RESUMO
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFß signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Indóis , Complexo Mediador/genética , Complexo Mediador/metabolismo , Morfolinas , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonamidas , Sulfóxidos/farmacologia , Sulfóxidos/uso terapêutico , Fator de Crescimento Transformador beta/metabolismoRESUMO
Non-melanoma skin cancer (NMSC) is the most common form of cancer. Ultraviolet-B (UVB) radiation has been shown to be a complete carcinogen in the development of NMSC. The mammalian target of rapamycin complex 1 (mTORC1) is upregulated by UVB. Ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, is also upregulated in response to UVB. However, the interplay between these two pathways after UVB exposure remains unclear. The studies described here compare mRNA stability between normal human keratinocytes (HaCaT cells) and HaCaT cells with low levels of raptor to investigate whether the induction of ODC by UVB is dependent on mTORC1. We show that the knockdown of mTORC1 activity led to decreased levels of ODC protein both before and after exposure to 20 mJ/cm2 UVB. ODC mRNA was less stable in cells with decreased mTORC1 activity. Polysome profiles revealed that the initiation of ODC mRNA translation did not change in UVB-treated cells. We have shown that the ODC transcript is stabilized by the RNA-binding protein human antigen R (HuR). To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability in human keratinocytes exposed to UVB. We show an increased cytoplasmic localization of HuR after UVB exposure in wild-type cells. The ablation of HuR via CRISPR/Cas9 did not alter the stability of the ODC message, suggesting the involvement of other trans-acting factors. These data suggest that in human keratinocytes, ODC mRNA stability is regulated, in part, by an mTORC1-dependent mechanism after UVB exposure.
Assuntos
Queratinócitos/enzimologia , Queratinócitos/efeitos da radiação , Ornitina Descarboxilase/metabolismo , Proteína Regulatória Associada a mTOR/genética , Linhagem Celular , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ornitina Descarboxilase/genética , Biossíntese de Proteínas/efeitos da radiação , Estabilidade de RNA/efeitos da radiação , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Raios UltravioletaRESUMO
Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS.