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The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.
Assuntos
Neoplasias , Monoéster Fosfórico Hidrolases , Via de Sinalização Hippo , Genes Reguladores , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a molecular imaging method that can be used to elucidate the small-molecule composition of tissues and map their spatial information using two-dimensional ion images. This technique has been used to investigate the molecular profiles of variety of tissues, including within the central nervous system, specifically the brain and spinal cord. To our knowledge, this technique has yet to be applied to tissues of the peripheral nervous system (PNS). Data generated from such analyses are expected to advance the characterization of these structures. The study aimed to: (i) establish whether DESI-MSI can discriminate the molecular characteristics of peripheral nerves and distinguish them from surrounding tissues and (ii) assess whether different peripheral nerve subtypes are characterized by unique molecular profiles. Four different nerves for which are known to carry various nerve fiber types were harvested from a fresh cadaveric donor: mixed, motor and sensory (sciatic and femoral); cutaneous, sensory (sural); and autonomic (vagus). Tissue samples were harvested to include the nerve bundles in addition to surrounding connective tissue. Samples were flash-frozen, embedded in optimal cutting temperature compound in cross-section, and sectioned at 14 µm. Following DESI-MSI analysis, identical tissue sections were stained with hematoxylin and eosin. In this proof-of-concept study, a combination of multivariate and univariate statistical methods was used to evaluate molecular differences between the nerve and adjacent tissue and between nerve subtypes. The acquired mass spectral profiles of the peripheral nerve samples presented trends in ion abundances that seemed to be characteristic of nerve tissue and spatially corresponded to the associated histology of the tissue sections. Principal component analysis (PCA) supported the separation of the samples into distinct nerve and adjacent tissue classes. This classification was further supported by the K-means clustering analysis, which showed separation of the nerve and background ions. Differences in ion expression were confirmed using ANOVA which identified statistically significant differences in ion expression between the nerve subtypes. The PCA plot suggested some separation of the nerve subtypes into four classes which corresponded with the nerve types. This was supported by the K-means clustering. Some overlap in classes was noted in these two clustering analyses. This study provides emerging evidence that DESI-MSI is an effective tool for metabolomic profiling of peripheral nerves. Our results suggest that peripheral nerves have molecular profiles that are distinct from the surrounding connective tissues and that DESI-MSI may be able to discriminate between nerve subtypes. DESI-MSI of peripheral nerves may be a valuable technique that could be used to improve our understanding of peripheral nerve anatomy and physiology. The ability to utilize ambient mass spectrometry techniques in real time could also provide an unprecedented advantage for surgical decision making, including in nerve-sparing procedures in the future.
Assuntos
Nervos Periféricos , Espectrometria de Massas por Ionização por Electrospray , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1007597.].
RESUMO
Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.
Assuntos
Cryptococcus neoformans/metabolismo , Dinoprostona/análogos & derivados , Eicosanoides/metabolismo , Animais , Animais Geneticamente Modificados , Técnicas de Cultura de Células , Criptococose/metabolismo , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/patogenicidade , Dinoprostona/metabolismo , Dinoprostona/fisiologia , Modelos Animais de Doenças , Eicosanoides/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Macrófagos/microbiologia , PPAR gama/metabolismo , Virulência/fisiologia , Peixe-Zebra/microbiologiaRESUMO
Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that represent an effective class of insulin-sensitizing agents; however, clinical use is associated with weight gain and peripheral edema. To elucidate the role of PPARγ expression in endothelial cells (ECs) in these side effects, EC-targeted PPARγ knockout (Pparg ΔEC) mice were placed on a high-fat diet to promote PPARγ agonist-induced plasma volume expansion, and then treated with the TZD rosiglitazone. Compared with Pparg-floxed wild-type control (Pparg f/f) mice, Pparg ΔEC treated with rosiglitazone are resistant to an increase in extracellular fluid, water content in epididymal and inguinal white adipose tissue, and plasma volume expansion. Interestingly, histologic assessment confirmed significant rosiglitazone-mediated capillary dilation within white adipose tissue of Pparg f/f mice, but not Pparg ΔEC mice. Analysis of ECs isolated from untreated mice in both strains suggested the involvement of changes in endothelial junction formation. Specifically, compared with cells from Pparg f/f mice, Pparg ΔEC cells had a 15-fold increase in focal adhesion kinase, critically important in EC focal adhesions, and >3-fold significant increase in vascular endothelial cadherin, the main component of focal adhesions. Together, these results indicate that rosiglitazone has direct effects on the endothelium via PPARγ activation and point toward a critical role for PPARγ in ECs during rosiglitazone-mediated plasma volume expansion.
