CaSki Cells Research Articles

Single-cell and spatial transcriptome analysis revealed cellular heterogeneity of glycosyltransferases in cervical cancer, and identified GALNT3-negative epithelial cells as a protective factor: a retrospective cohort study based on public database.

Cervical cancer (CC) is a common and deadly malignancy among women worldwide, characterized by high mortality and poor prognosis. Glycosyltransferases (GTs) are enzymes that catalyze glycosyl transfer reactions and play significant roles in cancer development and progression. However, their role in cervical cancer remains unclear. This study combined single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (stRNA-seq) to explore the role of GTs in cervical cancer. We utilized comprehensive scoring algorithms to evaluate GT expression, conducted cell communication analysis, and identified differentially expressed GT-related genes in tumor-associated epithelial cells. Functional assays were performed to assess the impact of GALNT3 expression on cervical cancer cell proliferation and invasiveness. Our analysis revealed that GT-related genes are highly enriched in cervical cancer epithelial cells. We identified 11 differentially expressed GT-related genes, including GALNT3, which showed distinct characteristics in epithelial cells. GALNT3-negative epithelial cells were found to be a protective factor for cervical cancer patients, showing a significant negative correlation with tumor-associated macrophages and myeloid-derived suppressor cells. Knockdown or overexpression of GALNT3 significantly affected cell proliferation and invasiveness in C-33A and Caski cell lines. This study highlights the critical role of GTs, particularly GALNT3, in the progression and prognosis of cervical cancer. GALNT3-negative epithelial cell infiltration could be a favorable factor for cervical cancer prognosis, providing new insights for therapeutic strategies targeting GTs in cervical cancer.

Biomolecular Affinities and Cytotoxicity of Copper(I) and Silver(I) Phosphine–Pyridinyl Complexes against CACO‐2 and CASKI Cell Lines

A series of three copper (I) and three silver (I) complexes with the general formula [M L(PPh3)2]NO3, (M = Cu for complexes 1 – 3 and Ag for complexes 4 – 6) were synthesized by reacting copper(I) or silver(I) ‐nitrate and triphenylphosphine with the bidentate ligands, (E)‐1‐(pyridin‐2‐yl)‐N‐(o‐tolyl)methanimine L1, (E)‐N‐isopropyl‐1‐(pyridine‐2‐yl)methanimine L2, or (E)‐N‐(2,6‐dimethylphenyl)‐1‐(pyridine‐2‐yl)methanimine L3. The structures were elucidated using NMR spectroscopy, FTIR, UV‐visible, mass spectrometry, elemental analysis, and single‐crystal X‐ray diffraction. Structural analysis revealed that the Schiff bases coordinate to the metal centres in a bidentate fashion, with triphenylphosphine occupying the remaining coordination sites in complexes 1, 2, and 5. In contrast, in complexes 3, 4, and 6, one coordination site is occupied by a nitrate anion instead of triphenylphosphine. All six complexes exhibit a distorted tetrahedral geometry around the metal centre, as confirmed by τ4 values ranging from 0.54 to 0.87. Binding studies with calf‐thymus DNA (CT‐DNA) demonstrated that complexes 1 – 6 interact via intercalation, with complex 5 exhibiting the highest binding constant. Furthermore, all complexes showed strong binding affinity toward bovine serum albumin (BSA). Biological studies revealed significant cytotoxicity of complexes 1 – 6 against Human Colon Adenocarcinoma (CACO‐2) and Human Cervical Epidemoid Carcinoma (CASKI) cell lines.

ZINC1797251, a novel natural product small molecule targets viral oncoprotein E6 in human papillomavirus-16 positive cervical cancer cells

