A Role of Bit1 in the Apoptosis Resistance, Anoikis Insensitvity, and Chemoresist

描述(申请人提供)：非小细胞肺癌(NSCLC)是一种高度侵袭性的恶性肿瘤，与转移和化疗反应差有关。非小细胞肺癌细胞对凋亡表现出很高的抵抗力，至少部分原因是由于凋亡机制被禁用。特别是，NSCLC细胞中caspase依赖的通路的部分缺陷对DNA损伤剂如依托泊苷或3-射线诱导的凋亡具有抵抗力(9，14)。因此，对抗非小细胞肺癌侵袭性和化疗耐药性的一个主要治疗途径是通过另一条caspase非依赖的细胞死亡途径有效地诱导细胞凋亡。该项目的目标是通过获取所需的信息来解决NSCLC治疗的关键障碍，以了解新的caspase非依赖性失巢蛋白效应因子Bcl2转录抑制因子(Bit1)在NSCLC细胞凋亡抵抗和恶性转化中的作用。一些观察表明，Bit1通路在肺癌中没有功能：i)Bit1在各种类型的非小细胞肺癌组织中的表达显著低于相应的正常肺组织(图1A和1B)，以及ii)Bit1功能的抑制剂TLE1作为一种肺特异的癌基因发挥作用，并在人类肺癌中过表达(1)。虽然胞质定位的Bit1在NCI-H460细胞中的异位表达诱导细胞凋亡(图1D-F)，但线粒体定位的Bit1在H460细胞中的恢复增强了NSCLC细胞的失巢凋亡敏感性，并减弱了锚定非依赖性生长和对依托泊苷诱导的凋亡的抵抗力(图2)。基于已发表的数据和我们的初步结果，我们的目标是验证这样的假设，即恶性NSCLC细胞可能绕过Bit1凋亡途径成为失巢耐药和锚定非依赖性的，并且Bit1途径的激活可能在诱导NSCLC细胞凋亡和减弱侵袭性表型方面有效。其具体目的是：1)研究Bit1凋亡通路在非小细胞肺癌细胞存活和凋亡抵抗中的调节作用和相关性；2)检验Bit1凋亡通路通过改变线粒体Bit1表达来调节NSCLC细胞侵袭性表型的假说及其对NSCLC细胞的失巢抵抗、锚定非依赖性生长潜能或化疗耐药的影响；3)以Bit1为工具了解和剖析NSCLC细胞的凋亡机制。这项研究的结果将阐明抑制或阻断Bit1凋亡通路在NSCLC获得失巢抵抗和锚定独立性方面的重要性。我们相信，通过Bit1的凋亡功能激活caspase非依赖的细胞死亡通路将导致成功地消除非小细胞肺癌的化疗耐药。因此，BIT-1可能成为治疗这种侵袭性癌症的新靶点。PHS398(5/01版)页面延续格式页面 公共卫生相关性：Bit1在非小细胞肺癌(NSCLC)细胞的凋亡抵抗、失巢不敏感和化疗耐药中的作用非小细胞肺癌(NSCLC)是一种高度侵袭性疾病，预后差，死亡率高。预后惨淡的部分原因是其对常规化疗的抗药性以及扩散到远处器官的倾向。为了克服非小细胞肺癌的侵袭性和化疗耐药性，寻找可能增强非小细胞肺癌细胞凋亡敏感性的新的细胞死亡途径(S)势在必行。

DESCRIPTION (provided by applicant): Non-small cell lung cancer (NSCLC) is a highly aggressive form of malignancy that is associated with metastasis and poor response to chemotherapy. Non-small cell lung cancer cells exhibit high resistance to undergo apoptosis, at least in part due to a disabled apoptotic machinery. In particular, partial defects in the caspase-dependent pathway in NSCLC cells conferred resistance to apoptosis induced by DNA-damaging agents such as etoposide or 3-radiation (9,14). Hence, a major therapeutic avenue in combating the aggressiveness and chemotherapy resistance of NSCLC is through effective induction of apoptosis by the alternative caspase-independent cell death pathway. The goal of this project is to address the critical barrier to NSCLC treatment by obtaining the information needed to understand the role of the novel caspase- independent anoikis effector, Bcl2-inhibitor of transcription (Bit1), in the apoptosis resistance and malignant transformation of NSCLC cells. Several observations indicate the nonfunctionality of the Bit1 pathway in lung cancer: i) Bit1 expression is significantly suppressed in various types of NSCLC tissue relative to counterpart normal lung tissue (Fig. 1A and 1B) and ii) an inhibitor of Bit1 function, TLE1, functions as a lung specific oncogene and is overexpressed in human lung tumors (1). While ectopic expression of cytoplasmic localized Bit1 in the NSCLC cell line NCI-H460 induces apoptosis (Fig 1D-F), restoration of mitochondrial localized Bit1 in H460 cells enhances anoikis sensitivity and attenuates anchorage independent growth and resistance to etoposide-induced apoptosis (Fig. 2). Based on the published data and our preliminary results, our objective is to test the hypothesis that malignant NSCLC cells are likely to bypass the Bit1 apoptotic pathway to become anoikis resistant and anchorage-independent, and that activation of the Bit1 pathway may be effective in inducing apoptosis and attenuating the aggressive phenotypes in NSCLC cells. The specific aims are: 1) to examine the regulation and relevance of the Bit1 apoptosis pathway in the survival and apoptotic resistance of NSCLC cells, 2) to test the hypothesis that the Bit1 apoptotic pathway regulates the aggressive phenotypes of NSCLC cells by alteration of mitochondrial Bit1 expression and its impact on anoikis resistance, anchorage- independent growth potential, or chemoresistance of NSCLC cells, and 3) to understand and dissect the apoptotic machinery in NSCLC using Bit1 as a tool. The outcome of this study will delineate the importance of the suppression or blockage of the Bit1 apoptotic pathway in the acquisition of anoikis resistance and anchorage-independence by the NSCLC. We believe that activation of the caspase-independent cell death pathway via the Bit1 apoptotic function will result in a successful eradication of chemoresistance in NSCLC. Thus, Bit-1 may serve as a novel therapeutic target for the treatment of this aggressive cancer. PHS398 (Rev. 5/01) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: A Role of Bit1 in the Apoptosis Resistance, Anoikis Insensitivity, and Chemoresistance in Non-small cell lung cancer (NSCLC) Cells Non-small cell lung cancer (NSCLC) is a highly aggressive disease with poor prognosis and high mortality. The dismal prognosis is in part due to its resistance to conventional chemotherapy and its propensity to disseminate to distant organs. To circumvent the aggressiveness and chemoresistance of NSCLC, it is imperative to identify novel cell death pathway(s) that may enhance the apoptotic sensitivity of NSCLC cells.