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1.
Gastric Cancer ; 24(4): 897-912, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33755862

RESUMO

BACKGROUND: Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. METHODS: We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. RESULTS: Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. CONCLUSION: These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

2.
Clin Cancer Res ; 27(11): 3126-3140, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33542076

RESUMO

PURPOSE: Gastric and gastroesophageal adenocarcinomas represent the third leading cause of cancer mortality worldwide. Despite significant therapeutic improvement, the outcome of patients with advanced gastroesophageal adenocarcinoma is poor. Randomized clinical trials failed to show a significant survival benefit in molecularly unselected patients with advanced gastroesophageal adenocarcinoma treated with anti-EGFR agents. EXPERIMENTAL DESIGN: We performed analyses on four cohorts: IRCC (570 patients), Foundation Medicine, Inc. (9,397 patients), COG (214 patients), and the Fondazione IRCCS Istituto Nazionale dei Tumori (206 patients). Preclinical trials were conducted in patient-derived xenografts (PDX). RESULTS: The analysis of different gastroesophageal adenocarcinoma patient cohorts suggests that EGFR amplification drives aggressive behavior and poor prognosis. We also observed that EGFR inhibitors are active in patients with EGFR copy-number gain and that coamplification of other receptor tyrosine kinases or KRAS is associated with worse response. Preclinical trials performed on EGFR-amplified gastroesophageal adenocarcinoma PDX models revealed that the combination of an EGFR mAb and an EGFR tyrosine kinase inhibitor (TKI) was more effective than each monotherapy and resulted in a deeper and durable response. In a highly EGFR-amplified nonresponding PDX, where resistance to EGFR drugs was due to inactivation of the TSC2 tumor suppressor, cotreatment with the mTOR inhibitor everolimus restored sensitivity to EGFR inhibition. CONCLUSIONS: This study underscores EGFR as a potential therapeutic target in gastric cancer and identifies the combination of an EGFR TKI and a mAb as an effective therapeutic approach. Finally, it recognizes mTOR pathway activation as a novel mechanism of primary resistance that can be overcome by the combination of EGFR and mTOR inhibitors.See related commentary by Openshaw et al., p. 2964.

3.
ESMO Open ; 5(5): e000937, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33122354

RESUMO

PURPOSE: Overexpression of miR-100 in stem cells derived from basal-like breast cancers causes loss of stemness, induction of luminal breast cancer markers and response to endocrine therapy. We, therefore, explored miR-100 as a novel biomarker in patients with luminal breast cancer. METHODS: miR-100 expression was studied in 90 patients with oestrogen-receptor-positive/human-epidermal growth factor receptor 2-negative breast cancer enrolled in a prospective study of endocrine therapy given either preoperatively, or for the treatment of de novo metastatic disease. Response was defined as a Ki67 ≤2.7% after 21±3 days of treatment. The prognostic role of miR-100 expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) breast cancer datasets. Additionally, we explored the correlation between miR-100 and the expression its targets reported as being associated with endocrine resistance. Finally, we evaluated whether a signature based on miR-100 and its target genes could predict the luminal A molecular subtype. RESULTS: Baseline miR-100 was significantly anticorrelated with baseline and post-treatment Ki67 (p<0.001 and 0.004, respectively), and independently associated with response to treatment (OR 3.329, p=0.047). In the METABRIC dataset, high expression of miR-100 identified women with luminal A tumours treated with adjuvant endocrine therapy with improved overall survival (HR 0.55, p<0.001). miR-100 was negatively correlated with PLK1, FOXA1, mTOR and IGF1R expression, potentially explaining its prognostic effect. Finally, a miR-100-based signature developed in patients enrolled in the prospective study outperformed Ki67 alone in predicting the luminal A phenotype. CONCLUSIONS: Our findings suggest that miR-100 should be further explored as a biomarker in patients with luminal breast cancer.