Cervical cancer burden due to recurrent human papillomavirus (HPV) infections necessitates the urgent need to impede viral proliferation targeting the oncogene E6 of the high-risk serotype HPV16. This study aims to identify a small molecule from a natural product library that could prevent a tumorigenic complex of E6 with p53 in HPV16-positive cervical cancer cells. In silico methods such as high-throughput virtual screening (HTVS) of natural product like library ZINC database followed by atomistic molecular dynamics (MD) simulations were performed to identify lead natural compound. This was validated with in vitro analysis using HPV16 positive SiHa cells and CaSki cells by MTT and flow cytometry assays. Virtual screening identified top 10 compounds with high affinity for HPV16 E6. The docking scores, Protein-Ligand Interaction Profiler analysis, MD simulation and molecular mechanics Poisson Boltzmann surface area-based binding energy estimation narrowed down the search to ZINC1797251, a molecule with stable binding, low energy scores and consistent H-bonds, establishing that it could prevent interaction of p53 and E6. ZINC1797251 inhibited the proliferation of SiHa and CaSki cells with a GI50 values of 615.40 and 417.30 nM, respectively. The compound reduced HPV16 E6, while increased p53 positive populations in SiHa and CaSki cells. Treatment with ZINC1797251 induced the G1 cell phase arrest and promoted early and late phase apoptosis in these cells. The restoration of tumor inhibitory activity of p53 in HPV-infected cervical cancer cells to promote apoptosis could be achieved using the ZINC molecule—ZINC1797251. However, further studies are deemed essential for further developments.

Abstract 4107: CircZFR-YTHDF3 complex drives lymph nodes metastasis in cervical cancer via facilitating FASN translation

Introduction: Lymph node metastasis is a key driver of poor outcomes in cervical cancer. Our study reveals that elevated fatty acid synthase (FASN) protein is closely linked to metastasis and reduced survival in cervical cancer, yet its regulatory mechanisms remained unclear. Methods: We compared the expression of FASN in the cervical cancer samples with or without lymph nodes, long or short disease-free survival (DFS) via qRT-PCR, western blot assay, and tissue array. We investigated and validated the combination between circZFR and YTH N6-methyladenosine RNA binding protein F3 (YTHDF3) protein via circRNA pulldown assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay. The m6A sites on FASN mRNA were tested via MeRIP-qPCR. Protein co-immunoprecipitation (Co-IP) assay was used to validate the combination between YTHDF3 and eukaryotic translation initiation factor 4A3 (eIF4A3) protein. The siRNAs for YTHDF3 or FASN were transfected into HeLa, C33A, and Caski cells. Proliferation ability of cells was tested via CCK-8 and EdU assay. Migration and invasion ability of cells were tested via transwell assay. The function of circZFR on promoting lymph nodes metastasis was validated by animal assay. Results: The test in clinical samples showed that the overexpression of circZFR and FASN is associated with lymph nodes metastasis and shorter DFS of cervical cancer patients. The results of circRNA pulldown and RIP assay demonstrated the combination between circZFR and YTHDF3 protein. The results of MeRIP-qPCR assay validated the m6A sites in FASN mRNA. The results of Co-IP assay showed that upregulated circZFR could increase the combination between YTHDF3 and eIF4A3 proteins. The results of proliferation, migration, and invasion assay suggested that knocking down FASN will inhibit the proliferation, migration, and invasion of cervical cancer cells. The results of animal assay validated the promotion of lymph nodes metastasis by circZFR. Conclusions: We identified circular RNA circZFR as a crucial regulator, significantly enhancing FASN protein expression. CircZFR overexpression accelerates lymph node metastasis and shortens DFS in patients. Mechanistically, circZFR binds to the m6A reader protein YTHDF3, facilitating m6A recognition on FASN mRNA and recruiting the translation initiator eIF4A3, thereby boosting FASN translation. These findings establish circZFR as a pivotal driver of cervical cancer progression and highlight its inhibition as a promising therapeutic strategy. Citation Format: Mingyi Zhou, Yong Zhang, Lian He, Bo Gao, Francois Claret, George Calin, Danbo Wang. CircZFR-YTHDF3 complex drives lymph nodes metastasis in cervical cancer via facilitating FASN translation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4107.

