RESUMO
Neuroblastoma (NB) is a deadly childhood disease that carries a 50% chance of relapse for anyone in remission and similar level of 5-year survival. We investigated the value of our proprietary approach-cell surface vimentin (CSV) positive circulating tumor cells (CTC) to monitor treatment response and predict relapse in NB patients under remission in a Phase II long-term preventative clinical trial. We longitudinally analyzed peripheral blood samples from 93 patients for 27 cycles (~25 months) and discovered that the presence of CSV+ CTCs in the first two sequential samples (baseline, cycle 4 [month 3-4]) was a significant indicator of earlier relapse. We observed strong correlation between relapse-free survival (RFS) and lack of CSV+ CTCs in first 4 cycles of therapy (95%). There was sensitivity reaching 100% in predicting RFS in patients who had neither CSV+ CTCs nor MycN amplification. Of note, the low number of CSV+ CTCs seems equivalent to low tumor load because the prevention therapy difluoromethylornithine yields faster reduction of relapse risk when none or only 1-2 CSV+ CTCs (every 6 mL) are present in the blood samples compared to >3 CSV+ CTCs. To the best of our knowledge, this is the first study that directly observes CTCs in under remission NB patients for relapse prediction and the first to gather sequential CSV+ CTC data in any study in a long-term longitudinal manner.
Assuntos
Recidiva Local de Neoplasia/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Neuroblastoma/diagnóstico , Vimentina/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Ensaios Clínicos Fase II como Assunto , Detecção Precoce de Câncer , Eflornitina/uso terapêutico , Transição Epitelial-Mesenquimal , Feminino , Humanos , Estudos Longitudinais , Masculino , Recidiva Local de Neoplasia/metabolismo , Neuroblastoma/metabolismo , Sensibilidade e Especificidade , Análise de SobrevidaRESUMO
Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related deaths worldwide with a poor survival rate. As many as 40% of HCCs are clonal, with alteration of key tumor-suppressor pathways in stem cells as the primary cause of HCC initiation. However, mechanisms that generate metastatic stem cells in preneoplastic liver tissue are not well understood. We hypothesized that chronic inflammation is a major driver of the transformation of genetically defective liver stem cells (LSCs) into highly metastatic liver cancer cells in premalignant liver tissue. We developed models of chronic inflammation in wild-type (WT) and ß2-spectrin (ß2SP)+/- (SPTBN1) mice. CD133+ LSCs derived from preneoplastic livers of ß2SP+/- mice treated with interleukin-6 (pIL6; IL6 ß2SP+/- LSCs) were highly tumorigenic and metastatic, whereas those derived from WT mice treated with pIL6 (IL6 WT LSCs) had significantly less proliferation and no tumorigenic properties. IL6 ß2SP+/- LSCs not only exhibited nuclear localization of Twist and Slug, markers of epithelial-mesenchymal transition (EMT), but also constitutive activation of nuclear factor kappa B (NFκB; RELA). Knockdown of NFκB decreased the EMT phenotypes and metastatic capacity of these cells. NFκB in IL6 ß2SP+/- LSCs was activated by transforming growth factor ß (TGFß)-activated kinase 1 (TAK1; MAP3K7), which is associated with poor survival in HCC and interleukin-6 (IL6) expression. The amount of constitutively activated NFκB increased dramatically from normal to cirrhotic to HCC tissues from human patients. CONCLUSION: IL6-mediated inflammation programs constitutive activation of the TAK1-NFκB signaling cascade in CD133+ LSCs, and this program interacts with deficient TGFß signaling, thereby accelerating the transformation of normal LSCs to metastatic cancer stem cells (mCSCs). Indeed, this study delineates the development of EMT-positive mCSCs in HCC-free liver tissue upon chronic inflammation. (Hepatology 2017;65:1222-1236).
