RESUMO
In advanced cancer, including glioblastoma, the transforming growth factor ß (TGF-ß) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-ß signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-ß receptor (TßR-I), leading to an enhanced TGF-ß signal. High expression of USP15 correlates with high TGF-ß activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-ß activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-ß signaling. Our results show that USP15 regulates the TGF-ß pathway and is a key factor in glioblastoma pathogenesis.
Assuntos
Neoplasias Encefálicas/metabolismo , Transformação Celular Neoplásica/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Glioblastoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Células HEK293 , Humanos , Imageamento por Ressonância Magnética , Camundongos , Fosforilação , Prognóstico , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta/genética , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2(+) patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclina E/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Amplificação de Genes/efeitos dos fármacos , Proteínas Oncogênicas/genética , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , TrastuzumabRESUMO
Small molecule inhibitors of HER2 are clinically active in women with advanced HER2-positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach, we performed a genome wide loss-of-function short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that phosphatidylinositol 3-kinase (PI3K)-induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance, which can be effectively reversed by NVP-BEZ235.