RESUMO
Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (â¼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor-dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in â¼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Succinato Desidrogenase/metabolismo , 5-Metilcitosina/química , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Mutação , Invasividade Neoplásica , Prognóstico , Succinato Desidrogenase/genética , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.
Assuntos
Neoplasias Renais/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Carcinoma de Células Renais , Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Inibidores de Proteases/farmacologia , Receptor Notch1/genéticaRESUMO
Diffuse large B-cell lymphomas (DLBCLs) contain 2 major molecular subtypes; namely, the germinal center B-cell-like (GCB) and the activated B-cell-like (ABC) DLBCLs. It is well documented that ABC-DLBCL cases have a significantly poorer survival response than GCB-DLBCLs in both the CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisone) and the rituximab (R)-CHOP eras. However, the underlying cause of this subtype disparity is poorly understood. Nevertheless, these clinical observations raise the possibility for an ABC-DLBCL-specific resistance mechanism that is directed toward 1 of the CHOP components and is inadequately addressed by rituximab. Here, we report that the main cytotoxic ingredient in CHOP, doxorubicin (Dox), has subtype-specific mechanisms of cytotoxicity in DLBCLs resulting from differences in the subcellular distribution pattern. Specifically, in cell line models of ABC-DLBCL, Dox is often enriched in the cytoplasm away from the nuclear DNA. As a result, Dox-induced cytotoxicity in ABC-DLBCLs is often dependent on oxidative stress, rather than DNA damage response. These findings are corroborated by gene signature analysis, which demonstrates that basal oxidative stress status predicts treatment outcome among patients with ABC-DLBCL, but not patients with GCB-DLBCL. In terms of redox-related resistance mechanism, our results suggest that STAT3 confers Dox resistance in ABC-DLBCLs by reinforcing an antioxidant program featuring upregulation of the SOD2 gene. Furthermore, a small-molecule STAT3 inhibitor synergizes with CHOP to trigger oxidative stress and kill ABC-DLBCL cells in preclinical models. These results provide a mechanistic basis for development of novel therapies that target either STAT3 or redox homeostasis to improve treatment outcomes for ABC-DLBCLs.
Assuntos
Linfócitos B/patologia , Doxorrubicina/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Linfócitos B/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Compostos Clorados/farmacologia , Dano ao DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Humanos , Compostos de Platina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Resultado do TratamentoRESUMO
Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell-derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism.
Assuntos
Anticorpos Antifosfolipídeos/química , Neoplasias/metabolismo , Neovascularização Patológica , Tromboplastina/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Endotoxinas/química , Feminino , Regulação da Expressão Gênica , Humanos , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia de Fluorescência , Transplante de NeoplasiasRESUMO
BACKGROUND: Aerosolized Azacitidine has been shown to inhibit orthotopic lung cancer growth and induce re-expression of methylated tumor suppressor genes in murine models. We hypothesized that inhaled Azacitidine is safe and effective in reversing epigenetic changes in the bronchial epithelium secondary to chronic smoking. PATIENTS AND METHODS: We report the first in human study of inhaled Azacitidine. Azacitidine in aqueous solution was used to generate an aerosol suspension of 0.25-5 µm particle size. Main inclusion criteria: Stage IV or recurrent NSCLC with predominantly lung involvement, ≥1 prior systemic therapy, ECOG PS 0-1, and adequate pulmonary function. Patients received inhaled Azacitidine daily on days 1-5 and 15-19 of 28-day cycles, at 3 escalating doses (15, 30 and 45 mg/m2 daily). The primary objective was to determine the feasibility and tolerability of this new therapeutic modality. The key secondary objectives included pharmacokinetics, methylation profiles and efficacy. RESULTS: From 3/2015 to 2/2018, eight patients received a median number of 2 (IQR = 1) cycles of inhaled Azacitidine. No clinically significant adverse events were observed, except one patient treated at the highest dose developed an asymptomatic grade 2 decreased DLCO which resolved spontaneously. One patient receiving 12 cycles of therapy had an objective and durable partial response, and two patients had stable disease. Plasma Azacitidine was only briefly detectable in patients treated at the higher doses. Moreover, in 2 of 3 participants who agreed and underwent pre- and post-treatment bronchoscopy, the global DNA methylation in the bronchial epithelium decreased by 24 % and 79 % post-therapy, respectively. The interval between last inhaled treatment and bronchoscopy was 3 days. CONCLUSIONS: Inhaled Azacitidine resulted in negligible plasma levels compared to the previously reported subcutaneous administration and was well-tolerated. The results justify the continued development of inhaled Azacitidine at non-cytotoxic doses for patients with lung-confined malignant and/or premalignant lesions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Azacitidina/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Metilação de DNA , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Recidiva Local de Neoplasia , Resultado do TratamentoRESUMO
BACKGROUND: The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied. METHODS: Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3-9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug. RESULTS: Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD. CONCLUSIONS: The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.
