RESUMO
The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction.
Assuntos
Complicações Pós-Operatórias/prevenção & controle , Sirolimo/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/cirurgia , Idoso , Animais , Antígeno B7-H1/antagonistas & inibidores , Proliferação de Células , Cistectomia/efeitos adversos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Complicações Pós-Operatórias/imunologia , Receptor de Morte Celular Programada 1 , Proteína S6 Ribossômica/metabolismo , Sirolimo/administração & dosagem , Sirolimo/efeitos adversos , Sirolimo/farmacocinética , Linfócitos T/imunologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
p53 is a transcriptional regulator that responds to cellular stresses to suppress oncogenesis, but some of these responses can have unintended consequences that influence non-cancer-related aging processes. The impact of these consequences is not well understood-partly due to the many complex processes that influence p53 function and partly due to the vast array of processes that p53 affects. p53 has the potential to both accelerate and hinder cellular aging processes, which would likely have antithetical biological outcomes with regard to organismal aging. To accelerate aging, p53 induces apoptosis or cell cycle arrest as a prerequisite to cellular senescence; both can impair the mobilization of stem and progenitor cell populations. To suppress aging, p53 inhibits unregulated proliferation pathways that could lead to cellular senescence and a senescence-associated secretory phenotype (SASP), which creates a pro-inflammatory and degenerative tissue milieu. A review of mouse models supports both possibilities, highlighting the complexity of the p53 influence over organismal aging. A deeper knowledge of how p53 integrates and is integrated with various biological processes will improve our understanding of its influence over the aging process.
RESUMO
Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic.
Assuntos
Sirolimo/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Sirolimo/sangue , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)alpha-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERalpha-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERalpha transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERalpha in the presence of arzoxifene. In this altered conformation, ERalpha still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERalpha-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERalpha antagonist into an agonist. This can be explained by a stabilization of the ERalpha/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERalpha upon binding to antiestrogen are at the basis of resistance to antiestrogens.
Assuntos
Ciclina D1/biossíntese , Resistência a Medicamentos , Moduladores de Receptor Estrogênico/farmacologia , Regulação da Expressão Gênica , Piperidinas/farmacologia , Tiofenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia Confocal/métodos , Modelos QuímicosRESUMO
We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor alpha (ERalpha) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERalpha- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in k(off). ERalpha-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERalpha DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/fisiologia , Ligantes , Transporte Ativo do Núcleo Celular , Sequência de Bases , Proteínas de Transporte/metabolismo , Diagnóstico por Imagem , Células HeLa , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Prolactina/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Polimerase II/metabolismo , Análise Serial de Tecidos/métodos , Fator de Transcrição Pit-1/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
The development of live cell and biochemical analysis methods has led to an increase in our understanding of the dynamic regulation of transcription. Live single cell studies using photobleaching techniques indicate that many proteins have a high nuclear mobility. Pioneering work using promoter array systems based on the lac operon or the mouse mammary tumor virus promoter enabled the study of chromatin structure, promoter occupancy and protein-chromatin interaction dynamics in relation to transcription. Chromatin immunoprecipitation (ChIP)-based assays allow an exhaustive analysis of the temporal recruitment of proteins to an endogenous promoter and provide evidence of cyclic protein-protein and protein-promoter interactions. Although reflecting different timescales, both ChIP and live cell studies indicate a highly dynamic control of transcription that until now has gone undetected and unappreciated.
Assuntos
Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/fisiologia , Cromatina/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação , Proteínas Nucleares/genéticaRESUMO
Pit-1, a POU-class nuclear DNA-binding transcription factor, specifies three of the parenchymal cell types in anterior pituitary ontogeny. Using fluorescent fusions and live cell imaging, we have compared the dynamic behavior of wild-type and inactivating Pit-1 point mutations. Fluorescence recovery after photobleaching (FRAP) and real-time extraction data indicate that wild-type Pit-1 has a dynamic mobility profile, with t(1/2s) approximately 5-7 s when expressed from low to high amounts, respectively. Biochemically, Pit-1 is approximately 50% retained according to direct observation during extraction, indicating a dynamic interaction with nuclear structure. An analysis of transiently expressed Pit-1 carrying two different debilitating mutations reveals that they translocate normally to the nucleus, but exhibit two different levels of mobility, both clearly distinguishable from wild-type Pit-1. At low expression levels, the t(1/2s) of Pit(W261C) and Pit(A158P) are extremely rapid (0.3 and 0.6 s t(1/2s), respectively). At higher expression levels, unlike wild-type Pit-1, both mutant proteins become immobilized and insoluble, and fractionate completely with the insoluble nuclear matrix. Relative to wild-type, over expression of mutated Pit-1 elicits a nuclear stress response indicated by increased levels of heat shock inducible heat shock protein 70 (Hsp70), and reorganization of heat shock factor-1. The decreased mobility of Pit(A158P) relative to Pit(W261C) at low expression levels correlates with its ability to partially activate when expressed at low levels and its ability to bind cognate DNA. At high expression levels, lower Pit(A158P) activation correlates with its immobilization and insolubility. These data suggest a link between specific rates of intranuclear mobility and Pit-1 transcription function, perhaps to insure sufficient interactions with chromatin, or in the case of non-DNA binding Pit-1, interaction as a repressor. These data imply inactivating mutations can lead to an intranuclear sorting away from transcription related pathways, and at least in part to a misfolded protein pathway. Taken together, caution is suggested when interpreting point (or other) mutational analyses of transactivator function, as new compartmentation, especially in the context of expression levels, may cloud the distinction between defining functional molecular domains and intranuclear processing of misfolded proteins.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mutação Puntual/genética , Dobramento de Proteína , Fatores de Transcrição/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico , Proteínas de Homeodomínio/genética , Humanos , Mutagênese , Fotodegradação , Fator de Transcrição Pit-1 , Transcrição GênicaRESUMO
The efficacy of dietary restriction in retarding tumor growth is well established in rodents. However, gene and cell lineage specificity of dietary restriction effects is far less defined. Mice with a single copy of the retinoblastoma susceptibility gene (Rb) develop a well-established syndrome of mouse neuroendocrine neoplasia associated with Rb deficiency. Thus, if DR represses tumor growth in this model, it should be unambiguously attributed to the Rb defect in neuroendocrine cell lineages. To address this possibility, Rb(+/-) mice were entered into a diet restriction study. Surprisingly, 40-50% reductions in dietary intake, relative to an ad libitum group, started on either postnatal day 28 or 42 had little to no effect on either the frequency or growth of pituitary tumors either during the latency period (postnatal day 224) or at the time of their natural death. Consistent with cross-section data, survival of 65 diet restricted Rb(+/-) mice was almost identical to that of 67 Rb(+/-) mice fed ad libitum (AL); median life span was 414 and 436 days for AL and DR groups, respectively. These findings indicate that diet restriction provides no significant benefit in delaying growth and progression of neuroendocrine tumors exhibiting loss of RB function. They also introduce the possibility that RB is required for the tumor-repressive effects of DR.