E2 Protein Levels Research Articles

Cationic Liposomes Carrying HPV16 E6-siRNA Inhibit the Proliferation, Migration, and Invasion of Cervical Cancer Cells.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5705.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

Sinomenine Hydrochloride Protects IgA Nephropathy Through Regulating Cell Growth and Apoptosis of T and B Lymphocytes.

Sinomenine hydrochloride (SH) is used to treat chronic inflammatory diseases such as rheumatoid arthritis and may also be efficacious against Immunoglobulin A nephropathy (IgAN). However, no trial has investigated the molecular mechanism of SH on IgAN. Therefore, this study aims to investigate the effect and mechanism of SH on IgAN. The pathological changes and IgA and C3 depositions in the kidney of an IgAN rat model were detected by periodic acid-Schiff (PAS) and direct immunofluorescence staining. After extracting T and B cells using immunomagnetic beads, we assessed their purity, cell cycle phase, and apoptosis stage through flow cytometry. Furthermore, we quantified cell cycle-related and apoptosis-associated proteins by Western blotting. SH reduced IgA and C3 depositions in stage 4 IgAN, thereby decreasing inflammatory cellular infiltration and mesangial injury in an IgAN model induced using heteroproteins. Furthermore, SH arrested the cell cycle of lymphocytes T and B from the spleen of IgAN rats. Regarding the mechanism, our results demonstrated that SH regulated the Cyclin D1 and Cyclin E1 protein levels for arresting the cell cycle and it also regulated Bax and Bcl-2 protein levels, thus increasing Cleaved caspase-3 protein levels in Jurkat T and Ramos B cells. SH exerts a dual regulation on the cell cycle and apoptosis of T and B cells by controlling cell cycle-related and apoptosis-associated proteins; it also reduces inflammatory cellular infiltration and mesangial proliferation. These are the major mechanisms of SH in IgAN.

#236 : Alleviating Effects of EGCG on Ovarian Hyperstimulation Syndrome and the Potential Modulating Mechanism

Background and Aims: Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. Increased capillary permeability is a hallmark of the pathophysiology of OHSS, vascular endothelial growth factor (VEGF) is an important mediator in OHSS, and studies have established a correlation between serum VEGF levels and the severity of the OHSS. (-)-epigallocatechin-3-gallate (EGCG), the most prevalent and biologically active polyphenolic catechin in green tea, has been reported to produce a number of beneficial effects on humans. Numerous studies have shown that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Methods: We investigated the role of EGCG in OHSS both in vitro and in vivo. Primary human granulosa-lutein (hGL) cells and a human granulosa-like tumor (KGN) cell line were cultured and treated for 24 hours with various concentrations of EGCG. In SD rats, an animal OHSS model was established by injecting pregnant mare serum gonadotropin (PMSG) and randomly assigning them to receive vehicle only or EGCG for three days. For comparison, all experiments were repeated 3-8 times. The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size and weight were measured after removal. Evans-blue was used to determine permeability. ELISA was used to measure serum estrogen and VEGF levels in rats. RNA and protein expressions of VEGF, VEGFR-2, TGF-[Formula: see text]1, and T[Formula: see text]R II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Results: Our research found that giving rats EGCG prevented the development of OHSS, as evidenced by histological examination, ovarian weight, and morphology. When compared to the OHSS group, the EGCG treatment group had significantly lower ovary weight (147.9 vs 206.5 mg) (Fig. 1). Furthermore, when compared to OHSS rats, EGCG-treated rats have lower ovarian VEGF expression as measured by IHC and RT-qPCR. VEGF and E2 protein levels in the serum of the EGCG treatment group are significantly lower. EGCG exerted inhibitory effects on cell growth only in high dose (50 uM) and longtime (48 h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-[Formula: see text] at the RNA and protein levels. Furthermore, EGCG inhibits TGF-[Formula: see text]1-induced VEGF production and secretion in KGN cells by suppressing TGF-[Formula: see text] expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway (Fig. 2). Conclusions: EGCG inhibited VEGF via the TGF-[Formula: see text]1 classical-SMAD pathway and the 67LR-mediated CREB pathway, reducing ovarian inflammation and slowing the progression of OHSS in a rat model.

