RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues
基于 RNA 的高危型 HPV 基因分型及宫颈组织中高危型 HPV 转录活性的鉴定
- 作者列表："Heuvel, Corina N. A. M.","Loopik, Diede L.","Ebisch, Renée M. F.","Elmelik, Duaa","Andralojc, Karolina M.","Huynen, Martijn","Bulten, Johan","Bekkers, Ruud L. M.","Massuger, Leon F. A. G.","Melchers, Willem J. G.","Siebers, Albert G.","Leenders, William P. J.
Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues ( n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.
几乎所有的宫颈癌都是由一种高危型人乳头瘤病毒 (高危型 HPV) 持续感染引起的。高危型 HPV DNA 检测高度敏感，但不能区分活动性、生产性感染和休眠感染或仅仅沉积的病毒。针对这一缺点的解决方案可能是检测病毒癌基因的转录活性，而不是仅仅存在高危 HPVs。在这项研究中，新鲜冷冻的宫颈组织 (n = 22) 使用 line 探针检测进行高危型 HPV DNA 检测，使用单分子倒位探针进行靶向 RNA 新一代测序。应用靶向 RNA 测序进行 (1) 基于 RNA 的高危型 HPV 基因分型，给出特定 HPV 亚型的信息 (2) 区分 E2 、 E6 、和 E7 转录本和 (3) 发现可能的非 HPV 癌症生物标志物。使用计算生物学分析数据。靶向 RNA 测序能够对高危型 HPV 亚型进行可靠的基因分型，并能够定量检测 E2 、 E6 和 E7 病毒基因表达，从而区分宫颈病变和正常宫颈组织。此外，靶向 RNA 测序确定了高危型 HPV 以外可能的宫颈癌生物标志物。有趣的是，靶向 RNA 测序也提供了来自宫颈刮片样本的高质量转录谱，即使在干燥储存或保存 1 周后。这项概念验证研究表明，靶向 RNA 测序可用于高危 HPV 基因分型和同时检测高危 HPV 基因活性。基于宫颈擦伤的分子分析，未来的研究有必要调查靶向 RNA 测序用于宫颈病变发展风险评估的潜力。
METHODS::Altered aerobic glycolysis is an important feature of cancer cell energy metabolism, known as the Warburg effect. Cervical cancer is one of the most common causes of cancer death in females. However, the roles of aerobic glycolysis in the development of cervical cancer are still poorly defined. Here, we identified a transcription factor (TF), ETS-related gene (ERG), as a new regulator of cancer progression and the glycolysis process in cervical cancer. In this study, we found that ectopic expression of ERG enhanced the capacity of aerobic glycolysis and increased glucose uptake, lactate production, and ATP generation. ERG overexpression increased and ERG knockdown decreased the anchorage independent cell growth and cell invasion in cervical cancer cells. Mechanistically, we propose that ERG regulates the expression of hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1) in the glycolytic pathway by directly binding to their promoters. A gain-of-function study showed that the knockdown or overexpression of HK2 and PGK1 abolished the increased or decreased aerobic glycolysis and cervical cancer progression induced by stable ectopic expression or depletion of ERG, respectively. Taken together, our findings suggest that ERG plays a potential role in the progression of cervical cancer, and could serve as a novel biomarker and potential therapeutic target in cervical cancer.
METHODS:INTRODUCTION:Australia has recently implemented major changes in cervical cancer prevention policies including introduction of primary human papillomavirus (HPV) screening starting at age 25, and replacement of the quadrivalent HPV vaccine with the nonavalent vaccine in the national school-based program. We assessed the feasibility and utility of conducting HPV testing in residual clinical specimens submitted for routine Chlamydia trachomatis screening, as a means of tracking HPV vaccine program impact among young sexually active women. METHODS:De-identified residual specimens from women aged 16-24 years submitted for chlamydia testing were collected from three pathology laboratories in Victoria and New South Wales. Limited demographic information, and chlamydia test results were also collected. Patient identifiers were sent directly from the laboratories to the National HPV Vaccination Program Register, to obtain HPV vaccination histories. Samples underwent HPV genotyping using Seegene Anyplex II HPV 28 assay. RESULTS:Between April and July 2018, 362 residual samples were collected, the majority (60.2%) of which were cervical swabs. Demographic data and vaccination histories were received for 357 (98.6%) women (mean age 21.8, SD 2.0). Overall, 65.6% of women were fully vaccinated, 9.8% partially, and 24.7% unvaccinated. The majority (86.0%) resided in a major city, 35.9% were classified in the upper quintile of socioeconomic advantage and chlamydia positivity was 7.8%.The prevalence of quadrivalent vaccine-targeted types (HPV6/11/16/18) was 2.8% (1.5-5.1%) overall with no differences by vaccination status (p = 0.729). The prevalence of additional nonavalent vaccine-targeted types (HPV31/33/45/52/58) was 19.3% (15.6-23.8%). One or more oncogenic HPV types were detected in 46.8% (95% CI 41.6-52.0%) of women. CONCLUSIONS:HPV testing of residual chlamydia specimens provides a simple, feasible method for monitoring circulating genotypes. Applied on a larger scale this method can be utilised to obtain a timely assessment of nonavalent vaccine impact among young women not yet eligible for cervical screening.
METHODS::Human papillomavirus (HPV) type 58 is the third most commonly detected HPV type in cervical cancer among Eastern Asians. Our previous international epidemiological studies revealed that a HPV58E7 natural variant, T20I/G63S (designated as V1), was associated with a higher risk of cervical cancer. We recently showed that V1 possesses a greater ability to immortalise and transform primary cells, as well as degrading pRB more effectively than the prototype and other common variants. In this study, we performed a series of phenotypic and molecular assays using physiologically relevant in vitro and in vivo models to compare the oncogenicity of V1 with that of the prototype and other common natural variants. Through activation of AKT and K-Ras/ERK signalling pathways, V1 consistently showed greater oncogenicity compared with prototype and other variants, as demonstrated by increased cell proliferation, migration and invasion, as well as induction of larger tumours in athymic nude mice. This study complements our previous epidemiological and molecular observations pinpointing the higher oncogenicity of V1 compared with prototype and all other common variants. Since V1 is more commonly found in Eastern Asia, our report provides insight into the design of HPV-screening assays and selection of components for HPV vaccines in this region.IMPORTANCE Epidemiological studies have revealed that a wild type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of its greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of AKT and K-Ras/ERK signalling pathways, thereby, explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumours, all to a greater extent than prototype HPV58 and other common variants.