HPV E6 and NFX1-123 in differentiation, cell regulation, and cancer

描述(申请人提供)：人乳头瘤病毒(HPV)影响高达75%的成年人，根据其与癌症的关系被归类为高风险(HR)或低风险(LR)。虽然有两种针对两种(HR HPV16，18)或四种(HR HPV16，18和LRHPV6，11)HPV基因型的预防性疫苗，但美国的疫苗接种率和完成率很低。这使得许多女性和男性继续面临患HPV相关癌症的风险，其中一些癌症的发病率正在上升。HR HPV癌基因E6和E7驱动细胞永生化；HR E6特异性地与几种内源性蛋白配对，以失调上皮细胞。在我们自己的研究中，我们发现16E6与NFX1-123相互作用。这些蛋白质共同作用于转录后，增加端粒酶催化亚单位hTERT的表达。端粒酶激活是细胞永生化的关键，是癌症发生的关键步骤，在HPV相关的癌症中普遍存在。我们现在准备研究受16E6和NFX1-123协同作用调控的其他基因和细胞通路。在全基因组表达芯片和验证研究中，我们鉴定了几个分化基因和一个主要的分化调节因子Notch1，它是由NFX1-123和16E6上调的。有趣的是，这种分化途径基因的增加并没有导致细胞生长停滞。这些数据使我们在研究16E6和NFX1-123在病毒生命周期、细胞、癌症发生和进展中的联合作用方面处于有利地位。我们的具体目标是：(1)确定16E6和NFX1-123对基因的调控机制。我们已经确定hTERT的转录后基因调控是16E6和NFX1-123的一个新的关键角色。我们假设NFX1-123和16E6共同发挥作用，以hTERT调控为工作模型，对其他基因进行失调。(2)探讨HPV和NFX1-123对上皮细胞分化平衡和持续增殖的影响。我们发现带有16E6的NFX1-123细胞分化基因和Notch1的表达增加，并且这些细胞在培养中继续增殖。我们将定义NFX1-123、16E6和Notch1是如何调节上皮结构的，并假设分化和生长抑制通路是解偶联的，以允许细胞生长并支持高效和长期的HPV感染，从而随着时间的推移导致恶变。(3)确定NFX1-123的表达如何改变并驱动HPV相关癌症的发生和发展。我们发现，在宫颈癌细胞株和30%的患者肿瘤样本中，NFX1-123表达增加。因此，我们假设NFX1-123及其下游基因靶点的增加有利于肿瘤的进展。使用宫颈不典型增生模型和患者样本，我们将量化NFX1-123的变化，并确定在HPV相关癌症中需要增加NFX1-123的临界点。这些研究将扩大我们对HPV驱动的肿瘤发生的理解，并有助于确定HPV相关癌症的生物标志物和治疗靶点。

DESCRIPTION (provided by applicant): Human papillomavirus (HPV) affects up to 75% of adults and is categorized as high risk (HR) or low risk (LR) based on its association with cancer. Although there are two preventive vaccines against two (HR HPV 16, 18) or four (HR HPV 16, 18 and LR HPV 6, 11) HPV genotypes, immunizations in the USA have had poor uptake and completion. This leaves many women and men at continued risk of HPV-associated cancers, some of which are increasing in incidence. The HR HPV oncogenes E6 and E7 drive cellular immortalization; HR E6 specifically partners with several endogenous proteins to dysregulate epithelial cells. In our own studies, we found that 16E6 interacts with the NFX1-123. Together, these proteins act post-transcriptionally to increase the expression of hTERT, the catalytic subunit of telomerase. Telomerase activation is critical for cellular immortalization, is a key ste in cancer development, and is universally detected in HPV-associated cancers. We are now prepared to study other genes and cellular pathways regulated by the concerted actions of 16E6 and NFX1-123. In whole-genome expression microarray and validation studies, we identified several differentiation genes and a master differentiation regulator, Notch1, as upregulated by NFX1-123 and 16E6. Interestingly, this increase in differentiation pathway genes did not lead to cellular growth arrest. These data put us in a strong position to study the combined roles of 16E6 and NFX1-123 in the viral life cycle, the cell, cancer development and progression. Our specific aims are to: (1) Determine the mechanism of gene regulation by 16E6 and NFX1-123. We have identified post-transcriptional gene regulation of hTERT as a new and critical role for 16E6 and NFX1-123. We hypothesize that NFX1-123 and 16E6 function together to dysregulate other genes, with hTERT regulation as our working model. (2) Determine how HPV and NFX1-123 affect the balance of differentiation and continued cellular proliferation in epithelium. We found NFX1-123 with 16E6 increased expression of differentiation genes and Notch1, and these same cells continued to proliferate in culture. We will define how NFX1-123, 16E6, and Notch1 modulate epithelial architecture, and hypothesize the differentiation and growth arrest pathways are uncoupled to allow cellular growth and support a productive and long-lived HPV infection that leads to malignant changes over time. (3) Determine how NFX1-123 expression changes and drives HPV-associated cancer development and progression. We found increased NFX1-123 in cervical cancer cell lines and in 30% of patient tumor samples. Therefore, we hypothesize that increased NFX1-123, and its downstream gene targets, favors oncogenic progression. Using cervical dysplasia models and patient samples, we will quantify changes in NFX1-123 and identify critical points where increased NFX1-123 is needed in HPV-associated cancers. These studies will expand our understanding of HPV-driven oncogenesis and help to identify biomarkers and therapeutic targets for HPV-associated cancers