ROLE OF CYP24 IN NON-SMALL CELL LUNG CANCER

描述（由申请人提供）：我们的研究已经确定了非小细胞肺癌（NSCLC）的一种新的生长调节途径，该途径涉及CYP24，该酶可降解内源性抗增殖剂1,25-二羟基维生素D3 （1,25(OH)2D3）。125 (OH)2D3在组织中的水平和生物活性通常是通过维持CYP27B1合成速率和CYP24降解速率之间的精确平衡来控制的。我们发现这种平衡在非小细胞肺癌中是失调的。具体来说，我们确定CYP24在人类非小细胞肺癌细胞和大多数人类肺肿瘤中过表达。我们假设CYP24过表达通过允许肿瘤细胞逃避局部产生的1,25(OH)2D3的抗增殖作用来促进肺癌的生长。因此，我们发现CYP24抑制揭示了CYP27B1+CYP24+ NSCLC细胞产生的1,25(OH)2D3，并允许足够数量的激素积累以抑制生长。我们的数据进一步表明，Ets-1和C/EBP蛋白增加了非小细胞肺癌细胞中CYP24的转录。这些转录因子的激活先前被认为与肺癌的致癌信号有关。我们的目的是确定CYP24异常表达对肺癌生长的影响以及CYP24过表达在肺癌中的机制。在Aim 1中，我们将通过降低1,25(OH)2D3的稳定性和抗肿瘤活性来验证CYP24过表达促进NSCLC生长的预测。我们将量化调节CYP24表达对非小细胞肺癌细胞系和肿瘤异种移植模型中1,25(OH)2D3分解代谢和抗肿瘤活性的影响。我们还将使用一种创新的离体实验来确定CYP24表达与人肺肿瘤中125 (OH)2D3分解代谢之间的关系。在Aim 2中，我们将测试CYP24过表达拮抗非小细胞肺癌中维生素D3自分泌生长调节的预测。我们将利用一种新的CYP24抑制剂来证明CYP24对CYP27B1+CYP24+ NSCLC细胞合成1,25(OH)2D3的影响，并证明局部产生的激素引发生物反应的能力。我们还将测试CYP24过表达和CYP27B1缺失是否与肺癌表型独立相关。在Aim 3中，我们将测试癌基因激活的Ets-1和C/EBP转录因子调节非小细胞肺癌中CYP24表达的预测。Ets-1和C/EBP调节对CYP24转录和活性的影响将被确定。Ets和C/EBP蛋白募集到内源性CYP24启动子（作为ras激活的功能）将通过染色质免疫沉淀进行评估。拟议的研究将确定NSCLC发病机制的新机制，其中癌基因激活的转录因子通过增加分解内源性生长抑制因子（1,25(OH)2D3）的蛋白质（CYP24）的表达来促进肿瘤的发生。一旦证实，这种模式的治疗意义是，抑制CYP24的药物可用于恢复维生素d3介导的非小细胞肺癌的生长控制。

DESCRIPTION (provided by applicant): Our studies have led to the identification of a novel growth regulatory pathway in non-small cell lung cancer (NSCLC) that involves CYP24, the enzyme that degrades the endogenous anti-proliferative agent, 1,25- dihydroxyvitamin D3 (1,25(OH)2D3). The level and biological activity of 1,25(OH)2D3 in tissues is normally controlled by maintaining a precise balance between the rates of its synthesis by CYP27B1 and degradation by CYP24. We discovered that this balance is dysregulated in NSCLC. Specifically, we determined that CYP24 is overexpressed in human NSCLC cells and a majority of human lung tumors. We hypothesize that CYP24 overexpression facilitates lung cancer growth by allowing neoplastic cells to escape the anti- proliferative effects of locally-produced 1,25(OH)2D3. Accordingly, we found that CYP24 inhibition uncovers 1,25(OH)2D3 production by CYP27B1+CYP24+ NSCLC cells and allows for accumulation of hormone in amounts sufficient for growth suppression. Our data further indicate that Ets-1 and C/EBP proteins increase CYP24 transcription in NSCLC cells. Activation of these transcription factors has previously been linked to oncogenic signaling in lung cancer. Our objective is to establish the effect of aberrant CYP24 expression on lung cancer growth and the mechanism for CYP24 overexpression in lung cancer. In Aim 1 we will test the prediction that CYP24 overexpression facilitates NSCLC growth by decreasing the stability and anti- tumor activity of 1,25(OH)2D3. We will quantify the effect of modulating CYP24 expression on 1,25(OH)2D3 catabolism and anti-tumor activity in NSCLC cell lines and tumor xenograft models. We will also use an innovative ex vivo assay to determine the association between CYP24 expression and 1,25(OH)2D3 catabolism in human lung tumors. In Aim 2 we will test the prediction that CYP24 overexpression antagonizes autocrine growth regulation by vitamin D3 in NSCLC. We will utilize a novel CYP24 inhibitor to demonstrate the effect of CYP24 on 1,25(OH)2D3 synthesis by CYP27B1+CYP24+ NSCLC cells and demonstrate the ability of locally- produced hormone to elicit biological responses. We will also test whether CYP24 overexpression and CYP27B1 loss are independently correlated with the lung cancer phenotype. In Aim 3 we will test the prediction that oncogene-activated Ets-1 and C/EBP transcription factors regulate CYP24 expression in NSCLC. The effect of Ets-1 and C/EBP modulation on CYP24 transcription and activity will be established. Recruitment of Ets and C/EBP proteins to the endogenous CYP24 promoter (as a function of ras activation) will be evaluated by chromatin immunoprecipitation. The proposed studies will identify a novel mechanism for the pathogenesis of NSCLC in which oncogene-activated transcription factors facilitate tumorigenesis by increasing the expression of a protein (CYP24) that catabolizes an endogenous growth suppressor (1,25(OH)2D3). The therapeutic implication of this paradigm, once confirmed, is that agents that inhibit CYP24 can be used to restore vitamin D3-mediated growth control in NSCLC.