Abstract B169: Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A

Abstract Background: Lunresertib (RP-6306) is a first-in-class membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) inhibitor that disrupts the G2/M checkpoint leading to premature mitosis and catastrophic DNA damage in cells harboring synthetic lethal genomic alterations. The safety and tolerability of lunresertib alone and in combination with camonsertib (RP-3500) is being investigated in the Phase 1 MYTHIC study in patients with advanced solid tumors with either CCNE1 amplifications (amp) or deleterious alterations in FBXW7 or PPP2R1A (NCT04855656). Here, we present a comprehensive retrospective biomarker analysis aimed at understanding concordance between local vs. central and tissue- vs. plasma-based next generation sequencing (NGS). The correlation between CCNE1 copy number assessment by NGS and fluorescence in situ hybridization (FISH), as well as the relationship between CCNE1 amp and Cyclin E1 protein levels, was investigated. Methods: Patients were enrolled based on results from sponsor-approved local tumor NGS and plasma circulating tumor DNA (ctDNA) tests. To ensure molecular eligibility criteria were met, local NGS reports were centrally reviewed and functionally annotated prior to enrollment. Tumor tissue and plasma samples collected at screening were retrospectively analyzed by SNiPDx™ and Tempus xF(+), respectively. Tissue samples were further evaluated by FISH and immunohistochemistry. Results: As of June 1, 2023, 145 patients were screened with uterine (23%), colorectal (CRC, 15%), ovarian (13%), and upper GI (6%) as the most frequent cancers. Ninety-four percent (137/145) of patients screened had local NGS results available (98% tissue, 2% plasma). CCNE1 amp (40.1%) were most frequently observed in ovarian, uterine, and upper GI malignancies. FBXW7 (39.4%) and PPP2R1A (16.0%) alterations were enriched in CRC and uterine cancer, respectively. TP53 alterations were frequent across all biomarker groups (76% CCNE1, 54% FBXW7, and 88% PPP2R1A). Central tissue NGS confirmed 94% (47/50) of the enrollment alterations. Central plasma ctDNA confirmed 83.0% (39/47) of FBXW7 and PPP2R1A mutations and had high positive predictive value (100%, 17/17) but low sensitivity (42.5%; 17/40) for CCNE1 amp. CCNE1 amp call and copy number estimation by tissue NGS and FISH were highly correlated (Spearman ρ=0.65, p=0.006). Seventy-five percent of tumors with CCNE1 amp had Cyclin E1 overexpression (H-score ≥100; mean=168; range [0, 300]). Conclusion: In this study, local NGS tests were an efficient and robust strategy for directing patients into a molecularly defined Phase 1 trial. Plasma ctDNA reliably detected alterations in all 3 genes, but caution should be taken when interpreting CCNE1 amplification negative results due to a high false negative rate. FISH and genomics are appropriate methods to estimate CCNE1 amp copy number. These retrospective analyses enable the testing of the synthetic lethal hypothesis of lunresertib and CCNE1, PPP2R1A and FBXW7, assuring reliable interpretation of the clinical data. Citation Format: Elia Aguado-Fraile, Sunantha Sethuraman, Adam Petrone, Emeline Bacque, Alison Schram, Stephanie Lheureux, Elizabeth K Lee, Maria Koehler, Ian M Silverman, Victoria Rimkunas, Timothy A Yap. Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B169.

P-631 Alleviating effects of EGCG on ovarian hyperstimulation syndrome and the potential modulating mechanism through the VEGF pathway

