E6 Expression Research Articles

Neutrophils insert elastase into hepatocytes to regulate calcium signaling in alcohol-associated hepatitis.

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

The Mechanism of HPV-mediated DNA Methylation in the Promoter Region of Long-chain Non-coding RNA MAGI2-AS3 in the Occurrence and Development of Cervical Cancer

Background: Cervical cancer, a malignancy of gynecological origin, typically necessitates a therapeutic approach combining surgery and chemoradiotherapy as the primary intervention. However, the 5-year survival rate remains suboptimal, prompting researchers to explore novel strategies for the early diagnosis and treatment of cervical cancer. This study delves into the investigation of human papillomavirus (HPV)-mediated DNA methylation modifications within the promoter region of the long non-coding RNA MAGI2-AS3 during cervical cancer development. This paper is an experimental study, laboratory based, using cell lines. Methods: A lentivirus overexpression vector encoding HPV16 E6/E7 proteins was established for transfecting cervical epithelial cells. The methylation status of DNA in the MAGI2-AS3 promoter region was assessed using MassARRAY, and the MAGI2-AS3 gene expression was measured through quantitative real time polymerase chain reaction (qRT-PCR). Subsequently, the correlation between methylation levels and gene expression in cervical cancer was analyzed. Results: (1) Relative to the HPV-negative cervical cancer cell line C33A, MAGI2-AS3 expression significantly decreased in the HPV-positive cervical cancer cell line Siha. (2) The methylation rate of 16 CpG sites in the HPV-positive cervical cancer cell line Siha was notably higher compared to the HPV-negative cervical cancer cell line C33A. (3) To further substantiate the regulatory impact on DNA methylation and expression within the MAGI2-AS3 promoter region, we silenced the expression of HPV16 E6 in the HPV-positive cervical cancer cell line Siha using HPV16 E6 siRNA. The ensuing qRT-PCR analysis revealed a significant up-regulation of MAGI2-AS3 expression in the HPV16 E6 siRNA group when contrasted with the negative control group. MassARRAY analysis was employed to gauge the DNA methylation levels in the promoter region of the MAGI2-AS3 gene in HPV-positive cervical cancer cells (Siha) following HPV16 E6 silencing. The results demonstrated a significantly lower methylation rate at the CpG_29 site in the HPV16 E6 siRNA group compared to the HPV16 E6 siRNA group. Conclusions: The study establishes a close association between HPV infection and elevated methylation levels coupled with diminished expression of MAGI2-AS3. The influence of HPV infection on the malignant transformation of cervical epithelial cells is potentially mediated through the regulatory modulation of MAGI2-AS3 expression via DNA methylation.

Atractylenolide I Alleviates Indomethacin-Induced Gastric Ulcers in Rats by Inhibiting NLRP3 Inflammasome Activation.

Atractylodes macrocephala Koidz, a traditional Chinese medicine, contains atractylenolide I (ATR-I), which has potential anticancer, anti-inflammatory, and immune-modulating properties. This study evaluated the therapeutic potential of ATR-I for indomethacin (IND)-induced gastric mucosal lesions and its underlying mechanisms. Noticeable improvements were observed in the histological morphology and ultrastructures of the rat gastric mucosa after ATR-I treatment. There was improved blood flow, a significant decrease in the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and IL-18, and a marked increase in prostaglandin E2 (PGE2) expression in ATR-I-treated rats. Furthermore, there was a significant decrease in the mRNA and protein expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteinyl aspartate specific proteinase-1 (caspase-1), and nuclear factor-κB (NF-κB) in rats treated with ATR-I. The results show that ATR-I inhibits the NLRP3 inflammasome signaling pathway and effectively alleviates local inflammation, thereby improving the therapeutic outcomes against IND-induced gastric ulcers in rats.

Morroniside promotes skin wound re-epithelialization by facilitating epidermal stem cell proliferation through GLP-1R-mediated upregulation of β-catenin expression.

