RESUMO
PURPOSE: The EGFR and PI3K/mTORC1/2 pathways are frequently altered in glioblastoma (GBM), but pharmacologic targeting of EGFR and PI3K signaling has failed to demonstrate efficacy in clinical trials. Lack of relevant models has rendered it difficult to assess whether targeting these pathways might be effective in molecularly defined subgroups of GBMs. Here, human brain tumor-initiating cell (BTIC) lines with different combinations of endogenous EGFR wild-type, EGFRvIII, and PTEN mutations were used to investigate response to the EGFR inhibitor gefitinib, mTORC1 inhibitor rapamycin, and dual mTORC1/2 inhibitor AZD8055 alone and in combination with temozolomide (TMZ) EXPERIMENTAL DESIGN: In vitro growth inhibition and cell death induced by gefitinib, rapamycin, AZD8055, and TMZ or combinations in human BTICs were assessed by alamarBlue, neurosphere, and Western blotting assays. The in vivo efficacy of AZD8055 was assessed in subcutaneous and intracranial BTIC xenografts. Kaplan-Meier survival studies were performed with AZD8055 and in combination with TMZ. RESULTS: We confirm that gefitinib and rapamycin have modest effects in most BTIC lines, but AZD8055 was highly effective at inhibiting Akt/mTORC2 activity and dramatically reduced the viability of BTICs regardless of their EGFR and PTEN mutational status. Systemic administration of AZD8055 effectively inhibited tumor growth in subcutaneous BTIC xenografts and mTORC1/2 signaling in orthotopic BTIC xenografts. AZD8055 was synergistic with the alkylating agent TMZ and significantly prolonged animal survival. CONCLUSION: These data suggest that dual inhibition of mTORC1/2 may be of benefit in GBM, including the subset of TMZ-resistant GBMs.
Assuntos
Neoplasias Encefálicas/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/administração & dosagem , Dacarbazina/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Mutação , PTEN Fosfo-Hidrolase/genética , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Temozolomida , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The current standard of care for glioblastoma (GBM) involves a combination of surgery, radiotherapy, and temozolomide chemotherapy, but this regimen fails to achieve long-term tumor control. Resistance to temozolomide is largely mediated by expression of the DNA repair enzyme MGMT; however, emerging evidence suggests that inactivation of MSH6 and other mismatch repair proteins plays an important role in temozolomide resistance. Here, we investigate endogenous MSH6 mutations in GBM, anaplastic oligodendroglial tumor tissue, and corresponding brain tumor-initiating cell lines (BTIC). EXPERIMENTAL DESIGN: MSH6 sequence and MGMT promoter methylation were determined in human tumor samples and BTICs. Sensitivity to temozolomide was evaluated in vitro using BTICs in the absence and presence of O(6)-benzylguanine to deplete MGMT. The influence of MGMT and MSH6 status on in vivo sensitivity to temozolomide was evaluated using intracranial BTIC xenografts. RESULTS: We identified 11 previously unreported mutations in MSH6 in nine different glioma samples and six paired BTIC lines from adult patients. In addition, MSH6 mutations were documented in three oligodendrogliomas and two treatment-naïve gliomas, both previously unreported findings. These mutations were found to influence the sensitivity of BTICs to temozolomide both in vitro and in vivo, independent of MGMT promoter methylation status. CONCLUSIONS: These data demonstrate that endogenous MSH6 mutations may be present before alkylator therapy and occur in at least two histologic subtypes of adult glial neoplasms, with this report serving as the first to note these mutations in oligodendroglioma. These findings broaden our understanding of the clinical response to temozolomide in gliomas.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Oligodendroglioma/genética , Regiões Promotoras Genéticas , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Alquilantes/uso terapêutico , Western Blotting , Neoplasias Encefálicas/tratamento farmacológico , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Feminino , Genótipo , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Oligodendroglioma/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temozolomida , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Investigating the biology of oligodendroglioma and its characteristic combined deletion of chromosomal arms 1p and 19q, mediated by an unbalanced translocation, t(1;19)(q10;p10), has been hampered by the lack of cell lines that harbor these traits. We grew cells from 2 anaplastic oligodendrogliomas in serum-free conditions. Serial propagation and expansion led to the establishment of permanent cell lines that maintained the genetic signature of the parent oligodendrogliomas and displayed features of brain tumor stem cells in vitro. One line was established from a treatment-naïve tumor and the other from a temozolomide resistant recurrent tumor. These lines may be important tools for understanding the biology of oligodendrogliomas and the function of their defining genetic traits.