Abstract Study question What are the underlying mechanisms of EGCG to alleviate Ovary hyperstimulation syndrome (OHSS)? Summary answer EGCG inhibited VEGF via TGF-β1 classical-SMAD pathway and 67LR-mediated CREB pathway and could reduce the ovarian inflammatory effect and attenuated OHSS progress in rat model. What is known already Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. The pathophysiology of OHSS is characterized by increased capillary permeability, VEGF is an important mediator in OHSS, and serum VEGF levels have been shown to correlate with OHSS severity. (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active polyphenolic catechin in green tea and has been reported to have multiple effects in humans. Many research indicated that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Study design, size, duration We investigated the role of EGCG in OHSS in vitro and in vivo. The primary human granulosa-lutein(hGL) cells and human granulosa-like tumor (KGN) cell line were cultured and treated with different concentrations of EGCG for 24 hours. Animal OHSS model was established in SD rats by injection of pregnant mare serum gonadotropin (PMSG), and randomly assigned to receive vehicle only or EGCG for 3 days. All experiments were performed 3-8 times for comparisons. Participants/materials, setting, methods The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size was measured after removal, the permeability was determined by Evans-blue. The serum estrogen and VEGF level in rats were detected by ELISA. RNA and protein expressions of VEGF, VEGFR-2, TGF-β, and TβR II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Main results and the role of chance Our study demonstrated that administration of EGCG attenuated the development of OHSS in rats, as shown by histological examination and ovarian weight and morphology. The ovary weight was significantly decreased in the EGCG treatment group compared with the OHSS group (147.9 vs 206.5 mg). Additionally, compared to OHSS rats, EGCG treated rats exhibit downregulated ovarian VEGF expression determined by IHC and RT-qPCR. VEGF and E2 protein levels significantly decrease in the serum of the EGCG treatment group. EGCG exerted inhibitory effects on cell growth only in high dose (50uM) and longtime (48h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-β at the RNA and protein levels. Furthermore, EGCG inhibits TGF-β1-induced VEGF production and secretion in KGN cells by suppressing TGF-β expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway. Limitations, reasons for caution EGCG has been reported to employ a wide range of biological effects. Therefore, its suppressive effects on VEGF and TGF-β signaling pathway might be part of the pharmacological mechanisms to alleviate Ovary hyperstimulation syndrome. Further studies are also needed to explore the therapeutic mechanisms of melatonin from other perspectives. Wider implications of the findings Our findings add mechanistic insight in support of using EGCG as an adjuvant therapy in the management of OHSS. EGCG shows the potential to act as a novel alternative therapeutic drug to treat OHSS. Daily intake of green tea might be beneficial for women under COH to prevent OHSS. Trial registration number Not applicable

Abstract 2132: Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression

Abstract Amplification of CCNE1 is a recurrent genetic alteration that has been associated with chemoresistance and poor prognosis in gynecological malignancies. We identified Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) as a novel synthetic lethal target in CCNE1 amplified cancers. In this genomic setting, PKMYT1 inhibition causes premature mitotic entry, mitotic catastrophe and, in turn, cell death. In pre-clinical models, correlation between CCNE1 copy number, cyclin E1 expression and sensitivity to PKMYT1 inhibition is imperfect. Thus, a deep understanding of CCNE1 amplification as a predictive biomarker for PKMYT1 inhibition is critical for refining patient selection strategies and strengthening our understanding of the mechanism of action. These learnings will be applied to correlative biomarker analyses in clinical trials evaluating RP-6306, a first-in-class, potent, and highly selective PKMYT1 inhibitor, in tumors with CCNE1 amplifications (NCT04855656, NCT05147272, NCT05147350). In this study, multiple features of CCNE1 were examined using a combination of whole genome and targeted next generation sequencing (NGS), FISH, and immunohistochemistry (IHC). Cyclin E1 levels were evaluated across tumor tissue microarrays spanning 5 tumor indications: triple-negative breast (n=100), colorectal (n=100), gastric (n=100), uterine (n=49), ovarian (n=100) cancers. The highest cyclin E1 protein levels were found in ovarian and uterine malignancies, consistent with indications with high prevalence of CCNE1 amplification (median H-score ovarian = 125; uterine = 100; gastric = 45; TNBC = 48 and CRC = 24). To explore CCNE1 amplification on a single cell level, we established a novel FISH assay using a subtelomeric q-arm control probe. The subtelomeric q-arm band is more frequently diploid than other regions on chromosome 19. Analytical validation of the FISH assay demonstrated sensitivity of 95%, specificity of 95.2%, and overall concordance of 95.1% when compared to a targeted sequencing panel (SNIPDx®). Within the uterine and ovarian cohorts, cancers harboring CCNE1 amplification were found to display higher cyclin E1 protein levels than non-amplified tumors (p-val=0.0002) and cyclin E1 expression was positively correlated with CCNE1 copy number (Spearman ρ=0.67; p-value=4.1e-05). Substantial intratumoral spatial heterogeneity of CCNE1 copy number when measured by FISH was observed across ovarian and uterine tumors. The potential impact of CCNE1 amplification heterogeneity, cyclin E1 protein levels and CCNE1 amplification fragment size on response to RP-6306 in preclinical models are currently being investigated and will be reported. Citation Format: Adam Petrone, Elia Aguado-Fraile, Sunantha Sethuraman, Adrienne Johnson, Ian Silverman, Gary Marshall, Jorge S. Reis-Filho, John Iafrate, Artur Veloso, Victoria Rimkunas. Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2132.

