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1.
EMBO Mol Med ; 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30377213

RESUMO

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2-related cancers. A test to identify additional HRR-deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2-related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi-sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2-related cancers.

2.
Genome Med ; 8(1): 88, 2016 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-27553366

RESUMO

BACKGROUND: Cancer patients often show no or only modest benefit from a given therapy. This major problem in oncology is generally attributed to the lack of specific predictive biomarkers, yet a global measure of cancer cell activity may support a comprehensive mechanistic understanding of therapy efficacy. We reasoned that network analysis of omic data could help to achieve this goal. METHODS: A measure of "cancer network activity" (CNA) was implemented based on a previously defined network feature of communicability. The network nodes and edges corresponded to human proteins and experimentally identified interactions, respectively. The edges were weighted proportionally to the expression of the genes encoding for the corresponding proteins and relative to the number of direct interactors. The gene expression data corresponded to the basal conditions of 595 human cancer cell lines. Therapeutic responses corresponded to the impairment of cell viability measured by the half maximal inhibitory concentration (IC50) of 130 drugs approved or under clinical development. Gene ontology, signaling pathway, and transcription factor-binding annotations were taken from public repositories. Predicted synergies were assessed by determining the viability of four breast cancer cell lines and by applying two different analytical methods. RESULTS: The effects of drug classes were associated with CNAs formed by different cell lines. CNAs also differentiate target families and effector pathways. Proteins that occupy a central position in the network largely contribute to CNA. Known key cancer-associated biological processes, signaling pathways, and master regulators also contribute to CNA. Moreover, the major cancer drivers frequently mediate CNA and therapeutic differences. Cell-based assays centered on these differences and using uncorrelated drug effects reveals novel synergistic combinations for the treatment of breast cancer dependent on PI3K-mTOR signaling. CONCLUSIONS: Cancer therapeutic responses can be predicted on the basis of a systems-level analysis of molecular interactions and gene expression. Fundamental cancer processes, pathways, and drivers contribute to this feature, which can also be exploited to predict precise synergistic drug combinations.


Assuntos
Antineoplásicos/farmacologia , Drogas em Investigação/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Proteínas de Neoplasias/genética , Medicamentos sob Prescrição/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Anotação de Sequência Molecular , Mutação , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
3.
Cancer Res ; 76(8): 2301-13, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-27020857

RESUMO

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.


Assuntos
Neoplasias da Mama/enzimologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Receptores Estrogênicos/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Xenoenxertos , Humanos , Camundongos , Piperazinas/uso terapêutico , Piridinas/uso terapêutico
4.
Clin Cancer Res ; 21(24): 5499-5510, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26272063

RESUMO

PURPOSE: PI3K pathway activation occurs in concomitance with RAS/BRAF mutations in colorectal cancer, limiting the sensitivity to targeted therapies. Several clinical studies are being conducted to test the tolerability and clinical activity of dual MEK and PI3K pathway blockade in solid tumors. EXPERIMENTAL DESIGN: In the present study, we explored the efficacy of dual pathway blockade in colorectal cancer preclinical models harboring concomitant activation of the ERK and PI3K pathways. Moreover, we investigated if TP53 mutation affects the response to this therapy. RESULTS: Dual MEK and mTORC1/2 blockade resulted in synergistic antiproliferative effects in cell lines bearing alterations in KRAS/BRAF and PIK3CA/PTEN. Although the on-treatment cell-cycle effects were not affected by the TP53 status, a marked proapoptotic response to therapy was observed exclusively in wild-type TP53 colorectal cancer models. We further interrogated two independent panels of KRAS/BRAF- and PIK3CA/PTEN-altered cell line- and patient-derived tumor xenografts for the antitumor response toward this combination of agents. A combination response that resulted in substantial antitumor activity was exclusively observed among the wild-type TP53 models (two out of five, 40%), but there was no such response across the eight mutant TP53 models (0%). Interestingly, within a cohort of 14 patients with colorectal cancer treated with these agents for their metastatic disease, two patients with long-lasting responses (32 weeks) had TP53 wild-type tumors. CONCLUSIONS: Our data support that, in wild-type TP53 colorectal cancer cells with ERK and PI3K pathway alterations, MEK blockade results in potent p21 induction, preventing apoptosis to occur. In turn, mTORC1/2 inhibition blocks MEK inhibitor-mediated p21 induction, unleashing apoptosis. Clin Cancer Res; 21(24); 5499-510. ©2015 AACR.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
5.
Sci Transl Med ; 7(283): 283ra51, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25877889

RESUMO

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.


Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Projetos de Pesquisa , Transdução de Sinais , Tiazóis/farmacologia
6.
PLoS Genet ; 10(10): e1004721, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329316

RESUMO

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Animais Recém-Nascidos , Apoptose/genética , Apoptose/efeitos da radiação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/genética , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Camundongos Transgênicos , Neoplasias de Células Escamosas/etiologia , Neoplasias de Células Escamosas/patologia , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
7.
Cancer Discov ; 3(11): 1238-44, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23950206

RESUMO

UNLABELLED: Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline TP53 mutation (Li-Fraumeni Syndrome) identified oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifications, an EGFR-exon 20 insertion, and the first-in-humans HER2V659E mutation showing a phenotypic convergent evolution toward HER2 and EGFR alterations. Following the identification of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A symptomatic and radiologic clinical response was achieved. HER2V659E sensitivity to lapatinib was confirmed in the laboratory. SIGNIFICANCE: The precise knowledge of the genomic alterations present in tumors is critical to selecting the optimal treatment for each patient. Here, we report the molecular characterization and clinical response to a lapatinib-based therapy for the tumors of a Li-Fraumeni patient showing prevalence of HER2 and EGFR genomic alterations.


Assuntos
Síndrome de Li-Fraumeni/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Receptor ErbB-2/genética , Adulto , Exoma , Feminino , Humanos , Lapatinib , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/metabolismo , Síndrome de Li-Fraumeni/patologia , Mutação , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
8.
Cancer Discov ; 2(11): 1036-47, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22915752

RESUMO

UNLABELLED: PARP inhibitors are active in tumors with defects in DNA homologous recombination (HR) due to BRCA1/2 mutations. The phosphoinositide 3-kinase (PI3K) signaling pathway preserves HR steady state. We hypothesized that in BRCA-proficient triple-negative breast cancer (TNBC), PI3K inhibition would result in HR impairment and subsequent sensitization to PARP inhibitors. We show in TNBC cells that PI3K inhibition leads to DNA damage, downregulation of BRCA1/2, gain in poly-ADP-ribosylation, and subsequent sensitization to PARP inhibition. In TNBC patient-derived primary tumor xenografts, dual PI3K and PARP inhibition with BKM120 and olaparib reduced the growth of tumors displaying BRCA1/2 downregulation following PI3K inhibition. PI3K-mediated BRCA downregulation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of an active form of MEK1 resulted in ERK activation and downregulation of BRCA1, whereas the MEK inhibitor AZD6244 increased BRCA1/2 expression and reversed the effects of MEK1. We subsequently identified that the ETS1 transcription factor was involved in the ERK-dependent BRCA1/2 downregulation and that knockdown of ETS1 led to increased BRCA1/2 expression, limiting the sensitivity to combined BKM120 and olaparib in 3-dimensional culture. SIGNIFICANCE: Treatment options are limited for patients with TNBCs. PARP inhibitors have clinical activity restricted to a small subgroup of patients with BRCA mutations. Here, we show that PI3K blockade results in HR impairment and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine PI3K and PARP inhibitors in this indication. Our findings could greatly expand the number of patients with breast cancer that would benefit from therapy with PARP inhibitors. On the basis of our findings, a clinical trial with BKM120 and olaparib is being initiated in patients with TNBCs.