Epidermal stem cells (EpSCs) play a vital role in skin wound healing through re-epithelialization. Identifying chemicals that can promote EpSC proliferation is helpful for treating skin wounds. This study investigates the effect of morroniside on cutaneous wound healing in mice and explores the underlying mechanisms. Application of 10‒50 μg/mL of morroniside to the skin wound promotes wound healing in mice. In vitro studies demonstrate that morroniside stimulates the proliferation of mouse and human EpSCs in a time- and dose-dependent manner. Mechanistic studies reveal that morroniside promotes the proliferation of EpSCs by facilitating the cell cycle transition from the G1 to S phase. Morroniside increases the expression of β-catenin via the glucagon-like peptide-1 receptor (GLP-1R)-mediated PKA, PKA/PI3K/AKT and PKA/ERK signaling pathways, resulting in an increase in cyclin D1 and cyclin E1 expression, either directly or by upregulating c-Myc expression. This process ultimately leads to EpSC proliferation. Administration of morroniside to mouse skin wounds increases the phosphorylation of AKT and ERK, the expressions of β-catenin, c-Myc, cyclin D1, and cyclin E1, as well as the proliferation of EpSCs, in periwound skin tissue, and accelerates wound re-epithelialization. These effects of morroniside are mediated by the GLP-1R. Overall, these results indicate that morroniside promotes skin wound healing by stimulating the proliferation of EpSCs via increasing β-catenin expression and subsequently upregulating c-Myc, cyclin D1, and cyclin E1 expressions through GLP-1R signaling pathways. Morroniside has clinical potential for treating skin wounds.

Apolipoprotein E2 Expression Alters Endosomal Pathways in a Mouse Model With Increased Brain Exosome Levels During Aging.

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.

A critical suppression feedback loop determines soybean photoperiod sensitivity

Photoperiod sensitivity is crucial for soybean flowering, adaptation, and yield. In soybean, photoperiod sensitivity centers around the evening complex (EC) that regulates the transcriptional level of the core transcription factor E1, thereby regulating flowering. However, little is known about the regulation of the activity of EC. Our study identifies how E2/GIGANTEA (GI) and its homologs modulate photoperiod sensitivity through interactions with the EC. During long days, E2 interacts with the blue-light receptor flavin-binding, kelch repeat, F box 1 (FKF1), leading to the degradation of J/ELF3, an EC component. EC also suppresses E2 expression by binding to its promoter. This interplay forms a photoperiod regulatory loop, maintaining sensitivity to photoperiod. Disruption of this loop leads to losing sensitivity, affecting soybean's adaptability and yield. Understanding this loop's dynamics is vital for molecular breeding to reduce soybean's photoperiod sensitivity and develop cultivars with better adaptability and higher yields, potentially leading to the creation of photoperiod-insensitive varieties for broader agricultural applications.

Differential cyclin-E1 expression in CIC-rearranged sarcoma

CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Syringic acid guards against indomethacin-induced gastric ulcer by alleviating inflammation, oxidative stress and apoptosis

ABSTRACT The purpose of this study was to evaluate the effects of syringic acid, an anti-oxidant, on indomethacin induced gastric ulcers in rats. Experimental groups were control, ulcer, ulcer treated with 20 mg/kg esomeprazole (a proton pump inhibitor that reduces acid secretion), and ulcer treated with 100 mg/kg syringic acid. Rats were pretreated with esomeprazole or syringic acid two weeks before ulcer induction. Our histopathological observations showed that either syringic acid or esomeprazole attenuated the severity of gastric mucosal damage. Moreover, syringic acid and esomeprazole pretreatments alleviated indomethacin-induced damage by regulating oxidative stress, inflammatory response, the level of transforming growth factor-β (TGF-β), expressions of COX and prostaglandin E2, cell proliferation, apoptosis and regulation of the NF-κB signaling pathway. We conclude that either esomeprazole or syringic acid administration protected the gastric mucosa from harmful effects of indomethacin. Syringic acid might, therefore be a potential therapeutic agent for preventing and treating indomethacin-induced gastric damage.