Effects of hypoxia on antigen presentation and T cell-based immune recognition of HPV16-transformed cells.

Attempts to develop a therapeutic vaccine against human papillomavirus (HPV)-induced malignancies have mostly not been clinically successful to date. One reason may be the hypoxic microenvironment present in most tumors, including cervical cancer. Hypoxia dysregulates the levels of human leukocyte antigen (HLA) class I molecules in different tumor entities, impacts the function of cytotoxic T cells, and leads to decreased protein levels of the oncoproteins E6 and E7 in HPV-transformed cells. Therefore, we investigated the effect of hypoxia on the presentation of HPV16 E6- and E7-derived epitopes in cervical cancer cells and its effect on epitope-specific T cell cytotoxicity. Hypoxia induced downregulation of E7 protein levels in all analyzed cell lines, as assessed by Western blotting. However, contrary to previous reports, no perturbation of antigen processing and presentation machinery (APM) components and HLA-A2 surface expression upon hypoxia treatment was detected by mass spectrometry and flow cytometry, respectively. Cytotoxicity assays performed in hypoxic conditions showed differential effects on the specific killing of HPV16-positive cervical cancer cells by epitope-specific CD8+ Tcell lines in a donor- and peptide-specific manner. Effects of hypoxia on the expression of PD-L1 were ruled out by flow cytometry analysis. Altogether, our results under hypoxia show a decreased expression of E6 and E7, but an intact APM, and epitope- and donor-dependent effects on T cell cytotoxicity towards HPV16-positive target cells. This suggests that successful immunotherapies can be developed for hypoxic HPV-induced cervical cancer, with careful choice of target epitopes, and ideally in combination with hypoxia-alleviating measures.

Pentagalloylglucose suppresses the growth and migration of human nasopharyngeal cancer cells via the GSK3β/β-catenin pathway in vitro and in vivo

BackgroundNasopharyngeal carcinoma (NPC) is a type of malignant squamous cell tumour originating from the nasopharynx epithelium. Pentagalloylglucose (PGG) is a natural polyphenolic compound that exerts anticancer effects in many types of tumours. However, the role and underlying mechanism of PGG in NPC cells have not been fully defined. PurposeThis study aimed to investigate the anticancer activity of PGG as well as the potential mechanism in NPC cells. MethodsThe effects of PGG on the proliferation, apoptosis and cell cycle distribution of CNE1 and CNE2 cells were assessed by MTT and flow cytometry assays. Cell migration was evaluated using wound healing and transwell assays. The expression of microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Western blotting was used to explore the levels of related proteins and signalling pathway components. Furthermore, the effects of PGG on NPC cell growth were analysed in a xenograft mouse model in vivo using cisplatin as a positive control. ResultsPGG dose-dependently inhibited the proliferation of CNE1 and CNE2 cells. PGG regulated the cell cycle by altering p53, cyclin D1, CDK2, and cyclin E1 protein levels. PGG induced apoptosis and autophagy in NPC cells and elevated the Bax/Bcl-2 ratio and the protein levels of LC3B. Moreover, PGG decreased NPC cell migration by increasing E-cadherin and decreasing N-cadherin, vimentin and CD44 protein levels. Mechanistically, PGG treatment downregulated p-mTOR and β-catenin expression but upregulated p-p38 MAPK and p-GSK3β expression. In addition, PGG significantly inhibited NPC cell tumour growth and lung metastasis in vivo. ConclusionPGG may suppress cell proliferation, induce apoptosis and autophagy, and decrease the metastatic capacity of NPC cells through the p38 MAPK/mTOR and Wnt/β-catenin pathways. The present study provides evidence for PGG as a potential therapy for NPC.

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.

Cyclic AMP-Dependent Protein Kinase Exhibits Antagonistic Effects on the Replication Efficiency of Different Human Papillomavirus Types.

