RESUMO
Bile duct cancer (BDC) frequently invades the nerve fibers, making complete surgical resection difficult. A single tumor mass contains cells of variable malignancy and cell-differentiation states, with cancer stem cells (CSCs) considered responsible for poor clinical outcomes. This study aimed to investigate the contribution of autosynthesized dopamine to CSC-related properties in BDC. Sphere formation assays using 13 commercially available BDC cell lines demonstrated that blocking dopamine receptor D1 (DRD1) signaling promoted CSC-related anchorage-independent growth. Additionally, we newly established four new BDC patient-derived organoids (PDOs) and found that blocking DRD1 increased resistance to chemotherapy and enabled xenotransplantation in vivo. Single-cell analysis revealed that the BDC PDO cells varied in their cell-differentiation states and responses to dopamine signaling. Further, DRD1 inhibition increased WNT7B expression in cells with bile duct-like phenotype, and it induced proliferation of other cell types expressing Wnt receptors and stem cell-like signatures. Reagents that inhibited Wnt function canceled the effect of DRD1 inhibition and reduced cell proliferation in BDC PDOs. In summary, in BDCs, DRD1 is a crucial protein involved in autonomous CSC proliferation through the regulation of endogenous WNT7B. As such, inhibition of the DRD1 feedback signaling may be a potential treatment strategy for BDC.
Assuntos
Neoplasias dos Ductos Biliares , Via de Sinalização Wnt , Humanos , Neoplasias dos Ductos Biliares/patologia , Dopamina , Fenótipo , Receptores Dopaminérgicos/genéticaRESUMO
BACKGROUND AIMS: This prospective clinical study aimed to determine the efficacy and prognostic factors of adoptive activated αßT lymphocyte immunotherapy for various refractory cancers. The primary endpoint was overall survival (OS), and the secondary endpoint was radiological response. METHODS: The authors treated 96 patients. Activated αßT lymphocytes were infused every 2 weeks for a total of six times. Prognostic factors were identified by analyzing clinical and laboratory data obtained before therapy. RESULTS: Median survival time (MST) was 150 days (95% confidence interval, 105-191), and approximately 20% of patients achieved disease control (complete response + partial response + stable disease). According to the multivariate Cox proportional hazards model with Akaike information criterion-best subset selection, sex, concurrent therapy, neutrophil/lymphocyte ratio, albumin, lactate dehydrogenase, CD4:CD8 ratio and T helper (Th)1:Th2 ratio were strong prognostic factors. Using parameter estimates of the Cox analysis, the authors developed a response scoring system. The authors then determined the threshold of the response score between responders and non-responders. This threshold was able to significantly differentiate OS of responders from that of non-responders. MST of responders was longer than that of non-responders (317.5 days versus 74 days). The validity of this response scoring system was then confirmed by internal validation. CONCLUSIONS: Adoptive activated αßT lymphocyte immunotherapy has clinical efficacy in certain patients. The authors' scoring system is the first prognostic model reported for this therapy, and it is useful for selecting patients who might obtain a better prognosis through this modality.
Assuntos
Linfócitos , Neoplasias , Humanos , Estudos Prospectivos , Neoplasias/terapia , Imunoterapia Adotiva , Resultado do Tratamento , Estudos Retrospectivos , ImunoterapiaRESUMO
Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC.
Assuntos
Neoplasias Colorretais , Sistema de Sinalização das MAP Quinases , Animais , Apoptose , Linhagem Celular Tumoral , Cromatina , Neoplasias Colorretais/patologia , DNA Helicases , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares , Fatores de TranscriçãoRESUMO
BACKGROUND: Pancreatic cancer is one of the most lethal tumors. The aim of this study is to provide an effective therapeutic discovery platform for pancreatic cancer by establishing and characterizing patient-derived organoids (PDOs). METHODS: PDOs were established from pancreatic tumor surgical specimens, and the mutations were examined using a panel sequence. Expression of markers was assessed by PCR, immunoblotting, and immunohistochemistry; tumorigenicity was examined using immunodeficient mice, and drug responses were examined in vitro and in vivo. RESULTS: PDOs were established from eight primary and metastatic tumors, and the characteristic mutations and expression of cancer stem cell markers and CA19-9 were confirmed. Tumorigenicity of the PDOs was confirmed in subcutaneous transplantation and in the peritoneal cavity in the case of PDOs derived from disseminated nodules. Gemcitabine-sensitive/resistant PDOs showed consistent responses in vivo. High throughput screening in PDOs identified a compound effective for inhibiting tumor growth of a gemcitabine-resistant PDO xenograft model. CONCLUSIONS: This PDO-based platform captures important aspects of treatment-resistant pancreatic cancer and its metastatic features, suggesting that this study may serve as a tool for the discovery of personalized therapies.