Assuntos
Neoplasias da Mama/dietoterapia , Neoplasias da Mama/tratamento farmacológico , Dieta Cetogênica/métodos , Sirolimo/farmacologia , Ácido 3-Hidroxibutírico/metabolismo , Animais , Antineoplásicos/farmacologia , Glicemia/efeitos dos fármacos , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Modelos Animais de Doenças , Feminino , Insulina/sangue , Cetose/fisiopatologia , CamundongosRESUMO
BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
PURPOSE: To evaluate therapeutic activity of PAK inhibition in ATLL and to characterize the role of PAK isoforms in cell proliferation, survival, and adhesion of ATLL cells in preclinical models. EXPERIMENTAL DESIGN: Frequency and prognostic impact of PAK2 amplification were evaluated in an ATLL cohort of 370 cases. Novel long-term cultures and in vivo xenograft models were developed using primary ATLL cells from North American patients. Two PAK inhibitors were used to block PAK kinase activity pharmacologically. siRNA-based gene silencing approach was used to genetically knockdown (KD) PAK1 and PAK2 in ATLL cell lines. RESULTS: PAK1/2/4 are the three most abundantly expressed PAK family members in ATLL. PAK2 amplifications are seen in 24% of ATLLs and are associated with worse prognosis in a large patient cohort. The pan-PAK inhibitor PF-3758309 (PF) has strong in vitro and in vivo activity in a variety of ATLL preclinical models. These activities of PF are likely attributed to its ability to target several PAK isoforms simultaneously because genetic silencing of either PAK1 or PAK2 produced more modest effects. PAK2 plays a major role in CADM1-mediated stromal interaction, which is an important step in systemic dissemination of the disease. This finding is consistent with the observation that PAK2 amplification is more frequent in aggressive ATLLs and correlates with inferior outcome. CONCLUSIONS: PAK2, a gene frequently amplified in ATLL, facilitates CADM1-mediated stromal interaction and promotes survival of ATLL cells. Taken together, PAK inhibition may hold significant promise as a targeted therapy for aggressive ATLLs.
Assuntos
Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirróis/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Adulto , Animais , Adesão Celular/efeitos dos fármacos , Molécula 1 de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Amplificação de Genes , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Cultura Primária de Células , RNA Interferente Pequeno/genética , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genéticaRESUMO
Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.
Assuntos
5-Metilcitosina/análogos & derivados , Oxirredutases do Álcool/biossíntese , Ácido Ascórbico/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , 5-Metilcitosina/metabolismo , Adulto , Oxirredutases do Álcool/genética , Animais , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , CamundongosRESUMO
Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.
