PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. AbbreviationsAKTV-Akt Murine Thymoma Viral OncogeneCACancer adjacentCDK1Cyclin dependent kinase 1CENPC-CCentromere Protein CChIPChromatin Immunoprecipitationco-IPCo-immunoprecipitationCOSMICCatalog of Somatic Mutations In CancerCREBcAMP Responsive Element Binding ProteinC-tailCarboxy terminal tailE2F1E2F Transcription Factor 1ECISElectric Cell-substrate Impedance SensingEGFREpidermal Growth Factor ReceptorGSIGamma Secretase InhibitorHDAC1Histone Deacetylase 1HP1Heterochromatin protein 1KAP1/TRIM28KRAB-Associated Protein 1/Tripartite Motif Containing 28MAF1Repressor of RNA polymerase III transcription MAF1 homologMCM2Minichromosome Maintenance Complex Component 2miRNAmicro RNAMTF1Metal-Regulatory Transcription Factor 1PARPPoly(ADP-Ribose) PolymerasePD-1Programmed Cell Death 1PD-L1Programmed Cell Death 1 Ligand 1PI3KPhosphatidylinositol-4,5-Bisphosphate 3-KinasePLKPolo-like KinasepPTENPhosphorylated PTENPTENPhosphatase and Tensin Homolog deleted on chromosome tenPTMPost Translational ModificationRad51RAD51 RecombinaseRad52RAD52 RecombinaseRPA1Replication protein ASILACStable Isotope Labeling with Amino Acids in Cell CultureSRFSerum Response FactorTKITyrosine Kinase inhbitorsTMATissue MicroarrayTOP2ADNA Topoisomerase 2A V-Akt Murine Thymoma Viral Oncogene Cancer adjacent Cyclin dependent kinase 1 Centromere Protein C Chromatin Immunoprecipitation Co-immunoprecipitation Catalog of Somatic Mutations In Cancer cAMP Responsive Element Binding Protein Carboxy terminal tail E2F Transcription Factor 1 Electric Cell-substrate Impedance Sensing Epidermal Growth Factor Receptor Gamma Secretase Inhibitor Histone Deacetylase 1 Heterochromatin protein 1 KRAB-Associated Protein 1/Tripartite Motif Containing 28 Repressor of RNA polymerase III transcription MAF1 homolog Minichromosome Maintenance Complex Component 2 micro RNA Metal-Regulatory Transcription Factor 1 Poly(ADP-Ribose) Polymerase Programmed Cell Death 1 Programmed Cell Death 1 Ligand 1 Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Polo-like Kinase Phosphorylated PTEN Phosphatase and Tensin Homolog deleted on chromosome ten Post Translational Modification RAD51 Recombinase RAD52 Recombinase Replication protein A Stable Isotope Labeling with Amino Acids in Cell Culture Serum Response Factor Tyrosine Kinase inhbitors Tissue Microarray DNA Topoisomerase 2A

第10号染色体缺失的磷酸酶及张力蛋白同源物（Phosphatase and Tensin Homolog deleted on chromosome ten，以下简称PTEN）在其C端尾部（C-tail）丝氨酸/苏氨酸簇的磷酸化负向调控其肿瘤抑制功能。然而，这种磷酸化抑制作用的具体后果及其在驱动肺癌发生过程中的下游效应仍未被阐明。本研究阐明了磷酸化损害PTEN功能、进而促进肺癌发生的分子机制。将PTEN的丝氨酸/苏氨酸残基替换为丙氨酸，可生成磷酸化缺陷型PTEN突变体PTEN-4A，该突变体能够抑制肺癌细胞的增殖与迁移。PTEN-4A优先定位于细胞核，并在此处抑制E2F转录因子1（E2F Transcription Factor 1，以下简称E2F1）介导的细胞周期基因转录。PTEN-4A可与转录因子E2F1发生物理相互作用，并结合于带有E2F1 DNA结合位点的基因启动子区域的染色质上，这可能是其转录抑制功能的潜在分子机制。缺失分析显示，PTEN的C2结构域对于抑制E2F1介导的转录是不可或缺的。进一步研究发现，癌症相关的C2结构域突变蛋白丧失了抑制E2F1介导转录的能力，这支持了这类突变在患者体内具有致癌性的观点。与上述发现一致，我们在肺癌患者样本中观察到PTEN磷酸化水平升高、核内PTEN水平降低，证实磷酸化是PTEN在肺癌中真实存在的失活机制。因此，使用阻碍PTEN磷酸化的小分子抑制剂是激活PTEN功能、用于肺癌治疗的可行策略。

缩写说明：
AKT：Akt鼠胸腺瘤病毒致癌基因（Akt Murine Thymoma Viral Oncogene）
CA：癌旁组织（Cancer adjacent）
CDK1：细胞周期蛋白依赖性激酶1（Cyclin dependent kinase 1）
CENP-C：着丝粒蛋白C（Centromere Protein C）
ChIP：染色质免疫共沉淀（Chromatin Immunoprecipitation）
co-IP：免疫共沉淀（Co-immunoprecipitation）
COSMIC：体细胞突变癌症目录（Catalog of Somatic Mutations In Cancer）
CREB：cAMP应答元件结合蛋白（cAMP Responsive Element Binding Protein）
C-tail：羧基末端尾部（Carboxy terminal tail）
E2F1：E2F转录因子1（E2F Transcription Factor 1）
ECIS：细胞-基质阻抗传感（Electric Cell-substrate Impedance Sensing）
EGFR：表皮生长因子受体（Epidermal Growth Factor Receptor）
GSI：γ分泌酶抑制剂（Gamma Secretase Inhibitor）
HDAC1：组蛋白去乙酰化酶1（Histone Deacetylase 1）
HP1：异染色质蛋白1（Heterochromatin protein 1）
KAP1/TRIM28：KRAB相关蛋白1/含三结构域蛋白28（KRAB-Associated Protein 1/Tripartite Motif Containing 28）
MAF1：RNA聚合酶Ⅲ转录抑制因子MAF1同源物（Repressor of RNA polymerase III transcription MAF1 homolog）
MCM2：微小染色体维持复合体组分2（Minichromosome Maintenance Complex Component 2）
miRNA：微小RNA（micro RNA）
MTF1：金属调控转录因子1（Metal-Regulatory Transcription Factor 1）
PARP：聚(ADP-核糖)聚合酶（Poly(ADP-Ribose) Polymerase）
PD-1：程序性死亡蛋白1（Programmed Cell Death 1）
PD-L1：程序性死亡蛋白配体1（Programmed Cell Death 1 Ligand 1）
PI3K：磷脂酰肌醇-4,5-二磷酸3-激酶（Phosphatidylinositol-4,5-Bisphosphate 3-Kinase）
PLK：Polo样激酶（Polo-like Kinase）
pPTEN：磷酸化PTEN（Phosphorylated PTEN）
PTM：翻译后修饰（Post Translational Modification）
Rad51：RAD51重组酶（RAD51 Recombinase）
Rad52：RAD52重组酶（RAD52 Recombinase）
RPA1：复制蛋白A1（Replication protein A）
SILAC：细胞培养氨基酸稳定同位素标记（Stable Isotope Labeling with Amino Acids in Cell Culture）
SRF：血清反应因子（Serum Response Factor）
TKI：酪氨酸激酶抑制剂（Tyrosine Kinase inhibitors，原文拼写为inhbitors，应为笔误）
TMA：组织微阵列（Tissue Microarray）
TOP2A：DNA拓扑异构酶2A（DNA Topoisomerase 2A）