RESUMO
Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay.
Assuntos
Medula Óssea , Leucemia Monocítica Aguda , Humanos , Projetos Piloto , Interleucina-10 , Linhagem Celular , Células EstromaisRESUMO
Bone marrow-derived stromal cells or mesenchymal stromal cells (BMSCs or MSCs, as we will call them in this work) are multipotent progenitor cells that can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs have been shown to modulate the function of a variety of immune cells. Donor age has been shown to affect the regenerative potential, differentiation, proliferation and anti-inflammatory potency of MSCs; however, the impact of donor age on their immunosuppressive activity is unknown. In this study, we evaluated the ability of MSCs derived from very young children and adults on T-cell suppression and cytokine secretion by monocytes/macrophages. MSCs were obtained from extra digits of children between 10 and 21 months and adults between 28 and 64 years of age. We studied cell surface marker expression, doubling time, lineage differentiation potential and immunosuppressive function of the MSCs. Young MSCs double more quickly and differentiate into bone and fat cells more efficiently than those from older donors. They also form more and dense colonies of fibroblasts (colony forming unit-fibroblast [CFU-F]). MSCs from both young and adult subjects suppressed T-cell proliferation in a mitogen-induced assay at 1:3 and 1:30 ratios. At a 1:30 ratio, however, MSCs from adults did not, but MSCs from infants did suppress T-cell proliferation. In the mixed lymphocyte reaction assay, MSCs from infants produced similar levels of suppression at all three MSC/T-cell ratios, but adult MSCs only inhibited T-cell proliferation at a 1:3 ratio. Cytokine analyses of co-cultures of MSCs and macrophages showed that both adult and young MSCs suppress tumor necrosis factor alpha (TNF-α) and induce interleukin-10 (IL-10) production in macrophage co-culture assay in a similar manner. Overall, this work shows that developing MSCs display a higher level of immunosuppression than mature MSCs.
Assuntos
Interleucina-10/biossíntese , Células-Tronco Mesenquimais/imunologia , Polidactilia/cirurgia , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Fatores Etários , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células do Tecido Conjuntivo/fisiologia , Feminino , Humanos , Lactente , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Polidactilia/patologiaRESUMO
BACKGROUND AIMS: Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to suppress T-cell proliferation and used to alleviate the symptoms of graft-versus-host disease (GVHD). MSCs are a mixed cell population and at this time there are no tools to isolate the cells responsible for the T-cell suppression. We wanted to find a way to enhance the immune-modulatory actions of MSCs and tried varying the temperature at which they were cultured. METHODS: We cultured human MSCs derived from healthy volunteers at different temperatures and tested their ability to switch macrophage character from pro-inflammatory to anti-inflammatory (M1 type to M2 type). Using an enzyme-linked immunosorbent assay (ELISA), we showed that when MSCs are cultured at higher temperatures their ability to induce co-cultured macrophages to produce more interleukin-10, (IL-10) (an anti-inflammatory cytokine) and less tumor necrosis factor alpha, (TNFα) (a pro-inflammatory cytokine) is increased. We performed Western blots and immunocytochemistry to screen for changes that might underlie this effect. RESULTS: We found that in hyperthermia the heat shock protein, HSF1, translocated into the nucleus of MSCs. It appears to induce the COX2/PGE2 (Cyclooxygenase2/Prostaglandin E2) pathway described earlier as a major mechanism of MSC-directed immune-suppression. CONCLUSION: Hyperthermia increases the efficacy of MSC-driven immune-suppression. We propose that changing the time of MSC administration to patients to mid-to-late afternoon when the body temperature is naturally highest might be beneficial. Warming the patient could also be considered.
Assuntos
Hipertermia Induzida/métodos , Macrófagos/metabolismo , Células-Tronco Mesenquimais/imunologia , Medula Óssea , Técnicas de Cocultura , Dinoprostona/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Interleucina-10/metabolismo , Macrófagos/citologia , Células-Tronco Mesenquimais/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. CONCLUSION: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).