Several types of widespread human papillomaviruses (HPVs) may induce the transformation of infected cells, provoking the development of neoplasms. Two main genera of HPVs are classified as mucosatropic alphapapillomaviruses and cutaneotropic betapapillomaviruses (α- and β-HPVs, respectively), and they both include high-risk cancer-associated species. The absence of antiviral drugs has driven investigations into the details of the molecular mechanisms of the HPV life cycle. HPV replication depends on the viral helicase E1 and the transcription factor E2. Their biological activities are controlled by numerous cellular proteins, including protein kinases. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of α-HPV11, α-HPV18, and β-HPV5. PKA stimulates the replication of both α-HPVs studied but has a more profound effect on the replication of high-risk α-HPV18. However, the replication of β-HPV5 is inhibited by activated PKA in human primary keratinocytes and U2OS cells. We show that the activation of PKA signaling by different pharmacological agents induces the rapid proteasomal degradation of the HPV5 E2 protein, which in turn leads to the downregulation of E2-dependent transcription. In contrast, PKA-stimulated induction of HPV18 replication is the result of the downregulation of the E8^E2 transcript encoding a potent viral transcriptional inhibitor together with the rapid upregulation of E1 and E2 protein levels. IMPORTANCE Several types of human papillomaviruses (HPVs) are causative agents of various types of epithelial cancers. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of various types of HPVs during the initial amplification and maintenance phases of the viral life cycle. The replication of the skin cancer-related pathogen HPV5 is suppressed, whereas the replication of the cervical cancer-associated pathogen HPV18 is activated, in response to elevated PKA activity. To inhibit HPV5 replication, PKA targets the viral transcriptional activator E2, inducing its rapid proteasomal degradation. PKA-dependent stimulation of HPV18 replication relies on the downregulation of another E2 gene product, E8^E2, which encodes a potent transcriptional repressor. Our findings highlight, for the first time, protein kinase-related mechanistic differences in the regulation of the replication of mucosal and cutaneous HPV types.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

Simple SummaryHigh-risk strains of human papillomavirus (HPV) such as HPV16 cause anogenital and oropharyngeal cancers, with HPV-associated cervical cancer being the fourth most common cancer worldwide and a major cause of death for women. Anti-HPV vaccines are available but coverage is low, and while the vaccines prevent infection they are not therapeutic for treating cancers in individuals already infected with high-risk HPV. Instead, current treatments for HPV-associated cancers include surgery, chemotherapy, and radiation, and affect diseased and healthy tissues alike. Accordingly, there is a dire need for better, molecularly targeted strategies. Herein, we report the novel finding that at least four protease inhibitor drugs currently used to treat HIV/AIDS—lopinavir, ritonavir, nelfinavir, and saquinavir—cause marked and selective depletion of HPV16 E6 and E7 oncoproteins in HPV-positive cancer cells and a 3-D cell culture model for HPV16-positive epithelial pre-cancer (NIKS16 cells). E6 and E7 depletion correlated with anticancer phenotypes including increased p53 protein levels and induction of cell growth arrest in CaSki cells as well as slowed epithelium growth and reversal of HPV16-driven phenotypes consistent with inhibition of the cellular autophagy pathway in the NIKS16 model. Based on this progress in vitro, we propose that HIV protease inhibitors, already known to have anticancer properties, be studied further in the context of developing a targeted therapy for HPV16-driven cancers and pre-cancers.High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16′s effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.

Abstract 578: Investigation of the effect of hypoxia on presentation of HPV16-derived antigens - implications for therapeutic vaccine design

