STAT1 Activation and HPP1 Tumor Suppression

摘要 HPP1是一种新近发现的基因，最初是由于其显著下调而被发现的 在来自增生性息肉病患者的大肠肿瘤中。使HPP1失活，这是 在启动子高甲基化的介导下，后来发现发生在结肠和结肠癌中 直肠、胃、小肠、胰腺、胆、前列腺和肺。高甲基化的HPP1 DNA可以 在胃肠道恶性肿瘤患者的血清和粪便中也可检测到。尽管不断增长 感兴趣的是HpP1的S作为表观遗传靶点和肿瘤生物标记物的作用，其生物学知识 行动机制一直受到限制。 HPP1被预测编码一个带有EGF样结构域的跨膜蛋白，这表明它在 ErbB受体信号。我们已经证明了在结肠癌细胞系中过表达HPP1的结果 在减少增殖和增加凋亡，以及在体外和体内的显著减少 致瘤性。随后，我们发现这些表型变化与一种基因有关 表达谱以STAT1和干扰素诱导基因的显著上调为特征。 有趣的是，在结肠癌细胞系中激活K-ras突变与几乎完全相同的 与STAT1和干扰素调节元件下调的情况相反。我们已经证明了 STAT1的减弱取消了HPP1的肿瘤抑制作用。 这项赠款提案将涉及两个非常重要的机制问题：1)虽然HPP1和K- RAS对STAT1有相反的作用，在HPP1启动子中存在c-myc抑制结合部位， 没有证据表明HPP1、c-myc和/或K-RAS的作用有机械联系。因此，我们 提出以下假设：K-ras通过依赖c-myc抑制HPP1下调STAT1。2) HPP1被认为是EGF样配体，在其胞外结构域中含有EGF样结构域， 具有可能与ErbB受体家族结合的His/Arg改变。然而，没有直接证据表明 该EGF样结构域对HPP1介导的STAT1激活和肿瘤抑制至关重要。因此，我们将 检验第二个假说：HPP1通过EGF样结构域刺激erb B抑制肿瘤生长 STAT1激活。这些假设将通过以下具体目标进行检验：1)确定 STAT1介导HPP1相关肿瘤抑制的机制。2)确定EGF样蛋白是否 HPP1结构域可激活erB家族受体，进而激活STAT1。3)确定K-ras是否- 相关的HPP1下调是由c-myc介导的。

SUMMARY HPP1 is a recently identified gene that was initially discovered by virtue of its significant downregulation in colorectal neoplasms derived from patients with Hyperplastic Polyposis. Inactivation of HPP1, which is mediated by promoter hypermethylation, has subsequently been found to occur in tumors of the colon and rectum, stomach, small bowel, pancreas, gallbladder, prostate and lung. Hypermethylated HPP1 DNA can also be detected in both the serum and stools of patients with gastrointestinal malignancies. Despite growing interest in HPP1's role as an epigenetic target and as a tumor biomarker, knowledge of its biologic mechanisms of action has been limited. HPP1 is predicted to encode a transmembrane protein with an EGF-like domain, suggesting a role in erbB receptor signaling. We have demonstrated that overexpression of HPP1 in a colon cancer cell line results in reduced proliferation and increased apoptosis, as well as a marked reduction in in-vitro and in-vivo tumorigenicity. Subsequently, we have found that these phenotypic alterations are associated with a gene expression profile characterized by a dramatic upregulation of STAT1 and interferon-inducible genes. Interestingly, activating mutations of K-ras in colon cancer cell lines are associated with nearly an exact opposite profile with downregulation of STAT1 and interferon-regulatory elements. We have shown that attenuation of STAT1 abrogates the tumor suppressive effects of HPP1. Two very important mechanistic issues will be addressed in this grant proposal: 1) While HPP1 and K- ras have opposite effects on STAT1, and a c-myc repressive binding site is present in the HPP1 promoter, there is no evidence that the actions of HPP1, c-myc and/or K-Ras are mechanistically linked. Therefore, we propose the following hypothesis: K-ras downregulates STAT1 by a c-myc dependent suppression of HPP1. 2) HPP1 is postulated to be an EGF-like ligand and contains an EGF-like domain in its extracellular domain that has a His/Arg alteration that may bind to the ErbB receptor family. However, there is no direct evidence that this EGF-like domain is critical to HPP1-mediated STAT1 activation and tumor suppression. Therefore, we will test a second hypothesis: HPP1 suppresses tumor growth by EGF-like domain stimulation of erbB-mediated STAT1 activation. These hypotheses will be tested by the following specific aims: 1) To determine the mechanisms by which STAT1 mediates HPP1-associated tumor suppression. 2) To determine if the EGF-like domain of HPP1 activates erbB family receptors and subsequently, STAT1. 3) To determine whether K-ras- associated downregulation of HPP1 is mediated by c-myc.