Targeted demethylation of the EphA7 promoter inhibits tumorigenesis via the SP1/DNMT1 and PI3K/AKT axes and improves the response to multiple therapies in cervical cancer

Aberrant methylation of the EphA7 promoter has been observed in cervical cancer (CC); however, its precise function and role in CC remain largely unknown. In this study, we investigated the role and molecular mechanisms of EphA7 promoter methylation in cervical carcinogenesis. First, our results indicated that the reactivation of EphA7 expression via a CRISPR demethylation tool (dCas9-Tet1) had antitumor effects. It restrained tumor proliferation and invasion while promoting apoptosis via the PI3K/AKT signaling pathway in both CaSki and SiHa cells. The upstream interacting factors were subsequently captured by CRISPR-mediated pull-down in situ, and the result revealed that SP1 and MAZ interacted with the promoter of EphA7. However, the perturbation results revealed that EphA7 expression was associated with SP1/DNMT1 but not MAZ. Furthermore, 17-β-estradiol (E2) can upregulate EphA7 expression through demethylation via the SP1/DNMT1 axis. A rescue experiment revealed that interference with SP1 expression could restore the effect of E2 on increasing the expression of EphA7 by upregulating estrogen receptor expression. In addition, EphA7 demethylation reduced the half-maximal inhibitory concentration (IC50) of cisplatin and paclitaxel. Pooled analysis revealed that EphA7 promoter hypermethylation was positively correlated with tumor purity but negatively correlated with immune cell infiltration, cytotoxic T lymphocyte (CTL) and immune checkpoint (IC) activity, and the expression of EphA7 was significantly positively correlated with tumor mutational burden (TMB), microsatellite instability (MSI) and the presence of single nucleotide variant (SNV) neoantigens, suggesting a better prognosis for patients with EphA7 promoter hypomethylation and high expression. Collectively, these findings indicate that targeted demethylation of the EphA7 promoter and restoration of endogenous EphA7 expression by dCas9-Tet1 are promising therapeutic approaches and are favorable for the prognosis of CC patients.

Abstract 1332: Major histocompatibility complex class I chain-related gene A and B expression induced by heavy ion beam irradiation in cervical cancer cells

Major histocompatibility complex class I chain-related gene A and B (MIC) are transmembrane glycoproteins to function as a ligand for human NKG2D that is a main activating receptor of NK cells. MIC is minimally expressed on normal cells, but frequently upregulated via viral infection, cellular stress signals, and malignant transformation. NKG2D recognizes MIC, resulting in the activation of NK cells. Recent progress on radiation biophysics has shown that photon or particle radiation can modulate anti-tumor immunity in host irradiated via regulation of immune recognition protein expression on cancer cells. The aim of this study is to investigate the effects of heavy ions beam irradiation on membrane MIC expression in human cervical cancer cells. We used HEK293 cells stably overexpressing HPV-E6 or E7 oncoproteins and 5 human cervical cancer cell lines in this study. Carbon ions beam irradiation (CIR, 290 MeV/n; LET, ∼70 keV/μm, mono peak), a beam of heavy ion beams, was performed at the in-house synchrotron accelerator that is able to produce carbon ion beams. Irradiated doses ranged from 0 to 10 Gy of carbon-ions. Cell membrane MIC expression was evaluated by flowcytometry using an anti-MIC antibody. MIC expression was upregulated in HPV-E6 or E7 overexpressing HEK293 cells and 5 human cervical cancer cells in a dose-dependent manner. HPV16-positive human cervical cancer Caski cells showed a remarkable upregulation of MIC expression after CIR. Other cervical cell lines (ME-180, HeLa, SiHa, and C33A) showed a modest MIC upregulation. E6- or E7-overexpressing HEK293 cells exhibited a considerable induction of MIC expression after CIR. We investigated the impact of DNA damage responses on HIR-mediated MIC upregulation using drug inhibitors of proteins that are involved in DNA damage responses. Both pharmacological Chk1 inhibition and ATM inhibition cancelled CIR-mediated MIC upregulation in HPV oncoprotein-positive HEK293 cells. The results indicated that ATM/ATR-Chk1 pathways were involved in MIC upregulation on HPV-positive cells including cervical cancer cells. These findings support the favorable combination of MIC-targeted therapy with heavy ion therapy against cervical cancers. Citation Format: Sumitaka Hasegawa, Nakako Nakajima. Major histocompatibility complex class I chain-related gene A and B expression induced by heavy ion beam irradiation in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1332.