Abstract 1649: Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications

Abstract Cyclin E1 is a key regulator of the G1/S transition and is thought to cause excessive entry into the cell cycle. CCNE1 is amplified in a range of tumor indications, and its amplification is associated with poorer prognosis and resistance to chemotherapy. Cyclin E1 is also being investigated as a potential biomarker for inhibitors targeting the replication stress response. Understanding the best way to quantify cyclin E1 overexpression is fundamental to support patient selection approaches for these inhibitors. In many cases, gene amplification correlates with increased cyclin E1 protein expression; however, there are a proportion of high-grade serous ovarian cancer and basal-like breast cancer cases where cyclin E1 protein overexpression does not correlate with gene amplification. In this study, we investigate if this phenomenon is observed in other cancers and some of the potential mechanisms causing cyclin E1 overexpression. We used a multi-omics approach to assess cyclin E1 expression in 1417 formalin-fixed paraffin-embedded (FFPE) patient tumor resections across 6 indications (SCCHN, Bladder, NSCLC, TNBC, Ovarian &amp; SCLC). We used copy number variation (CNV) determined by next generation sequencing (NGS), NanoString nCounter RNA analysis and protein expression by immunohistochemistry (IHC). 830 were evaluable for all 3 methods and demonstrated CCNE1 gene amplification by NGS (CNV normalized log2 ratio ≥1.5) in 3.3% cases. Using a threshold of mRNA z-score &amp;gt;2, 17% cases had high CCNE1 mRNA and 29% had high cyclin E1 protein (H-score ≥100). 15.7% (130/830) cases had high protein expression without gene amplification or gain, crucially this was found across all 6 indications (n=20/102 SCCHN, n=29/166 Bladder, n=34/232 NSCLC, n=15/76 SCLC, n=15/143 TNBC &amp; n=17/118 Ovarian). Impaired protein degradation has been proposed as a mechanism that may result in high Cyclin E1 expression. From a panel of 1600 genes, we assessed the relationship between cyclin E1 and mRNA expression of 57 genes involved in the ubiquitin-proteasome pathway in 1239 samples across the 6 indications. We assessed mutations which could impair ubiquitination in 915 samples across the 6 indications. We also assessed the methylation status of 893 genes associated with ubiquitin ligases or deubiquitinases in a cohort of 170 TNBC samples. Further investigation into other mechanisms causing cyclin E1 overexpression is required. IHC provides an ideal platform to build our understanding of cyclin E1 expression spatially. Our results indicate that assessing Cyclin E1 protein overexpression instead of CCNE1 gene amplification could greatly broaden the population of patients who could benefit from replication stress response kinase inhibitors, as cyclin E1 overexpression correlates with genomic instability and replication stress. Citation Format: Sophie E. Willis, Gemma N. Jones, Shaan Gill, Emma V. Jones, Amelia Raymond, Barrett Nuttall, Sophie Kirschner, Sakshi Gulati, Paola Marco-Casanova, Benedetta Lombardi, David Jenkins, Felicia S. Ng, James Hadfield, Paul Waring, Helen K. Angell, Heike Laman. Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications [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 1649.

Abstract 2955: HPV E6* and senescence-related gene expression in cervical cancer

Abstract Previous work in our lab found that increased expression of the E6* variant of the HPV E6 viral protein was significantly correlated with worse response to standard-of-care chemoradiation (CRT) and progression-free survival, independently of HPV genotype. In vitro validation showed that overexpression of E6* induces senescence in an HPV 16-positive cervical cancer cell line. Given these findings, we hypothesized that increased E6* expression mediates resistance to CRT by inducing a reversible state of tumor cell senescence. We created a senescence score using ten genes that have been consistently reported in the literature as associated with senescence. Using an institutional cohort of 68 patients with cervical cancer with prospectively collected clinical outcome data, we performed bulk RNA sequencing to assess senescence scores before CRT and determine whether there is an association between senescence scores and E6* expression. Using a second institutional cohort of 35 patients with cervical cancer treated with CRT with pre and posttreatment tumor biopsies, we performed bulk RNA sequencing to evaluate the same relationship and determine whether expression of senescence-related genes was affected by CRT. Nine cervical cancer cell lines stratified into low- and high-E6* expression groups will be assessed regarding their senescence score, beta-galactosidase staining, and SASP expression before and after CRT. In our first cohort, there was no correlation between E6*:E6 expression ratio and senescence score, regardless of HPV genotype. In our second cohort, CRT increased senescence score in CRT-treated samples (p &amp;lt; 0.01), but p16INK4a expression significantly decreased compared to pre-treatment samples (p &amp;lt; 0.01). Increased senescence score was not associated with cancer recurrence. There was no significant difference in senescence scores between different HPV genotypes before therapy. Tumors that were positive for HPV 16 or HPV 18 had significantly lower senescence scores compared to HPV negative tumors (p = 0.037 and p = 0.038, respectively) after CRT. Among HPV 16-positive tumors (n = 12), there was no correlation between E6*:E6 expression ratio and senescence score before or after CRT. We found no association between senescence score and E6* expression, and between senescence score and cancer recurrence after CRT in our dataset. Additionally, our results suggest that p16INK4a might not be an appropriate senescence marker in tumors that have intrinsically high p16INK4a expression, such as in cervical cancer, reinforcing the need for a cervical cancer-specific senescence signature. We are currently testing beta-galactosidase and SASP expression in vitro and in vivo before and after CRT. Citation Format: Louise Medina Bengtsson, Matthew Inkman, Naoshad Muhammad, Leahan Castillo, Naomi Msengi, Tyler McKinnish, Nishanth Gabriel, Jin Zhang, Julie Schwarz. HPV E6* and senescence-related gene expression in cervical cancer [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 2955.

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.

“Keep on ROCKIn”: Repurposed ROCK inhibitors to boost corneal endothelial regeneration

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

PAD-mediated citrullination is a novel candidate diagnostic marker and druggable target for HPV-associated cervical cancer.

Citrullination is an emerging post-translational modification catalyzed by peptidyl-arginine deiminases (PADs) that convert peptidyl-arginine into peptidyl-citrulline. In humans, the PAD family consists of five isozymes (PADs 1-4, 6) involved in multiple diseases, including cancer. Given that high-risk (hr) human papillomaviruses (HPVs) are the etiological agents of cervical cancer, in this study, we sought to determine whether PAD-mediated protein citrullination would play a functional role in the HPV-driven transformation of epithelial cells. Here we show that both total protein citrullination and PAD4 expression levels are significantly associated with cervical cancer progression. Specifically, epithelial immunostaining for PAD4 revealed an increasingly higher histoscore from low-grade (CIN1) to high-grade (CIN2, CIN3) cervical intraepithelial neoplasia, and invasive squamous cell carcinoma (SCC) lesions, raising the attractive possibility that PAD4 may be used as tumor staging markers. Furthermore, taking advantage of the epidermoid cervical cancer cell line CaSki, which harbors multiple copies of the integrated HPV16 genome, we show that the expression of E6 and E7 HPV oncoproteins is impaired by treatment with the pharmacological pan-PAD inhibitor BB-Cl-amidine. Consistently, p53 and p21, two targets of HPV oncoproteins, are upregulated by the PAD inhibitor, which undergoes cell growth arrest and apoptosis. Altogether, these findings highlight a novel mechanism by which hrHPVs alter host regulatory pathways involved in cell cycle and survival to gain viral fitness, raising the possibility that PADs may represent an attractive target for developing novel host-targeting antivirals effective in preventing cervical cancer progression.

The POU-HD TFs impede the replication efficiency of several human papillomavirus genomes

Human papillomavirus (HPV) is a double-stranded DNA virus that infects cutaneous and mucosal epithelial cells. HPV replication initiates at the origin (ori), located within a noncoding region near the major early promoter. Only two viral proteins, E1 and E2, are essential for replication, with the host cell contributing other necessary factors. However, the role of host cell proteins in regulating HPV replication remains poorly understood. While several binding sites for cellular transcription factors (TFs), such as POU-HD proteins, have been mapped in the regulatory region, their functional importance is unclear. Some POU-HD TFs have been shown to influence replication in a system where E1 and E2 are provided exogenously. In this study, we investigated the impact of several POU-HD TFs on the replication of the HPV5, HPV11, and HPV18 genomes in U2OS cells and human primary keratinocytes. We demonstrated that OCT1, OCT6, BRN5A, and SKN1A are expressed in HPV host cells and that their overexpression inhibits HPV genome replication, whereas knocking down OCT1 had a positive effect. Using the replication-deficient HPV18-E1− genome, we demonstrated that OCT1-mediated inhibition of HPV replication involves modulation of HPV early promoters controlling E1 and E2 expression. Moreover, using Oct6 mutants deficient either in DNA binding or transcriptional regulation, we showed that the inhibition of HPV18 replication is solely dependent on Oct6’s DNA binding activity. Our study highlights the complex regulatory roles of POU-HD factors in the HPV replication.

Abstract A017: Prevalence and significance of common molecular alterations in Tp53-mutated uterine serous carcinoma