Assuntos
Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Proteínas Serina-Treonina Quinases/fisiologia , Junções Íntimas/fisiologia , Junções Íntimas/ultraestrutura , Proteínas Quinases Ativadas por AMP , Animais , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
Bone marrow stromal cells (BMSCs) have immunomodulatory activities in numerous species and have been used in clinical trials. BMSCs also make antibacterial agents. Since hepcidin is known to have antimicrobial effects in fish, we wondered if it might also be used as an antimicrobial agent by mammalian BMSCs. In the present study, we show hepcidin expression in both mouse (mBMSC) and human BMSCs (hBMSC). We observed a hBMSC hepcidin-dependent degradation of ferroportin in HEK-293 reporter cells in vitro. In human and mouse bone marrows (BM) we detected hepcidin-positive BMSCs in close proximity to hematopoietic progenitors. The conditioned culture medium of hBMSCs significantly reduced bacterial proliferation that was partially blocked by a hepcidin-neutralizing antibody. Similarly, medium in which hepcidin-deficient (Hamp-/-) mouse BMSCs had been grown was significantly less effective in reducing bacterial counts than the medium of wild-type cells. In a zymosan-induced peritonitis mouse model we found that mBMSC-derived hepcidin reduced the number of invading polymorphonuclear (PMN) cells in the peritoneal cavity. Our results show that BMSC-derived hepcidin has antimicrobial properties in vitro and also reduces inflammation in vivo. We conclude that hepcidin should be added to the expanding arsenal of agents available to BMSCs to fight infections and inflammation.
Assuntos
Anti-Infecciosos , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Hepcidinas/metabolismo , Células HEK293 , Anti-Infecciosos/farmacologia , Inflamação/metabolismo , Células da Medula Óssea , MamíferosRESUMO
We have developed a mouse model for ovarian carcinoma by using an avian retroviral gene delivery technique for the introduction of multiple genes into somatic ovarian cells of adult mice. Ovarian cells from transgenic mice engineered to express the gene encoding the avian receptor TVA were efficiently infected in vitro with multiple vectors carrying coding sequences for oncogenes and marker genes. When target cells were derived from TVA transgenic mice deficient for p53, the addition of any two of the oncogenes c-myc, K-ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected at subcutaneous, intraperitoneal, or ovarian sites. We demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells. The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread.
Assuntos
Carcinoma/genética , Modelos Animais de Doenças , Genes myc/genética , Genes ras/genética , Neoplasias Ovarianas/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Actinas/genética , Actinas/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Proteínas Aviárias , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma/patologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt , Receptores Virais/genética , Receptores Virais/metabolismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections result in the temporary loss of smell and taste in about one third of confirmed cases. METHODS: We used immunohistochemistry to confirm the presence of ACE2, NRP1 and TMPRSS2 in two cranial nerves (IX and X) that mediate taste where they leave/join the medulla. Samples from three (two paraffin embedded and one frozen) postmortem samples were studied (facial (VII) nerve was not available). We also performed immunohistochemistry using the same antibodies in two human cell lines (oligodendrocytes and fibroblasts), and we isolated RNA from one nerve and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. FINDINGS: All three of the proteins (ACE-2, NRP1 and TMPRSS2) required for SARS-CoV-2 infections appear to be present in all cellular components (Schwann cells, axons, vascular endothelium, and connective tissue) of the human IXth and Xth nerves near the medulla. We also found their mRNAs in the nerve and in human oligodendrocytes and fibroblasts which were stained by antibodies directed at the three proteins examined. INTERPRETATION: Infection of the IXth and Xth nerves by the SARS-CoV-2 virus is likely to cause the loss of taste experienced by many Covid patients. Migration of the virus from the oral cavity through these nerves to brainstem respiratory centers might contribute to the problems that patients experience. FUNDING: This study was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research (NIDCR), NIH (intramural project no. ZDE000755-01), and the Human Brain Tissue Bank, Semmelweis University, Budapest, Hungary from the Hungarian Brain Research Program (2017-1.2.1-NKP-2017-00002).
Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Humanos , Internalização do VírusRESUMO
Sarcoidosis is a devastating inflammatory disease affecting many organs, especially the lungs and lymph nodes. Bone marrow-derived mesenchymal stromal cells (MSCs) can "reprogram" various types of macrophages towards an anti-inflammatory phenotype. We wanted to determine whether alveolar macrophages from sarcoidosis subjects behave similarly by mounting an anti-inflammatory response when co-cultured with MSCs. Fifteen sarcoidosis and eight control subjects underwent bronchoscopy and bronchoalveolar lavage (BAL). Unselected BAL cells (70-94% macrophages) were isolated and cultured with and without MSCs from healthy adults. Following stimulation of the cultured cells with lipopolysaccharide, the medium was removed to measure interleukin 10 and tumor necrosis factor alpha (IL-10 and TNF-α). In two additional sarcoidosis subjects, flow cytometry was used to study intracellular cytokines and surface markers associated with alveolar macrophages to confirm the results. Unselected BAL cells from sarcoidosis subjects co-cultured with MSCs showed a reduction in TNF-α (pro-inflammatory M1) and an increase in IL-10 (anti-inflammatory M2) in 9 of 11 samples studied. Control subject samples showed few, if any, differences in cytokine production. Unselected BAL cells from two additional patients analyzed by flow cytometry confirmed a switch towards an anti-inflammatory state (i.e., M1 to M2) after co-culture with MSCs. These results suggest that, similarly to other macrophages, alveolar macrophages also respond to MSC contacts by changing towards an anti-inflammatory phenotype. Based on our results, we hypothesize that mesenchymal stromal cells applied to the airways might alleviate lung inflammation and decrease steroid need in patients with sarcoidosis.
RESUMO
Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Animais , Benzoxazóis/farmacologia , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Prognóstico , Pirimidinas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
PURPOSE: We studied the impact of mTOR signaling inhibition with rapamycin in head and neck squamous cell carcinoma (HNSCC) in the neoadjuvant setting. The goals were to evaluate the mTOR pathway as a therapeutic target for patients with advanced HNSCC, and the clinical safety, antitumor, and molecular activity of rapamycin administration on HNSCC. PATIENTS AND METHODS: Patients with untreated stage II-IVA HNSCC received rapamycin for 21 days (day 1, 15 mg; days 2-12, 5 mg) prior to definitive treatment with surgery or chemoradiation. Treatment responses were assessed clinically and radiographically with CT and FDG-PET. Pre- and posttreatment biopsies and blood were obtained for toxicity, immune monitoring, and IHC assessment of mTOR signaling, as well as exome sequencing. RESULTS: Sixteen patients (eight oral cavity, eight oropharyngeal) completed rapamycin and definitive treatment. Half of patients were p16 positive. One patient had a pathologic complete response and four (25%) patients met RECIST criteria for response (1 CR, 3 PR, 12 SD). Treatment was well tolerated with no grade 4 or unexpected toxicities. No significant immune suppression was observed. Downstream mTOR signaling was downregulated in tumor tissues as measured by phosphorylation of S6 (P < 0.0001), AKT (P < 0.0001), and 4EBP (P = 0.0361), with a significant compensatory increase in phosphorylated ERK in most patients (P < 0.001). Ki67 was reduced in tumor biopsies in all patients (P = 0.013). CONCLUSIONS: Rapamycin treatment was well tolerated, reduced mTOR signaling and tumor growth, and resulted in significant clinical responses despite the brief treatment duration, thus supporting the potential role of mTOR inhibitors in treatment regimens for HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Sirolimo/administração & dosagem , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Serina-Treonina Quinases TOR/genética , Animais , Apoptose , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Fosforilação , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.
