STAT1 Activation and HPP1 Tumor Suppression

描述(申请人提供)：HPP1是一个最近发现的基因，最初是由于它在来自增生性息肉病患者的结直肠肿瘤中显著下调而被发现的。HPP1的失活是由启动子高甲基化介导的，随后在结肠和直肠、胃、小肠、胰腺、胆囊癌、前列腺癌和肺癌中发现了HPP1的失活。在胃肠道恶性肿瘤患者的血清和粪便中也可以检测到HPP1DNA的高甲基化。尽管人们对HPP1的S作为表观遗传靶点和肿瘤生物标志物的作用越来越感兴趣，但对其生物学作用机制的了解一直很有限。 PP1被预测编码一个带有EGF样结构域的跨膜蛋白，这表明PP1在erbB受体信号转导中发挥作用。我们已经证明，在结肠癌细胞系中过表达HPP1会导致增殖减少和细胞凋亡增加，并显著降低体外和体内的致瘤性。随后，我们发现这些表型改变与以STAT1和干扰素诱导基因显著上调为特征的基因表达谱有关。有趣的是，在结肠癌细胞系中，K-ras的激活突变与STAT1和干扰素调节元件的下调几乎是相反的。我们已经证明，STAT1的减弱可以消除HPP1的肿瘤抑制作用。 在这项赠款提案中将解决两个非常重要的机制问题：1)虽然HPP1和K-RAS对STAT1具有相反的作用，并且在HPP1启动子中存在c-myc抑制结合部位，但没有证据表明HPP1、c-myc和/或K-RAS的作用是机械联系的。因此，我们提出如下假设：K-ras通过依赖c-myc抑制HPP1下调STAT1。2)HPP1被认为是一种EGF样配体，在其胞外区含有EGF样结构域，具有His/Arg改变，可能与ErbB受体家族结合。然而，没有直接证据表明这个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设：HPP1通过EGF样域刺激erbB介导的STAT1激活来抑制肿瘤生长。这些假说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HPP1的EGF样结构域是否激活erbB家族受体，进而激活STAT1。3)确定K-ras相关的HPP1下调是否由c-myc介导。 与公共卫生的相关性：2008年，将诊断出约149,000例结直肠癌病例，并将导致近50,000人死亡。我们的目标是进一步鉴定HPP1的生物学功能，这是一种可能的肿瘤抑制基因，具有潜在的生物标志物作用，可在结直肠癌患者的血清和粪便中检测到。我们的发现可能有助于确定促进结直肠癌发生的新途径，此外，还可能鼓励进一步发展HPP1作为早期诊断和判断预后的生物标记物。

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.