低氧条件下结肠癌细胞K-ras突变对HIF-1ɑ和下游Adrenomedullin基因的分子调控机制

Tumor cells with mutant K-ras survival in hypoxic condition was the major cause of chemotherapy-resistant and poor prognosis in patients with colon cancer. We have previously identified Adrenomedullin(ADM) as one of key downstream gene in colon cancer cells harboring mutant K-ras, and demonstrated that ADM was highly expressed in colon cancer tissues with mutational K-ras. Exogenous administration with ADM could promote tumor cell invasion.Therefore, we proposed that ADM is the key secrected protein which contributes to the growth and invasion of K-ras mutation cells. In this proposal, we aim to determine whether the induction of HIF-1ɑ and ADM by mutant K-ras was mediated through PI3K/Akt/mTOR or MEK/Erk pathways, and the expression of ADM in hypoxia was dependent upon HIF-1ɑ. We further clone and validate the hypoxia response elements in the promoter of ADM gene.The functional studies including tumor growth, invasion and angiogenesis after siliencing expression of ADM with shRNA plasmid were evaluated in vitro and in vivo. The relationship between ADM and VEGF was evaluated to understand the key mechanisms of anti-angiogenesis by ADM in hypoxic conditions. Summarily, we aim to demonstrate the key mechanisms of the regulation of HIF-1ɑ and ADM by mutant K-ras in hypoxia in colon cancer. We proposed that the molecular target with the secreted protein-ADM could be a potential therapeutic strategy of colon cancer with mutant K-ras.

K-ras突变和肿瘤低氧环境生长是结肠癌患者化疗耐受和预后差的重要因素。我们前期筛选到Adrenomedullin (ADM)是低氧下K-ras突变型结肠癌细胞的关键下游基因,.发现ADM在K-ras突变型结肠癌组织中高表达，并促进结肠癌细胞侵袭。因此，我们推测ADM是K-ras突变型结肠癌在低氧条件下生长和侵袭的关键分泌蛋白。本课题拟系统研究低氧下PI3K/Akt/mTOR和raf/MEK/Erk等K-ras下游信号通路对结肠癌细胞低氧诱导因子（HIF-1ɑ）和ADM的分子调控机制；明确ADM低氧表达是否依赖于HIF-1ɑ，并鉴定ADM低氧反应元件；体内外试验评价shRNA干扰ADM对K-ras突变型结肠癌生长，侵袭和肿瘤血管生成的影响和分子机理。研究将阐明结肠癌低氧环境下K-ras突变对HIF-1ɑ和ADM的分子调控机制，ADM"靶向"干预有望为K-ras突变型结肠癌治疗提供新途径。

KRAS基因影响结直肠癌的血管形成、转移和化疗耐药在低氧环境下这些恶性行为得到增强。为明确低氧环境下，突变型KRAS的功能，我们在KRAS分别突变和野生的DKs5和DKO3结肠癌细胞中进行microarray分析，筛选到ADM是低氧下DKs5中表达明显上调的基因之一。在低氧下KRAS野生的Caco-2 细胞中过表达突变型KRAS后，ADM的表达明显增加；而KRAS突变的HCT116, DLD1和SW480细胞中KRAS被干扰后，ADM的表达明显受到抑制。结肠癌细胞中ADM被干扰后，细胞侵袭明显受到抑制，ADM表达增加或外源性添加ADM后，细胞的侵袭明显增多。ADM被稳定敲低后建立的裸鼠皮下移植瘤生长明显受限，免疫组化检测发现血管形成明显被抑制，细胞凋亡明显增强。进一步在体外研究中发现，ADM可以调节结肠癌细胞的侵袭过程。56例结直肠癌的患者，KRAS突变的样本中ADM的表达显著增高。有淋巴结转移的结肠癌组织中，ADM, ADMR和CRLR 的表达明显增加，ADMR和CRLR表达被抑制后，结肠癌细胞的增殖、迁移、侵袭等均明显受限， 尤其是在ADMR和 CRLR共同干扰组。KRAS和ADM有望作为结肠癌的诊断标记物及治疗靶点，具有重要的理论指导意义和潜在应用价值。