Assuntos
Fibroblastos Associados a Câncer/patologia , Reprogramação Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Ácido Láctico/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Reprogramação Celular/genética , Metilação de DNA/efeitos dos fármacos , Humanos , Ácidos Cetoglutáricos/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Invasividade Neoplásica , Receptores CXCR4/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Transcriptoma/genética , Neoplasias PancreáticasRESUMO
INTRODUCTION: Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. Identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The forkhead box class O (FOXO) transcription factor FOXO3a has been suggested to function as a tumor suppressor in breast cancer. Using protein-protein interaction screening, we found that FOXO3a interacted with ER-alpha and ER-beta proteins in the human breast carcinoma cell line MCF-7, suggesting that there exists a crosstalk between the FOXO3a and ER signaling pathways in estrogen-dependent breast cancer cells. METHODS: The interaction between FOXO3a and ER was investigated by using co-immunoprecipitation and immunoblotting assays. Inhibition of ER-alpha and ER-beta transactivation activity by FOXO was determined by luciferase reporter assays. Cell proliferation in culture was evaluated by counting cell numbers. Tumorigenesis was assessed in athymic mice that were injected with MCF-7 cell lines over-expressing FOXO3a. Protein expression levels of cyclin-dependent kinase inhibitors, cyclins, ERs, FOXM1, and the proteins encoded by ER-regulated genes in MCF-7 cell lines and breast tumors were examined by immunoblotting analysis and immunohistochemical staining. RESULTS: We found that FOXO3a interacted with ER-alpha and ER-beta proteins and inhibited 17beta-estradiol (E2)-dependent, ER-regulated transcriptional activities. Consistent with these observations, expression of FOXO3a in the ER-positive MCF-7 cells decreased the expression of several ER-regulated genes, some of which play important roles in cell proliferation. Moreover, we found that FOXO3a upregulated the expression of the cyclin-dependent kinase inhibitors p21Cip1, p27Kip1, and p57Kip2. These findings suggest that FOXO3a induces cell growth arrest to effect tumor suppression. FOXO3a repressed the growth and survival of MCF-7 cells in cell culture. In an orthotopic breast cancer xenograft model in athymic mice, over-expression of FOXO3a in MCF-7 cells suppressed their E2-induced tumorigenesis, whereas knockdown of FOXO3a in MCF-7 resulted in the E2-independent growth. CONCLUSION: Functional interaction between FOXO3a and ER plays a critical role in suppressing estrogen-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that agents that activate FOXO3a may be novel therapeutic agents that can inhibit and prevent tumor proliferation and development in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Proteína Forkhead Box O3 , Humanos , Camundongos , Neoplasias Hormônio-Dependentes/metabolismo , Transdução de SinaisRESUMO
PURPOSE: To develop an optimal nonviral aerosol formulation for locoregional treatment of early lung cancer. EXPERIMENTAL DESIGN: The formulation was made of polylysine/protamine combination (AND) as the carrier and p53 gene (p53sm) as therapeutic agent. To estimate the aerosol deposition, the aerodynamic size of the AND-p53sm was measured with extrusion-precipitation method. To accurately determine the dose, the aerosol efficiency in mice was measured with a fluorescent dye. The transfection efficiency and DNA protection function of the aerosolized formulation in cultured cells and mouse lungs were detected with reporter gene assays and/or reverse transcription-PCR. The preclinical safety and efficacy of AND-p53sm were studied in healthy mice and mice bearing orthotopic human non-small-cell lung cancer (NSCLC) xenograft. RESULTS: After aerosolization, AND is 3- to 17-fold more effective than commonly used PEI or cationic lipid formulations in transfecting the NSCLC cells (relative light units, 1,494 versus 534 and 86; P < 0.003). Aerodynamic size of AND-p53sm ranged 0.2 to 3 mum is the optimal aerosol droplets for deposition in the entire human respiratory tract. Significant gene expression was detected in the lungs of mice given aerosolized AND-p53sm and AND-luciferase. Aerosolized AND-p53sm significantly prolonged the life of mice bearing orthotopic human NSCLC xenografts, and it was more effective than an optimal i.v. cisplatin chemotherapy (increased life span, 93% versus 25%; P = 0.014). Inhalation of AND produced low and reversible pulmonary toxicity and no systemic toxicity. CONCLUSIONS: This optimal formulation is suitable for delivering biological materials to human lung with aerosol administration. This therapeutic strategy is an option for patients with early lung cancer and bronchoalveolar carcinoma.