EFFICACY AND ANTITUMOR MECHANISMS OF EGFR ANTISENSE GENE THERAPY

我们已经证明，口腔鳞状细胞癌中EGFR的上调是由于激活的基因转录，而EGFR的下调导致口腔鳞癌细胞增殖减少，而不是正常细胞。我们实验室的进一步研究发现，以U6小核RNA启动子为基础构建的EGFR反义表达载体与DC-CHOL脂质体复合后，体内肿瘤生长受到抑制。这种抗肿瘤作用伴随着肿瘤中EGFR蛋白表达的减少和细胞凋亡的增加。初步结果表明，口腔鳞癌中的EGFR信号涉及Stat3α-β的结构性激活，反义寡核苷酸或显性负性突变体下调STAT3可抑制口腔鳞癌的增殖。我们的发现强调了这种自分泌途径的重要性，即在原发口腔鳞癌肿瘤中EGFR的蛋白表达水平是一个重要的和独立的生存率下降的预测因子。因此，我们建议验证这一假设，即口腔鳞状细胞癌生长控制的丧失是通过获得EGFR自分泌信号通路来调节的，该信号通路可以使用反义基因治疗方法来靶向。在具体目标1中，我们建议通过确定：a)EGFR表达水平与抗肿瘤活性之间的关系；以及b)最佳效果的剂量、程序和时间依赖的参数，来表征EGFR反义治疗在体外和小鼠异种移植模型中的效果。在特定目的2中，我们建议通过以下方法来确定EGFR反义基因治疗在体外和小鼠移植瘤模型中的抗肿瘤作用机制：a)表征治疗对特定STAT蛋白亚型表达和激活的影响；b)检测治疗对细胞凋亡的影响。在具体目标3中，我们建议在I期环境中确定口腔鳞癌患者的最大耐受量(MTD)和肿瘤内脂质体介导的EGFR反义基因治疗的生物学效应。

We have demonstrated that EGFR up-regulation in OSCC is due to activated gene transcription and that down-modulation of EGFR results in decreased proliferation of OSCC but not normal cells. Further investigation in our laboratory revealed inhibition of tumor growth in vivo following intratumoral inoculation of an EGFR antisense expression construct based on the U6 small nuclear RNA promoter in complexed with DC-chol liposomes. This anti-tumor effect was accompanied by decreased EGFR protein expression in the tumors and increased apoptosis. Preliminary results suggest that EGFR signaling in OSCC involves constitutive activation of Stat3alpha-beta and that down-modulation of Stat3 using antisense oligonucleotides or dominant negative mutants result in inhibition of OSCC proliferation. The importance of this autocrine pathway is underscore by our finding that protein expression levels of EGFR in the primary OSCC tumor is a significant and independent predictor of decreased survival. Therefore, we propose to test the hypothesis that the loss of growth control in OSCC is mediated through acquisition of an EGFR autocrine signaling pathway, which can be targeted using an antisense gene therapy approach. In specific aim 1 we propose to characterize the effects of EGFR antisense therapy in OSCC in vitro and in murine xenograft models by determining: a) the association between EGFR expression levels and anti-tumor activity; and b) the dose, schedule, and time-dependent parameters for optimal effect. In specific aim 2 we propose to determine the mechanism of the anti-tumor effects of EGFR antisense gene therapy in vitro and in murine xenograft models by: a) characterizing the impact of treatment on expression and activation of specific STAT protein isoforms; and b) examination the consequences of therapy on apoptosis. In specific aim 3 we propose to determine in the phase I setting, the maximally tolerated dose (MTD) and biologic effects of intratumoral liposome-mediated EGFR antisense gene therapy in OSCC patients.