STAT1 Activation and HPP1 Tumor Suppression

描述（由申请人提供）：HPP 1是最近鉴定的基因，最初是由于其在增生性息肉病患者的结直肠肿瘤中的显著下调而发现的。随后发现，由启动子超甲基化介导的HPP 1失活发生在结肠和直肠、胃、小肠、胰腺、胆囊、前列腺和肺的肿瘤中。在胃肠道恶性肿瘤患者的血清和粪便中也可以检测到高甲基化的HPP 1 DNA。尽管HPP 1作为表观遗传靶点和肿瘤生物标志物的作用越来越受到关注，但对其生物学作用机制的了解仍然有限。 PP 1被预测为编码具有EGF样结构域的跨膜蛋白，表明在erbB受体信号传导中的作用。我们已经证明，HPP 1在结肠癌细胞系中的过表达导致增殖减少和凋亡增加，以及体外和体内致瘤性的显著降低。随后，我们发现这些表型改变与STAT 1和干扰素诱导基因显著上调的基因表达谱相关。有趣的是，结肠癌细胞系中K-ras的激活突变与STAT 1和干扰素调控元件的下调几乎完全相反。我们已经表明，STAT 1的衰减消除了HPP 1的肿瘤抑制作用。 在该资助提案中将解决两个非常重要的机制问题：1）虽然HPP 1和K-Ras对STAT 1具有相反的作用，并且在HPP 1启动子中存在c-myc抑制性结合位点，但没有证据表明HPP 1、c-myc和/或K-Ras的作用在机制上相关联。因此，我们提出以下假设：K-ras下调STAT 1的c-myc依赖性抑制HPP 1。2)HPP 1被假定为EGF样配体，并且在其细胞外结构域中含有EGF样结构域，其具有可能与ErbB受体家族结合的His/Arg改变。然而，没有直接证据表明这种EGF样结构域对HPP 1介导的STAT 1激活和肿瘤抑制至关重要。因此，我们将测试第二个假设：HPP 1抑制肿瘤生长的EGF样结构域刺激erbB介导的STAT 1激活。这些假设将通过以下具体目的进行检验：1）确定STAT 1介导HPP 1相关肿瘤抑制的机制。2)确定HPP 1的EGF样结构域是否激活erbB家族受体，随后激活STAT 1。3)确定K-ras相关的HPP 1下调是否由c-myc介导。 公共卫生相关性：2008年，将诊断出约149，000例结肠直肠癌，并将导致近50，000例死亡。我们的目标是进一步表征HPP 1的生物学功能，HPP 1是一种可能的肿瘤抑制基因，具有作为生物标志物的潜力，可在结直肠癌患者的血清和粪便中检测到。我们的研究结果可能会导致识别有助于结直肠癌发展的新途径，此外，可能会鼓励HPP 1作为早期诊断和预后确定的生物标志物的进一步发展。

DESCRIPTION (provided by applicant): 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. PP1 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. PUBLIC HEALTH RELEVANCE: In 2008, approximately 149,000 cases of colorectal cancer will be diagnosed and will account for close to 50,000 deaths. Our goals are to further characterize the biologic function of HPP1, a possible tumor suppressor gene with potential as a biomarker that is detectable in the serum and stool of patients with colorectal cancer. Our findings may lead to the identification of novel pathways that contribute to colorectal cancer development and additionally, may encourage the further development of HPP1 as a biomarker for early diagnosis and determination of prognosis.