Assuntos
Carcinoma Hepatocelular/patologia , Interleucina-6/farmacologia , Neoplasias Hepáticas/patologia , MAP Quinase Quinase Quinases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Biópsia por Agulha , Western Blotting , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , MAP Quinase Quinase Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Distribuição Aleatória , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta1/efeitos dos fármacosRESUMO
UNLABELLED: The cellular and molecular etiology of unresolved chronic liver inflammation remains obscure. Whereas mutant p53 has gain-of-function properties in tumors, the role of this protein in liver inflammation is unknown. Herein, mutant p53(R172H) is mechanistically linked to spontaneous and sustained liver inflammation and steatosis when combined with the absence of interleukin-27 (IL27) signaling (IL27RA), resembling the phenotype observed in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) patients. Indeed, these mice develop, with age, hepatocyte necrosis, immune cell infiltration, fibrosis, and micro- and macrosteatosis; however, these phenotypes are absent in mutant p53(R172H) or IL27RA(-/-) mice. Mechanistically, endothelin A receptor (ETAR)-positive macrophages are highly accumulated in the inflamed liver, and chemical inhibition of ETAR signaling reverses the observed phenotype and negatively regulates mutant p53 levels in macrophages. CONCLUSION: The combination of mutant p53 and IL27RA(-/-) causes spontaneous liver inflammation, steatosis, and fibrosis in vivo, whereas either gene alone in vivo has no effects on the liver.
Assuntos
Genes p53 , Macrófagos/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Receptor de Endotelina A/metabolismo , Receptores de Citocinas/deficiência , Animais , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores de InterleucinaRESUMO
BACKGROUND & AIMS: Sepsis is an acute systemic inflammatory response to infection associated with high patient mortality (28-40%). We hypothesized that interleukin (IL)-30, a novel cytokine protecting mice against liver injury resulting from inflammation, would generate a protective effect against systemic inflammation and sepsis-induced death. METHODS: Sepsis was induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). The inhibitory effects of IL-30 on septic inflammation and associated therapeutic effects were determined in wild-type, IL30 (p28)(-/-), IL10(-/-), and CD1d(-/-) mice. RESULTS: Mice treated with pIL30 gene therapy or recombinant IL-30 protein (rIL30) were protected from LPS-induced septic shock or CLP-induced polymicrobial sepsis and showed markedly less liver damage and lymphocyte apoptosis than control septic mice. The resulting reduction in mortality was mediated through attenuation of the systemic pro-inflammatory response and augmentation of bacterial clearance. Mice lacking IL-30 were more sensitive to LPS-induced sepsis. Natural killer-like T cells (NKT) produced much higher levels of IL-10 and lower levels of interferon-gamma and tumor necrosis factor-alpha in IL-30-treated septic mice than in control septic mice. Likewise, deficiency in IL-10 or NKT cells abolished the protective role of IL-30 against sepsis. Furthermore, IL-30 induced IL-10 production in purified and LPS-stimulated NKT cells. Blocking IL-6R or gp130 inhibited IL-30 mediated IL-10 production. CONCLUSIONS: IL-30 is important in modulating production of NKT cytokines and subsequent NKT cell-mediated immune regulation of other cells. Therefore, IL-30 has a role in prevention and treatment of sepsis via modulation of cytokine production by NKT.