Assuntos
Organoides , Neoplasias Pancreáticas , Animais , Descoberta de Drogas , Humanos , Camundongos , Organoides/patologia , Pâncreas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias PancreáticasRESUMO
Tumor cells capable of self-renewal and continuous production of progeny cells are called tumor stem cells (TSCs) and are considered to be potential therapeutic targets. However, the mechanisms underlying the survival and function of TSCs are not fully understood. We previously reported that chromatin remodeling regulator Brg1 is essential for intestinal stem cells in mice and Dclk1 is an intestinal TSC marker. In this study, we investigated the role of Brg1 in Dclk1+ intestinal tumor cells for the maintenance of intestinal tumors in mice. Specific ablation of Brg1 in Dclk1+ intestinal tumor cells reduced intestinal tumors in ApcMin mice, and continuous ablation of Brg1 maintained the reduction of intestinal tumors. Lineage tracing in the context of Brg1 ablation in Dclk1+ intestinal tumor cells revealed that Brg1-null Dclk1+ intestinal tumor cells did not give rise to their descendent tumor cells, indicating that Brg1 is essential for the self-renewal of Dclk1+ intestinal tumor cells. Five days after Brg1 ablation, we observed increased apoptosis in Dclk1+ tumor cells. Furthermore, Brg1 was crucial for the stemness of intestinal tumor cells in a spheroid culture system. BRG1 knockdown also impaired cell proliferation and increased apoptosis in human colorectal cancer (CRC) cells. Microarray analysis revealed that apoptosis-related genes were upregulated and stem cell-related genes were downregulated in human CRC cells by BRG1 suppression. Consistently, high BRG1 expression correlated with poor disease-specific survival in human CRC patients. These data indicate that Brg1 plays a crucial role in intestinal TSCs in mice by inhibiting apoptosis and is critical for cell survival and stem cell features in human CRC cells. Thus, BRG1 represents a new therapeutic target for human CRC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neoplasias Colorretais/patologia , DNA Helicases/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , CamundongosRESUMO
Inactivating mutations of Arid1a, a subunit of the Switch/sucrose nonfermentable chromatin remodeling complex, have been reported in multiple human cancers. Intestinal deletion of Arid1a has been reported to induce colorectal cancer in mice; however, its functional role in intestinal homeostasis remains unclear. We investigated the functional role of Arid1a in intestinal homeostasis in mice. We found that intestinal deletion of Arid1a results in loss of intestinal stem cells (ISCs), decreased Paneth and goblet cells, disorganized crypt-villous structures, and increased apoptosis in adult mice. Spheroids did not develop from intestinal epithelial cells deficient for Arid1a Lineage-tracing experiments revealed that Arid1a deletion in Lgr5+ ISCs leads to impaired self-renewal of Lgr5+ ISCs but does not perturb intestinal homeostasis. The Wnt signaling pathway, including Wnt agonists, receptors, and target genes, was strikingly down-regulated in Arid1a-deficient intestines. We found that Arid1a directly binds to the Sox9 promoter to support its expression. Remarkably, overexpression of Sox9 in intestinal epithelial cells abrogated the above phenotypes, although Sox9 overexpression in intestinal epithelial cells did not restore the expression levels of Wnt agonist and receptor genes. Furthermore, Sox9 overexpression permitted development of spheroids from Arid1a-deficient intestinal epithelial cells. In addition, deletion of Arid1a concomitant with Sox9 overexpression in Lgr5+ ISCs restores self-renewal in Arid1a-deleted Lgr5+ ISCs. These results indicate that Arid1a is indispensable for the maintenance of ISCs and intestinal homeostasis in mice. Mechanistically, this is mainly mediated by Sox9. Our data provide insights into the molecular mechanisms underlying maintenance of ISCs and intestinal homeostasis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Animais , Células Epiteliais/metabolismo , Homeostase/fisiologia , Intestinos/fisiologia , Camundongos , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição , Via de Sinalização Wnt/fisiologiaRESUMO
