RESUMEN
Epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial-mesenchymal plasticity and gene expression.
RESUMEN
The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended "spikes" in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.
Asunto(s)
Antineoplásicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Mamarias Animales/patología , Mesodermo/patología , Organoides/patología , Animales , Azacitidina/farmacología , Benzamidas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Procesamiento de Imagen Asistido por Computador , Neoplasias Mamarias Animales/genética , Ratones Endogámicos BALB C , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Organoides/efectos de los fármacos , Pirimidinas/farmacología , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Cancer-induced immune responses affect tumour progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages that define 'immune subtypes' of triple-negative breast cancer (TNBC), including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumour-intrinsic pathways and mutual regulation between macrophages (or monocytes) and neutrophils contribute to the development of a dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells), is resistant to ICB, and contains a minority of macrophages that seem to be unaffected by CCR2 knockout. A MES-to-NES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.
Asunto(s)
Inmunoterapia , Células Mieloides/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Microambiente Tumoral/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Granulocitos/inmunología , Inmunoterapia/métodos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Células Supresoras de Origen Mieloide/inmunología , Neutrófilos/inmunología , Neutrófilos/patología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Puberty is characterized by dynamic tissue remodeling in the mammary gland involving ductal elongation, resolution into the mature epithelial bilayer, and lumen formation. To decipher the cellular mechanisms underlying these processes, we studied the fate of putative stem cells, termed cap cells, present in terminal end buds of pubertal mice. Employing a p63CreERT2-based lineage-tracing strategy, we identified a unipotent fate for proliferative cap cells that only generated cells with basal features. Furthermore, we observed that dislocated "cap-in-body" cells underwent apoptosis, which aided lumen formation during ductal development. Basal lineage-specific profiling and genetic loss-of-function experiments revealed a critical role for FOXO transcription factors in mediating these proliferative versus apoptotic fates. Importantly, these studies revealed a mode of WNT signaling-mediated FOXO1 inhibition, potentially mediated through AKT. Together, these data suggest that the WNT pathway confers proliferative and survival advantages on cap cells via regulation of FOXO1 localization.
Asunto(s)
Apoptosis/fisiología , Mama/metabolismo , Proteína Forkhead Box O1/metabolismo , Homeostasis/fisiología , Células Madre/citología , Vía de Señalización Wnt/fisiología , Animales , Mama/citología , Linaje de la Célula/fisiología , Proliferación Celular/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones TransgénicosRESUMEN
Histone modification alters chromatin architecture to regulate gene transcription. KDM3A is a histone demethylase in the JmjC domain-containing protein family. It removes di- and mono- methyl residues from di- or mono-methylated lysine 9 of histone H3 (H3K9me2/me1). Recent studies have shown that Kdm3a plays an important role in self-renewal of embryonic stem cells, spermatogenesis, metabolism, sex determination and tumor angiogenesis. However, its role in mammary gland development and breast carcinogenesis remains unclear. In this study, we found that Kdm3a is expressed in the mouse mammary gland epithelial cells. Knockout of Kdm3a significantly increased H3K9me2/me1 levels in these epithelial cells, which correlated with markedly decreased mammary gland ductal elongation and branching in the intact knockout virgin mice. Furthermore, estrogen replacement in the ovariectomized Kdm3a knockout mice couldn't rescue the retarded ductal growth. Moreover, transplantation of KO mammary gland pieces to wild type recipient mice showed slower ductal growth compared with that of WT gland pieces. Consistently, knockout of Kdm3a also reduced the proliferation rates and cyclin D1 expression in the mammary gland epithelial cells. In addition, Kdm3a knockout did not significantly change the latency of the polyoma middle T oncogene-induced mammary gland tumorigenesis. Tumor growth, however, was slowed which might be due to the decrease in cyclin D1 expression and tumor cell proliferation. We also found that Kdm3a binds and activates the cyclin D1 promoter. These results demonstrate that Kdm3a plays an important intrinsic role in promoting mammary gland ductal growth and tumor growth probably through enhancing cyclin D1 expression and cell proliferation.
RESUMEN
While most breast cancers are thought to arise from the luminal layer of the breast tissue, it remains unclear which specific cells in the luminal layer are the cells of origin of breast cancer. We have previously reported that WAP-positive luminal epithelial cells are at increased susceptibility to tumor initiation by ErbB2 compared to the bulk population, while the mammary cells with canonical Wnt signaling activity fail to evolve into tumors upon ErbB2 activation. Here, we used retrovirus to introduce ErbB2 into the Krt6a-positive mammary progenitor subset of the luminal epithelium and, for comparison, into the mammary luminal epithelium indiscriminately. Tumors developed from both groups of cells with a similar latency. These data indicate that the Krt6a-positive subset of mammary epithelial cells can be induced to form cancer by ErbB2 but it is not more susceptible to tumorigenesis initiated by ErbB2 than the bulk population of the luminal epithelium.