Assuntos
Citocinas/metabolismo , Interleucinas/farmacologia , Fígado/patologia , Células T Matadoras Naturais/imunologia , Choque Séptico/tratamento farmacológico , Animais , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Choque Séptico/metabolismo , Choque Séptico/patologiaRESUMO
Recent advances in cancer stem cell biology have shown that cancer stem-like cells with epithelial-mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem-like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem-like cells, but none has been identified for isolating cancer stem-like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem-like cancer cells with EMT phenotype, by using a specific CSV-binding antibody, 84-1. Using this antibody, we purified the CSV-positive, CD133-negative (csVim(+) CD133(-) ) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim(+) CD133(-) cells have stem-like properties similar to csVim(-) CD133(+) population. Our investigation further revealed that the csVim(+) CD133(-) cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E-cadherin. The csVimentin-negative CD133-positive stem cells do not have any EMT phenotypes. csVim(+) CD133(-) cells exhibited more aggressively metastatic in livers than csVim(-) CD133(+) cells. Our findings indicate that csVim(+) CD133(-) cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal , Glicoproteínas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Vimentina/metabolismo , Antígeno AC133 , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Membrana Celular/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Confocal , Neoplasias Experimentais/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica/imunologia , Fatores de Transcrição SOXB1/metabolismo , Células Tumorais Cultivadas , Vimentina/imunologiaRESUMO
BACKGROUND: Detection, isolation, and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an epithelial cell adhesion molecule (EpCAM)-based method that is used to isolate epithelial CTCs, has gained prominence, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns regarding its utility in clinical management. METHODS: To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma tumors. In the present study, we tested the sensitivity and specificity of detecting CTCs from blood collected at a random time during therapy from each of 58 patients with metastatic breast cancer by use of 84-1 (a monoclonal antibody against CSV to detect epithelial/mesenchymal-transition CTCs) and CellSearch methods. Additionally, we tested the possibility of improving the sensitivity and specificity of detection by use of additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios. RESULTS: CTC counts with CSV were significant (P = 0.0053) in differentiating populations responsive and nonresponsive to treatment compared with CTC counts with CellSearch (P = 0.0564). The specificity of CTC detection was found to be highest when the sum of CTC counts from the 2 methods was above a threshold of 8 CTCs/7.5 mL. CONCLUSIONS: The sum of CTC counts from the CellSearch and CSV methods appears to provide new insights for assessment of therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.
Assuntos
Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Moléculas de Adesão Celular/sangue , Membrana Celular/metabolismo , Transição Epitelial-Mesenquimal , Células Neoplásicas Circulantes/patologia , Vimentina/metabolismo , Anticorpos Monoclonais/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Contagem de Células , Molécula de Adesão da Célula Epitelial , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Metástase Neoplásica , Células Neoplásicas Circulantes/efeitos dos fármacos , Projetos Piloto , Estudos Retrospectivos , Resultado do Tratamento , Vimentina/imunologiaRESUMO
UNLABELLED: Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus-mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine-based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)-30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha-smooth muscle actin (α-SMA) protein indicated that IL-30-based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL-30 recruits natural-killer-like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody-mediated neutralization studies showed that liver NKT cells up-regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T-cell-deficient mice showed reduction of fibrosis upon IL-30 administration. CONCLUSIONS: Highly target-specific liver NKT cells selectively remove activated HSCs through an NKG2D-Rae1 interaction to ameliorate liver fibrosis after IL-30 treatment.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Interleucinas/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células T Matadoras Naturais/efeitos dos fármacos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Animais , Tetracloreto de Carbono , Avaliação Pré-Clínica de Medicamentos , Feminino , Células Estreladas do Fígado/metabolismo , Interleucinas/farmacologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/imunologia , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , PiridinasRESUMO
Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.
RESUMO
Signal transducer and activator of transcription 5b (Stat5b) is a critical node in the signaling network downstream of external (cytokines or growth factors) or internal (oncogenic tyrosine kinases) stimuli. Maximum transcriptional activation of Stat5b requires both tyrosine and serine phosphorylation. Although the mechanisms governing tyrosine phosphorylation and activation of Stat5b have been extensively studied, the role of serine phosphorylation remains to be fully elucidated. Using mass spectrometry and phospho-specific antibodies, we identified Ser-193 as a novel site of cytokine-induced phosphorylation within human Stat5b. Stat5b Ser(P)-193 was detected in activated primary human peripheral blood mononuclear cells or lymphoid cell lines in response to several γ common (γc) cytokines, including interleukin (IL)-2, IL-7, IL-9, and IL-15. Kinetic and spatial analysis indicated that Stat5b Ser-193 phosphorylation was rapid and transient and occurred in the cytoplasmic compartment of the cell prior to Stat5b translocation to the nucleus. Moreover, inducible Stat5b Ser-193 phosphorylation was sensitive to inhibitors of mammalian target of rapamycin (mTOR), whereas inhibition of protein phosphatase 2A (PP2A) induced phosphorylation of Ser-193. Reconstitution assays in HEK293 cells in conjunction with site-directed mutagenesis, EMSA, and reporter assays indicated that Ser(P)-193 is required for maximal Stat5b transcriptional activity. Indeed, Stat5b Ser-193 was found constitutively phosphorylated in several lymphoid tumor cell lines as well as primary leukemia and lymphoma patient tumor cells. Taken together, IL-2 family cytokines tightly control Stat5b Ser-193 phosphorylation through a rapamycin-sensitive mechanism. Furthermore, constitutive Ser-193 phosphorylation is associated with Stat5b proto-oncogenic activity and therefore may serve as a novel therapeutic target for treating hematopoietic malignancies.