Assuntos
Proteína BRCA1/biossíntese , Proteína BRCA2/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Inibidores Enzimáticos/uso terapêutico , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Prognóstico , Transdução de Sinais
9.
Clin Cancer Res ; 18(9): 2603-12, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22407832

RESUMO

PURPOSE: The PI3K/Akt/mTOR pathway is an attractive target in HER2-positive breast cancer that is refractory to anti-HER2 therapy. The hypothesis is that the suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. EXPERIMENTAL DESIGN: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR, and extracellular signal-regulated kinase (ERK) signaling pathways was evaluated by Western blotting. Tumor growth inhibition after treatment with lapatinib, INK-128, or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed on trastuzumab-based therapy. RESULTS: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade produced synergistic induction of cell death in five different HER2-positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line-based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models. CONCLUSIONS: The simultaneous blockade of both PI3K/Akt/mTOR and ERK pathways obtained by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer models refractory to anti-HER2 therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteínas/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzoxazóis/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Lapatinib , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Complexos Multiproteicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Trastuzumab , Células Tumorais Cultivadas
10.
BMC Cancer ; 10: 265, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20529342

RESUMO

BACKGROUND: Melanoma is the most deadly form of skin cancer without effective treatment. Methylthioadenosine (MTA) is a naturally occurring nucleoside with differential effects on normal and transformed cells. MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types. In this study we have assessed the therapeutic potential of MTA in melanoma treatment. METHODS: To investigate the therapeutic potential of MTA we performed in vitro proliferation and viability assays using six different mouse and human melanoma cell lines wild type for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in vivo in a xenograft mouse melanoma model and using variety of molecular techniques and tissue culture we investigated its anti-proliferative and pro-apoptotic properties. RESULTS: In vitro experiments showed that MTA treatment inhibited melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumor growth. The molecular analysis of tumor samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1. CONCLUSIONS: MTA inhibits melanoma cell proliferation and in vivo tumor growth particularly in BRAF mutant melanoma cells. These data reveal a naturally occurring drug potentially useful for melanoma treatment.


Assuntos
Adenosina/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Tionucleosídeos/farmacologia , Adenosina/análogos & derivados , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Genes ras , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Tempo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Cancer ; 126(7): 1549-61, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19810100

RESUMO

Melanoma is the most lethal human skin cancer. If metastatic, it becomes very aggressive and resistant to standard modalities of anticancer treatment. During the last 10 years, several therapeutic strategies have been tested including the use of single and combined small drugs. Experimental results indicate that RAS and PI3K pathways are important for the development and maintenance of melanoma. In this study, we assessed the in vitro and in vivo inhibition potential of PI-103, a PI3K (p110alpha)/mTOR inhibitor and sorafenib, a BRAF inhibitor, as single agents and in combination in primary melanoma cell lines. Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway appeared to be more effective. The combination of the two agents in in vitro showed a synergistic effect inhibiting RAS and PI3K pathways in a cell line dependent manner. However, no cooperative effect was observed in blocking in vivo tumor growth in immunocompetent mice. In contrary to the expected, the data indicate that PI-103 induced immunosuppression promoting in vivo tumor growth and inhibiting apoptosis. Furthermore, in vitro studies examining the effects of the PI3K/mTOR inhibitor in tumor derived cell lines indicated that PI-103 induced the anti-apoptotic BH3 family proteins Mcl1, Bcl2 and Bcl(xL) favoring, the in vitro survival of sorafenib treated melanoma cells. These data certainly makes an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models prior to conducting clinical studies.


Assuntos
Benzenossulfonatos/farmacologia , Furanos/farmacologia , Imunossupressão , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Melanoma/patologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Niacinamida/análogos & derivados , Compostos de Fenilureia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Taxa de Sobrevida , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas , Proteínas ras/genética , Proteínas ras/metabolismo
12.
Mol Cell Biol ; 23(14): 5078-89, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12832491

RESUMO

The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Matriz Extracelular/metabolismo , Humanos , Carioferinas/metabolismo , Leucina/metabolismo , Camundongos , Dados de Sequência Molecular , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Serina/metabolismo , Fatores de Transcrição da Família Snail , Frações Subcelulares , Células Tumorais Cultivadas
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