Tumor gene expression in vitro and in vivo

我们的生长调控研究一直关注癌基因作为正常和肿瘤生长的正调控基因和抑癌基因的负调控基因。目前的主要项目集中在肿瘤抑制基因DLC1的分子生物学上，包括调节它的靶点和它调节的靶点。DLC1在多种肿瘤中经常失活，但其许多方面的作用机制尚不清楚。它通过其Rho-Gap活性负向调节Rho，但必须编码其他活动，因为其他Rho-Gap在癌症中尚不被激活。我们的一个主要假设是，DLC1在癌症中经常失活，因为它编码一种多功能蛋白质。为了支持这种可能性，我们先前已经确定DLC1通过DLC1的N末端区域与张力蛋白家族的成员相互作用；2)通过与粘着斑激酶(FAK)和talin的8个氨基酸基序相互作用，与Paxlin中与张力蛋白结合的蛋白质区域附近的LD基序同源；以及3)通过DLC1 C末端附近的星相关脂转移(START)结构域与小窝蛋白1(CAV-1)相互作用。对各种DLC1突变体的分析表明，每个相互作用的结合部位都有助于DLC1的生长抑制功能，但这种结合并不影响DLC1体内的Rho-Gap活性。这些研究证实了DLC1是一种多功能蛋白质的假设，其生物学活性既取决于其RhoGAP活性，也取决于其结合各种信号分子的能力。为了研究内源性DLC1在RhoGTP控制和细胞转化中的作用，我们用编码Cre重组酶的腺病毒破坏了基因工程的内源性DLC1等位基因，使DLC1在小鼠胚胎成纤维细胞(MEF)中失活。DLC1的失活导致RhoGTP的增加而没有细胞转化，这表明内源DLC1对Rho的调节是有限的，但DLC1的失活不足以驱动转化。然而，与继续表达内源性DLC1的对照MEF相比，额外传代的带有失活DLC1的细胞确实进展到细胞转化和锚定无关的生长。对导致进展表型的基因的评估导致了p15-Ink4b和p16-Ink4a表达的下调，以及CDK4和CDK6活性的上调。在公开的具有基因表达谱的人类肿瘤数据集中，这些基因与肺腺癌和结直肠癌中DLC1的低表达一起被确定为预后较差。因此，一些基因和生化活动与DLC1的失活协同作用，导致MEF的细胞转化，所鉴定的基因与DLC1低表达的人类肿瘤相关。此外，我们还发现CDK5是DLC1的主要激活剂，它是一种细胞质蛋白，具有促进分化的生理作用。这种激活是因为DLC1有4个丝氨酸被CDK5磷酸化。当这些位于DLC1的Rho-Gap结构域的N端的丝氨酸没有被磷酸化时，N端与Rho-Gap结构域结合，从而使DLC1处于闭合的、不活跃的构象。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，从而使DLC1处于开放的、活跃的构象中。在癌症中，CDK5表现为促癌因子，可能是因为它比DLC1等抗癌靶点刺激更多的促癌靶点。与这一假设一致，DLC1的下调极大地增加了癌细胞中CDK5的致癌活性。

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 it mechanism of action remain poorly understood. It negatively regulates Rho, via its Rho-GAP activity, but must encode other activities, as other Rho-GAPs are not known to be inactivated in cancer. One of our main hypotheses is that DLC1 is frequently inactivated in cancer because it encodes a multifunctional protein. In support of this possibility, we have previously determined that DLC1 interacts with: 1) members of the tensin gene family, via an N-terminal region of DLC1 for which no function had been previously identified; 2) with focal adhesion kinase (FAK) and with talin, via a shared 8 amino acid motif with homology to LD motifs in paxillin near the region of the protein that binds tensin; and 3) with caveolin-1 (CAV-1), via the StAR-related lipid transfer (START) domain near the C-terminus of DLC1. Analysis of various DLC1 mutants indicated that the binding sites for each of these interactions contributed to the growth suppressor function of DLC1, but that this binding 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 RhoGAP activity and its ability to bind a variety of signaling molecules. To examine the role of endogenous DLC1 in the control of RhoGTP and cell transformation, we inactivated DLC1 in mouse embryo fibroblasts (MEFs) by disrupting the genetically engineered endogenous floxed DLC1 alleles with an adenovirus encoding the Cre recombinase. Inactivation of DLC1 resulted in an increase in RhoGTP without cell transformation, indicating that endogenous DLC1 is limiting for the regulation of Rho, but that inactivation of DLC1 is not sufficient to drive transformation. However, additional passage of the cells with inactivated DLC1 did progress to cell transformation and anchorage-independent growth, in contrast to control MEFs that continued to express endogenous DLC1. An evaluation of genes that contributed to the progressed phenotype led to the identification of the down-regulation of p15-Ink4b and p16-Ink4a expression, and the up-regulation of CDK4 and CDK6 activity. In publicly available human tumor datasets with gene expression profiles, a poor prognosis was identified for these genes in conjunction with low DLC1 expression in lung adenocarcinoma and colorectal cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression. In addition, we have identified CDK5, which is a cytoplasmic protein whose physiologic role promotes differentiation, as a major activator of DLC1. This activation occurs because DLC1 has 4 serines that are phosphorylated by CDK5. 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. In cancer, CDK5 behaves as a pro-oncogenic factor, presumably because it stimulates more pro-oncogenic targets than anti-oncogenic targets, such as DLC1. Consistent with that hypothesis, down-regulation of DLC1 greatly increases the pro-oncogenic activity of CDK5 in cancer cells.