Assuntos
Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/terapia , Imunoterapia/métodos , Ipilimumab/uso terapêutico , Neoplasias Bucais/terapia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma de Células Escamosas/induzido quimicamente , Linhagem Celular Tumoral , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/induzido quimicamente , Humanos , Camundongos , Neoplasias Bucais/induzido quimicamente , Nicotiana/efeitos adversosRESUMO
Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Caderinas/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/genética , Caderinas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Fator de Crescimento de Hepatócito/metabolismo , Via de Sinalização Hippo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.
Assuntos
Anemia/metabolismo , Vasopressinas/metabolismo , Vasopressinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , Ratos , Receptores de Vasopressinas/metabolismoRESUMO
Ras genes are the most frequently mutated oncogenes in human cancer. However, the contribution of ras to tumor initiation still is unclear because ras expression in primary cells can cause cell cycle arrest and even cell death by apoptosis. Furthermore, when expressed in the epidermis of mice, mutant ras promotes the formation of benign papillomas, only few of which will progress into carcinomas. However, in these cases, ras-transgene expression often is restricted to suprabasal or follicular epithelial cells that may lack self-renewal capacity. Thus, it still is conceivable that expression of active ras in other epithelial compartments may exert a distinct ability to promote malignant progression. To address this possibility, transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium, which includes the epithelial stem cells, were engineered and crossed with mice expressing the K-ras(G12D) oncogene under the control of tet-regulated responsive elements. On doxycycline administration, proliferative lesions ranging from hyperplasias, papillomas, and dysplasias to metastatic carcinomas developed in squamous epithelia of the skin, oral mucosa, salivary glands, tongue, esophagus, forestomach, and uterine cervix within just 10 to 20 days. The most noticeable lesions were invasive squamous carcinomas of the skin and oral mucosa. These findings suggest that the expression of oncogenes in an epithelial compartment that includes the stem cells may be sufficient to promote squamous carcinogenesis. They also provide a molecularly defined conditional animal model system in which the mechanisms responsible for cancer initiation, maintenance, and metastatic spread can be readily investigated.
Assuntos
Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Genes ras/fisiologia , Neoplasias Cutâneas/genética , Células-Tronco/fisiologia , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epidérmicas , Epiderme/metabolismo , Epiderme/fisiologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/citologia , Transgenes , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
Most squamous cell carcinomas of the head and neck (HNSCC) exhibit a persistent activation of the PI3K-mTOR signaling pathway. We have recently shown that metformin, an oral antidiabetic drug that is also used to treat lipodystrophy in HIV-infected (HIV(+)) individuals, diminishes mTOR activity and prevents the progression of chemically induced experimental HNSCC premalignant lesions. Here, we explored the preclinical activity of metformin in HNSCCs harboring PIK3CA mutations and HPV oncogenes, both representing frequent HNSCC alterations, aimed at developing effective targeted preventive strategies. The biochemical and biologic effects of metformin were evaluated in representative HNSCC cells expressing mutated PIK3CA or HPV oncogenes (HPV(+)). The oral delivery of metformin was optimized to achieve clinical relevant blood levels. Molecular determinants of metformin sensitivity were also investigated, and their expression levels were examined in a large collection of HNSCC cases. We found that metformin inhibits mTOR signaling and tumor growth in HNSCC cells expressing mutated PIK3CA and HPV oncogenes, and that these activities require the expression of organic cation transporter 3 (OCT3/SLC22A3), a metformin uptake transporter. Coexpression of OCT3 and the mTOR pathway activation marker pS6 were observed in most HNSCC cases, including those arising in HIV(+) patients. Activation of the PI3K-mTOR pathway is a widespread event in HNSCC, including HPV(-) and HPV(+) lesions arising in HIV(+) patients, all of which coexpress OCT3. These observations may provide a rationale for the clinical evaluation of metformin to halt HNSCC development from precancerous lesions, including in HIV(+) individuals at risk of developing HPV(-) associated cancers.