Abstract 3482: Enhanced targeting of HPV positive cancers using gamma delta 1 T cells armored with an HPV E7-specific TCR: a novel approach to overcome HLA downregulation

Abstract T-cell receptor (TCR) engineered T cells have emerged as a promising cancer therapeutic. While the clinical trial using ab T cells targeting the HPV E7 protein showed promise, it also highlighted a concerning issue: tumor escape due to the acquisition of defects in peptide-HLA presentation. As an alternative cellular platform, Gamma Delta One T cells (DOT) offer several advantages over alpha/beta T cells. These include their tissue-homing abilities, their use as allogeneic cells without GVHD risk, and their potential to target cells through both TCR and NK-like receptors (which can kill HLA Class-I deficient cells). The overall purpose of this study is to establish DOTs as an effective platform for TCR-T therapy targeting solid tumors, demonstrating enhanced therapeutic efficacy in overcoming tumor escape through mutations in the antigen-processing machinery (APM). We developed a novel protocol to isolate and expand DOTs from peripheral blood using a Delta 1 TCR-specific antibody and EasySep positive selection. By day 4, DOTs reached 37.3% (SD ± 10.3%) enrichment, increasing to 91.85% (SD ± 2.15%) by the end of culture. From transduction day, cells expanded over 100-fold by day 15, demonstrating scalability for therapeutic use. Efficiently transduced with an E7-specific TCR engineered to minimize mispairing (murine constant alpha and beta chains), we achieved transduction levels comparable to alpha/beta T cells. DOTs expressed high levels of NK-like receptors, including NKG2D (94.85% SD ± 0.55%), DNAM-1 (99.1% SD ± 0.3%), and NKp30 (76% SD ± 11%), crucial for targeting HLA-negative tumors. Using HPV+ E7+ SCC90 and CaSki cell lines with varying NKG2D ligand expression, we performed CRISPR/Cas9-mediated B2M knockout to generate HLA class I-deficient variants. Cytotoxicity was evaluated using in vitro chromium release (short-term) and IncuCyte (long-term) assays, where DOTs consistently outperformed alpha/beta T cells in killing HLA-heterogeneous tumors. DOT TCR-T cells exhibited comparable cytotoxicity to alpha/beta TCR-T cells against HLA-A*02:01+ E7+ targets while excelling against HLA-negative variants. These findings establish DOTs as a robust platform for TCR-engineered therapies, capable of overcoming tumor immune evasion across diverse HLA settings. We are currently validating these results in vivo using murine models. In summary, DOTs offer a promising platform for TCR-engineered therapies, excelling in targeting HLA-heterogeneous tumor populations through their dual recognition mechanisms. This work highlights their potential for developing off-the-shelf TCR-T therapies to overcome HLA downregulation and tumor heterogeneity in solid tumors. Citation Format: Andres Mosquera, Diego F. Chamorro, Paul Shafer, Lauren K. Somes, Emily Madaras, Valentina Hoyos. Enhanced targeting of HPV positive cancers using gamma delta 1 T cells armored with an HPV E7-specific TCR: a novel approach to overcome HLA downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3482.

Detection and isolation of viable cancer cells mediated by spytag and spycatcher using conditionally replicative adenovirus and magnetic microbeads

Circulating tumor cells (CTCs) are critical biomarkers for cancer diagnosis, prognosis, and therapy monitoring, but their rarity and reliance on surface markers limit detection and isolation. While conditionally replicative adenoviruses (crADs) enable tumor-selective targeting, their use has been limited to fluorescence-based detection without robust isolation of viable cells. To overcome this, we developed a crAD-based platform integrating SpyTag/SpyCatcher technology with SpyCatcher-decorated magnetic microbeads for marker-independent CTC detection and isolation. The engineered adenovirus (CR-Ad5-ST-GFP) selectively replicates in telomerase-positive tumor cells, expressing green fluorescent protein (GFP) and SpyTag under independent promoters. By leveraging the SpyTag/SpyCatcher interaction, our platform isolates CTCs without relying on surface markers, addressing epithelial-to-mesenchymal transition (EMT) and phenotype variations. In proof-of-concept experiments, A-549 and Ca Ski cells spiked into peripheral blood mononuclear cells (PBMCs) at 1:10,000 were detected and isolated with over 80% efficiency. The isolated cells remained viable and were successfully re-cultured, demonstrating their potential for downstream applications such as molecular profiling and drug sensitivity testing. This method advances crAD-based approaches by combining tumor-selective viral targeting with marker-independent, viable CTC isolation. Its compatibility with microfluidic systems makes it a promising tool for tumor monitoring and personalized cancer treatment.