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Genes p53 , Terapia Genética , Neoplasias Pulmonares/terapia , Aerossóis , Animais , Química Farmacêutica , Feminino , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Plasmídeos , Polilisina/administração & dosagem , Protaminas/administração & dosagemRESUMO
Small cell lung cancer (SCLC) is the most aggressive lung-cancer subtype and so far, no favorable therapeutic strategy has been established for chemo-resistant SCLC. Cisplatin is one of the most important components among all standard poly-chemotherapeutic regimens for SCLC; therefore, this study focused on revealing Cisplatin-resistance mechanism(s) in this disease. Cisplatin-resistant SCLC cells were generated in the NCI-H69 xenograft model in nude mice by continuous intravenous administration of Cisplatin; Cisplatin resistance of the tumor cells was confirmed by in vitro and in vivo tests, and the gene expression profile of the resistant cells was determined using microarray analysis. A significantly higher expression of tribbles pseudokinase 2 (TRIB2) mRNA in the Cisplatin-resistant cells was found compared to parental H69 cells. Further, the Cisplatin-resistance level was decreased when TRIB2 expression was knocked down. The mRNA and protein levels of CCAAT/enhancer binding protein alpha (CEBPA), known to be a transcription factor regulating cell differentiation and a target for degradation by TRIB2, as well as selected cancer stem cell makers in the Cisplatin-resistant cells, were measured. We found that CEBPA protein levels could be upregulated by knocking down the overexpressed TRIB2, which also reversed the Cisplatin-resistance of these cells; further, the Cisplatin-resistant SCLC cells demonstrated certain cancer stem cell-like properties. Similar patterns were also observed in limited human tumor specimens of chemo-resistant SCLC patients: namely, overexpressed TRIB2 and undetected CEBPA proteins. Our study revealed a possible molecular mechanism for Cisplatin-resistant SCLC involving induced TRIB2 overexpression and downregulation of CEBPA protein. We propose that this mechanism is a potential therapeutic target to circumvent chemo-resistance in SCLC.
RESUMO
BACKGROUND: The epigenetic combinations of DNA demethylating agents and histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits for non-small cell lung cancer (NSCLC) treatment, however, there are few studies uncovering the underlying molecular mechanism of the combinations. Our previous study showed that DNA demethylating agent Azacitidine (Aza) demethylated CpG sites in paired box gene 5 (pax5) promoter region, but did not induce pax5 mRNA or protein expression. METHODS: In this study we used epigenetic combination of Aza and HDAC inhibitor Vorinostat (SAHA) to treat NSCLC cells and to elucidate the underlying molecular mechanism. We treated pax5- silenced NSCLC H460 cells with Aza+SAHA combination at sub-toxic concentration and detected the re-expression of pax5 mNRA and protein. RESULTS: The results showed demethylation of CpG sites in pax5 promoter region by Aza treatment and increased DNA accessibility for protein binding by SAHA treatment. The combination of Aza+SAHA significantly increased p53 protein binding to DNA in pax5 promoter region (p<0.01). More efficient binding of the transcription factor p53 to pax5 promoter region is likely because SAHA increased accessibility of the chromatin conformation and Aza-demethylated DNA was more permissive, allowing transcription factors to bind. CONCLUSION: Our study not only explained an underlying mechanism, that pax5 re-expression was induced by Aza+SAHA combination in H460 cells via p53, but also demonstrated a pattern showing that the combination of demethylating agent and HDAC inhibitor can re-activate tumor suppressor gene (TSG) which is associated with the enhancement of transcription factors binding to the promoter region of the TSG.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Fator de Transcrição PAX5/genética , Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azacitidina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Fator de Transcrição PAX5/metabolismo , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , VorinostatRESUMO