Abstract Background: Uterine Serous Carcinoma (USC) is an aggressive subtype of endometrial cancer characterized by ubiquitous TP53 mutations and high replication stress. The resulting dependency on DNA damage repair and cell cycle checkpoints triggers sensitivity to the abrogation of the G2 checkpoint using Wee1 inhibitors (Wee1i) such as Azenosertib. Wee1i sensitivity has been associated with high levels of Cyclin E1, which may result from gene amplification of CCNE1 or the alteration of its ubiquitin ligase FBXW7 1. In addition, signaling activity of Wee1 is controlled by the PP2A protein phosphatase2, the regulatory subunit of which, PPP2R1A, is frequently mutated in USC. Comprehensive molecular profiling has only been performed in a limited number of cohorts and the distribution and relationship of these candidate biomarkers to one another and other molecular alterations is unclear. Methods: Genomic and phenotypic data from TP53-mutated USC were collected from TCGA-pancancer (N=95), AACR-GENIE v13 (N=796) or Azenosertib studies (N=46). The mutational status of 6 mutated genes and 1 gene set (HRR; N=14 genes) was determined from pathogenic alterations. The prevalence of altered genes was measured accounting for variable gene inclusion for each assay. Cross-cohort prevalence or gene-pair mutual exclusivity and co-occurrence were measured using Fisher exact test. Results: The prevalence of alterations in TCGA, AACR-GENIE and Azenosertib cohorts were similar for PIK3CA (37%, 34%, 28%), FBXW7 (21%, 17%, 8%), PPP2R1A (37%, 30%, 29%), PIK3R1 (13%, 12%, 8%), HRR genes (7%, 7%, 15%), or amplification of ERBB2 (19%, 14%, 9%). In contrast, prevalence of CCNE1 amplification in TCGA was significantly higher (27% vs 16% and 7%), which could reflect differences in patient population or methodology. Of all 28 combinations, no gene pairs were significantly co-mutated and only FBXW7 and PPP2R1A were mutually exclusive in both TCGA and AACR-GENIE. The prevalence of the corresponding co-alterations was similar between cohorts: mutated in both (3% in both), neither (45% and 57%), FBXW7 only (18% and 13%) or PPP2R1A only (34% and 27%). Furthermore, alterations in genes encoding components of the PI3K enzyme (PIK3CA or PIK3R1) affected 45% of the patients in either cohort. None of the mutational status or derived subtypes had prognostic value (TCGA). Similarly, apart from CCNE1 amplification, none of the subtypes evaluated were associated with Cyclin E1 expression as measured by reverse phase protein array (TCGA) or immunohistochemistry (Azenosertib studies). Conclusion: Important processes related to CCNE1, FBXW7 and PPP2R1A function have been associated with high replication stress and Wee1i sensitivity. Our analyses suggest that alterations in these genes are common and sometimes mutually exclusive, leading to broad genomic instability across USC and suggesting an explanation for the clinical efficacy that has been seen with Azenosertib and other Wee1i. Citation Format: Olivier Harismendy, Jianhui Ma, Adrian M. Jubb, Carrie Brownstein, Mark R. Lackner, Ramez N. Eskander, Joyce Liu, Shannon N. Westin. Prevalence and significance of common molecular alterations in Tp53-mutated uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A017.

Zishen Yutai pills restore fertility in premature ovarian failure through regulating arachidonic acid metabolism and the ATK pathway

Ethnopharmacological relevanceThe Zishen Yutai pills (ZYP), a Chinese medicinal formulation derived from the Qing Dynasty prescription “Shou Tai pills", have been documented to exhibit beneficial effects in clinical observations treating premature ovarian failure (POF). However, the anti-POF effects and its comprehensive systemic mechanism have not yet been clarified. Aim of the reviewTherapeutic effects and systemic mechanism of ZYP in POF were evaluated. Materials and methodsAfter pulverization, sieving, and stirring, ZYP was administered intragastrically to cisplatin-induced POF mice at a dose of 1.95 mg/kg/d for 14 days. The anti-POF effects of ZYP were investigated by assessing the number of ovarian follicles at different developmental stages, as well as measuring serum estradiol (E2) levels and ovarian-expressed anti-Müllerian hormone (AMH). Reproductive performance and offspring health were evaluated to predict fertility restoration. Furthermore, a combination of proteomic and metabolomic profiling was employed to elucidate the underlying molecular mechanism of ZYP in treating POF. Western blot (WB) analyses and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to explore the mechanisms through which ZYP exerted its anti-POF effects. ResultsWe have demonstrated that oral administration of ZYP reversed the reduction in follicles at different developmental stages and stimulated the expressions of serum E2 and ovarian-expressed AMH in a cisplatin-induced POF model. Additionally, ZYP ameliorated follicle apoptosis in ovaries affected by cisplatin-induced POF. Furthermore, treatment with ZYP restored the quantity and quality of oocytes, as well as enhanced fertility. Our results revealed 62 differentially expressed proteins (DEPs) through proteomic analyses and identified 26 differentially expressed metabolites (DEMs) through metabolomic analyses. Both DEPs and DEMs were highly enriched in the arachidonic acid (AA) metabolism pathway. ZYP treatment effectively upregulated the protein and mRNA expression of critical targets in AA metabolism and the AKT pathway, including CYP17α1, HSD3β1, LHR, STAR, and AKT, in cisplatin-induced POF mice. ConclusionsThese results indicated that ZYP exerted protective effects against POF and restored fertility from cisplatin-induced apoptosis. ZYP could be a satisfying alternative treating POF.

CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured invitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated invitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.

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.