Abstract Human papillomavirus type 16 (HPV16) is a causal factor of ~50% of cervical cancers, and of ~95% of extra-cervical (other anogenital and oropharyngeal) HPV-mediated cancers. The aim of our group is to develop a therapeutic vaccine against HPV16-induced malignancies. To find optimal vaccine antigens, it is important to assess the effect of the tumor microenvironment on viral antigen presentation. Hypoxia has been reported to regulate HLA class-I expression levels in the context of various tumors. Thus, the aim of the present study was to explore the effect of hypoxia on various components of the antigen processing machinery (APM) in HPV16-positive tumor cells and on the HPV16 E6- and E7-derived HLA class-I epitope repertoire. We are investigating the effect of hypoxia on cervical cancer cells transformed with HPV16 variant lineages (European, Asian-American and African) correlated with differences in oncogenic potential. This is achieved by comparing cells cultured under hypoxia (1.2% O2) or normoxia (21% O2). We investigate E6 protein levels by immunoblotting and HLA-A2 surface expression by flow cytometry. Subsequently, quantitative PCR and immunoblotting are used to assess whether components of the APM are affected by hypoxia. Cervical cancer cell lines transformed by sublineages of the HPV16 European variant (CaSki and SNU17) showed a decrease in E6 protein levels upon hypoxic treatment. HLA-A2 levels were increased in SNU17 cells (HPV16 European Asian prototype), but no change was observed in CaSki cells (European Prototype 2). First qPCR experiments of SNU17 cells showed changes in additional components of the APM at the mRNA level, which still have to be assessed at the protein level. Cell lines transformed by HPV16 variants other than European will be subjected to the same analysis. Potential changes in the HPV16 epitope repertoire will be investigated using a targeted mass spectrometry-based epitope detection strategy established in the lab. These investigations may yield novel insights into HPV16 immune evasion pathways. Furthermore, the identification of epitopes that are presented under both normoxic and hypoxic conditions will provide optimal candidates for therapeutic vaccine design against HPV-induced malignancies. Citation Format: Nitya Mohan, Maria Bonsack, Jonas Förster, Alina Steinbach, Renata Blatnik, Mogjiborahman Salek, Angelika B. Riemer. Investigation of the effect of hypoxia on presentation of HPV16-derived antigens - implications for therapeutic vaccine design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 578.

Abstract 1739: Diminished SKP1 or CUL1 expression induces chromosome instability in high-grade serous ovarian cancer precursor cells

Abstract High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype as well as the most lethal. High mortality rates associated with HGSOC are in part due to a lack of reliable early detection methods and effective therapies. Importantly, chromosome instability (CIN; an increased rate of chromosome gains or losses), is causally implicated in cancer development, progression, and resistance to treatment, and is just beginning to be evaluated in an HGSOC context. Overexpression of Cyclin E1 protein induces CIN and genomic amplification contributes to HGSOC pathogenesis in 20% of HGSOC patients. Cyclin E1 protein levels are normally regulated in a cell cycle-dependent manner by the SCF (SKP1-CUL1-FBOX) complex, an E3 ubiquitin ligase that targets substrates for proteolytic degradation. In cases where genomic amplification is absent, we hypothesize that loss of SCF complex function stemming from diminished expression of individual SCF complex components underlies increases in Cyclin E1 levels and induces CIN. The current study focuses on the SCF complex components SKP1 and CUL1 and characterizes their role in early HGSOC pathogenesis within a fallopian tube secretory epithelial cell model (FT; a cell of origin for HGSOC). Using siRNA-based approaches, we show that diminished SKP1 and CUL1 expression results in increased Cyclin E1 protein levels in FT cells. Using quantitative imaging microscopy techniques, we further identify statistically significant changes in CIN-associated phenotypes within the silenced populations, including changes in nuclear areas, increases in micronucleus formation (i.e. small DNA-containing bodies outside of the primary nucleus), and increases in numerical chromosome abnormalities identified in mitotic chromosome spreads. Thus, the current study explores the early origins of HGSOC and identifies SKP1 and CUL1 as two promising new molecular players that may contribute to CIN to drive FT cell transformation, drug resistance and disease recurrence. Citation Format: Chloe C. Lepage, Mark W. Nachtigal, Kirk J. McManus. Diminished SKP1 or CUL1 expression induces chromosome instability in high-grade serous ovarian cancer precursor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1739.

Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus

Single-stranded circular RNAs (circRNAs), generated through ‘backsplicing’, occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.