Assuntos
Tecido Adiposo/metabolismo , Células Endoteliais/metabolismo , Hipoglicemiantes/farmacologia , PPAR gama/deficiência , Rosiglitazona/farmacologia , Remodelação Vascular/fisiologia , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/efeitos dos fármacos , Animais , Células Endoteliais/efeitos dos fármacos , Deleção de Genes , Masculino , Camundongos , Camundongos Transgênicos , PPAR gama/genética , Volume Plasmático/efeitos dos fármacos , Volume Plasmático/fisiologia , Remodelação Vascular/efeitos dos fármacosRESUMO
BACKGROUND: Among women worldwide, breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates genes involved in insulin sensitivity and adipogenesis. Previously, we showed, using 7,12-dimethylbenz [a] anthracene (DMBA)-treated haploinsufficient PPARγ mice, that PPARγ suppresses breast tumour progression; however, the PPARγ expressing cell types and mechanisms involved remain to be clarified. Here, the role of PPARγ expression and activation in mammary epithelial cells (MG) with respect to DMBA-mediated breast tumourigenesis was investigated. METHODS: PPARγ MG knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated once a week for six weeks by oral gavage with 1 mg DMBA dissolved in corn oil and maintained on a normal chow diet. At week 7, mice were randomly divided into those maintained on a normal chow diet (DMBA Only; PPARγ-WT: n = 25 and PPARγ-MG KO: n = 39) or those receiving a diet supplemented with the PPARγ ligand, rosiglitazone (ROSI, 4 mg/kg/day) (DMBA + ROSI; PPARγ-WT: n = 34 and PPARγ-MG KO: n = 17) for the duration of the 25-week study. RESULTS: Compared to DMBA Only-treated PPARγ-WTs, both breast tumour susceptibility and serum levels of proinflammatory and chemotactic cytokines, namely IL-4, eotaxin, GM-CSF, IFN-γ, and MIP-1α, were decreased among PPARγ-MG KOs. Cotreatment with ROSI significantly reduced breast tumour progression among PPARγ-WTs, correlating with increased BRCA1 and decreased VEGF and COX-2 protein expression levels in breast tumours; whereas, surprisingly DMBA + ROSI-treated PPARγ-MG KOs showed increased breast tumourigenesis, correlating with activation of COX-2. CONCLUSION: These novel data suggest MG-specific PPARγ expression and signaling is critical during breast tumourigenesis, and may serve as a strong candidate predictive biomarker for response of breast cancer patients to the use of therapeutic strategies that include PPARγ ligands.
Assuntos
Progressão da Doença , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , PPAR gama/metabolismo , Transdução de Sinais , 9,10-Dimetil-1,2-benzantraceno , Animais , Proteína BRCA1/metabolismo , Citocinas/sangue , Células Epiteliais/patologia , Feminino , Deleção de Genes , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/sangue , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/sangue , Neoplasias Mamárias Experimentais/patologia , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos , Carga TumoralRESUMO
Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator-activated receptor (PPAR)γ((+/-)) mice, we showed normal expression of PPARγ was critical to stop 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE-specific loss of PPARγ was hypothesized to enhance DMBA-mediated breast tumorigenesis. To test this, MSE cell-specific PPARγ knockout (PPARγ-MSE KO) and control (PPARγ-WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ-WT, n = 15; PPARγ-MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ-WT, n = 17; PPARγ-MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ-MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ-WT controls. PPARγ activation significantly reduced DMBA-mediated malignant mammary tumor volumes irrespective of genotype. MSE-specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down-regulating Cox-1, Cox-2 and cyclin D1. Collectively, these studies highlight a protective role of MSE-specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.