Assuntos
Núcleo Celular/metabolismo , Citocinas/metabolismo , Neoplasias Hematológicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT5/metabolismo , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/patologia , Citocinas/genética , Células HEK293 , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Fosforilação/genética , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição STAT5/genética , Serina/genética , Serina/metabolismoRESUMO
RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Deleção de Genes , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Fosforilação , RNA Interferente Pequeno , Proteínas Smad/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/genética , Espectrina/genética , Espectrina/metabolismo , Células-Tronco/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para Cima , Sequenciamento do ExomaRESUMO
BACKGROUND: Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated. RESULTS: Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFkappaB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFkappaB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFkappaB activity independent of IL-2. CONCLUSION: These results suggest that the NFkappaB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Biblioteca Genômica , Humanos , Interleucina-2/farmacologia , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/metabolismo , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Bases de Mannich/farmacologia , Oligodesoxirribonucleotídeos Antissenso/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Liver is the second most transplanted organ according to United network for organ sharing. Due to shortage of compatible donors, surgical difficulties, immunological hindrance, and high postoperative cost, stem cell therapy is an attractive substitute of liver transplant for millions of patients suffering from hepatic failure. Due to several technical limitations such as viral integration, inefficient differentiation, and adult phenotypes and epigenetic memory of fibroblasts, induced pluripotent stem cells, mesenchymal stem cells, or induced hepatocyte may not present a great clinical substitute for liver transplant. We pioneered a novel technology for robust expansion of quiescent liver stem cells (LSCs) from mice via utilizing of Hedgehog agonist HhAg1.5 for 3 weeks. These expanded LSCs retained stem-like properties after multiple passaging and differentiated to hepatocytes and cholangiocytes. Grafting of ex vivo expanded LSCs in Fah-/- Rag2-/- Il2rg-/- knockout mice, significantly increased life span compared to control group (P < 0.001). Thus in this study, we provide a promising viable substitute for primary hepatocytes for regenerative medicine and for life-threatening metabolic liver diseases.
Assuntos
Células-Tronco Adultas/citologia , Proteínas Hedgehog/agonistas , Falência Hepática/terapia , Fígado/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antígeno AC133/imunologia , Células-Tronco Adultas/imunologia , Animais , Sistema Biliar/citologia , Diferenciação Celular , Hepatócitos/citologia , Antígenos Comuns de Leucócito/imunologia , Fígado/imunologia , Longevidade , Camundongos , Camundongos Knockout , Medicina RegenerativaRESUMO
There is a growing need for a more accurate, real-time assessment of tumor status and the probability of metastasis, relapse, or response to treatment. Conventional means of assessment include imaging and tissue biopsies that can be highly invasive, may not provide complete information of the disease's heterogeneity, and not ideal for repeat analysis. Therefore, a less-invasive means of acquiring similar information at greater time points is necessary. Liquid biopsies are samples of a patients' peripheral blood and hold potential of addressing these criteria. Ongoing research has revealed that a tumor can release circulating cells, genetic materials (DNA or RNA), and exosomes into circulation. These potential biomarkers can be captured in a liquid biopsy and analyzed to determine disease status. To achieve these goals, numerous technologies have been developed. In this review, we discuss both prominent and newly developed technologies for circulating tumor cell capture and analysis and their clinical impact.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/diagnóstico , Neoplasias/patologia , Células Neoplásicas Circulantes , Humanos , MicrofluídicaRESUMO