Among cancer cells, there are specific cell populations of whose activities are comparable to those of stem cells in normal tissues, and for whom the levels of cell dedifferentiation are reported to correlate with poor prognosis. Information concerning the mechanisms that modulate the stemness like traits of cancer cells is limited. Therefore, we examined five gastric cancer cell lines and isolated gastric oncospheres from three gastric cancer cell lines. The gastric cancer cells that expanded in the spheres expressed relatively elevated proportion of CD44, which is a marker of gastric cancer stem cells (CSCs), and displayed many properties of CSCs, for example: chemoresistance, tumorigenicity and epithelial-mesenchymal transition (EMT) acquisition. SNAIL, which is a key factor in EMT, was highly expressed in the gastric spheres. Microarray analysis in gastric cancer cell line HGC27 showed that CCN3 and NEFL displayed the greatest differential expression by knocking down of SNAIL; the former was upregulated and the latter downregulated, respectively. Downregulation of CCN3 and upregulation of NEFL gene expression impaired the SNAIL-dependent EMT activity: high tumorigenicity, and chemoresistance in gastric cancer cells. Thus, approach that disrupts SNAIL/CCN3/NEFL axis may be credible in inhibiting gastric cancer development.
Assuntos
Carcinogênese/genética , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteínas de Neurofilamentos/genética , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Gástricas/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Proteínas de Neurofilamentos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/genética , Estômago/patologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)-targeted therapy is considered a promising approach for this disease. Epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT-TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin-like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer.
Assuntos
Antineoplásicos/uso terapêutico , Carcinogênese/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Neoplasias Pancreáticas/genética , Fatores de Transcrição da Família Snail/genética , Animais , Carcinogênese/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Interferência de RNA , Fatores de Transcrição da Família Snail/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
OBJECTIVE: Nardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA). DESIGN: We analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC. RESULTS: We found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D -driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA. CONCLUSION: Nrdc inhibits pancreatitis and suppresses PDA initiation in mice.
Assuntos
Carcinoma Ductal Pancreático/prevenção & controle , Metaloendopeptidases/fisiologia , Neoplasias Pancreáticas/prevenção & controle , Pancreatite/prevenção & controle , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Pancreatite/patologiaRESUMO
Natural killer (NK) cells are innate lymphoid cells important for host defense against patho- gens, and are also thought to play an important role in the immunosurveillance against can- cers. NK cells express surface receptors that interact with target cells and determine their response to infected or transformed cells without prior sensitization or gene rearrangement. Activated NK cells can kill target cells by producing effector molecules and transmit impor- tant information to the rest of the immune system subsequently by producing cytokines. Here, we review the mechanism of cytotoxic function of NK cells and a recent development in NK cell biology, and highlight therapeutic approaches for NK cell-based immunotherapy and targeting NK cells in the treatment of cancer. //.
Assuntos
Imunoterapia , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Humanos , Neoplasias/imunologiaRESUMO
OBJECTIVE: Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. DESIGN: We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. RESULTS: Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5(neg or low) cell population but was distinct from typical antral Lgr5(high) stem cells. Treatment with progastrin interconverts Lgr5(neg or low) CCK2R+ cells into Lgr5(high) cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. CONCLUSIONS: CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.
Assuntos
Carcinogênese , Antro Pilórico/citologia , Receptor de Colecistocinina B/fisiologia , Células-Tronco/fisiologia , Animais , Células Cultivadas , Gastrinas/fisiologia , Camundongos , Precursores de Proteínas/fisiologiaRESUMO
Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Produtos do Gene tax/imunologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/terapia , Fragmentos de Peptídeos/imunologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Citocinas/metabolismo , Feminino , Produtos do Gene tax/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Imunoterapia , Leucemia-Linfoma de Células T do Adulto/diagnóstico , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.