Benzo[a]pyrene diol epoxide (BPDE, a carcinogen present in tobacco smoke and environmental pollution) has been shown to suppress retinoic acid receptor-beta2 (RAR-beta(2)) and induce cyclooxygenase-2 (COX-2) expression. Restoration of RAR-beta(2) inhibited growth and colony formation of esophageal cancer cells, which was correlated with COX-2 suppression. In this study, we investigated the molecular mechanisms for RAR-beta(2)-mediated suppression of COX-2 expression using BPDE as a tool. We found that BPDE-induced COX-2 expression was through inhibition of RAR-beta(2) and consequently, induction of epidermal growth factor receptor (EGFR), extracellular signal-regulated protein kinases 1/2 (Erk1/2) phosphorylation, and c-Jun expression. Esophageal cancer cells that do not express RAR-beta(2) did not respond to BPDE for induction of COX-2. BPDE was also unable to induce COX-2 expression after RAR-beta(2) expression was manipulated in these esophageal cancer cells. Furthermore, BPDE induced time-dependent methylation of RAR-beta(2) gene promoter in esophageal cancer cells. Transfection of RAR-beta(2) expression vector into esophageal cancer cells suppressed expression of EGFR, Erk1/2 phosphorylation, c-Jun, and COX-2. In addition, co-treatment of RAR-beta(2)-positive cells with BPDE and the MEK1/2 inhibitor U0126 caused little change in c-Jun and COX-2 expression. This study demonstrated that BPDE-suppressed expression of RAR-beta(2) results in COX-2 induction and restoration of RAR-beta(2) expression reduces COX-2 protein in esophageal cancer cells, thereby further supporting our previous finding that RAR-beta(2) plays an important role in suppressing esophageal carcinogenesis.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/farmacologia , Ciclo-Oxigenase 2/biossíntese , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/biossíntese , Animais , Butadienos/farmacologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Metilação de DNA/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Nus , Família Multigênica/fisiologia , Nitrilas/farmacologia , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores do Ácido Retinoico/fisiologia , Fator de Transcrição AP-1/fisiologiaRESUMO
PURPOSE: Erlotinib (Tarceva, OSI-774) is a potent and specific inhibitor of the HER1/epidermal growth factor receptor (EGFR) tyrosine kinase. In phase II clinical studies, oral erlotinib monotherapy has shown antitumor activity in patients with advanced non-small cell lung cancer, head and neck cancer, and ovarian cancer after the failure of standard chemotherapy. We hypothesized that some tumors treated with multiple cytotoxic therapies may become more dependent on the HER1/EGFR signaling pathways for survival. EXPERIMENTAL DESIGN: The growth-inhibitory effect of erlotinib was tested on 10 pairs of chemosensitive, parental, and chemoresistant tumor cell lines. RESULTS: Enhanced sensitivity to erlotinib was observed in the doxorubicin-resistant human breast cancer cell line MCF-7, paclitaxel-resistant human ovarian carcinoma cell line A2780, and cisplatin-resistant human cervical carcinoma cell line ME180. The IC(50) values of erlotinib in the resistant cell lines were 2- to 20-fold lower than those in the corresponding parental cell lines. This enhanced sensitivity to erlotinib correlated with higher HER1/EGFR and phospho-HER1/EGFR expression when compared with the corresponding parental cell lines. Acquired resistance to cytotoxic agents was not associated with cross-resistance to erlotinib. AE-ME180/CDDP-resistant xenografts showed greater sensitivity to erlotinib than parental ME180 xenografts did. CONCLUSIONS: Our findings suggest that acquired resistance to cytotoxic therapy in some tumors is associated with enhanced sensitivity to HER1/EGFR inhibitors, which correlates with increased HER1/EGFR expression. These data may explain some of the observed clinical activity of HER1/EGFR inhibitors in patients previously treated with multiple therapies. HER1/EGFR tyrosine kinase inhibitors may be more effective as second- or third-line treatment for certain patients with tumors that were previously treated with multiple chemotherapy regimens.
