Dissecting the metastasis suppressor complex to identify colon cancer biomarkers

描述（由申请人提供）：散发性结肠癌的杂合性缺失（LOH）分析和后续研究使我们证明SMAD4是主要的靶肿瘤抑制因子，定位于与晚期疾病相关的染色体18q21的最小缺失区域。其他人随后的研究证实了这些最初的观察结果，并确定SMAD4突变/缺失的频率随着癌症从腺瘤发展到转移性疾病而增加。总的来说，这些发现与转移性结肠癌中Smad4功能的丧失是一致的。为了描述SMAD4缺陷与转移性结肠癌之间的分子基础，我们已经开始使用适当的工程模型系统。我们的初步研究表明，SMAD4缺陷导致VEGF水平升高、MEK-Erk和p38-MAPK辅助通路过度激活、结肠癌细胞迁移增强（MMP9相应增加）、缺氧下GLUT1过表达、有氧糖酵解增加以及对5- fu介导的细胞凋亡的抵抗。我们还发现Smad4在Smad4缺失的模型结肠癌细胞中过表达抑制VEGF报告因子活性，Smad4与特异性转录因子（tf）如HIF1？潜在地调节结肠癌的转移进展。虽然分子特征与Smad4可能在形成由各种转录因子和辅助因子组成的结肠癌转移抑制复合体中发挥核心作用以共同阻断结肠癌转移的观点一致，但直接靶向Smad4功能的丧失只能解释大约30%在18q21处携带LOH的肿瘤。因此，保留完整Smad4的转移性结肠癌也可能通过抑制转移程序的复合物的其他组分失活而进展。在本提案中，我们将验证保留Smad4的转移性结肠癌在结肠癌转移抑制复合体的其他成分中表现出改变的假设。因此，我们预测，通过揭示结肠癌转移抑制复合体的组成，人们将发现新的预后生物标志物，这些生物标志物可作为转移性结肠癌失活的替代靶向。在这里，我们概述了一种使用模型细胞系分离和表征由Smad4组成的结肠癌转移抑制复合物组分的策略：(1)利用蛋白质组学分析确定TFs和辅助因子作为候选预后生物标志物，研究在完整和缺陷的Smad4信号条件下组装和溶解的高阶蛋白质复合物的性质；(2)检查转移抑制复合体的这些其他因素的失调是否会破坏其功能；(3)确定是否

DESCRIPTION (provided by applicant): Loss of heterozygosity (LOH) analysis and follow up studies of sporadic colon cancer enabled us to show that SMAD4 is the primary target tumor suppressor, localized to the minimally lost region at chromosome 18q21-linked to an advanced stage disease. Subsequent studies by others have confirmed these initial observations and have established that the frequency of SMAD4 mutations/deletions increases as the cancer progresses from adenomas to the metastatic disease. Overall, these findings are consistent with loss of Smad4 function in metastatic colon cancer. In an effort to delineate a molecular basis for an association between SMAD4 deficiency and metastatic colon cancer, we have begun to use appropriately engineered model systems. Our preliminary studies showed that the SMAD4 defect was responsible for an increase in the levels of VEGF, overactivation of MEK-Erk and p38-MAPK auxiliary pathways, enhanced migration of colon cancer cells with a corresponding increase in MMP9, overexpression of GLUT1 under hypoxia, increased aerobic glycolysis and resistance to 5-FU-mediated apoptosis. We also found that overexpression of Smad4 in the model colon cancer cells with SMAD4 deficiency inhibited VEGF reporter activity and Smad4 physically interacts with specific transcription factors (TFs) such as HIF1? to potentially regulat metastatic progression of colon cancer. While the molecular characterizations are consistent with the notion that Smad4 may play a central role in forming a colon cancer metastasis suppressor complex consisting of various transcription factors and cofactors to collectively block colon cancer metastasis, direct targeting for loss of Smad4 function could only account for approximately 30% of tumors that harbor LOH at 18q21. Therefore, metastatic colon cancers that retained intact Smad4 could also progress through inactivation of the other components of the complex that suppresses the metastatic program. In this proposal, we will test the hypothesis that metastatic colon cancers which retain Smad4 exhibit alterations in the other components of the colon cancer metastasis suppressor complex. Thus, we predict that by unraveling the composition of the colon cancer metastasis suppressor complex, one would uncover novel prognostic biomarkers that are alternatively targeted for inactivation in metastatic colon cancer. Here, we outline a strategy using the model cell lines to isolate and characterize the components of the colon cancer metastasis suppressor complex consisting of Smad4 to (1) investigate the nature of the higher order protein complexes that are assembled and dissolved under the conditions of intact and defective Smad4 signaling using proteomic analysis to identify TFs and co-factors as candidate prognostic biomarkers; (2) examine if dysregulation of these other factors of the metastasis suppressor complex disrupts its functionality; and (3) determine if defects/deficiency in the alternate targets of Smad4 metastasis suppressor complex could serve as prognostic biomarkers for colon cancer. In summary, the proposed studies may unravel novel prognostic biomarkers for metastatic colon cancer and it could aid the development of personalized therapy.