Pro-Oncogenic Role of LKB1 in NSCLC

描述（由申请人提供）：EGFR激活突变经常出现在非吸烟相关的肺癌中。这些肿瘤大多对EGFR信号依赖，可以用酪氨酸激酶抑制剂（TKI）如吉非替尼有效治疗。肺癌患者对吉非替尼的高敏感性与EGFR信号应答依赖AKT激活密切相关，吉非替尼治疗抑制AKT激活。最近发现，AKT功能的恢复是EGFR突变肺肿瘤获得TKI耐药的关键步骤，其中一个潜在机制涉及c-MET的扩增，其绕过对EGFR的需求，使EGFR抑制剂无效。LKB1通常被认为是一种肿瘤抑制因子，因为体细胞LKB1失活突变是吸烟相关肺癌中最常见的突变基因之一。然而，在EGFR突变的肺癌中很少发现LKB1突变。我们发现LKB1在依赖EGFR信号的肺癌中具有一种新的、潜在的致癌作用。我们发现LKB1表达的抑制导致了EGFR或AKT组成活性的6个细胞系的细胞凋亡，但在没有EGFR或AKT激活的2个细胞系中没有。更重要的是，通过两种已知机制获得tki抗性的EGFR突变细胞仍然容易发生LKB1缺失诱导的凋亡。在机制上，我们发现EGFR/AKT激活导致LKB1野生型细胞中FoxO3A苏氨酸32 （Thr32）磷酸化增加，但在LKB1无细胞中没有。在野生型LKB1细胞中，LKB1的缺失导致激活的EGFR/AKT抑制FoxO3A的磷酸化，而在LKB1缺失的细胞中，LKB1功能的恢复重建了EGFR/AKT介导的FoxO3A磷酸化。将我们的分析扩展到其他AKT靶点，使用三种不同的等基因LKB1敲低细胞系对和磷酸化特异性抗体芯片，我们观察到其他AKT下游靶点的磷酸化需要LKB1，包括BAD (Ser136), fox01 (Ser319), FoxO4 （Ser197）和GSK32 （Ser9）。由于AKT对这些位点的磷酸化抑制细胞凋亡，LKB1的需求提示LKB1可能在依赖EGFR信号的肿瘤细胞中具有促凋亡作用。综上所述，我们认为LKB1在肺癌的发展中起双重作用。虽然它在吸烟引起的肺癌中是一种肿瘤抑制因子，但我们假设LKB1/AMPK通路的完整性是嗜EGFR信号的非吸烟相关肺癌中促凋亡蛋白磷酸化和失活的关键决定因素。更好地了解LKB1/AMPK通路在NSCLC中潜在的促癌作用及其在化疗敏感性中的作用，可能会对现有疗法（如EGFR抑制剂）的靶向临床应用产生直接影响，并可能为未来实施“个性化”治疗提供分子基础。

DESCRIPTION (provided by applicant): EGFR activation mutations are frequently present in non-smoking related lung cancer. Most of these tumors are addicted to EGFR signaling and can be effectively treated with tyrosine kinase inhibitors (TKI), such as gefitinib. High sensitivity to gefitinib in lung cancer is closely correlated with dependence on AKT activation in response to EGFR signaling, and gefitinib treatment suppresses AKT activation. The restoration of AKT function was recently found to be a key step in EGFR mutant lung tumors that acquired resistance to TKI, and one potential mechanism involves the amplification of c-MET which bypasses the requirement for EGFR, rendering EGFR inhibitors ineffective. LKB1 is commonly known as a tumor suppressor because somatic LKB1 inactivation mutation is one of the most frequently mutated genes in smoking-related lung cancer. However, LKB1 mutations were rarely found in lung cancers with EGFR mutations. We have discovered a novel, potentially oncogenic role for LKB1 in lung cancers that are addicted to EGFR signaling. We found the suppression of LKB1 expression led to apoptosis in six cell lines in which either EGFR or AKT is constitutively active, but not in two cell lines without EGFR or AKT activation. More importantly, EGFR mutant cells with acquired TKI-resistance through two known mechanisms are still prone to apoptosis induced by LKB1 depletion. Mechanistically, we found that EGFR/AKT activation led to increased phosphorylation of FoxO3A at threonine 32 (Thr32) in LKB1 wild-type cells, but not in LKB1-null cells. Depletion of LKB1 in the cells with wild- type LKB1 resulted in attenuation of that phosphorylation of FoxO3A by activated EGFR/AKT, while the restoration of LKB1 function in LKB1-null cells re-established EGFR/AKT mediated FoxO3A phosphorylation. Upon expanding our analysis to other AKT targets, using three different isogenic LKB1 knockdown cell line pairs and a phospho-specific antibody microarray, we observed that there was a requirement for LKB1 in the phosphorylation of other AKT down-stream targets, including BAD (Ser136), FoxO1 (Ser319), FoxO4 (Ser197) and GSK32 (Ser9). Because the phosphorylation of these sites by AKT suppresses apoptosis, the requirement of LKB1 suggests that LKB1 may have a pro-apoptotic role in tumor cells that are addicted to EGFR signaling. In summary, we believe LKB1 plays dual roles in lung cancer development. While it is a tumor-suppressor in lung cancer caused by smoking, we hypothesize that the integrity of the LKB1/AMPK pathway is a critical determinant of the phosphorylation and inactivation of pro-apoptotic proteins in non-smoking related lung cancers that are addicted to EGFR signaling. A better understanding of this novel, potential pro-oncogenic role of the LKB1/AMPK pathway in NSCLC and its role in chemosensitivity may have a direct impact on the targeted clinical use of existing therapies, such as EGFR inhibitors, and may provide a molecular basis for future implementation of "personalized" therapy.