BACKGROUND: Although accumulated evidence provides a strong scientific premise for using immune profiles to predict survival in patients with cancer, a universal immune profile across tumor types is still lacking, and how to achieve a survival-associated immune profile remains to be evaluated. METHODS: We analyzed datasets from The Cancer Genome Atlas to identify an immune profile associated with prolonged overall survival in multiple tumor types and tested the efficacy of tumor cell-surface vimentin-targeted interleukin 12 (ttIL-12) in inducing that immune profile and prolonging survival in both mouse and patient-derived xenograft tumor models. RESULTS: We identified an immune profile (IFNγHiCD8HiFOXP3LowCD33Low) associated with prolonged overall survival across several human tumor types. ttIL-12 in combination with surgical resection of the primary tumor transformed tumors to this immune profile. Intriguingly, this immune profile transformation led to inhibition of metastasis and to prolonged survival in both mouse and patient-derived xenograft malignant models. Wild-type IL-12 combined with surgery was significantly less effective. In the IL-12-sensitive C3H mouse strain, in fact, wild-type IL-12 and surgery resulted in shorter overall survival than in mice treated with control pDNA; this surprising result is believed to be attributable to IL-12 toxicity, which was absent in the mice treated with ttIL-12. The ttIL-12-induced immune profile associated with longer overall survival was also associated with a greater accumulation of CD8+ T cells and reduced infiltration of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The underlying mechanism for this transformation by ttIL-12 treatment was induction of expression of CXCL9 and reduction of expression of CXCL2 and CCL22 in tumors. CONCLUSIONS: ttIL-12 when combined with surgery led to conversion to the IFNγHiCD8HiFOXP3LowCD33Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.
Assuntos
Interleucina-12/farmacologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-12/imunologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Terapia de Alvo Molecular , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Osteossarcoma/genética , Osteossarcoma/imunologia , Osteossarcoma/patologia , Osteossarcoma/terapia , Análise de Sobrevida , Vimentina/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: p53 is mutated in about 50% of human cancers, mostly through missense mutations. Expression of mutant p53 is associated with poor clinical outcomes or metastasis. Although mutant p53 is inherently instable, various stressors such as DNA damage or expression of the oncogenic Kras or c-myc affect the oncogenic properties of mutant p53. However, the effects of inflammation on mutant p53 are largely unknown. IL27 is an important immunomodulatory cytokine, but its impact on mutant p53-driven tumorigenesis has not been reported. EXPERIMENTAL DESIGN: IL27RA(-/-) mice were bred with mutant p53 heterozygous (p53(R172H/+)) mice to obtain IL27RA(-/-)p53(H/+) and IL27RA(-/-)p53(H/H) mice. Mouse survival and tumor spectra for the cohort were analyzed. Stability of p53 protein was analyzed via IHC and Western blot analysis. RESULTS: This study unraveled that lack of IL27 signaling significantly shortened the survival duration of mice with tumors expressing both copies of the mutant p53 gene (Li-Fraumeni mouse model). Interestingly, in mice that were heterozygous for mutant p53, lack of IL27 signaling not only significantly shortened survival time but also doubled the incidence of osteosarcomas. Furthermore, lack of IL27 signaling is closely associated with increased mutant p53 stability in vivo from early age. CONCLUSIONS: These results suggest that IL27 signaling modulates the oncogenic properties of mutant p53 in vivo Clin Cancer Res; 22(15); 3876-83. ©2016 AACR.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Imunomodulação , Interleucina-27/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Genótipo , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Receptores de Interleucina/metabolismo , Transdução de Sinais , Microtomografia por Raio-XRESUMO
Establishment of a permissive pre-malignant niche in concert with mutant stem are key triggers to initiate skin carcinogenesis. An understudied area of research is finding upstream regulators of both these triggers. IL27, a pleiotropic cytokine with both pro- and anti-inflammatory properties, was found to be a key regulator of both. Two step skin carcinogenesis model and K15-KRASG12D mouse model were used to understand the role of IL27 in skin tumors. CD11b-/- mice and small-molecule of ETAR signaling (ZD4054) inhibitor were used in vivo to understand mechanistically how IL27 promotes skin carcinogenesis. Interestingly, using in vivo studies, IL27 promoted papilloma incidence primarily through IL27 signaling in bone-marrow derived cells. Mechanistically, IL27 initiated the establishment of the pre-malignant niche and expansion of mutated stem cells in K15-KRASG12D mouse model by driving the accumulation of Endothelin A receptor (ETAR)-positive CD11b cells in the skin-a novel category of pro-tumor inflammatory identified in this study. These findings are clinically relevant, as the number of IL27RA-positive cells in the stroma is highly related to tumor de-differentiation in patients with squamous cell carcinomas.
