RESUMO
The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Óxidos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Trióxido de Arsênio , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Cetonas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Regulação para Cima , Vimblastina/farmacologia , Vincristina/farmacologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.
Assuntos
Furanos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Embrião de Galinha , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system [Formula: see text] that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.
RESUMO
Rhabdomyosarcoma (RMS) frequently exhibits concomitant activation of the PI3K/Akt/mTOR and the Ras/MEK/ERK pathways. Therefore, we investigated whether pharmacological cotargeting of these two key survival pathways suppresses RMS growth. Here, we identify a synthetic lethal interaction between PI3K/Akt/mTOR and Ras/MEK/ERK pathway inhibition in RMS. The dual PI3K/mTOR inhibitor PI103 and the MEK inhibitor UO126 synergize to trigger apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1), whereas either agent alone induces minimal cell death. Similarly, genetic knockdown of p110α and MEK1/2 cooperates to induce apoptosis. Molecular studies reveal that cotreatment with PI103/UO126 cooperates to suppress PI3K/Akt/mTOR and Ras/MEK/ERK signaling, whereas either compound alone is not only less effective to inhibit signaling, but even cross-activates the other pathway. Accordingly, PI103 alone increases ERK phosphorylation, while UO126 enhances Akt phosphorylation, consistent with negative crosstalks between these two signaling pathways. Furthermore, PI103/UO126 cotreatment causes downregulation of several antiapoptotic proteins such as XIAP, Bcl-xL and Mcl-1 as well as increased expression and decreased phosphorylation of the proapoptotic protein BimEL, thus shifting the balance towards apoptosis. Consistently, PI103/UO126 cotreatment cooperates to trigger Bax activation, loss of mitochondrial membrane potential, caspase activation and caspase-dependent apoptosis. This identification of a synthetic lethal interaction between PI3K/mTOR and MEK inhibitors has important implications for the development of novel treatment strategies in RMS.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Rabdomiossarcoma/enzimologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Furanos/farmacologia , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Terapia de Alvo Molecular , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Proteínas ras/metabolismoRESUMO
Abstract Breast cancer patients with disseminated metastatic disease still have a very unfavorable prognosis. Investigations into the molecular mechanisms that underlie metastasis formation have a high priority and can possibly result in improved therapeutic interventions. The process of oncogenic epithelial to mesenchymal transition (EMT) has recently become a focus in cancer research because it encompasses many of the phenotypic traits characteristic of metastatic cells, e.g., increased motility, invasion, anoikis resistance, immunosuppression, and cancer stem cell potential. A number of central cellular signaling pathways and transcription factors have been implied in the control of EMT and metastasis formation, among them signal originating from the activation of the transforming growth factor ß (TGFß), epithelial growth factor, Wnt, Notch, and Hedgehog pathways. We have investigated the contribution of TGFß signaling to metastasis-related cellular properties. TGFß signaling can have tumor-suppressive and -promoting effects depending on the tumor type and the stage of tumor progression. TGFß can inhibit the proliferation of mammary epithelial cells (MECs), but it can also induce EMT, invasion, and metastasis, possibly through Smad-independent signaling events. We investigated the effects of TGFß pathway inhibition on the proliferation, differentiation, and invasion of both normal and malignant MECs. shRNA-mediated downregulation of the Smad4 protein in non-tumorigenic HC11 and tumorigenic 4T1 cells promotes the invasiveness of both cell lines. Mammary gland reconstitution studies, with primary MECs expressing shSmad4, resulted only in subtle effects on the glandular morphogenesis. Orthotopic transplantation of shSmad4-transduced 4T1 tumor cells caused the accelerated growth of mammary tumors and enhanced colonization and macroscopic lung metastases when compared to control cells. Surprisingly, the expression of Smad4 was restored, and a strong activation of Stat3 was found in the metastatic lesions present in the lungs. These lesions express metastatic factors, such as angiopoietin-like-4 and the inhibitor of DNA binding/differentiation 1. We suggest that the downregulation of Smad4 inhibits the tumor-suppressive effects of TGFß signaling and enhances tumor growth. The downregulation, however, was only transient, and the reactivation of Smad4 expression caused the reversal of EMT, mesenchymal to epithelial transition, and thereby promoted metastasis formation in the lungs.
RESUMO
We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.