Assuntos
Carcinoma de Células Escamosas/prevenção & controle , Metformina/farmacologia , Neoplasias Bucais/prevenção & controle , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Western Blotting , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Ciclo Celular , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/prevenção & controle , Humanos , Hipoglicemiantes/farmacologia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Neoplasias Bucais/etiologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Papillomaviridae/fisiologia , Infecções por Papillomavirus/etiologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Análise Serial de Tecidos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Hepatócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/deficiência , Transporte Proteico , Transdução de Sinais , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Cetuximab, a monoclonal blocking antibody against the epidermal growth factor receptor EGFR, has been approved for the treatment of squamous cell carcinomas of the head and neck (HNSCC). However, only few patients display long-term responses, prompting the search for cetuximab resistance mechanisms and new therapeutic options enhancing cetuximab effectiveness. METHODS: Cetuximab-sensitive HNSCC cells were retro-engineered to express PIK3CA and RAS oncogenes. These cells and HNSCC cells harboring endogenous PIK3CA and RAS oncogenes were xenografted into mice (n = 10 per group) and studied for their biochemical, antitumor, antiangiogenic, and antilymphangiogenic responses to cetuximab and mTOR targeting agents. All P values are two-sided. RESULTS: Cetuximab treatment of PIK3CA- and RAS-expressing HNSCC xenografts promoted an initial antitumor response, but all tumors relapsed within few weeks. In these tumors, cetuximab did not decrease the activity of mTOR, a downstream signaling target of EGFR, PIK3CA, and RAS. The combined administration of cetuximab and mTOR inhibitors exerted a remarkably increased antitumor activity, particularly in HNSCC cells that are resistant to cetuximab as a single agent. Indeed, cotargeting mTOR together with cetuximab caused a rapid tumor collapse of both PIK3CA- and RAS-expressing HNSCC xenografts (P < .001), concomitant with reduced proliferation (P < .001) and lymphangiogenesis (P < .001). CONCLUSION: The presence of PIK3CA and RAS mutations and other alterations affecting the mTOR pathway activity in HNSCC could be exploited to predict the potential resistance to cetuximab, and to select the patients that may benefit the most from the concomitant administration of cetuximab and PI3K and/or mTOR inhibitors as a precision molecular therapeutic option for HNSCC patients.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas ras/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Classe I de Fosfatidilinositol 3-Quinases , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/genética , Humanos , Linfangiogênese/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-ß in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Papillomaviridae/genética , Transcriptoma/genética , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Caderinas/genética , Caspase 8/genética , Linhagem Celular Tumoral , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Quinase 2 Dependente de Ciclina/genética , Variações do Número de Cópias de DNA/genética , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Humanos , PTEN Fosfo-Hidrolase/genética , Infecções por Papillomavirus/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Medicina de Precisão , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Notch1/genética , Análise de Sequência de DNA , Proteína Smad4/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/biossínteseRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. The recent identification of the mTOR complex 1 (mTORC1) signaling pathway as a highly prevalent molecular signature underlying HNSCC pathogenesis has provided the foundation to search for novel therapeutic approaches to prevent and treat HNSCC. Here, we asked whether metformin, the most widely used medication for the treatment of type II diabetes, which acts in part by stimulating the AMP-activated protein kinase (AMPK) signaling pathway thereby reducing mTORC1 activity, may lower the risk of HNSCC development. Indeed, we show that metformin reduces the growth of HNSCC cells and diminishes their mTORC1 activity by both AMPK-dependent and -independent mechanisms. We also optimized an oral-specific carcinogenesis mouse model that results in the accumulation of multiple oral premalignant lesions at the end of the carcinogen exposure, some of which then spontaneously progress into HNSCC. Using this mouse model, we observed that metformin specifically inhibits mTORC1 in the basal proliferating epithelial layer of oral premalignant lesions. Remarkably, metformin prevented the development of HNSCC by reducing significantly the size and number of carcinogen-induced oral tumoral lesions and by preventing their spontaneous conversion to squamous cell carcinomas. Collectively, our data underscore the potential clinical benefits of using metformin as a targeted chemopreventive agent in the control of HNSCC development and progression.