Assuntos
Antígeno CD11b/genética , Interleucinas/metabolismo , Pirrolidinas/farmacologia , Neoplasias Cutâneas/patologia , Animais , Antígeno CD11b/metabolismo , Camundongos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor de Endotelina A/metabolismo , Receptores de Citocinas/genética , Receptores de Interleucina , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Nicho de Células-TroncoRESUMO
Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of ß2-spectrin (ß2SP, encoded by SPTBN1), a SMAD adaptor for TGF-ß signaling, is causally associated with BWS; however, a role of TGF-ß deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-ß signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-ß-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-ß inducible and facilitates TGF-ß-mediated repression of TERT transcription via interactions with ß2SP and SMAD3. This regulation was abrogated in TGF-ß-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-ß pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.
Assuntos
Síndrome de Beckwith-Wiedemann/metabolismo , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Síndrome de Beckwith-Wiedemann/genética , Fator de Ligação a CCCTC , Proteínas de Transporte/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Células Hep G2 , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Telomerase/biossíntese , Telomerase/genética , Telomerase/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Chronic liver diseases (CLDs) such as hepatitis, alcoholic liver disease, nonalcoholic fatty liver, and their downstream effect cancer affect more than a billion of people around the world both symptomatically and asymptomatically. The major limitation for early detection and suitable medical management of CLDs and liver cancer is either the absent of symptoms or their similar manifestations as other diseases. This detection impediment has led to a steady increase in the number of people suffering from CLDs with an ultimate outcome of liver failure and undergoing transplantation. A better understanding of CLD pathogenesis has helped us to develop novel therapies for patients who are at greatest risk for CLD progression to the most serious disease cancer. With the discovery of aberrant molecular pathways in CLDs, it is now possible to delineate a road map for selecting targeted therapies for CLDs. Technological advances in imaging as well as the availability of several stable, sensitive, early, noninvasive biomarkers for distinguishing different stages of CLDs and cancer have greatly facilitated both drug target identification and real-time monitoring of response to therapy. Biomarkers are the most useful in clinical practice for liver diseases like hepatocellular carcinoma (HCC), which is associated with secretion of various tumor-related proteins or nucleotides in peripheral circulation. The need for the identification of CLD biomarkers remains high. This article reviews the etiologies of CLDs, the results of recent clinical trials of treatments for CLDs, and development of noninvasive methodologies for detecting CLDs and monitoring their progression toward HCC.
Assuntos
Biomarcadores/metabolismo , Hepatopatias , Humanos , Hepatopatias/diagnóstico , Hepatopatias/tratamento farmacológico , Hepatopatias/etiologia , Hepatopatias/metabolismoRESUMO
Tumor relapse and metastasis are the primary causes of poor survival rates in patients with advanced cancer despite successful resection or chemotherapeutic treatment. A primary cause of relapse and metastasis is the persistence of cancer stem cells (CSCs), which are highly resistant to chemotherapy. Although highly efficacious drugs suppressing several subpopulations of CSCs in various tissue-specific cancers are available, recurrence is still common in patients. To find more suitable therapy for relapse, the mechanisms underlying metastasis and drug-resistance associated with relapse-initiating CSCs need to be identified. Recent studies in circulating tumor cells (CTCs) of some cancer patients manifest phenotypes of both CSCs and epithelial-mesenchymal transition (EMT). These patients are unresponsive to standard chemotherapies and have low progression free survival, suggesting that EMT-positive CTCs are related to co-occur with or transform into relapse-initiating CSCs. Furthermore, EMT programming in cancer cells enables in the remodeling of extracellular matrix to break the dormancy of relapse-initiating CSCs. In this review, we extensively discuss the association of the EMT program with CTCs and CSCs to characterize a subpopulation of patients prone to relapses. Identifying the mechanisms by which EMT-transformed CTCs and CSCs initiate relapse could facilitate the development of new or enhanced personalized therapeutic regimens.