Assuntos
Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Animais , Antineoplásicos/farmacologia , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastatic breast cancer requires systemic treatment. We have developed a systemic gene therapy approach for breast cancer, consisting of a nonviral gene delivery system (SN) and a proapoptotic gene, bik. The transfection efficiency of SN carrying a reporter gene was 5-10 times higher than the common nonviral agents Fugene-6 and Lipofectamine in the presence of serum. The SN-bik gene complex induced significant apoptosis in four breast cancer cell lines in vitro as well as in orthotopic tumor tissues in nude mice. Systemically administrated SN-bik significantly inhibited the growth and metastasis of human breast cancer cells implanted in nude mice and prolonged the life span of the treated animals. This study demonstrates that SN-bik is a promising approach for further development as a potential therapeutic agent of cancer.
Assuntos
Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , DNA/administração & dosagem , Terapia Genética/métodos , Proteínas de Membrana , Proteínas/genética , Animais , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Cátions , Divisão Celular/genética , DNA/genética , Feminino , Proteínas de Fluorescência Verde , Lipossomos/administração & dosagem , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Nus , Proteínas Mitocondriais , Biossíntese de Proteínas , Proteínas/fisiologia , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Azacitidine as an effective epigenetic therapeutic agent has not been used as an aerosol form to treat lung cancer patients. We aerosolized clinical grade azacitidine (Aza), optimized the formulation, and studied its pharmacokinetics and toxicity in mice. METHODS: Extrusion-precipitation method and DNA methyltransferase inhibition rate were used to measure the aerodynamic size and aerosolized Aza activity. In the single dose pharmacokinetic study, Aza concentrations in peripheral blood and lungs were measured by LC-MS method. In the multiple-dose toxicity studies, histo-pathological evaluation was used to determine the organ and bone marrow toxicities. RESULTS: In pharmacokinetic study, aerosolized Aza was found to deposit mainly into the lung with very little drug detected in the circulation. In contrast, intravenously injected (IV) Aza resulted in a high Aza concentration in the peripheral blood, with trace amounts of drug in the lung, and it was associated with significant myelosuppression. No significant myelosuppression, pulmonary toxicity, hepatotoxicity, or nephrotoxicity were observed at a daily dose of 2.5 mg/m(2) for 7 days. Reversible lung inflammation was found in mice treated with 7.5 mg/m(2) aerosolized Aza at 3 but not 6 weeks after treatment. CONCLUSIONS: Aerosol Aza aerodynamic size favors deposition of the drug to the human lower airways. The aerosol process do not compromise the drug activity. Aerosolized Aza has higher lung deposition and much less systemic toxicity than IV drug. The safe starting dose for clinical phase I trials should be 2.5 mg/m(2) for 5 to 7 days.
Assuntos
Administração por Inalação , Azacitidina/farmacocinética , Leucócitos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/metabolismo , Administração Intravenosa , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. Cancer Res; 76(16); 4841-9. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Indazóis/farmacologia , Leucemia Mieloide Aguda/patologia , Síndromes Mielodisplásicas/patologia , Receptor TIE-2/antagonistas & inibidores , Ureia/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Angiopoietina-1/metabolismo , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Modelos de Riscos Proporcionais , Ureia/farmacologiaRESUMO
The adenomatous polyposis coli (APC) or beta-catenin genes are frequently mutated in colorectal cancers, leading to activation of downstream genes with beta-catenin/T-cell factor (Tcf)-responsive promoters. We have developed a gene therapy approach selectively targeting colorectal cancer cells in which beta-catenin/Tcf4 pathway is activated by using a recombinant adenovirus AdTOP-CMV-TK, which carries a herpes simplex virus thymidine kinase gene (HSV TK) under the control of a beta-catenin/Tcf-response promoter linking to a minimum CMV promoter. AdTOP-CMV-TK and ganciclovir (GCV) treatment significantly suppressed the growth of human DLD-1 colon cancer cells in nude mice. Furthermore, no significant tumor suppression effect was observed in human hepatoma cell line SK-HEP-1, in which the beta-catenin/Tcf pathway is not activated, as a control experiment. In summary, we demonstrated the selective targeting of colorectal cancers with activated beta-catenin by AdTOP-CMV-TK and GCV treatment in animal models, as well as its therapeutic potential for colon cancer metastasized to liver.