RESUMO
Breast cancer is a heterogeneous disease at both the clinical and molecular levels. This heterogeneity may give rise to different therapy responses. Molecular profiling has facilitated identification of signatures for stratifying patients who would potentially benefit from given therapies. Previously, we reported on a subset of genes with the potential for predicting response of primary breast cancer to neoadjuvant chemotherapy. Herein, we report that patients with luminal (estrogen receptor α [ERα]-expressing) breast cancer were enriched for nonresponders. To identify novel factors that contribute to the survival of breast cancer cells, a loss-of-function screen was performed with a subset of genes overexpressed in patients with disease resistant to chemotherapy. This approach led us to identify protein phosphatase 1, regulatory subunit 15B (PPP1R15B) as a factor with a potentially essential role in the survival of ERα-positive breast cancer cells. Functional analyses showed that PPP1R15B depletion results in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle, and apoptosis induction. Moreover, our data revealed a regulatory role for PPP1R15B in activating ERα. Furthermore, a high level of PPP1R15B mRNA expression was associated with poor outcome following tamoxifen-based therapy. Accordingly, knockdown of PPP1R15B expression sensitized tamoxifen-resistant MCF-7 breast cancer cells to tamoxifen while reducing ERα abundance in these cells. Our findings reveal a novel role for PPP1R15B in the survival and therapy response of ERα-positive breast cancer and may open new avenues for tumor subtype-specific therapeutic strategies in the era of personalized medicine.
Assuntos
Apoptose , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Fosfatase 1/metabolismo , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Luciferases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
In recent years, RNA interference (RNAi) has been widely used to uncover gene function or pathway context of novel genes. In our study, we describe a short-hairpin RNA-based RNAi screening of a set of functionally uncharacterized human genes for their possible capability to inhibit apoptosis. We thereby identified a new antiapoptotic function for CHMP5 (charged multivesicular body protein 5), which was confirmed by overexpression and rescue assays. Furthermore, caspase assays showed that CHMP5 silencing induced caspase cascade activation mainly through extrinsic apoptosis pathway. Based on genome-wide expression array profiling, a possible regulatory role of CHMP5 on apoptosis-associated genes and different signaling pathways including nuclear factor kappa B was revealed. In addition, we found significantly higher CHMP5 mRNA levels in acute myeloid leukemia patients. This observation together with the antiapoptotic feature of CHMP5 suggests a possible oncogenic function for this gene in leukemogenesis.
Assuntos
Apoptose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Leucemia Mieloide Aguda/genética , Western Blotting , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Inativação Gênica , Humanos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Application of mathematical algorithms to sequenced whole genomes revealed a large number of predicted genes, requiring functional assays for their characterization in a high-throughput manner. Here, we report on the development of a screening assay, which is based on reverse transfection of cellular arrays and subsequent analysis of cell morphology to identify novel proapoptotic genes. Expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pEYFP to screen for apoptotic body formation, based on EYFP fluorescence. The assay was validated and applied to 382 human sequence-verified full-length open reading frames, most of them of unknown function. In this initial screening, proapoptotic effects could be demonstrated for 10 of these genes. For 6 of them apoptosis induction could be confirmed both by TUNEL assay and by FACS analysis of cells stained according to Nicoletti: 1 gene was not yet annotated for an apoptotic function (ST6GAL2), while 5 genes were without annotated function (FLJ20551, CXorf12, FAM105A, TMEM66, C19orf4). Our study demonstrates the potential of this method to characterize functionally genes of unknown function in a highly parallel format.