Assuntos
Transformação Celular Neoplásica/genética , Gastrinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Peptídeos/genética , Neoplasias Gástricas/prevenção & controle , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Modelos Animais de Doenças , Feminino , Gastrinas/deficiência , Gastrinas/genética , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter felis/patogenicidade , Histonas/metabolismo , Humanos , Masculino , Metilnitrosoureia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeos/deficiência , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Fatores de Tempo , Transfecção , Fator Trefoil-1 , Proteínas Supressoras de Tumor/metabolismoRESUMO
Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Gastrinas/metabolismo , Precursores de Proteínas/metabolismo , Proteína Supressora de Tumor p53/genética , Focos de Criptas Aberrantes/induzido quimicamente , Focos de Criptas Aberrantes/genética , Focos de Criptas Aberrantes/metabolismo , Animais , Azoximetano/toxicidade , Carcinógenos/toxicidade , Proliferação de Células , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MutaçãoRESUMO
Iron is essential in cellular proliferation and survival based on its crucial roles in DNA and ATP synthesis. Tumor cells proliferate rapidly even in patients with low serum iron, although their actual mechanisms are not well known. To elucidate molecular mechanisms of efficient tumor progression under the hypoferric condition, we studied the roles of six-transmembrane epithelial antigen of the prostate family member 3 (STEAP3), which was reported to facilitate iron uptake. Using Raji cells with low STEAP3 mRNA expression, human STEAP3-overexpressing cells were established. The impact of STEAP3 expression was analyzed about the amount of iron storage, the survival under hypoferric conditions in vitro and the growth of tumor in vivo. STEAP3 overexpression increased ferritin, an indicator of iron storage, in STEAP3-overexpressing Raji cells. STEAP3 gave Raji cells the resistance to iron deprivation-induced apoptosis. These STEAP3-overexpressing Raji cells preserved efficient growth even in hypoferric mice, while parental Raji cells grew less rapidly. In addition, iron deficiency enhanced STEAP3 mRNA expression in tumor cells. Furthermore, human colorectal cancer tissues exhibited more STEAP3 mRNA expression and iron storage compared with normal colon mucosa. These findings indicate that STEAP3 maintains iron storage in human malignant cells and tumor proliferation under the hypoferric condition.
Assuntos
Ferro/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Oncogênicas/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Ferritinas/metabolismo , Células HeLa , Humanos , Ferro/sangue , Deficiências de Ferro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas/genética , Oxirredutases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Células Secretoras de Gastrina/metabolismo , Gastrinas/genética , Proteínas de Fluorescência Verde/genética , Helicobacter felis/genética , Envelhecimento/metabolismo , Animais , Azoximetano , Carcinógenos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sulfato de Dextrana , Regulação para Baixo , Jejum , Feto/metabolismo , Ácido Gástrico/metabolismo , Gastrinas/deficiência , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Camundongos , Camundongos Transgênicos , Antro Pilórico/metabolismo , Antro Pilórico/patologia , Somatostatina/administração & dosagem , Distribuição Tecidual , Transcrição Gênica , Transgenes , Regulação para CimaRESUMO
BACKGROUND & AIMS: The nuclear factor kappaB (NF-kappaB)/IkappaB-kinase-beta (IKKbeta) pathway has been shown to represent a key link between inflammation and cancer, inducing pro-inflammatory cytokines in myeloid cells and anti-apoptotic pathways in epithelial cells. However, the role of NF-kappaB pathway in gastric carcinogenesis and injury has not been well-defined. We derived mice with a conditional knockout of IKKbeta in gastric epithelial cells (GECs) and myeloid cells, and examined responses to ionizing radiation (IR) and Helicobacter felis infection. METHODS: Ikkbeta(Deltastom) mice were generated by crossing Foxa3-Cre mice to Ikkbeta(F/F) mice. Cellular stress was induced with IR and H felis in Ikkbeta(Deltastom), Ikkbeta(F/F), and cis-NF-kappaB-enhanced green fluorescent protein (GFP) reporter mice. Gastric histopathology, apoptosis, proliferation, necrosis, reactive oxygen species, and expression of cytokines, chemokines, and anti-apoptotic genes were assessed. The role of myeloid IKKbeta in these models was studied by crosses with LysM-Cre mice. RESULTS: NF-kappaB activity was upregulated in myeloid cells with acute H felis infection, but in GECs by IR or long-term H felis infection during progression to dysplasia. Deletion of IKKbeta in GECs led to increased apoptosis, reactive oxygen species, and cellular necrosis, and resulted in up-regulation of interleukin-1alpha and down-regulation of anti-apoptotic genes. Loss of IKKbeta in GECs resulted in worse inflammation and more rapid progression to gastric preneoplasia, while loss of IKKbeta in myeloid cells inhibited development of gastric atrophy. CONCLUSIONS: The loss of IKKbeta/NF-kappaB signaling in GECs results in increased apoptosis and necrosis in response to cellular stress, and accelerated development of dysplasia by Helicobacter infection.
