Quantitative fragmentomics allow affinity mapping of interactomes

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here we measured the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complement protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within realistic reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus (HPV) E6 oncoprotein deeply impacts the host cell proteome way beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Effect of modified neoadjuvant chemotherapy on levels of E6 oncoprotein in HPV-infected cervical tumor tissue

. Background. Human papillomavirus (HPV) has been established to be the etiological factor of cervical cancer (CC). HPV infection and CC progression involve the direct participation of the E6 oncoprotein. Aim. An analysis of the E6 oncoprotein levels in tissues of the tumor and its perifocal area in HPV-associated cervical squamous cell cancer as an objective indicator of the effect of treatment depending on preoperative chemotherapy. Material and methods. The study included clinical and laboratory data of 237 patients with high-risk HPV infection of the cervix. The patients were divided into 4 groups: two main groups (CC T2а2–2bN0–1M0) and two control groups. Patients in the main group 1 (n=84) received standard neoadjuvant chemotherapy (NACT), in the main group 2 (n=93) — modified NACT with prior plasmapheresis session and a parallel course of nonspecific immunotherapy with Allokin-alpha. Control group 1 (n=40) included patients with CC T1b2–2a1N0–1M0, surgical treatment; control group 2 (n=20) — HPV-positive patients without CC. Levels of E6 were measured in samples of the cervical tumor and perifocal tissues. Results. The lowest levels of the E6 oncoprotein were registered in the group of HPV-positive patients without CC. After modified NACT, E6 levels in tumor tissues remained 4.6 times higher than in intact tissues, and even so, these patients demonstrated minimal E6 levels compared to other CC patients. E6 in tumor tissues was significantly lower than in main group 1 (by 3.3 times) and 8 times lower than in control group 1. E6 levels in the perifocal tissues of patients in main group 2 were 1.9 times lower than in the corresponding tissues of patients in main group 1 and 2.2 times lower than in control group 1. Conclusions. Inclusion of plasmapheresis and inducers of endogenous interferonogenesis into neoadjuvant treatment for cervical cancer can be considered pathogenetically justified, since it affects the key unit of cervical carcinogenesis.

Human DLG1 and SCRIB Are Distinctly Regulated Independently of HPV-16 during the Progression of Oropharyngeal Squamous Cell Carcinomas: A Preliminary Analysis

The major causative agents of head and neck squamous cell carcinomas (HNSCCs) are either environmental factors, such as tobacco and alcohol consumption, or infection with oncogenic human papillomaviruses (HPVs). An important aspect of HPV-induced oncogenesis is the targeting by the E6 oncoprotein of PDZ domain-containing substrates for proteasomal destruction. Tumor suppressors DLG1 and SCRIB are two of the principal PDZ domain-containing E6 targets. Both have been shown to play critical roles in the regulation of cell growth and polarity and in maintaining the structural integrity of the epithelia. We investigated how modifications in the cellular localization and protein expression of DLG1 and SCRIB in HPV16-positive and HPV-negative histologic oropharyngeal squamous cell carcinomas (OPSCC) might reflect disease progression. HPV presence was determined by p16 staining and HPV genotyping. Whilst DLG1 expression levels did not differ markedly between HPV-negative and HPV16-positive OPSCCs, it appeared to be relocated from cell–cell contacts to the cytoplasm in most samples, regardless of HPV16 positivity. This indicates that alterations in DLG1 distribution could contribute to malignant progression in OPSCCs. Interestingly, SCRIB was also relocated from cell–cell contacts to the cytoplasm in the tumor samples in comparison with normal tissue, regardless of HPV16 status, but in addition there was an obvious reduction in SCRIB expression in higher grade tumors. Strikingly, loss of SCRIB was even more pronounced in HPV16-positive OPSCCs. These alterations in SCRIB levels may contribute to transformation and loss of tissue architecture in the process of carcinogenesis and could potentially serve as markers in the development of OPSCCs.

Development of models for cervical cancer screening: construction in a cross-sectional population and validation in two screening cohorts in China

Background Current methods for cervical cancer screening result in an increased number of referrals and unnecessary diagnostic procedures. This study aimed to develop and evaluate a more accurate model for cervical cancer screening. Methods Multiple predictors including age, cytology, high-risk human papillomavirus (hrHPV) DNA/mRNA, E6 oncoprotein, HPV genotyping, and p16/Ki-67 were used for model construction in a cross-sectional population including women with normal cervix (N = 1085), cervical intraepithelial neoplasia (CIN, N = 279), and cervical cancer (N = 551) to predict CIN2+ or CIN3+. A base model using age, cytology, and hrHPV was calculated, and extended versions with additional biomarkers were considered. External validations in two screening cohorts with 3-year follow-up were further conducted (NCohort-I = 3179, NCohort-II = 3082). Results The base model increased the area under the curve (AUC, 0.91, 95% confidence interval [CI] = 0.88–0.93) and reduced colposcopy referral rates (42.76%, 95% CI = 38.67–46.92) compared to hrHPV and cytology co-testing in the cross-sectional population (AUC 0.80, 95% CI = 0.79–0.82, referrals rates 61.62, 95% CI = 59.4–63.8) to predict CIN2+. The AUC further improved when HPV genotyping and/or E6 oncoprotein were included in the base model. External validation in two screening cohorts further demonstrated that our models had better clinical performances than routine screening methods, yielded AUCs of 0.92 (95% CI = 0.91–0.93) and 0.94 (95% CI = 0.91–0.97) to predict CIN2+ and referrals rates of 17.55% (95% CI = 16.24–18.92) and 7.40% (95% CI = 6.50–8.38) in screening cohort I and II, respectively. Similar results were observed for CIN3+ prediction. Conclusions Compared to routine screening methods, our model using current cervical screening indicators can improve the clinical performance and reduce referral rates.

