Silencing CD44v6 Expression Prevents Intestinal Tumor Growth

描述（由申请人提供）：结肠癌是美国第二大常见的死亡和发病原因。尽管最近在结肠癌的治疗方面取得了进展，但目前还没有有效的化学预防治疗方法，而且对大量患者来说，这些可用策略的结果往往很差。本提案中描述的工作探索了一种新的化学预防方法，该方法针对阻止结肠癌发展的促癌性CD44v6蛋白表达。我们已经开发了一种新的纳米颗粒递送系统，可以选择性地将CD44v6shRNA定向到肠/结肠细胞并阻断腺瘤的形成。该方法得到了以下观察结果的支持：(1)与正常细胞相比，CD44v6在腺瘤细胞中过表达。(2) CD44v6是比总CD44特异性的肿瘤干细胞标志物，可与CD166或CD133联合用于分离肿瘤干细胞。(3)体内敲低CD44v6表达可减少Apc Min/+小鼠模型中腺瘤的大小和数量，使腺瘤自发生长。4)在分选的癌症干细胞中敲除CD44v6可以逆转它们的耐药性和在软琼脂中的生长。5)在腺瘤前Apc10.1细胞中敲低CD44v6可抑制HA与CD44v6结合的下游信号，以及HGF与CD44v6和c-Met的结合。这些观察结果表明，CD44v6是调节肠/结肠肿瘤细胞生长起始和进展的关键跨膜蛋白的假设：我们假设，通过纳米颗粒将CD44v6shRNA“早期”（在腺瘤发生之前）递送到断奶Apc Min/+小鼠中，阻断CD44v6的表达将有效防止腺瘤的形成。我们还假设CD44v6shRNA/纳米颗粒的组织特异性递送提供了一种新的组织靶向化学预防方法。我们有两个目的：目的1)利用断奶Apc Min/+小鼠模型确定CD44v6shRNA在肠腺瘤中的疗效和药代动力学。疗效将被定义为CD44v6shRNA对腺瘤大小/数量的影响。通过研究CD44v6shRNA和Cre质粒在靶肠腺瘤中的分布，并与正常肠/结肠组织进行比较，确定CD44v6shRNA的药代动力学。目的2)确定CD44v6shRNA对腺瘤组织中药效学标志物（CD44v6诱导的c-Met信号级联的组成部分）的影响；血清中腺瘤形成和进展的生物标志物（HGF、HA、PGE2和可溶性CD44v6）；肿瘤干细胞标志物（CD44v6、CD166、CD133和p- PTEN）在Apc Min/+小鼠早期肠腺瘤中的表达。拟议的研究将推进CD44v6shRNA作为化学预防剂改变癌症前驱病变行为的潜力。

DESCRIPTION (provided by applicant): Colon cancer is the second common cause of mortality and morbidity in the United States. Despite recent advances in the treatment of colon cancer, there are no effective chemopreventive treatments available at this time and the outcome tends to be poor in these available strategies for a high number of patients. The work described in this proposal explores a novel chemopreventive approach which targets pro-oncogenic CD44v6 protein expressions that block the development of colon cancer. We have developed a novel nanoparticle delivery system that selectively directs CD44v6shRNA to intestine/colon cells and blocks adenoma formation. This approach is supported by the following observations: (1) CD44v6 is overexpressed in adenoma cells compared to normal cells. (2) CD44v6 is a specific cancer stem cell marker than total CD44, and can be used in conjunction with CD166 or CD133 to isolate cancer stem cells. (3) Knocking down CD44v6 expression in vivo reduces the size and number of adenomas in the Apc Min/+ mouse model, in which adenomas grow spontaneously. 4) Knocking down CD44v6 in sorted cancer stem cells reverses their drug resistance and the growth in soft agar. 5) Knocking down CD44v6 in pre-adenoma Apc10.1 cells inhibits signaling downstream from the binding of HA to CD44v6, and the binding of HGF to CD44v6 and c-Met. These observations suggest the hypothesis that CD44v6 is a key transmembrane protein in the regulation of intestine/colon tumor cell growth initiation and progression: we postulate that blocking CD44v6 expression by CD44v6shRNA delivered "early" (before adenomas develop) via nanoparticles into weanling Apc Min/+ mice will effectively prevent adenoma formation. We also postulate that tissue-specific delivery of CD44v6shRNA/nanoparticles provide a novel tissue targeted chemopreventive approach. We have two aims: Aim 1) To determine the efficacy and pharmacokinetics of CD44v6shRNA in intestinal adenomas using a weanling Apc Min/+ mouse model. Efficacy will be defined as effects of CD44v6shRNA on adenoma size/number. Pharmacokinetics of CD44v6shRNA will be determined by studying the distribution of CD44v6shRNA as well as Cre plasmid in the targeted intestine adenomas in comparison with that of normal intestine/colon tissues. Aim 2) To determine the effect of CD44v6shRNA on: pharmacodynamic markers (components of the CD44v6- induced c-Met signaling cascade) in adenoma tissue; biomarkers of adenoma formation and progression (HGF, HA, PGE2 and soluble CD44v6) in serum; and cancer stem cell markers (CD44v6, CD166, CD133 and p- PTEN) in early intestinal adenomas in the Apc Min/+ mouse model. The proposed studies will advance the potential of CD44v6shRNA as a chemopreventive agent to alter the behavior of cancer precursor lesions.