Assuntos
Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/etiologia , PPAR gama/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Células Epiteliais/metabolismo , Feminino , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/análise , Proteína X Associada a bcl-2/análiseRESUMO
The dependency of cancer cells on glucose can be targeted with high-fat low-carbohydrate ketogenic diet (KD). However, hepatic ketogenesis is suppressed in IL-6 producing cancers, which prevents the utilization of this nutrient source as energy for the organism. In two IL-6 associated murine models of cancer cachexia we describe delayed tumor growth but accelerated onset of cancer cachexia and shortened survival when mice are fed KD. Mechanistically, we find this uncoupling is a consequence of the biochemical interaction of two simultaneously occurring NADPH-dependent pathways. Within the tumor, increased production of lipid peroxidation products (LPPs) and, consequently, saturation of the glutathione (GSH) system leads to ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impairs the biosynthesis of corticosterone, the main regulator of metabolic stress, in the adrenal glands. Administration of dexamethasone, a potent glucocorticoid, improves food intake, normalizes glucose homeostasis and utilization of nutritional substrates, delays onset of cancer cachexia and extends survival of tumor-bearing mice fed KD, while preserving reduced tumor growth. Our study highlights that the outcome of systemic interventions cannot necessarily be extrapolated from the effect on the tumor alone, but that they have to be investigated for anti-cancer and host effects. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.
RESUMO
Glucose dependency of cancer cells can be targeted with a high-fat, low-carbohydrate ketogenic diet (KD). However, in IL-6-producing cancers, suppression of the hepatic ketogenic potential hinders the utilization of KD as energy for the organism. In IL-6-associated murine models of cancer cachexia, we describe delayed tumor growth but accelerated cachexia onset and shortened survival in mice fed KD. Mechanistically, this uncoupling is a consequence of the biochemical interaction of two NADPH-dependent pathways. Within the tumor, increased lipid peroxidation and, consequently, saturation of the glutathione (GSH) system lead to the ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impair corticosterone biosynthesis. Administration of dexamethasone, a potent glucocorticoid, increases food intake, normalizes glucose levels and utilization of nutritional substrates, delays cachexia onset, and extends the survival of tumor-bearing mice fed KD while preserving reduced tumor growth. Our study emphasizes the need to investigate the effects of systemic interventions on both the tumor and the host to accurately assess therapeutic potential. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.
Assuntos
Dieta Cetogênica , Ferroptose , Neoplasias , Camundongos , Animais , Caquexia , Corticosterona , Interleucina-6 , NADP , Corpos Cetônicos , Glucose , Neoplasias/complicaçõesRESUMO
Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.
Assuntos
Adipócitos/metabolismo , Neoplasias Mamárias Experimentais/prevenção & controle , PPAR gama/fisiologia , Células Estromais/metabolismo , Animais , Sequência de Bases , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Knockout , PPAR gama/genética , Reação em Cadeia da Polimerase em Tempo Real , Rosiglitazona , Tiazolidinedionas/administração & dosagemRESUMO
Obesity is a global pandemic that is causally linked to many life-threatening diseases. Apart from some rare genetic conditions, the biological drivers of overeating and reduced activity are unclear. Here, we show that neurotensin-expressing neurons in the mouse interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a nucleus of the central extended amygdala, encode dietary preference for unhealthy energy-dense foods. Optogenetic activation of IPACNts neurons promotes obesogenic behaviors, such as hedonic eating, and modulates food preference. Conversely, acute inhibition of IPACNts neurons reduces feeding and decreases hedonic eating. Chronic inactivation of IPACNts neurons recapitulates these effects, reduces preference for sweet, non-caloric tastants and, furthermore, enhances locomotion and energy expenditure; as a result, mice display long-term weight loss and improved metabolic health and are protected from obesity. Thus, the activity of a single neuronal population bidirectionally regulates energy homeostasis. Our findings could lead to new therapeutic strategies to prevent and treat obesity.
Assuntos
Núcleo Central da Amígdala , Neurotensina , Camundongos , Animais , Neurotensina/metabolismo , Neurônios/fisiologia , Núcleo Central da Amígdala/metabolismo , Metabolismo Energético , Homeostase , Obesidade/metabolismoRESUMO
The chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) has been used for many decades to induce skin, mammary, and ovarian tumors in mice. There are however a wide range of doses and treatment regimens in the literature that sometimes confound comparative interpretations of different studies. Here we describe a proven method to generate in vivo DMBA-mediated murine mammary tumors to enable consistent studies of the cell targeted role of genes of interest during this process.