Tumor gene expression in vitro and in vivo

我们的生长调节研究一直关注癌基因作为正常和肿瘤生长的正调节因子和肿瘤抑制基因作为负调节因子。目前的主要项目集中在肿瘤抑制基因DLC1的分子生物学，包括调节它的靶点和它调节的靶点。DLC1在广泛的肿瘤中经常失活，但其作用机制的许多方面仍不完全清楚。DLC1通过其Rho-GAP活性负调控Rho，并且还编码其他活性。一个主要的假设是，DLC1在癌症中经常失活，因为它编码一种生物学上重要的多功能蛋白质。为了支持这种可能性，我们先前已经确定DLC1与以下蛋白相互作用：1）张力蛋白，通过DLC1的N-末端区域，其先前没有鉴定出功能; 2）粘着斑激酶（FAK）和talin，通过共享的8个氨基酸基序;和3）小窝蛋白-1（CAV-1），通过DLC1的C-末端附近的StAR相关脂质转移（START）结构域。DLC1突变体分析表明，这些相互作用的结合位点有助于DLC1的生长抑制功能，但与突变体的结合减少不影响DLC1的体内Rho-GAP活性。这些研究验证了DLC1是一种多功能蛋白质的假设，其生物活性取决于其Rho-GAP活性及其结合多种信号分子的能力。我们还在TCGA中评估了12种癌症类型中DLC1和9种其他Rho-GAP中点突变的存在。结果表明，DLC1是突变最频繁的Rho-GAP，在评估的五种肿瘤类型中，5 - 8%的肿瘤具有DLC1错义突变。此外，在这五种肿瘤类型中的四种中，20 - 26%的肿瘤在10个Rho-GAP中的至少一个中含有错义突变。DLC1突变体的实验分析表明，7个突变体的病变位于Rho-GAP结构域是缺乏Rho-GAP活性和抑制细胞迁移和锚定非依赖性生长。DLC1中的点突变分散在整个基因的编码序列中，这意味着重要的生物学功能存在于整个蛋白质中。这些结果表明，除了基因缺失和表观遗传学改变之外，DLC1的点突变是其在癌症中功能降低的重要机制。我们的特点是如何翻译后修饰DLC1，特别是磷酸化，可能会影响其活动。我们首先确定了CDK5，一种细胞质激酶，其生理作用促进分化，作为DLC1的主要激活剂，其通过DLC1中4个丝氨酸的CDK5磷酸化发生。当这些位于DLC1的RH0-GAP结构域的N-末端的丝氨酸未被磷酸化时，N-末端结合至Rh0-GAP结构域，这将DLC1置于封闭的无活性构象。当丝氨酸被磷酸化时，它减少了N-末端与Rho-GAP结构域的相互作用，这使得DLC1处于开放的活性构象。AKT是另一种直接磷酸化DLC1的激酶，位于Rho-GAP结构域N-末端的3个丝氨酸上。AKT的作用拮抗CDK5的作用，因为AKT磷酸化通过将DLC1从开放的活性构型改变为封闭的非活性构型来减弱DLC1的Rho-GAP和肿瘤抑制活性。AKT位于受体酪氨酸激酶（RTK）的下游，我们的数据表明，用RTK配体刺激细胞后Rho-GTP的增加可归因于AKT的激活及其对DLC1的减弱。我们还鉴定了SRC家族激酶（SFKs）作为在两个酪氨酸上磷酸化DLC1的直接靶点，这减弱了DLC1的RHO-GAP和肿瘤抑制活性。一个酪氨酸位于RHO-GAP结构域，其被SFKs磷酸化降低DLC1的RHO-GAP功能。另一个酪氨酸位于结合张力蛋白所需的序列附近，其磷酸化降低张力蛋白结合。AKT和SFK的研究结果可能具有治疗潜力，因为与不表达DLC1的同基因肿瘤相比，用AKT和/或SFK抑制剂治疗肿瘤异种移植物对表达DLC1的肿瘤具有更大的抗肿瘤活性。来自AKT和/或SFK抑制的高抗肿瘤活性与AKT和/或SFK对DLC1的磷酸化的丧失以及DLC1的RHO-GAP和肿瘤抑制活性的再活化相关。

Our growth regulation research has been concerned with oncogenes as positive regulators and tumor suppressor genes as negative regulators of normal and neoplastic growth. The main current project is focused on the molecular biology of the tumor suppressor gene DLC1, including the targets that regulate it and the targets that it regulates. DLC1 is inactivated frequently in a wide range of tumors, but many aspects of its mechanism of action remain incompletely understood. DLC1 negatively regulates Rho, via its Rho-GAP activity, and also encodes other activities. A major hypothesis is that DLC1 is frequently inactivated in cancer because it encodes a biologically important multifunctional protein. In support of this possibility, we have previously determined that DLC1 interacts with: 1) the tensin proteins, via an N-terminal region of DLC1 for which no function had been previously identified; 2) focal adhesion kinase (FAK) and talin, via a shared 8 amino acid motif; and 3) caveolin-1 (CAV-1), via the StAR-related lipid transfer (START) domain near the C-terminus of DLC1. DLC1 mutant analysis indicated that the binding sites for each of these interactions contributed to the growth suppressor function of DLC1, but that reduced binding seen with the mutants did not affect in vivo Rho-GAP activity of DLC1. These studies validate the hypothesis that DLC1 is a