Overexpression of miR-23b-3p+miR-218-5p+miR-124-3p differentially modifies the transcriptome of C-33A and CaSki cells and the regulation of cellular processes involved in the progression of cervical cancer.

Overexpression of miR-23b-3p+miR-218-5p+miR-124-3p differentially modifies the transcriptome of C-33A and CaSki cells and the regulation of cellular processes involved in the progression of cervical cancer.

Transcriptomic profile induced by calcitriol in CaSki human cervical cancer cell line.

The vitamin D endocrine system, primarily mediated by its main metabolite calcitriol and the vitamin D receptor (VDR), plays a critical role in numerous human physiological processes, ranging from calcium metabolism to the prevention of various tumors, including cervical cancer. In this study, we comprehensively investigated the genomic regulatory effects of calcitriol in a cervical cancer model. We examined the transcriptional changes induced by calcitriol in CaSki cells, a cervical cell line harboring multiple copies of HPV16, the primary causal agent of cervical cancer. Our microarray findings, revealed that calcitriol regulated over 1000 protein-coding genes, exhibiting a predominantly repressive effect on the CaSki cell transcriptome by suppressing twice as many genes as it induced. Calcitriol decreased EPHA2 and RARA expression while inducing KLK6 and CYP4F3 expression in CaSki cells, as validated by qPCR and Western blot. Functional analysis demonstrated that calcitriol effectively inhibited key processes involved in cancer progression, including cell proliferation and migration. This was further supported by the significant downregulation of MMP7 and MMP13 mRNA levels. Our microarray results also showed that, in addition to its effects on protein-coding genes, calcitriol significantly regulates non-coding RNAs, altering the expression of approximately 400 non-coding RNAs, including 111 microRNA precursors and 29 mature microRNAs, of which 17 were upregulated and 12 downregulated. Notably, among these calcitriol-regulated microRNAs are some involved in cervical cancer biology, such as miR-6129, miR-382, miR-655, miR-211, miR-590, miR-130a, miR-301a, and miR-1252. Collectively, these findings suggest that calcitriol exhibits a significant antitumor effect in this advanced cervical cancer model by blocking critical processes for tumor progression, underscoring the importance of maintaining adequate vitamin D nutritional status.

Inhibition of p16 and NF-κB Oncogenic Activity in Human Papillomavirus-Infected Cervical Cancer Cells: A New Role for Activating Transcription Factor-3.

Objective: Activating transcription factor 3 (ATF3) has attracted recent scientific attention as a novel mediator of tumor suppression, particularly within the context of cervical cancer (CC). Our prior research demonstrated that ATF3 overexpression induces cell cycle arrest and apoptosis in human papillomavirus (HPV)16- and HPV18-positive CC cells. The present study aims to examine the impact of ATF3 overexpression on the expression levels of p16 and NF-κB, two proteins with pro-tumorigenic roles in HPV-induced CC. Methods: Ca Ski and HeLa cells underwent transfection with pCMV6-AC-IRES-GFP plasmids containing the ATF3 gene. To establish the optimal plasmid DNA quantities for transfection, MTT assay was conducted. Furthermore, fluorescence microscopy and flow cytometric analysis were employed to assess the efficiency of transfection. The expression levels of p16 and NF-κB were evaluated by RT-qPCR and western blotting prior and subsequent to ATF3 overexpression. Results: The overexpression of ATF3 induced a decrease in p16 mRNA levels in both Ca Ski and HeLa cells (p<0.04), along with the concomitant reduction of p16 protein expression within both cellular populations (p<0.005). Additionally, it led to a reduction in NF-κB p65 protein levels in both cell lines (p<0.005), with no discernible impact on its mRNA expression. Conclusion: Given ATF3's demonstrated capability to downregulate p16 and NF-κB, both of which play important pro-tumorigenic roles in HPV-related CC, ATF3 emerges as a promising therapeutic candidate with the potential for application in the treatment of CC.