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Carcinogénesis/patología , Células Epiteliales/metabolismo , Queratina-6/metabolismo , Glándulas Mamarias Animales/patología , Receptor ErbB-2/metabolismo , Células Madre/metabolismo , Animales , Apoptosis , Carcinogénesis/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/patología , Femenino , Humanos , Virus del Tumor Mamario del Ratón/metabolismo , Ratones Transgénicos , Fosforilación , Retroviridae/metabolismo , Factor de Transcripción STAT5/metabolismo , Células Madre/patología , Virión/metabolismo , Latencia del VirusRESUMEN
In breast cancer, overexpression of the nuclear coactivator NCOA1 (SRC-1) is associated with disease recurrence and resistance to endocrine therapy. To examine the impact of NCOA1 overexpression on morphogenesis and carcinogenesis in the mammary gland (MG), we generated MMTV-hNCOA1 transgenic [Tg(NCOA1)] mice. In the context of two distinct transgenic models of breast cancer, NCOA1 overexpression did not affect the morphology or tumor-forming capability of MG epithelial cells. However, NCOA1 overexpression increased the number of circulating breast cancer cells and the efficiency of lung metastasis. Mechanistic investigations showed that NCOA1 and c-Fos were recruited to a functional AP-1 site in the macrophage attractant CSF1 promoter, directly upregulating colony-simulating factor 1 (CSF1) expression to enhance macrophage recruitment and metastasis. Conversely, silencing NCOA1 reduced CSF1 expression and decreased macrophage recruitment and breast cancer cell metastasis. In a cohort of 453 human breast tumors, NCOA1 and CSF1 levels correlated positively with disease recurrence, higher tumor grade, and poor prognosis. Together, our results define an NCOA1/AP-1/CSF1 regulatory axis that promotes breast cancer metastasis, offering a novel therapeutic target for impeding this process.
Asunto(s)
Neoplasias de la Mama/patología , Factor Estimulante de Colonias de Macrófagos/genética , Coactivador 1 de Receptor Nuclear/genética , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis Linfática , Células MCF-7 , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Macrófagos/inmunología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Recurrencia Local de Neoplasia/genética , Células Neoplásicas Circulantes/patología , Coactivador 1 de Receptor Nuclear/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Interferencia de ARN , ARN Interferente Pequeño , Factor de Transcripción AP-1/genéticaRESUMEN
The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.
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Diferenciación Celular , Células Epiteliales/patología , Glándulas Mamarias Animales/patología , Comunicación Paracrina , Receptores Acoplados a Proteínas G/metabolismo , Animales , Comunicación Celular , Proliferación Celular , Colágeno/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Femenino , Proteínas Hedgehog/metabolismo , Hiperplasia , Integrasas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Receptores Notch/metabolismo , Transducción de Señal , Receptor SmoothenedRESUMEN
Estrogen signaling is required for the proliferation of normal breast epithelial cells. However, prophylactic inhibition of estrogen signaling fails to prevent 56% of human breast cancer cases. The underlying mechanism is not well understood. Aberrant activation of growth factor signaling is known to provide alternative proliferation pathways in breast cells that are fully transformed, but it is not known whether activation of growth factor signaling can substitute for estrogen signaling in causing aberrant proliferation in the normal breast epithelium. Here, we report that in a retrovirus-based somatic mouse model (replication-competent ALV-LTR splice acceptor/tumor virus A) that closely mimics the evolution of sporadic human breast cancers, mammary epithelial cells harboring PyMT or activated ErbB2 evolve into tumors independent of estrogen or other ovarian functions in contrast to previous observations of estrogen-dependent cancer formation in germ line mouse models of ErbB2 activation. Importantly, ErbB2 activation in normal mammary cells causes estrogen-independent proliferation in both estrogen receptor (ER)-negative cells as well as in normally quiescent ER-positive cells. Therefore, aberrant activation of growth factor signaling contributes to estrogen-independent proliferation of both preneoplastic and cancerous mammary cells, and prophylactic therapy against both growth factor signaling and estrogen signaling may need to be considered in women with increased risk of breast cancer.
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Antígenos Virales de Tumores/genética , Carcinoma Ductal de Mama/genética , Estrógenos/farmacología , Genes erbB-2 , Neoplasias Mamarias Experimentales/genética , Poliomavirus/genética , Animales , Antígenos Virales de Tumores/metabolismo , Antígenos Virales de Tumores/fisiología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Activación Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Genes erbB-2/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Poliomavirus/inmunología , Poliomavirus/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/fisiología , TransfecciónRESUMEN
We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be approximately 2 x 10(3) cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse.