RESUMEN
Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we have developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are highest in liver tissue, which is also a hub for lipid production. While the loss of GSH does not cause liver failure, it decreases lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we find that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.
Asunto(s)
Glutamato-Cisteína Ligasa , Glutatión , Hígado , Factor 2 Relacionado con NF-E2 , Triglicéridos , Animales , Glutatión/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Hígado/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutamato-Cisteína Ligasa/genética , Ratones , Triglicéridos/metabolismo , Estrés Oxidativo , Masculino , Metabolismo de los Lípidos , Ratones Noqueados , Ratones Endogámicos C57BL , Oxidación-Reducción , Lipogénesis/genéticaRESUMEN
Although distinct epithelial cell types have been distinguished in glandular tissues such as the mammary gland, the extent of heterogeneity within each cell type and the degree of endocrine control of this diversity across development are incompletely understood. By combining mass cytometry and cyclic immunofluorescence, we define a rich array of murine mammary epithelial cell subtypes associated with puberty, the estrous cycle, and sex. These subtypes are differentially proliferative and spatially segregate distinctly in adult versus pubescent glands. Further, we identify systematic suppression of lineage programs at the protein and RNA levels as a common feature of mammary epithelial expansion during puberty, the estrous cycle, and gestation and uncover a pervasive enrichment of ribosomal protein genes in luminal cells elicited specifically during progesterone-dominant expansionary periods. Collectively, these data expand our knowledge of murine mammary epithelial heterogeneity and connect endocrine-driven epithelial expansion with lineage suppression.
Asunto(s)
Señales (Psicología) , Glándulas Mamarias Animales , Ratones , Animales , Glándulas Mamarias Animales/metabolismo , ARN/metabolismo , Proliferación Celular , Análisis Espacial , Células Epiteliales/metabolismoRESUMEN
Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.
RESUMEN
The breast is a dynamic organ whose response to physiological and pathophysiological conditions alters its disease susceptibility, yet the specific effects of these clinical variables on cell state remain poorly annotated. We present a unified, high-resolution breast atlas by integrating single-cell RNA-seq, mass cytometry, and cyclic immunofluorescence, encompassing a myriad of states. We define cell subtypes within the alveolar, hormone-sensing, and basal epithelial lineages, delineating associations of several subtypes with cancer risk factors, including age, parity, and BRCA2 germline mutation. Of particular interest is a subset of alveolar cells termed basal-luminal (BL) cells, which exhibit poor transcriptional lineage fidelity, accumulate with age, and carry a gene signature associated with basal-like breast cancer. We further utilize a medium-depletion approach to identify molecular factors regulating cell-subtype proportion in organoids. Together, these data are a rich resource to elucidate diverse mammary cell states.
Asunto(s)
Neoplasias de la Mama , Transcriptoma , Animales , Mama , Neoplasias de la Mama/genética , Femenino , Humanos , Glándulas Mamarias Animales , Embarazo , Proteómica , Transcriptoma/genéticaRESUMEN
Apoptosis of CD8 T cells is an essential mechanism that maintains immune system homeostasis, prevents autoimmunity, and reduces immunopathology. CD8 T cell death also occurs early during the response to both inflammation and costimulation blockade (CoB). In this article, we studied the effects of a combined deficiency of Fas (extrinsic pathway) and Bim (intrinsic pathway) on early T cell attrition in response to lymphocytic choriomeningitis virus infection and during CoB during transplantation. Loss of Fas and Bim function in Bcl2l11-/-Faslpr/lpr mice inhibited apoptosis of T cells and prevented the early T cell attrition resulting from lymphocytic choriomeningitis virus infection. Bcl2l11-/-Faslpr/lpr mice were also resistant to prolonged allograft survival induced by CoB targeting the CD40-CD154 pathway. These results demonstrate that both extrinsic and intrinsic apoptosis pathways function concurrently to regulate T cell homeostasis during the early stages of immune responses and allograft survival during CoB.
Asunto(s)
Apoptosis , Proteína 11 Similar a Bcl2/genética , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Inflamación/inmunología , Receptor fas/genética , Animales , Infecciones por Arenaviridae/inmunología , Linfocitos T CD8-positivos/virología , Regulación de la Expresión Génica , Homeostasis , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de PielRESUMEN
Autophagy is required for cellular homeostasis and can determine cell viability in response to stress. It is established that MTOR is a master regulator of starvation-induced macroautophagy/autophagy, but recent studies have also implicated an essential role for the MAPK8/cJun NH2-terminal kinase 1 signal transduction pathway. We found that MAPK8/JNK1 and MAPK9/JNK2 were not required for autophagy caused by starvation or MTOR inhibition in murine fibroblasts and epithelial cells. These data demonstrate that MAPK8/9 has no required role in starvation-induced autophagy. We conclude that the role of MAPK8/9 in autophagy may be context-dependent and more complex than previously considered. ABBREVIATIONS: AKT: thymoma viral proto-oncogene;ALB: albumin; ATG4: autophagy related 4; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CQ: chloroquine diphosphate; DMEM: Dulbecco's modified Eagle's medium; EDTA: ethylenediaminetetraacetic acid; EBSS: Earle's balanced salt solution; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HRAS: Harvey rat sarcoma virus oncogene; IgG: Immunoglobulin G; MAPK3/ERK1: mitogen-activated protein kinase 3; MAPK8/JNK1: mitogen-activated protein kinase 8; MAPK9/JNK2: mitogen-activated protein kinase 9; MAPK10/JNK3: mitogen-activated protein kinase 10; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; RPS6KB1/p70: ribosomal protein S6 kinase, polypeptide 1; PPARA: peroxisome proliferator activated receptor alpha; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; TRP53: transforming related protein 53; TUBA: tubulin alpha; UV: ultraviolet; WT: wild-type.
Asunto(s)
Aminoácidos/deficiencia , Autofagia , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Animales , Autofagia/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Naftiridinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Proteínas ras/metabolismoRESUMEN
Breast cancer is the most commonly diagnosed malignancy in women. Analysis of breast cancer genomic DNA indicates frequent loss-of-function mutations in components of the cJUN NH2-terminal kinase (JNK) signaling pathway. Since JNK signaling can promote cell proliferation by activating the AP1 transcription factor, this apparent association of reduced JNK signaling with tumor development was unexpected. We examined the effect of JNK deficiency in the murine breast epithelium. Loss of JNK signaling caused genomic instability and the development of breast cancer. Moreover, JNK deficiency caused widespread early neoplasia and rapid tumor formation in a murine model of breast cancer. This tumor suppressive function was not mediated by a role of JNK in the growth of established tumors, but by a requirement of JNK to prevent tumor initiation. Together, these data identify JNK pathway defects as 'driver' mutations that promote genome instability and tumor initiation.
Asunto(s)
Carcinogénesis/genética , Carcinogénesis/patología , Inestabilidad Genómica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Exoma/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/deficiencia , Ratones Endogámicos C57BL , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , FenotipoRESUMEN
Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glándulas Mamarias Animales/metabolismo , Transducción de Señal , Animales , Apoptosis , Catepsinas/genética , Catepsinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/deficiencia , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Lactancia , Glándulas Mamarias Animales/patología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Factor de Transcripción STAT3/metabolismo , Proteína Smad2/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
This corrects the article DOI: 10.1038/nature20609.
RESUMEN
Developmental morphogenesis, tissue injury, and oncogenic transformation can cause the detachment of epithelial cells. These cells are eliminated by a specialized form of apoptosis (anoikis). While the processes that contribute to this form of cell death have been studied, the underlying mechanisms remain unclear. Here, we tested the role of the cJUN NH2-terminal kinase (JNK) signaling pathway using murine models with compound JNK deficiency in mammary and kidney epithelial cells. These studies demonstrated that JNK is required for efficient anoikis in vitro and in vivo. Moreover, JNK-promoted anoikis required pro-apoptotic members of the BCL2 family of proteins. We show that JNK acts through a BAK/BAX-dependent apoptotic pathway by increasing BIM expression and phosphorylating BMF, leading to death of detached epithelial cells.
Asunto(s)
Anoicis , Células Epiteliales/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant. Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours. However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases. These findings, and those in pancreatic cancer and melanoma models, indicate that dissemination might occur during the early stages of tumour evolution. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown. Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelial-mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.
RESUMEN
Smac mimetics (SMs), a class of drugs that can promote tumor cell death, represent a potential therapeutic strategy for the treatment of cancer. In this issue of Cancer Cell, Lalaoui et al. (2016) report that SM efficacy can be potently increased by inhibition of the p38α MAPK/MK2 signaling pathway.
Asunto(s)
Antineoplásicos/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia/tratamiento farmacológico , Proteínas Mitocondriales/fisiología , Imitación Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , HumanosRESUMEN
We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1(-/-) or jnk2(-/-) mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo.
Asunto(s)
Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Células Epidérmicas , Epidermis/metabolismo , Células Epiteliales/citología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Ratones , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , FosforilaciónRESUMEN
cJun NH(2)-terminal kinase (JNK) signaling has been implicated in the developmental morphogenesis of epithelial organs. In this study, we employed a compound deletion of the murine Jnk1 and Jnk2 genes in the mammary gland to evaluate the requirement for these ubiquitously expressed genes in breast development and tumorigenesis. JNK1/2 was not required for breast epithelial cell proliferation or motility. However, JNK1/2 deficiency caused increased branching morphogenesis and defects in the clearance of lumenal epithelial cells. In the setting of breast cancer development, JNK1/2 deficiency significantly increased tumor formation. Together, these findings established that JNK signaling is required for normal mammary gland development and that it has a suppressive role in mammary tumorigenesis.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/enzimología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Movimiento Celular/fisiología , Femenino , Expresión Génica , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Proteína Quinasa 8 Activada por Mitógenos/deficiencia , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/deficiencia , Proteína Quinasa 9 Activada por Mitógenos/genéticaRESUMEN
The stress-activated protein kinase (SAPK) p38 can induce apoptosis, and its inhibition facilitates mammary tumorigenesis. We found that during mammary acinar morphogenesis in MCF-10A cells grown in three-dimensional culture, detachment of luminal cells from the basement membrane stimulated mitogen-activated protein kinase (MAPK) kinases 3 and 6 (MKK3/6) and p38α signaling to promote anoikis. p38α signaling increased transcription of the death-promoting protein BimEL by phosphorylating the activating transcription factor 2 (ATF-2) and increasing c-Jun protein abundance, leading to cell death by anoikis and acinar lumen formation. Inhibition of p38α or ATF-2 caused luminal filling reminiscent of that observed in ductal carcinoma in situ (DCIS). The mammary glands of MKK3/6 knockout mice (MKK3(-/-)/MKK6(+/- )) showed accelerated branching morphogenesis relative to those of wild-type mice, as well as ductal lumen occlusion due to reduced anoikis. This phenotype was recapitulated by systemic pharmacological inhibition of p38α and ß (p38α/ß) in wild-type mice. Moreover, the development of DCIS-like lesions showing marked ductal occlusion was accelerated in MMTV-Neu transgenic mice treated with inhibitors of p38α and p38ß. We conclude that p38α is crucial for the development of hollow ducts during mammary gland development, a function that may be crucial to its ability to suppress breast cancer.
Asunto(s)
Anoicis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Glándulas Mamarias Animales/embriología , Proteína Quinasa 11 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Morfogénesis/fisiología , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Animales , Línea Celular Tumoral , Femenino , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 11 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. To investigate the cellular origin(s) of this cancer, we determined the effect of PDAC-relevant gene mutations in distinct cell types of the adult pancreas. We show that a subpopulation of Pdx1-expressing cells is susceptible to oncogenic K-Ras-induced transformation without tissue injury, whereas insulin-expressing endocrine cells are completely refractory to transformation under these conditions. However, chronic pancreatic injury can alter their endocrine fate and allow them to serve as the cell of origin for exocrine neoplasia. These results suggest that one mechanism by which inflammation and/or tissue damage can promote neoplasia is by altering the fate of differentiated cells that are normally refractory to oncogenic stimulation.