Synthesis and Biological Evaluation of Some Coumarin–Triazole Conjugates as Potential Anticancer Agents

Despite the discovery of many chemotherapeutic drugs that prevent uncontrolled cell division processes, the development of compounds with higher anticancer efficacy and a lower level of side effects is an important task in modern pharmaceutical chemistry. Herein, a mild and convenient method for the preparation of N1-substituted 3-(1,2,3-triazolyl-methoxycarbonyl)coumarins or bis(coumarine-3-carboxylate)bis(triazole)alkandiyl by the copper(I)-catalyzed Huisgen cycloaddition reaction of readily available coumarin-3-carboxylic acid propynyl ester with azides or diazides has been presented. The synthesized compounds have been tested for their cytotoxicity on various cancer and noncancerous cell lines using the MTT assay. All new compounds were nontoxic on normal epithelial VERO cells. Two derivatives exhibited selectivity towards HPV-negative human cervical cancer cells, C33 A, with excellent activities in low concentrations (GI50 4.4–7.0 µM). In vitro mechanistic studies showed that bis(coumarine)bis(triazolylester) conjugate 3 induced time-dependent apoptosis in cervical cancer cell lines C33 A and CaSki, at the GI50 concentration, as measured by Annexin V-FITC/PI staining. The most active coumarin–triazolyl ester conjugate 2g possessed anticancer activities, as indicated by its ability to induce S/G2 phase cell cycle arrest at a low concentration and early apoptosis in CaSki cells. The obtained results revealed the potential of new compounds as anticancer agents, particularly against cervical cancer.

Abstract B018: Identifying a novel aggressive subtype of cervical cancer predominantly affecting minority populations through long-read whole genome sequencing

Abstract Purpose of the study: YAP1 is the most frequently amplified oncogene in cervical cancer. The Cancer Genome Atlas Research Network study showed that the YAP1 (11q22) oncogene is amplified in 11% of cervical cancer cases. Our lab recently identified that YAP1 oncogene amplification is associated with a 12-year earlier age of diagnosis, poorer survival, and defies a novel aggressive cervical cancer subtype that is more frequent in minority populations. The present study aims to characterize 19 YAP1 amplified cervical cancers from Guatemala and Venezuela via Oxford Nanopore technology (ONT) whole genome long-read sequencing. Methods: In this study, 380 cervical cancer patients were assessed for YAP1 amplification using a TaqMan assay, with 45 samples identified as positive for YAP1 amplification. These 45 samples were then subjected to whole genome sequencing with 5x coverage using Oxford Nanopore Technologies (ONT) to confirm the amplification status. A subset of 19 YAP1-amplified samples underwent deeper sequencing via ONT-LSK114 library preparation. The HPV integration status in these samples was determined by uploading the sequencing data (fastq_pass files) to the Cancer Genomics Cloud, where breakpoints were manually examined using the Integrated Genome Viewer (IGV). HPV and human DNA segments were further analyzed and identified using bioinformatics tools such as BLAT and BLAST. Results: In our study, we confirmed YAP1 amplification in 24 out of 45 tumor samples using barcode sequencing, and due to limited DNA availability (1 µg), only 19 of these samples underwent deep sequencing with 25-30x coverage. Notably, we observed a higher frequency of HPV integration (11/19) in YAP1-amplified tumors compared to those with episomal HPV status, suggesting a potential link between HPV integration and YAP1 amplification. We noticed that YAP1 amplification is driven by Breakage-Fusion-Bridge (BFB) events, which also lead to the co-amplification of the nearby BIRC2/3 genes, contributing to genomic instability and immune escape. This mechanism was further supported by a similar pattern observed in the TCGA whole genome sequence data, where 27 out of 31 YAP1-amplified cervical tumors also exhibited BFB events. Finally, treatment with the PIK3CA inhibitor Alpelisib resulted in a decrease in YAP1 protein levels in CaSki cells, a cervical cancer cell line with mutated PIK3CA and amplified YAP1 expression. Conclusions: These findings highlight a possible interplay between HPV integration and BFB-driven amplification of YAP1 and its neighboring genes, offering insights into potential therapeutic targets for this novel aggressive subtype of cervical cancer. Citation Format: Sonam Tulsyan, Hong Lou, Yi Xie, Emma Robinson, Danielle Kayembe, Eduardo Gharzouzi, Roberto Orozco, Enrique Alvirez, Michael Dean. Identifying a novel aggressive subtype of cervical cancer predominantly affecting minority populations through long-read whole genome sequencing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B018.

