Molecular Mechanisms of Gastrin Mediated Pathways in Gastric Cancer Cells

描述(由申请人提供)： 理论基础：胃癌是癌症死亡的主要原因，在全球癌症死亡率中排名第二。胃肠激素胃泌素(包括加工和未加工形式)被认为是胃肠道恶性肿瘤病理生理过程中的一个重要因素。对过度表达成熟的酰胺化形式的胃泌素的小鼠的研究表明，这会导致浸润性胃癌，这与幽门螺杆菌感染协同促进肿瘤的进展。胃泌素的这些影响构成了一个主要的临床问题，因为血清胃泌素水平的增加是使用质子泵抑制剂(PPI)或幽门螺杆菌感染的常见副作用。因此，胃泌素介导癌症进展的信号通路，尽管仍然知之甚少，但代表了抗击胃癌药物开发的一个重要靶点。本研究的主要目的是阐明胃泌素促进胃癌细胞肿瘤进展和转移的信号机制，并在小鼠胃癌模型中靶向这一信号轴。我们最近的研究表明：(I)酰胺化胃泌素(G17)可有效地诱导胃癌细胞的迁移和侵袭；(Ii)G17诱导的迁移与所有3条MAPK通路的激活有关，包括ERK、p38和JNK；(Iii)G17刺激导致MAP3K成员MLK3的激活，它是JNK的上游激活剂；(Iv)G17诱导的迁移在体外通过MLK3/JNK1轴进行；(V)G17通过释放某些因子激活c-jun下游的JNK；(Vi)G17处理的胃癌细胞获得的条件培养液显示G17增加了IL-10的释放。假设：基于这些初步数据，我们假设G17通过激活MLK3/JNK1信号途径诱导胃癌细胞的生长(增殖)、迁移和侵袭。具体目的：为了阐明G17诱导胃癌进展的信号机制，我们提出了以下三个具体目标：(1)确定G17诱导的迁移、侵袭、EMT和生长是否涉及MLK3/JNK1轴；(2)阐明G17激活MLK3/JNK1轴的机制和细胞因子在其中的中介作用；(3)确定高胃泌素血症在体内对胃癌进展的影响以及通过药物抑制或通过基因沉默抑制MLK3/JNK1通路的作用。使用程序：拟议的研究将使用广泛的技术方法，包括迁移、侵袭和细胞增殖分析、Western分析、DNA转染、荧光素酶和激酶分析、免疫沉淀、siRNA技术、异种皮下移植和尾静脉注射。潜在新发现的意义：高胃泌素血症促进肿瘤进展这一事实在PPI使用量增加和幽门螺杆菌感染的背景下提出了一个主要的临床问题。目前还没有关于MLK3/JNK轴参与促进胃癌生长和进展的信息。这些研究的成功完成将确定该轴是否促进胃泌素诱导的胃癌进展。这些研究的结果可以进一步促进对现有MLK/JNK途径抑制剂作为安全、非侵入性药物治疗胃和其他胃泌素依赖的胃肠道恶性肿瘤的潜在用途的详细调查。

DESCRIPTION (provided by applicant): Rationale: Gastric cancer is a leading cause of cancer death and ranks second in the overall cancer mortality worldwide. The GI (gastrointestinal) peptide hormone gastrin (both processed and unprocessed forms) is regarded as an important contributing factor towards the patho-physiology of GI malignancies. Studies with mice over-expressing the mature amidated form of gastrin were shown to produce invasive gastric carcinoma, which synergized with Helicobacter infection to promote carcinoma progression. These effects of gastrin pose a major clinical concern, since an increase in serum gastrin levels is a common side effect of the use of proton pump inhibitors (PPIs) or Helicobacter pylori infection. Thus the signaling pathways by which gastrin mediates cancer progression, though still poorly understood, represent an important target for the development of drugs to combat gastric cancer. The major focus of this proposal is to elucidate the signaling mechanism by which gastrin promotes tumor progression and metastasis in gastric cancer cells, and to target this signaling axis in mouse models of gastric cancer. Our recent studies indicated that: (i) treatment with amidated gastrin (G17) potently induces gastric cancer cell migration and invasion; (ii) G17-induced migration is associated with activation of all 3 MAPK pathways including ERK, p38 and JNK; (iii) G17 stimulation results in activation of the MAP3K member MLK3, which is an upstream activator of JNK, (iv) G17-induced migration operates via MLK3/JNK1 axis in vitro; (v) G17 activates the JNK downstream c-Jun via releasing some factor and (vi) conditioned media obtained from G17-treated gastric cancer cells showed increased IL-10 release by G17. Hypothesis: Based on these preliminary data we hypothesize that G17-induced gastric cancer cell growth (proliferation), migration and invasion operates through activation of MLK3/JNK1 signaling. Specific Objectives: To elucidate the signaling mechanism by which G17 induces gastric carcinoma progression, we propose the following 3 specific aims: (1) to determine whether G17-induced migration, invasion, EMT and growth involve the MLK3/JNK1 axis in vitro; (2) to elucidate the mechanism by which G17 activates MLK3/JNK1 axis and the role of cytokines in mediating this; (3) to determine the effect of hypergastrinemia on the progression of gastric cancer in vivo and the effect of inhibition of MLK3/JNK1 pathways, either by pharmacological inhibitors or via gene silencing on this progression. Procedures to be used: The proposed studies will be performed using a broad range of technical approaches, including migration, invasion and cell proliferation assays, Western analyses, DNA transfection, luciferase and kinase assays, immunoprecipitation, SiRNA technology, subcutaneous xenografts and tail vein injections. Significance of potential new findings: The fact that hypergastrinemia promotes tumor progression poses a major clinical concern in the setting of increased PPI use and H.pylori infection. No information is currently available regarding the involvement of MLK3/JNK axis in promoting gastric cancer growth and progression. The successful completion of these studies will determine whether this axis promotes gastrin-induced gastric carcinoma progression. The results from these studies can further promote the detailed investigation of the potential use of available MLK/JNK pathway inhibitors as safe, noninvasive drugs for the treatment of patients with gastric as well as other gastrin- dependent GI malignancies.