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DLC 1的分子生物学，包括调节它的靶点和它调节的靶点。DLC 1在广泛的肿瘤中经常失活，但其作用机制的许多方面仍知之甚少。它通过其Rho-GAP活性负调节Rho，但必须编码其他活性，因为其他Rho-GAP在癌症中不被灭活。我们的一个主要假设是，DLC 1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DLC 1与：1）张力蛋白基因家族的成员，通过DLC 1的N-末端区域，其功能先前未被鉴定; 2）与粘着斑激酶（FAK）和talin，通过与结合张力蛋白的蛋白质区域附近的桩蛋白中的LD基序同源的共享8个氨基酸基序;和3）与小窝蛋白-1（CAV-1），通过DLC 1的C-末端附近的StAR相关脂质转移（START）结构域。对各种DLC 1突变体的分析表明，这些相互作用中的每一个的结合位点都有助于DLC 1的生长抑制功能，但是这种结合并不影响DLC 1的体内Rho-GAP活性。这些研究验证了DLC 1是一种多功能蛋白质的假设，其生物活性取决于其RhoGAP活性及其结合多种信号分子的能力。为了研究内源性DLC 1在RhoGTP和细胞转化控制中的作用，我们通过用编码Cre重组酶的腺病毒破坏遗传工程化的内源性floxed DLC 1等位基因来灭活小鼠胚胎成纤维细胞（MEFs）中的DLC 1。DLC 1的失活导致RhoGTP增加而没有细胞转化，表明内源性DLC 1限制了Rho的调节，但DLC 1的失活不足以驱动转化。然而，与继续表达内源性DLC 1的对照MEF相比，具有失活DLC 1的细胞的额外传代确实进展到细胞转化和锚定非依赖性生长。对导致进展表型的基因的评估导致鉴定出p15-Ink 4 b和p16-Ink 4a表达的下调，以及CDK 4和CDK 6活性的上调。在具有基因表达谱的公开可用的人类肿瘤数据集中，在肺腺癌和结直肠癌中鉴定出这些基因与低DLC 1表达相关的不良预后。因此，几个基因和生物化学活性与DLC 1的失活协作以引起MEFs中的细胞转化，并且所鉴定的基因与DLC 1低表达的人类肿瘤相关。此外，我们已经确定了CDK 5，这是一种细胞质蛋白，其生理作用促进分化，作为DLC 1的主要激活剂。这种激活的发生是因为DLC 1有4个被CDK 5磷酸化的丝氨酸。当位于DLC 1的Rho-GAP结构域的N-末端的这些丝氨酸未被磷酸化时，N-末端与Rho-GAP结构域结合，这将DLC 1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DLC 1处于开放的活性构象。在癌症中，CDK 5表现为一种促癌因子，可能是因为它刺激的促癌靶点多于抗癌靶点，如DLC 1。与该假设一致，DLC 1的下调大大增加了癌细胞中CDK 5的促癌活性。

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.