Assuntos
Apoptose , Proliferação de Células , Mucosa Gástrica/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter felis/patogenicidade , Quinase I-kappa B/deficiência , Lesões Pré-Cancerosas/enzimologia , Neoplasias Gástricas/enzimologia , Animais , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/metabolismo , Atrofia , Proliferação de Células/efeitos da radiação , Modelos Animais de Doenças , Progressão da Doença , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Mucosa Gástrica/efeitos da radiação , Genes Reporter , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Fator 3-gama Nuclear de Hepatócito/genética , Quinase I-kappa B/genética , Mediadores da Inflamação/metabolismo , Integrases/genética , Interleucina-1alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/enzimologia , Células Mieloides/microbiologia , Células Mieloides/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Necrose , Estresse Oxidativo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Fatores de TempoRESUMO
We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.
Assuntos
Gastrinas/metabolismo , Infecções por Helicobacter/metabolismo , Lesões Pré-Cancerosas/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Gastrinas/genética , Gastrite/genética , Gastrite/imunologia , Gastrite/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/imunologia , Helicobacter felis , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells make up approximately 0.5%-1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony-forming units (CFU-F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells engrafted in the bone marrow long-term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells have immune modulatory effects, in vitro coculture with activated CD4+ T-cells resulted in decreased Th17 cell differentiation by Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells. Furthermore, serial infusions with Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells reduced the progression to low-grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)-17F, IL-22, and ROR-gammat gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17-dependent pathways.
Assuntos
Células da Medula Óssea/fisiologia , Helicobacter felis/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neoplasias Gástricas/prevenção & controle , Neoplasias Gástricas/terapia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Citometria de Fluxo , Infecções por Helicobacter/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco , Neoplasias Gástricas/microbiologiaRESUMO
Cancer stem cells (CSCs) have been defined as a unique subpopulation in tumors that possess the ability to initiate tumor growth and sustain tumor self-renewal. Although the evidence has been provided to support the existence of CSCs in various solid tumors, the identity of gastric CSCs has not been reported. In this study, we have identified gastric cancer-initiating cells from a panel of human gastric cancer cell lines using cell surface marker CD44. Among six gastric cancer cell lines, three lines MKN-45, MKN-74, and NCI-N87 had a sizeable subpopulation of CD44(+) cells, and these cells showed spheroid colony formation in serum-free media in vitro as well as tumorigenic ability when injected into stomach and skin of severe combined immunodeficient (SCID) mice in vivo. The CD44(+) gastric cancer cells showed the stem cell properties of self-renewal and the ability to form differentiated progeny and gave rise to CD44(-) cells. CD44 knockdown by short hairpin RNA resulted in much reduced spheroid colony formation and smaller tumor production in SCID mice, and the CD44(-) populations had significantly reduced tumorigenic ability in vitro and in vivo. Other potential CSC markers, such as CD24, CD133, CD166, stage-specific embryonic antigen-1 (SSEA-1), and SSEA-4, or sorting for side population did not show any correlation with tumorigenicity in vitro or in vivo. The CD44(+) gastric cancer cells showed increased resistance for chemotherapy- or radiation-induced cell death. These results support the existence of gastric CSCs and may provide novel approaches to the diagnosis and treatment of gastric cancer.