Tanshinone IIA inhibits proliferation and activates apoptosis in C4-1 cervical carcinoma cells in vitro

Tanshinone IIA (Tan IIA) is a natural product that has been identified to have anti-proliferative properties against cervical cancer. The study was designed to investigate the anti-tumor effects of Tan IIA at different concentrations on the proliferation and apoptosis of human cervical carcinoma C4-1 cells. Human cervical carcinoma C4-1 cells were treated with different doses of Tan IIA, and MTT assay was performed to determine effects on cell viability. Flow cytometry was used to detect apoptosis and Western blot analysis was carried out to determine alterations in the expression of key proteins. Tan IIA treatment (0.5, 1.0, 2.0, and 5.0 mg/mL) significantly inhibited the growth of C4-1 cells in a time- and dose-dependent manner. Significant differences were observed in the growth inhibition rate between Tan IIA group and the control group (0.5-1.0 mg/L Group: p < 0.05; 2.0–5.0 mg/L Group: p < 0.01). Flow cytometry showed that C4-1 cells became apoptotic after treatment with Tan IIA. In the control group, the apoptosis rate of C4-1 cells was 2.56 ± 0.21% whilst in the Tan IIA-treated groups, different concentrations markedly increased the rate of apoptosis (1.0 mg/L group, 7.9 ± 0.43% [p < 0.05]; 2.0 g/L group, 10.2 ± 0.42%; 5.0 mg/L, 20.44 ± 1.24% [p < 0.01]; except 0.5 mg/L group, 5.8 ± 0.32% [p > 0.05]). Western blot assays indicated that the Tan IIA decreased Bcl-2, HPV 16 E6 and E7 protein levels, but elevated Bax and cleaved-Caspase-3 expression in cells. Tan IIA may inhibit the proliferation and induce the apoptosis of C4-1 cells in a concentration-dependent manner.

Effects of palbociclib on oral squamous cell carcinoma and the role of PIK3CA in conferring resistance.

ObjectiveLack of effective therapies remains a problem in the treatment of oral squamous cell carcinoma (OSCC), especially in patients with advanced tumors. OSCC development is driven by multiple aberrancies within the cell cycle pathway, including amplification of cyclin D1 and loss of p16. Hence, cell cycle inhibitors of the CDK4/6-cyclin D axis are appealing targets for OSCC treatment. Here, we determined the potency of palbociclib and identified genetic features that are associated with the response of palbociclib in OSCC.MethodsThe effect of palbociclib was evaluated in a panel of well-characterized OSCC cell lines by cell proliferation assays and further confirmed by in vivo evaluation in xenograft models. PIK3CA-mutant isogenic cell lines were used to investigate the effect of PIK3CA mutation towards palbociclib response. ResultsWe demonstrated that 80% of OSCC cell lines are sensitive to palbociclib at sub-micromolar concentrations. Consistently, palbociclib was effective in controlling tumor growth in mice. We identified that palbociclib-resistant cells harbored mutations in PIK3CA. Using isogenic cell lines, we showed that PIK3CA mutant cells are less responsive to palbociclib as compared to wild-type cells with concurrent upregulation of CDK2 and cyclin E1 protein levels. We further demonstrated that the combination of a PI3K/mTOR inhibitor (PF-04691502) and palbociclib completely controlled tumor growth in mice. ConclusionsThis study demonstrated the potency of palbociclib in OSCC models and provides a rationale for the inclusion of PIK3CA testing in the clinical evaluation of CDK4/6 inhibitors and suggests combination approaches for further clinical studies.

Lactic acid induced microRNA-744 enhances motility of SiHa cervical cancer cells through targeting ARHGAP5

High-risk (hr) human papillomaviruses (HPV) infection and integration has caused the majority of cervical cancer, of which E6 and E7 oncogenes are invariably retained and expressed to immortalize cells probably via affecting cell migration and invasion, and tumor metastasis. However, the underlying mechanism that mediates the procedure such as motility of cervical cancer cells within the tumor microenvironment is not well understood. Herein, we examined one possible factor—extracellular lactic acid, an end up chemical in glycolytic tumor cells, on the motility in HPV16 positive SiHa cells. The results showed that lactic acid enhanced cell migration and invasion behavior via stimulating the expression of miR-744. ARHGAP5 was confirmed to be a target of miR-744, and silencing ARHGAP5 exhibited an inhibiting effect on cell migration and invasion as that observed by suppressing miR-744. In addition, lactic acid down-regulated E6 and E7 protein levels, and overexpression of either miR-744 or ARHGAP5 could also reduce E6 and E7 levels. Overall, our findings suggest that the miR-774/ARHGAP5 axis may provide a vital role in triggering lactic acid-induced migration and invasion in SiHa cells, regardless of the diminished effect due to the partial inhibition of E6 and E7 expression.

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

BackgroundGene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.MethodsHPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.ResultsAlthough ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.ConclusionsOur study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.