellular differentiation cannot occur without proper signals, we also describe how the tissue microenvironment influences mammary gland development.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia , Adipócitos/fisiologia , Animais , Células Epiteliais/fisiologia , Feminino , Fibroblastos/fisiologia , Humanos , Camundongos , Puberdade/fisiologiaRESUMO
Epithelial cell invasion through the extracellular matrix (ECM) is a crucial step in branching morphogenesis. The mechanisms by which the mammary epithelium integrates cues from the ECM with intracellular signaling in order to coordinate invasion through the stroma to make the mammary tree are poorly understood. Because the cell membrane-bound matrix metalloproteinase Mmp14 is known to play a key role in cancer cell invasion, we hypothesized that it could also be centrally involved in integrating signals for mammary epithelial cells (MECs) to navigate the collagen 1 (CL-1)-rich stroma of the mammary gland. Expression studies in nulliparous mice that carry a NLS-lacZ transgene downstream of the Mmp14 promoter revealed that Mmp14 is expressed in MECs at the tips of the branches. Using both mammary organoids and 3D organotypic cultures, we show that MMP activity is necessary for invasion through dense CL-1 (3 mg/ml) gels, but dispensable for MEC branching in sparse CL-1 (1 mg/ml) gels. Surprisingly, however, Mmp14 without its catalytic activity was still necessary for branching. Silencing Mmp14 prevented cell invasion through CL-1 and disrupted branching altogether; it also reduced integrin ß1 (Itgb1) levels and attenuated MAPK signaling, disrupting Itgb1-dependent invasion/branching within CL-1 gels. FRET imaging revealed that Mmp14 associates directly with Itgb1. We identified a domain of Mmp14 that is required for modulating the levels of Itgb1, MEC signaling and the rate of invasion within CL-1. These results shed light on hitherto undescribed non-proteolytic activities of Mmp14 that are necessary for the Itgb1-dependent biochemical and mechanical signals that regulate branching in the mammary epithelium.
Assuntos
Membrana Celular/metabolismo , Integrina beta1/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 14 da Matriz/metabolismo , Animais , Domínio Catalítico , Colágeno/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Inativação Gênica , Lentivirus/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , TransgenesRESUMO
Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear ß-actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear ß-actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear ß-actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular ß-actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and ß-actin in developing mammary end buds. These data indicate a novel role for nuclear ß-actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.
Assuntos
Actinas/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Actinas/genética , Animais , Núcleo Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Laminina/genética , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
Delineation of the mechanisms that establish and maintain the polarity of epithelial tissues is essential to understanding morphogenesis, tissue specificity and cancer. Three-dimensional culture assays provide a useful platform for dissecting these processes but, as discussed in a recent study in BMC Biology on the culture of mammary gland epithelial cells, multiple parameters that influence the model must be taken into account.
Assuntos
Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/fisiologia , Glândulas Mamárias Humanas/citologia , Animais , Apoptose/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Epitélio/fisiologia , Humanos , Glândulas Mamárias Animais/citologia , Transdução de Sinais/fisiologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual , Alicerces Teciduais/classificaçãoRESUMO
Patterning of developing tissues arises from a number of mechanisms, including cell shape change, cell proliferation, and cell sorting from differential cohesion or tension. Here, we reveal that differences in cell motility can also lead to cell sorting within tissues. Using mosaic engineered mammary epithelial tubules, we found that cells sorted depending on their expression level of the membrane-anchored collagenase matrix metalloproteinase (MMP)-14. These rearrangements were independent of the catalytic activity of MMP14 but absolutely required the hemopexin domain. We describe a signaling cascade downstream of MMP14 through Rho kinase that allows cells to sort within the model tissues. Cell speed and persistence time were enhanced by MMP14 expression, but only the latter motility parameter was required for sorting. These results indicate that differential directional persistence can give rise to patterns within model developing tissues.
Assuntos
Movimento Celular , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Células Epiteliais/enzimologia , Feminino , Hemopexina/genética , Hemopexina/metabolismo , Glândulas Mamárias Animais/enzimologia , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
Here, we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following 1-3 d. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.
Assuntos
Células Epiteliais/fisiologia , Neoplasias/metabolismo , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Animais , Linhagem Celular Tumoral , Colágeno Tipo I/química , Células Endoteliais/fisiologia , Humanos , Rim/citologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , CamundongosRESUMO
The treelike structures of many organs, including the mammary gland, are generated by branching morphogenesis, a reiterative process of branch initiation and invasion from a preexisting epithelium. Using a micropatterning approach to control the initial three-dimensional structure of mouse mammary epithelial tubules in culture, combined with an algorithm to quantify the extent of branching, we found that the geometry of tubules dictates the position of branches. We predicted numerically and confirm experimentally that branches initiate at sites with a local minimum in the concentration of autocrine inhibitory morphogens, such as transforming growth factor-beta. These results reveal that tissue geometry can control organ morphogenesis by defining the local cellular microenvironment, a finding that has relevance to control of invasion and metastasis.
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Células Epiteliais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Morfogênese , Organoides/crescimento & desenvolvimento , Algoritmos , Animais , Linhagem Celular , Difusão , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Feminino , Fator de Crescimento de Hepatócito/farmacologia , Glândulas Mamárias Animais/citologia , Camundongos , Técnicas de Cultura de Órgãos , Organoides/citologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Engenharia Tecidual , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
The mammary gland consists of an extensively branched ductal network contained within a distinctive basement membrane and encompassed by a stromal compartment. During lactation, production of milk depends on the action of the two epithelial cell types that make up the ductal network: luminal cells, which secrete the milk components into the ductal lumen; and myoepithelial cells, which contract to aid in the ejection of milk. There is increasing evidence that the myoepithelial cells also play a key role in the organizational development of the mammary gland, and that the loss and/or change of myoepithelial cell function is a key step in the development of breast cancer. In this review we briefly address the characteristics of breast myoepithelial cells from human breast and mouse mammary gland, how they function in normal mammary gland development, and their recently appreciated role in tumor suppression.