Pro-Oncogenic Role of LKB1 in NSCLC

描述(申请人提供)：EGFR激活突变经常出现在与非吸烟相关的肺癌中。这些肿瘤中的大多数都对EGFR信号上瘾，可以用酪氨酸激酶抑制剂(TKI)有效地治疗，如吉非替尼。肺癌对吉非替尼的高敏感性与EGFR信号对AKT激活的依赖性密切相关，而吉非替尼治疗可抑制AKT的激活。最近发现，AKT功能的恢复是获得对TKI耐药的EGFR突变肺癌的关键步骤，一个潜在的机制涉及c-met的扩增，它绕过了对EGFR的要求，使EGFR抑制剂无效。LKB1通常被认为是一种肿瘤抑制基因，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，在有EGFR突变的肺癌中很少发现LKB1突变。我们已经发现了LKB1在依赖于EGFR信号的肺癌中的一个新的、潜在的致癌作用。我们发现，抑制LKB1的表达导致了6个EGFR或AKT具有结构性活性的细胞株的凋亡，但在两个没有EGFR或AKT活性的细胞系中没有。更重要的是，通过两种已知机制获得TKI抗性的EGFR突变细胞仍然容易被LKB1耗尽诱导凋亡。在机制上，我们发现在LKB1野生型细胞中，EGFR/AKT的激活导致了32位苏氨酸(Thr32)处FOXO3a的磷酸化增加，但在LKB1缺失的细胞中却没有。野生型LKB1缺失导致EGFR/AKT激活的FOXO3a磷酸化减弱，而LKB1缺失细胞中LKB1功能的恢复重新建立了EGFR/AKT介导的FOXO3a磷酸化。将我们的分析扩展到其他AKT靶点，使用三个不同的等位基因LKB1敲除细胞系对和磷酸化特异性抗体微阵列，我们观察到其他AKT下游靶点的磷酸化需要LKB1，包括BAD(Ser136)、FoxO1(Ser319)、FOXO4(Ser197)和GSK32(Ser9)。由于AKT对这些位点的磷酸化抑制了细胞的凋亡，对LKB1的需求表明LKB1可能在依赖于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在肺癌的发生发展中起着双重作用。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，在依赖于EGFR信号的非吸烟相关肺癌中，LKB1/AMPK通路的完整性是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通路在非小细胞肺癌中潜在的致癌作用及其在化疗敏感性中的作用，可能会直接影响现有治疗方法(如EGFR抑制剂)的靶向临床应用，并可能为未来个性化治疗的实施提供分子基础。 公共卫生相关性：虽然EGFR抑制剂最初会在一些非小细胞肺癌患者中诱导临床反应，但所有这些药物最终都会产生耐药性。我们的研究结果表明，抑制LKB1信号通路可以促进这些肿瘤细胞的凋亡，尽管它们通过两种不同的机制产生耐药性。这项拟议研究的完成将导致开发一种新的治疗方法，用于治疗对EGFR抑制剂获得性耐药的肺癌。

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. PUBLIC HEALTH RELEVANCE: While EGFR inhibitors initially induce clinical responses in some non-small cell lung cancer patients, all of them eventually develop drug resistance. Our data indicated that the inhibition of LKB1 signaling can promote apoptosis in these tumors in spite the development of drug-resistance by two different mechanisms. The completion of this proposed study should result in the development of a novel therapeutic approach for the treatment of lung cancers with acquired resistance to EGFR inhibitors.