multifunctional protein whose biological activity depends both on its Rho-GAP activity and its ability to bind a variety of signaling molecules. We have also evaluated 12 cancers type in TCGA for the presence of point mutations in DLC1 and 9 other Rho-GAPs. The results indicate that DLC1 is the Rho-GAP that is mutated most frequently, with 5-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated seven of nine mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Point mutations in DLC1 were scattered throughout the coding sequences of the gene, implying that important biological functions are present throughout the protein. These results indicate that point mutation in DLC1 is an important mechanism for its decreased function in cancer, in addition to gene deletion and epigenetic changes. We have characterized how post-translational modifications of DLC1, especially phosphorylations, may affect its activity. We first identified CDK5, a cytoplasmic kinase whose physiologic role promotes differentiation, as a major activator of DLC1, which occurs via CDK5 phosphorylation of 4 serines in DLC1. When these serines, which are located N-terminal to the RHO-GAP domain of DLC1, are not phosphorylated, the N-terminus binds to the Rho-GAP domain, which places DLC1 in a closed, inactive conformation. When the serines are phosphorylated, it decreases the interaction of the N-terminus with the Rho-GAP domain, which places DLC1 in an open, active conformation. AKT is another kinase that directly phosphorylates DLC1, on 3 serines located N-terminal to the Rho-GAP domain. The effects of AKT antagonize those of CDK5, in that the AKT phosphorylations attenuate the Rho-GAP and tumor suppressor activities of DLC1 by changing DLC1 from an open, active configuration to a closed, inactive configuration. AKT lies downstream from receptor tyrosine kinases (RTKs), and our data indicate that increased Rho-GTP following stimulation of cells with RTK ligands is attributable to the activation of AKT and its attenuation of DLC1. We also identified SRC family kinases (SFKs) as direct targets for phosphorylating DLC1 on two tyrosines, which attenuate the RHO-GAP and tumor suppressor activities of DLC1. One tyrosine is located in the RHO-GAP domain, and its phosphorylation by SFKs reduces the RHO-GAP function of DLC1. The other tyrosine is located near the sequence required for binding tensin, and its phosphorylation reduces tensin binding. The findings with AKT and SFKs may have therapeutic potential, as treatment of tumor xenografts with AKT and/or SFK inhibitors has much greater antitumor activity against tumors that express DLC1 compared with isogenic tumors that do not express DLC1. The high antitumor activity from AKT and/or SFK inhibition is associated with loss of phosphorylation of the DLC1 by AKT and/or SFK together with reactivation of the RHO-GAP and tumor suppressor activities of DLC1.