Assuntos
Neoplasias da Mama , MicroRNAs , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Fator 3-alfa Nuclear de Hepatócito , Humanos , MicroRNAs/genética , Prognóstico , Estudos Prospectivos
4.
J Hepatol ; 72(6): 1159-1169, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31954205

RESUMO

BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

6.
Neoplasia ; 19(12): 1012-1021, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29136529

RESUMO

The Yes-associated protein (YAP) is a transcriptional co-activator upregulating genes that promote cell growth and inhibit apoptosis. The main dysregulation of the Hippo pathway in tumors is due to YAP overexpression, promoting epithelial to mesenchymal transition, cell transformation, and increased metastatic ability. Moreover, it has recently been shown that YAP plays a role in sustaining resistance to targeted therapies as well. In our work, we evaluated the role of YAP in acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in lung cancer. In EGFR-addicted lung cancer cell lines (HCC4006 and HCC827) rendered resistant to several EGFR inhibitors, we observed that resistance was associated to YAP activation. Indeed, YAP silencing impaired the maintenance of resistance, while YAP overexpression decreased the responsiveness to EGFR inhibitors in sensitive parental cells. In our models, we identified the AXL tyrosine kinase receptor as the main YAP downstream effector responsible for sustaining YAP-driven resistance: in fact, AXL expression was YAP dependent, and pharmacological or genetic AXL inhibition restored the sensitivity of resistant cells to the anti-EGFR drugs. Notably, YAP overactivation and AXL overexpression were identified in a lung cancer patient upon acquisition of resistance to EGFR TKIs, highlighting the clinical relevance of our in vitro results. The reported data demonstrate that YAP and its downstream target AXL play a crucial role in resistance to EGFR TKIs and suggest that a combined inhibition of EGFR and the YAP/AXL axis could be a good therapeutic option in selected NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Transcrição Genética
7.
Oncotarget ; 8(9): 15716-15731, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28157710

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo. On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development.


Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas Experimentais/genética , Alquilantes/farmacologia , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Análise por Conglomerados , Dietilnitrosamina/farmacologia , Perfilação da Expressão Gênica/métodos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia de Fluorescência , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
J Hepatol ; 64(4): 891-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26658681

RESUMO

BACKGROUND & AIMS: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. METHODS: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. RESULTS: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. CONCLUSION: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC.


Assuntos
Oxirredutases do Álcool/genética , Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/patologia , Oxirredutases do Álcool/fisiologia , Animais , Carcinoma Hepatocelular/mortalidade , Regulação para Baixo , Células Hep G2 , Humanos , Fígado/enzimologia , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Gradação de Tumores , Ratos , Especificidade da Espécie
9.
Hepatology ; 62(3): 851-62, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25783764

RESUMO

UNLABELLED: Hepatocellular carcinoma (HCC) develops through a multistage process, but the nature of the molecular changes associated with the different steps, the very early ones in particular, is largely unknown. Recently, dysregulation of the NRF2/KEAP1 pathway and mutations of these genes have been observed in experimental and human tumors, suggesting their possible role in cancer development. To assess whether Nrf2/Keap1 mutations are early or late events in HCC development, we investigated their frequency in the rat Resistant Hepatocyte model, consisting of the administration of diethylnitrosamine followed by a brief exposure to 2-acetylaminofluorene. This model enables the dissection of all stages of hepatocarcinogenesis. We found that Nrf2/Keap1 mutations were present in 71% of early preneoplastic lesions and in 78.6% and 59.3% of early and advanced HCCs, respectively. Mutations of Nrf2 were more frequent, missense, and located in the Nrf2-Keap1 binding region. Mutations of Keap1 occurred at a much lower frequency in both preneoplastic lesions and HCCs and were mutually exclusive with those of Nrf2. Functional in vitro and in vivo studies showed that Nrf2 silencing inhibited the ability of tumorigenic rat cells to grow in soft agar and to form tumors. Unlike Nrf2 mutations, those of Ctnnb1, which are frequent in human HCC, were a later event as they appeared only in fully advanced HCCs (18.5%). CONCLUSION: In the Resistant Hepatocyte model of hepatocarcinogenesis the onset of Nrf2 mutations is a very early event, likely essential for the clonal expansion of preneoplastic hepatocytes to HCC, while Ctnnb1 mutations occur only at very late stages. Moreover, functional experiments demonstrate that Nrf2 is an oncogene critical for HCC progression and development.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Fator de Transcrição de Proteínas de Ligação GA/genética , Neoplasias Hepáticas Experimentais/genética , Mutação , Análise de Variância , Animais , Carcinoma Hepatocelular/patologia , Progressão da Doença , Células HEK293 , Humanos , Neoplasias Hepáticas Experimentais/patologia , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , beta Catenina/genética
10.
Hepatology ; 61(1): 249-59, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25156012

