Pathogenic role of human papillomavirus (HPV) DNA Integration and clonal expansion in cervical tumorigenesis

人乳头瘤病毒 (HPV) DNA 整合和克隆扩增在宫颈肿瘤发生中的致病作用

基本信息

批准号：
10507371
负责人：
Anne Van Arsdale
金额：
$ 26.39万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-09 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10507371
关键词：
Award Bar Codes Biological Assay CRISPR-mediated transcriptional activation CRISPR/Cas technology Cancer Etiology Carcinoma Cell Culture Techniques Cell Cycle Cells Cervical Cervical Intraepithelial Neoplasia Cervical dysplasia Cervix Neoplasms Cervix carcinoma Circular DNA Clinical Clonal Evolution Clonal Expansion Clustered Regularly Interspaced Short Palindromic Repeats Collection Color Contact Inhibition Custom DNA DNA Integration DNA Structure DNA sequencing Development Disease Dysplasia Episome Epithelial Event Fluorescent in Situ Hybridization Funding Genes Genome Genomic DNA Genomic Instability Genomics Goals Growth Guide RNA HPV-High Risk Histologic Human Human Engineering Human Genome Human Papillomavirus Human papilloma virus 45 Human papilloma virus infection Human papillomavirus 16 Human papillomavirus 18 Individual Infection Interphase K-Series Research Career Programs Knowledge Label Laboratories Laboratory Research Lentivirus Lesion Location Malignant Neoplasms Malignant neoplasm of cervix uteri Maps Mentors Methodology Methods Modeling Molecular Molecular Structure Normal Cell Nucleotides Oncogenes Oncogenic Outcome PVT1 gene Pathogenicity Patients Phase Phenotype Plasmids Population Positioning Attribute Property Recombinants Recurrence Research Research Design Research Personnel Resolution Role Sampling Science Scientist Sequencing By Hybridizations Site System Technology Testing Time Training Transcriptional Activation Transfection Viral Viral Genome Viral Oncogene Woman base career ds-DNA genetic testing human DNA insight integration site interest keratinocyte laboratory experience migration next generation sequencing novel premalignant programs promoter skills spatiotemporal tool transcriptomics tumor tumorigenesis tumorigenic viral DNA

项目摘要

Summary/Abstract Infection with high-risk Human Papillomavirus (hrHPV) is a necessary, key event in cervical carcinoma. A major advance in understanding cervical cancer was the recognition that HPV DNA is integrated into the human genome in almost all advanced cervical tumors. Human genome integration of HPV DNA 1) stably associates the viral oncogenes with a host cell, 2) potentially drives expression of host oncogenes that flank the sites of HPV DNA insertions, and 3) also causes human genome rearrangements. HPV integration is thought to occur randomly in the human genome, but integrated HPV DNA in tumors is often inserted at one of dozens of common genes in independent tumors, which suggests a clonal proliferation and survival advantage of cells containing insertions at these positions. During the initial phase of my research laboratory training, I developed an extensive, nascent research program to 1) detect and map HPV-human DNA junctions at single nucleotide resolution (hybridization capture + next generation sequencing, HC+NGS), 2) thoroughly characterize integrated viral DNA structure, and 3) describe the transcriptomic landscape in both cervical dysplasia and carcinoma clinical samples. I also applied the HC+NGS assay to a recently developed cell culture model where HPV circular DNA episomes replicate in early passage, human primary keratinocytes. I found that HPV DNA integration occurs efficiently in this model, thus providing a potent system to study HPV DNA integration and its functional consequences. To my knowledge, this is the first system for performing this type of study. I detected HPV integrated into multiple sites in the human genome, some of which were detected multiple times indicating clonal expansion of cells containing them. I hypothesize that HPV DNA integration is a key molecular event that promotes clonal expansion of cells, and promotes the development of dysplasia and invasive cervical carcinoma. In this application, I seek to develop my laboratory skills by generating and using new tools to study the spatio-temporal consequences of HPV integration on clonal expansion in patient derived, progressive cervical dysplasia lesions using multi-color, interphase DNA FISH assays (Aim 1); assess tumorigenic properties of cultured keratinocytes (Aim 2) and finally to test the role of transcriptional activation of flanking human genes in the expanded clonal populations (AIM 3). I will utilize a novel barcoding system based on CRISPR activation of GFP in clones of interest combined with a custom recombinant HPV quasivirus approach to lineage trace expanded HPV DNA integrated clones. I will carry out the proposed research over a 5 year period as part of a comprehensive K08 Career Development Award. Along with my co-mentors, Dr. Cristina Montagna and Dr. Jack Lenz, we have developed a training plan focusing on laboratory science and methodology to achieve my career goal of becoming a fully independent and funded clinician scientist.

摘要/摘要高风险人乳头瘤病毒（HRHPV）感染是宫颈中必要的关键事件癌。理解宫颈癌的主要进步是认识到HPV DNA已整合几乎所有晚期宫颈肿瘤中的人类基因组。 HPV DNA的人类基因组整合1）稳定地将病毒性癌基因与宿主细胞相关联，2）可能驱动宿主癌基因的表达， HPV DNA插入的位点，3）也会引起人类基因组重排。 HPV集成是被认为是在人类基因组中随机发生的，但是肿瘤中综合的HPV DNA通常插入独立肿瘤中的数十个共同基因，这表明克隆增殖和生存优势在这些位置插入的细胞。在我的研究实验室培训的最初阶段，我开发了一个广泛的新生研究计划，以1）检测和映射单个单一的HPV-Human DNA连接核苷酸分辨率（杂交捕获 +下一代测序，HC + NGS），2）彻底表征整合病毒DNA结构，3）描述两个宫颈的转录组景观发育不良和癌临床样本。我还将HC+NGS分析应用于最近开发的细胞培养模型，HPV圆形DNA偶发在人类原发性角质形成细胞中复制。我发现HPV DNA积分在该模型中有效地发生，因此提供了一个有效的系统来研究HPV DNA整合及其功能后果。据我所知，这是执行此操作的第一个系统研究类型。我检测到集成到人类基因组中多个位点的HPV，其中一些被检测到多次表明包含它们的细胞的克隆扩展。我假设HPV DNA整合是一个促进细胞克隆扩张的关键分子事件，并促进发育不良的发展和侵入性宫颈癌。在此应用中，我试图通过生成和使用来发展我的实验室技能研究HPV整合对患者克隆扩张的时空后果的新工具使用多色，相间DNA鱼类测定法（AIM 1）的衍生，进行性宫颈发育不良病变；评估培养角质形成细胞的致瘤特性（AIM 2），最后测试转录激活的作用在扩展的克隆种群中的人类基因的侧翼（AIM 3）。我将利用一个新颖的条形码系统基于感兴趣克隆中GFP的CRISPR激活以及定制重组HPV准病毒谱系迹线扩展的HPV DNA积分克隆的方法。我将对 5年期限为综合K08职业发展奖的一部分。和我的副手博士克里斯蒂娜·蒙塔格纳（Cristina Montagna）和杰克·伦茨（Jack Lenz）博士，我们制定了一项培训计划，重点是实验室科学和实现我的职业目标的方法是成为一名完全独立且资助的临床医生。