Human Colon Cancer: Role of the Src Tyrosine Kinase

描述(由申请人提供)：我们有兴趣了解正常的肠道细胞如何调节它们的生长，以及这种调节的丧失如何导致恶变。我们的研究集中在Src酪氨酸激酶参与调控的分子机制上。我们和其他人发现，Src活性的下调对分化很重要，而上调对肠道细胞的恶性转化很重要。因此，致力于确定在肠道细胞成熟和恶变过程中调节Src活性的机制。我们最近发现RACK1是一种新的Src底物和结合伙伴，是Src活性和细胞生长的抑制因子。我们推测RACK1也是Src细胞转化的抑制因子。我们建议对此进行如下测试：目标1：我们将评估RACK1的S对Src转化细胞的影响。这些研究涉及分析NIH 3T3和SV40LT永生化的结肠上皮细胞，这些细胞表达v-Src或c-Src突变体以及野生型或突变型RACK1或抑制肽，以在软琼脂中锚定非定植生长、在低血清中生长、在正常细胞的单层上焦点形成以及在裸鼠体内形成肿瘤；完全转化的v-Src细胞具有而正常细胞缺乏的所有能力。目的2：我们将分析在人结肠癌细胞中激活Src的机制，以及激活的功能后果。这些研究包括确定Src和RACK1的突变、翻译后修饰、亚细胞定位和蛋白质-蛋白质相互作用，并评估它们对Src活性、细胞转化和肿瘤发生的影响。这些研究应该会产生关于一种新的src和细胞生长抑制物的重要新信息。了解有丝分裂信号抑制物如何调节肠道细胞生长，以及这种抑制作用的丧失如何导致不受控制的生长和恶变，应该会影响我们对结肠癌发生的理解，并导致结肠癌治疗新策略的开发。内源性或多肽的致癌激酶抑制剂是潜在的肿瘤抑制因子；它们代表了治疗干预的令人兴奋的新靶点。

DESCRIPTION (provided by applicant): We are interested in understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. Our research focuses on molecular mechanisms by which the Src tyrosine kinases contributes to the regulation. We and others showed that downregulation of Src activity is important for differentiation, and upregulation is important for malignant transformation of intestinal cells. Thus, effort is directed towards identifying mechanisms that modulate Src activity during intestinal cell maturation and malignant transformation. We recently identified RACK1 as a novel Src substrate and binding partner and an inhibitor of Src activity and cell growth. We hypothesize that RACK1 is also an inhibitor of cell transformation by Src. We propose to test this as follows: Aim 1: We will assess RACK1's influence on cell transformation by Src. These studies involve analyzing NIH 3T3 and SV40LT-immortalized colonic epithelial cells that express v-Src or c-Src mutants together with wild-type or mutant RACK1 or inhibitory peptides, for anchorage-independent growth in soft agar, growth in low serum, focus formation on monolayers of normal cells, and tumor formation in nude mice; all capabilities that fully transformed v-Src cells have and normal cells lack. Aim 2: We will analyze mechanisms by which Src is activated in human colon carcinoma cells, and the functional consequences of the activation. These studies involve identifying mutations, posttranslational modifications, subcellular localization and protein-protein interactions of Src and RACK1, and assessing their influence on Src activity, cell transformation and tumorigenesis. These studies should generate significant new information regarding a novel inhibitor of Src and cell growth. Understanding how inhibitors of mitogenic signals work to regulate intestinal cell growth and how loss of that inhibition results in uncontrolled growth and malignant transformation, should impact our understanding of colonic carcinogenesis and lead to development of novel strategies for colon cancer therapy. Endogenous or peptide inhibitors of oncogenic kinases are potentially tumor suppressors; they represent exciting new targets for therapeutic intervention.