RESUMO

UNLABELLED: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRß1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRß1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRß1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRß1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRß1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRß1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRß1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. CONCLUSION: Down-regulation of TRs, especially TRß1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor.


Assuntos
Carcinoma Hepatocelular/etiologia , Hipotireoidismo/complicações , Neoplasias Hepáticas Experimentais/etiologia , Lesões Pré-Cancerosas/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese , Proliferação de Células , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Hipotireoidismo/metabolismo , Cirrose Hepática/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Ratos Endogâmicos F344 , Receptores dos Hormônios Tireóideos/genética
11.
Oncotarget ; 6(4): 2315-30, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25537513

RESUMO

Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.


Assuntos
Neoplasias da Mama/genética , Diferenciação Celular/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Transplante Heterólogo
12.
Hepatology ; 59(1): 228-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23857252

RESUMO

UNLABELLED: Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant-hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19-positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results. CONCLUSION: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/etiologia , Proliferação de Células , Humanos , Neoplasias Hepáticas Experimentais/etiologia , Masculino , Ratos , Ratos Endogâmicos F344
13.
Mol Biol Cell ; 24(2): 129-44, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23154999

RESUMO

The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Lactamas Macrocíclicas/farmacologia , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Receptor ErbB-2/metabolismo , Transferrina/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Clatrina/fisiologia , Vesículas Revestidas por Clatrina/metabolismo , Dinaminas/metabolismo , Tomografia com Microscopia Eletrônica , Endocitose , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Camundongos , Microscopia de Fluorescência , Corpos Multivesiculares/efeitos dos fármacos , Corpos Multivesiculares/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Análise de Célula Única
14.
Clin Cancer Res ; 18(3): 737-47, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22179665

RESUMO

PURPOSE: MET, the tyrosine kinase receptor for hepatocyte growth factor, is frequently overexpressed in colon cancers with high metastatic tendency. We aimed to evaluate the role of its negative regulators, miR-1 and miR-199a*, and its transcriptional activator, the metastasis-associated in colon cancer 1 (MACC1), in controlling MET expression in human colon cancer samples. EXPERIMENTAL DESIGN: The expression of MET, miR-1, miR-199a*, and MACC1 was evaluated by real-time PCR in 52 matched pairs of colorectal cancers and nontumoral surrounding tissues. The biological role of miR-1 in controlling MET expression and biological activity was assessed in colon cancer cells either by its forced expression or by AntagomiR-mediated inhibition. RESULTS: MiR-1 was downregulated in 84.6% of the tumors and its decrease significantly correlated with MET overexpression, particularly in metastatic tumors. We found that concurrent MACC1 upregulation and miR-1 downregulation are required to elicit the highest increase of MET expression. Consistent with a suppressive role of miR-1, its forced in vitro expression in colon cancer cells reduced MET levels and impaired MET-induced invasive growth. Finally, we identified a feedback loop between miR-1 and MET, resulting in their mutual regulation. CONCLUSIONS: This study identifies an oncosuppressive role of miR-1 in colorectal cancer in which it acts by controlling MET expression through a feedback loop. Concomitant downregulation of miR-1 and increase of MACC1 can thus contribute to MET overexpression and to the metastatic behavior of colon cancer cells.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Idoso , Western Blotting , Neoplasias do Colo/genética , Regulação para Baixo , Feminino , Dosagem de Genes , Humanos , Imuno-Histoquímica , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-met/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores , Fatores de Transcrição/genética , Transfecção , Regulação para Cima
15.
Breast Cancer Res Treat ; 130(1): 29-40, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21153051

RESUMO

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Neoplasias da Mama/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Benzenossulfonatos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Lapatinib , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Niacinamida/análogos & derivados , Proteína Oncogênica v-akt/metabolismo , Compostos de Fenilureia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas/uso terapêutico , Quinazolinas/uso terapêutico , RNA Interferente Pequeno , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Survivina , Trastuzumab , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Expert Opin Pharmacother ; 10(4): 589-600, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19284362

RESUMO

Target therapies for the treatment of human cancers have revolutionized the concept of oncological medicine. This type of therapeutic approach is directed to the inhibition of molecular targets that play a pivotal role in tumor progression -- such as tyrosine kinase receptors (TKIs) controlling cell proliferation and survival -- mainly by means of compounds able to block their activity. In the beginning, the aim of target therapies was specifically to hit a single molecule expressed in neoplastic cells. Now the prevailing idea is that inhibiting both cancer cells and cells of the stroma supporting the tumor would gain better results in fighting the disease. Therefore, the single-target therapy is fading in favor of a multitarget approach and the new generation of TKIs is selected on the basis of their ability simultaneously to target different molecules. This review summarizes the molecular basis of multitarget therapies and the most relevant results obtained in different cancer types.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Desenho de Fármacos , Humanos , Oncologia/tendências , Neoplasias/enzimologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Células Estromais/efeitos dos fármacos
17.
Mol Biol Cell ; 20(9): 2495-507, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19297528

RESUMO

Hepatocyte growth factor/scatter factor (HGF/SF) acts through the membrane-anchored Met receptor tyrosine kinase to induce invasive growth. Deregulation of this signaling is associated with tumorigenesis and involves, in most cases, overexpression of the receptor. We demonstrate that Met is processed in epithelial cells by presenilin-dependent regulated intramembrane proteolysis (PS-RIP) independently of ligand stimulation. The proteolytic process involves sequential cleavage by metalloproteases and the gamma-secretase complex, leading to generation of labile fragments. In normal epithelial cells, although expression of cleavable Met by PS-RIP is down-regulated, uncleavable Met displayed membrane accumulation and induced ligand-independent motility and morphogenesis. Inversely, in transformed cells, the Met inhibitory antibody DN30 is able to promote Met PS-RIP, resulting in down-regulation of the receptor and inhibition of the Met-dependent invasive growth. This demonstrates the original involvement of a proteolytic process in degradation of the Met receptor implicated in negative regulation of invasive growth.


Assuntos
Membrana Celular/enzimologia , Regulação para Baixo , Presenilinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Anticorpos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Ligantes , Metaloproteases/antagonistas & inibidores , Camundongos , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteassoma , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo
18.
Cancer Res ; 68(24): 10128-36, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19074879