In Silico Approaches: A Way to Unveil Novel Therapeutic Drugs for Cervical Cancer Management

Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.

Human Papillomavirus Type 16 E6 induces cell competition

High risk human papillomavirus (HPV) infections induce squamous epithelial tumors in which the virus replicates. Initially, the virus-infected epithelial cells are untransformed, but expand in both number and area at the expense of normal squamous epithelial cells. How this occurs is unknown, but is presumed to be due to viral oncogene expression. We have developed an in vitro assay in which colonies of post-confluent HPV16 expressing cells outcompete confluent surrounding normal keratinocytes for surface area. The enhanced cell competition induced by the complete HPV16 genome is conferred by E6 expression alone, and not by individual expression of E5 or E7. In traditional oncogene assays, E7 is a more potent oncogene than E6, but such assays do not include interaction with normal surrounding cells. These new results separate classic oncogenicity that is primarily conferred by E7, from cell competition that we show is primarily conferred by E6, and provides a new biological role for E6 oncoproteins from high risk human papillomaviruses. Importance High risk papillomavirus infections induce epithelial tumors, some of which evolve into malignancies. The development and maintenance of cancer is due to the virally encoded E6 and E7 oncoproteins. How a virally infected keratinocyte out-competes normal uninfected keratinocytes has been unknown. The present work shows that the enhanced competition of HPV16-infected cells is primarily due to the expression of the E6 oncoprotein and not the E7 or E5 oncoproteins. This work shows the importance of measuring oncoprotein traits in the context of cell competition with uninfected cells, and shows the potential of papillomavirus oncoproteins to be novel genetic probes for the analysis of cell competition

Prognosis of endometrial cancer depends on intracrine ratio of sex steroids.

e17564 Background: Local imbalance of sex steroids (SS) plays a leading role in the development of gynecologic tumors. The purpose of the study was to investigate the effect of changes in SS and E6 protein levels on the course of endometrial cancer (EC). Methods: 50 patients with EC T1-2N0M0 (histologically - endometrioid adenocarcinoma, AC), aged 53.4±3.2 years, were recruited. Levels of SS and E6 protein were determined by standard ELISA systems in tumor samples. The coefficient of estrogens to the amount of testosterone and progesterone – C = (E1+E2):(T+P4) was calculated. Intact endometrium (IE) obtained in surgical treatment for uterine fibroids was used as the intact tissue. Results: AC patients demonstrated elevated estrone (E1) levels, compared to IE: by 2.2 times in 88% and by 5.9 times in 12% cases (p < 0.05). Levels of estradiol (E2) were similar in AC and IE. Progesterone (P4) levels in 32 patients were 1.5 times lower than in IE, and testosterone (T) 1.5 times higher (p < 0.05). P4 in 18 patients was 3 times lower than in IE, and T – 1.4 times lower in 12 women and 2.2 times lower in 6 women (p < 0.05). The C coefficient increased in 32 patients by 1.3 times (p< 0.05), in 12 patients by 2.7 times, in 6 patients by 8.2 times (p < 0.05). E6 protein was found in tumor tissue of 18 patients with elevated C. 6 of 18 women with C = 24.54±2.4 and E6 = 420±32 ng/g of tissue developed recurrence during 6 months, and 12 patients with C = 8.15±1.2 and E6 = 28±2.1 ng/g of tissue developed recurrence in a period of 6 months to 1 year. Women with C = 4.04±0.39 without E6 oncoprotein in tumor tissue had a relapse-free period for more than 1 year. Conclusions: An analysis of C and E6 oncoprotein in tumor tissues allows identification of high-risk patients and a personalized approach to adequate treatment.

PPI Modulators of E6 as Potential Targeted Therapeutics for Cervical Cancer: Progress and Challenges in Targeting E6

Advanced cervical cancer is primarily managed using cytotoxic therapies, despite evidence of limited efficacy and known toxicity. There is a current lack of alternative therapeutics to treat the disease more effectively. As such, there have been more research endeavors to develop targeted therapies directed at oncogenic host cellular targets over the past 4 decades, but thus far, only marginal gains in survival have been realized. The E6 oncoprotein, a protein of human papillomavirus origin that functionally inactivates various cellular antitumor proteins through protein–protein interactions (PPIs), represents an alternative target and intriguing opportunity to identify novel and potentially effective therapies to treat cervical cancer. Published research has reported a number of peptide and small-molecule modulators targeting the PPIs of E6 in various cell-based models. However, the reported compounds have rarely been well characterized in animal or human subjects. This indicates that while notable progress has been made in targeting E6, more extensive research is needed to accelerate the optimization of leads. In this review, we summarize the current knowledge and understanding of specific E6 PPI inhibition, the progress and challenges being faced, and potential approaches that can be utilized to identify novel and potent PPI inhibitors for cervical cancer treatment.