PDF(Pubmed)
Abstract：
UNASSIGNED: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in advanced EGFR-mutation non-small cell lung cancer (NSCLC) but the magnitude of tumor regression varies, and drug resistance is unavoidable. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) levels are reduced or lost and acts as a tumor suppressor in many cancers. Here, we hypothesized that PHLPP is a key regulator of EGFR-TKI sensitivity and a potential treatment target for overcoming resistance to EGFR-TKI in lung cancer.
UNASSIGNED: Cell proliferation and growth inhibition were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assay. PHLPP- knockdown stable cell lines were generated by lentivirus-mediated delivery of PHLPP shRNAs. The expression of PHLPP mRNA and protein levels was detected by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. Immunohistochemical (IHC) staining was performed to detect the PHLPP expression in clinical patient tissue samples. A transcriptomic assay of genome-wide RNA expressions of PHLPP in NSCLC cell lines according to gefitinib sensitivity was obtained from Gene Expression Omnibus (GEO) database. Murine xenograft model was established to verify the function of PHLPP in gefitinib resistance in vivo.
UNASSIGNED: PHLPP highly expressed in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines. In gefitinib-acquired resistance cell line HCC827-GR, PHLPP expression even dramatically reduced. Knockdown of PHLPP in NSCLC cells decreased cell death induced by the EGFR-TKI, while overexpression PHLPP in gefitinib-resistance NSCLC cells can enhance or restore EGFR-TKIs sensitivity. Mechanism study indicated that PHLPP downregulation attenuates the effect of EGFR-TKI on the both AKT and ERK pathway, thereby decreasing the cell death sensitivity to EGFR inhibitors. In xenograft mice, knockdown of PHLPP decreased tumor response to gefitinib and advanced tumor cells re-growth after gefitinib treatment. In clinical, PHLPP expression were reduced in the post-relapse tumor compared to that of pre-treatment, and lower pre-treatment PHLPP levels were significantly correlated with shorter progression-free survival (PFS) in patients with EGFR-mutant lung adenocarcinoma whom treated with EGFR-TKI.
UNASSIGNED: Our data strongly demonstrated that loss of PHLPP function was a key factor of EGFR-TKI resistance in NSCLC. Downregulated PHLPP expression activated PI3K-AKT and MAPK-ERK pathway which strengthened cell survival to EGFR-TKI. Therefore, PHLPP expression level was not only a potential biomarker to predict EGFR-TKIs sensitivity but also as a therapeutic target in EGFR-TKIs therapy, enhancing PHLPP expression may be a valuable strategy for delaying or overcoming EGFR-TKIs drug resistance.
UNASSIGNED: Cell proliferation and growth inhibition were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assay. PHLPP- knockdown stable cell lines were generated by lentivirus-mediated delivery of PHLPP shRNAs. The expression of PHLPP mRNA and protein levels was detected by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. Immunohistochemical (IHC) staining was performed to detect the PHLPP expression in clinical patient tissue samples. A transcriptomic assay of genome-wide RNA expressions of PHLPP in NSCLC cell lines according to gefitinib sensitivity was obtained from Gene Expression Omnibus (GEO) database. Murine xenograft model was established to verify the function of PHLPP in gefitinib resistance in vivo.
UNASSIGNED: PHLPP highly expressed in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines. In gefitinib-acquired resistance cell line HCC827-GR, PHLPP expression even dramatically reduced. Knockdown of PHLPP in NSCLC cells decreased cell death induced by the EGFR-TKI, while overexpression PHLPP in gefitinib-resistance NSCLC cells can enhance or restore EGFR-TKIs sensitivity. Mechanism study indicated that PHLPP downregulation attenuates the effect of EGFR-TKI on the both AKT and ERK pathway, thereby decreasing the cell death sensitivity to EGFR inhibitors. In xenograft mice, knockdown of PHLPP decreased tumor response to gefitinib and advanced tumor cells re-growth after gefitinib treatment. In clinical, PHLPP expression were reduced in the post-relapse tumor compared to that of pre-treatment, and lower pre-treatment PHLPP levels were significantly correlated with shorter progression-free survival (PFS) in patients with EGFR-mutant lung adenocarcinoma whom treated with EGFR-TKI.
UNASSIGNED: Our data strongly demonstrated that loss of PHLPP function was a key factor of EGFR-TKI resistance in NSCLC. Downregulated PHLPP expression activated PI3K-AKT and MAPK-ERK pathway which strengthened cell survival to EGFR-TKI. Therefore, PHLPP expression level was not only a potential biomarker to predict EGFR-TKIs sensitivity but also as a therapeutic target in EGFR-TKIs therapy, enhancing PHLPP expression may be a valuable strategy for delaying or overcoming EGFR-TKIs drug resistance.