RESUMO

MicroRNAs (miRNA) are a recently identified class of noncoding, endogenous, small RNAs that regulate gene expression, mainly at the translational level. These molecules play critical roles in several biological processes, such as cell proliferation and differentiation, development, and aging. It is also known that miRNAs play a role in human cancers where they can act either as oncogenes, down-regulating tumor suppressor genes, or as onco-suppressors, targeting molecules critically involved in promotion of tumor growth. One of such molecules is the tyrosine kinase receptor for hepatocyte growth factor, encoded by the MET oncogene. The MET receptor promotes a complex biological program named "invasive growth" that results from stimulation of cell motility, invasion, and protection from apoptosis. This oncogene is deregulated in many human tumors, where its most frequent alteration is overexpression. In this work, we have identified three miRNAs (miR-34b, miR-34c, and miR-199a*) that negatively regulate MET expression. Inhibition of these endogenous miRNAs, by use of antagomiRs, resulted in increased expression of MET protein, whereas their exogenous expression in cancer cells blocked MET-induced signal transduction and the execution of the invasive growth program, both in cells expressing normal levels of MET and in cancer cells overexpressing a constitutively active MET. Moreover, we show that these same miRNAs play a role in regulating the MET-induced migratory ability of melanoma-derived primary cells. In conclusion, we have identified miRNAs that behave as oncosuppressors by negatively targeting MET and might thus provide an additional option to inhibit this oncogene in tumors displaying its deregulation.


Assuntos
MicroRNAs/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Regiões 3' não Traduzidas , Animais , Células COS , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HT29 , Humanos , MicroRNAs/biossíntese , Invasividade Neoplásica , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Fatores de Crescimento/genética , Transfecção
19.
Proc Natl Acad Sci U S A ; 103(13): 5090-5, 2006 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-16547140

RESUMO

Targeting tyrosine kinase receptors (RTKs) with specific Abs is a promising therapeutic approach for cancer treatment, although the molecular mechanism(s) responsible for the Abs' biological activity are not completely known. We targeted the transmembrane RTK for hepatocyte growth factor (HGF) with a monoclonal Ab (DN30). In vitro, chronic treatment of carcinoma cell lines resulted in impairment of HGF-induced signal transduction, anchorage-independent growth, and invasiveness. In vivo, administration of DN30 inhibited growth and metastatic spread to the lung of neoplastic cells s.c. transplanted into immunodeficient nu/nu mice. This Ab efficiently down-regulates HGF receptor through a molecular mechanism involving a double proteolytic cleavage: (i) cleavage of the extracellular portion, resulting in "shedding" of the ectodomain, and (ii) cleavage of the intracellular domain, which is rapidly degraded by the proteasome. Interestingly, the "decoy effect" generated by the shed ectodomain, acting as a dominant negative molecule, enhanced the inhibitory effect of the Ab.


Assuntos
Anticorpos Monoclonais/imunologia , Regulação para Baixo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais
20.
Oncogene ; 24(18): 3002-10, 2005 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-15735715

RESUMO

The HER2 gene encodes a tyrosine kinase receptor overexpressed in 25-30% of human breast cancers. Clinical trials have shown the efficacy of the anti-HER2 monoclonal antibody Trastuzumab in metastatic breast cancer patients. Nevertheless, 70% of patients are unresponsive from start of treatment and nearly all become unresponsive during treatment. Possible mechanisms for these failures could depend on impairment of the machinery responsible for receptor downregulation. To test this hypothesis, we analysed the genomic sequences encoding regions known to be critical for HER2 downregulation, of both HER2 and of the ubiquitin ligase Cbl. We investigated 63 breast cancers, and found no mutations in these regions. We thus considered alternative mechanisms -- such as TGFalpha production -- possibly interfering with HER2 downregulation. In selected cases, by comparing breast cancer neoplastic tissue before and after Trastuzumab treatment, we found induction of TGFalpha expression. Moreover, by in vitro expression of exogenous TGFalpha in breast cancer cells, we observed a dramatic reduction in Trastuzumab-induced HER2 endocytosis, downregulation and cell growth inhibition. Our results suggest that unresponsiveness to Trastuzumab may not be due to intrinsic defects in the machinery responsible for HER2 downregulation, but can be associated with a TGFalpha-related mechanism of escape to HER2 downregulation.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Genes erbB-2 , Fator de Crescimento Transformador alfa/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/imunologia , Regulação para Baixo , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Estrutura Terciária de Proteína , Fator de Crescimento Transformador alfa/biossíntese , Trastuzumab , Células Tumorais Cultivadas
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