RESUMO
The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genótipo , Lipídeos , Protoplastos , Vitis , Vitis/genética , Edição de Genes/métodos , Protoplastos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Vinho , Genoma de Planta/genética , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
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.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medicina de Precisão/métodos , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Humanos , Imunoconjugados/uso terapêutico , Estudos Prospectivos , Proteínas Tirosina Quinases/antagonistas & inibidores , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Anticarcinógenos/administração & dosagem , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Progressão da Doença , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Tri-Iodotironina/administração & dosagem , Idoso , Animais , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Receptores beta dos Hormônios Tireóideos/metabolismo , Transcriptoma , Tri-Iodotironina/metabolismoRESUMO
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 EspécieRESUMO
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 , Carcinoma Hepatocelular , Neoplasias Hepáticas Experimentais , Mutação , Fator 2 Relacionado a NF-E2 , Animais , Humanos , Masculino , Ratos , Análise de Variância , beta Catenina/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Progressão da Doença , Células HEK293 , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Distribuição Aleatória , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Fator 2 Relacionado a NF-E2/genéticaRESUMO
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/genéticaRESUMO
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 F344RESUMO
Despite negative results of clinical trials conducted on the overall population of patients with gastric cancer, PARP inhibitor (PARPi) therapeutic strategy still might represent a window of opportunity for a subpopulation of patients with gastric cancer. An estimated 7% to 12% of gastric cancers exhibit a mutational signature associated with homologous recombination (HR) failure, suggesting that these patients could potentially benefit from PARPis. To analyze responsiveness of gastric cancer to PARPi, we exploited a gastroesophageal adenocarcinoma (GEA) platform of patient-derived xenografts (PDX) and PDX-derived primary cells and selected 10 PDXs with loss-of-function mutations in HR pathway genes. Cell viability assays and preclinical trials showed that olaparib treatment was effective in PDXs harboring BRCA2 germline mutations and somatic inactivation of the second allele. Olaparib responsive tumors were sensitive to oxaliplatin as well. Evaluation of HR deficiency (HRD) and mutational signatures efficiently stratified responder and nonresponder PDXs. A retrospective analysis on 57 patients with GEA showed that BRCA2 inactivating variants were associated with longer progression-free survival upon platinum-based regimens. Five of 7 patients with BRCA2 germline mutations carried the p.K3326* variant, classified as "benign." However, familial history of cancer, the absence of RAD51 foci in tumor cells, and a high HRD score suggest a deleterious effect of this mutation in gastric cancer. In conclusion, PARPis could represent an effective therapeutic option for BRCA2-mutated and/or high HRD score patients with GEA, including patients with familial intestinal gastric cancer. SIGNIFICANCE: PARP inhibition is a potential strategy for treating patients with gastric cancer with mutated BRCA2 or homologous repair deficiency, including patients with familial intestinal gastric cancer, for whom BRCA2 germline testing should be recommended.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Neoplasias Gástricas , Humanos , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutação em Linhagem Germinativa , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Estudos Retrospectivos , Proteína BRCA1/genética , Proteína BRCA2/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
BACKGROUND: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. METHODS: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. RESULTS: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. CONCLUSIONS: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate.
Assuntos
Retroelementos , Neoplasias de Mama Triplo Negativas , Antígeno B7-H1/metabolismo , Hormônios , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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 XenoenxertoRESUMO
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.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Anticorpos Monoclonais/uso terapêutico , Neoplasias Esofágicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animais , Estudos de Coortes , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais CultivadasRESUMO
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 ProspectivosRESUMO
[This corrects the article DOI: 10.18632/oncotarget.2962.].
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 Gênica , Receptor Tirosina Quinase AxlRESUMO
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 ReversaRESUMO
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 CultivadasRESUMO
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 , Quinase 1 Polo-LikeRESUMO
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 ÚnicaRESUMO
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 CimaRESUMO
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.