摘要：
表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）对晚期EGFR突变非小细胞肺癌（NSCLC）有效，但肿瘤消退的程度各不相同，耐药性是不可避免的。pleckstrin同源结构域富含亮氨酸的重复蛋白磷酸酶（PHLPP）水平降低或丢失，并在许多癌症中充当肿瘤抑制剂。这里,我们假设PHLPP是EGFR-TKI敏感性的关键调节因子,也是克服肺癌EGFR-TKI耐药的潜在治疗靶点.
通过溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑(MTT)和集落形成测定来测量细胞增殖和生长抑制。通过慢病毒介导的PHLPPshRNA的递送产生PHLPP-敲低稳定细胞系。通过实时定量聚合酶链反应（qPCR）和蛋白质印迹法检测PHLPPmRNA和蛋白的表达水平。进行免疫组织化学(IHC)染色以检测临床患者组织样品中的PHLPP表达。根据吉非替尼敏感性,从基因表达综合(GEO)数据库获得NSCLC细胞系中PHLPP的全基因组RNA表达的转录组学测定。建立小鼠异种移植模型,验证PHLPP在吉非替尼耐药中的作用。
■PHLPP在吉非替尼敏感的NSCLC细胞系中比吉非替尼耐药的NSCLC细胞系中高表达。在吉非替尼获得性耐药细胞系HCC827-GR中，PHLPP表达甚至显著降低。在NSCLC细胞中敲除PHLPP降低EGFR-TKI诱导的细胞死亡,而吉非替尼耐药NSCLC细胞中过度表达PHLPP可增强或恢复EGFR-TKIs敏感性。机制研究表明，PHLPP下调减弱EGFR-TKI对AKT和ERK通路的影响，从而降低对EGFR抑制剂的细胞死亡敏感性。在异种移植小鼠中，PHLPP敲除降低吉非替尼治疗后肿瘤对吉非替尼的反应和晚期肿瘤细胞的再生长.在临床上，与治疗前相比，复发后肿瘤中的PHLPP表达降低，在接受EGFR-TKI治疗的EGFR突变型肺腺癌患者中,较低的治疗前PHLPP水平与较短的无进展生存期(PFS)显著相关.
■我们的数据有力地表明，PHLPP功能的丧失是NSCLC中EGFR-TKI耐药的关键因素。下调的PHLPP表达激活了PI3K-AKT和MAPK-ERK途径，从而增强了细胞对EGFR-TKI的存活。因此,PHLPP表达水平不仅是预测EGFR-TKIs敏感性的潜在生物标志物，也是EGFR-TKIs治疗的治疗靶点。增强PHLPP表达可能是延缓或克服EGFR-TKIs耐药的有价值的策略.
通过溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑(MTT)和集落形成测定来测量细胞增殖和生长抑制。通过慢病毒介导的PHLPPshRNA的递送产生PHLPP-敲低稳定细胞系。通过实时定量聚合酶链反应（qPCR）和蛋白质印迹法检测PHLPPmRNA和蛋白的表达水平。进行免疫组织化学(IHC)染色以检测临床患者组织样品中的PHLPP表达。根据吉非替尼敏感性,从基因表达综合(GEO)数据库获得NSCLC细胞系中PHLPP的全基因组RNA表达的转录组学测定。建立小鼠异种移植模型,验证PHLPP在吉非替尼耐药中的作用。
■PHLPP在吉非替尼敏感的NSCLC细胞系中比吉非替尼耐药的NSCLC细胞系中高表达。在吉非替尼获得性耐药细胞系HCC827-GR中，PHLPP表达甚至显著降低。在NSCLC细胞中敲除PHLPP降低EGFR-TKI诱导的细胞死亡,而吉非替尼耐药NSCLC细胞中过度表达PHLPP可增强或恢复EGFR-TKIs敏感性。机制研究表明，PHLPP下调减弱EGFR-TKI对AKT和ERK通路的影响，从而降低对EGFR抑制剂的细胞死亡敏感性。在异种移植小鼠中，PHLPP敲除降低吉非替尼治疗后肿瘤对吉非替尼的反应和晚期肿瘤细胞的再生长.在临床上，与治疗前相比，复发后肿瘤中的PHLPP表达降低，在接受EGFR-TKI治疗的EGFR突变型肺腺癌患者中,较低的治疗前PHLPP水平与较短的无进展生存期(PFS)显著相关.
■我们的数据有力地表明，PHLPP功能的丧失是NSCLC中EGFR-TKI耐药的关键因素。下调的PHLPP表达激活了PI3K-AKT和MAPK-ERK途径，从而增强了细胞对EGFR-TKI的存活。因此,PHLPP表达水平不仅是预测EGFR-TKIs敏感性的潜在生物标志物，也是EGFR-TKIs治疗的治疗靶点。增强PHLPP表达可能是延缓或克服EGFR-TKIs耐药的有价值的策略.