TGF-β Induces the Secretion of Extracellular Vesicles Enriched with CD39 and CD73 from Cervical Cancer Cells.

The presence of TGF-β in the tumor microenvironment of cervical cancer (CC) is important for tumor progression. In this study, we analyzed the effect of TGF-β on the expression of the ectonucleotidases CD39 and CD73, which are involved in the generation of adenosine (Ado), in CC cells and in extracellular vesicles (EVs) secreted by these cells. Treatment of HeLa and CaSki cells for 72 h with recombinant human TGF-β increased the expression of CD39 and CD73 by 20 and 30% and by 40 and 100%, respectively. The addition of SB505124, an inhibitor of the TGF-β1 receptor, or GW4869, an inhibitor of exosome formation and release, reduced the expression and release of both ectonucleotidases in CC cells. Furthermore, TGF-β promoted the secretion of medium-large EVs (>130 nm) in HeLa cells (HeLa + TGF-β/EVs) and CaSki cells (CaSki + TGF-β/EVs), which increased the expression of CD39 (>20%) and CD73 (>60%), and EVs obtained from cells treated with TGF-β had a greater capacity to generate Ado than did EVs obtained from cells cultured in the absence of this factor (HeLa/EVs and CaSki/EVs). These findings suggest that the production of TGF-β in the CC TME can promote neoplastic progression through the secretion of EVs enriched with CD39 and CD73. Therefore, the inhibition of CD39+ CD73+ EVs could be a strategy for the treatment of CC.

Targeting the interplay between human papillomavirus oncoproteins and hedgehog signaling: assessment of chemopreventive potential of carvacrol in cervical cancer.

Cervical carcinoma is the fourth most frequently diagnosed cancer and is a serious cause of increased mortality among females globally. Hedgehog/GLI signaling has now been established to play a pivotal role in imparting tumor recurrence and promoting metastasis in cervical carcinoma. HPV associated oncoproteins particularly E6/E7 concomitantly with altered signaling pathways are key determinants of cervical cancer. Nevertheless, the nexus between HPV oncogenes and Hedgehog/GLI signaling till date remains unclear. In this study, we investigated the anticancer and apoptotic potential of carvacrol against cervical cancer cells in vitro by targeting the plausible nexus between HPV oncoproteins and Hedgehog signaling. The findings from cell proliferation, LDH cytotoxicity, and morphology analysis suggested that carvacrol treatment significantly decreased the number of viable CaSki cells in a concentration and time-related manner. Morphological trademarks of cell death, including fragmentation of CaSki cell nucleus were studied by DAPI/PI and Hoechst33342 staining. The cytotoxicity of carvacrol was mediated through apoptosis, as confirmed by the Annexin V/FITC assay and caspase activation. Cell cycle analysis showed that carvacrol exerted significant impeding effects on the proliferation of CaSki cells via G0/G1 arrest. Intriguingly, carvacrol mediated the downregulation of HPV E6 and E7 oncogenes indicated its plausible role as an anti-HPV agent against HPV16+ CaSki cells. Additionally, carvacrol further restored p53 expression implicating that carvacrol may protect E6 mediated p53 protein degradation in CaSki cells. Thus, carvacrol exhibited strong antiproliferative potential by inducing apoptosis in cervical carcinoma cells via mediating the crosstalk between the downregulation of HPV oncogenes and inhibition of the hedgehog signaling pathway.

Activating transcription factor 3 induces oxidative stress and genotoxicity, transcriptionally modulating metastasis-related gene expression in human papillomavirus-infected cervical cancer

BackgroundActivating Transcription Factor 3 (ATF3) is known for its tumor-suppressive properties in cervical cancer, particularly through its role in stress response and interactions with human papillomavirus (HPV) oncogenes. This study investigates ATF3’s regulatory impact on metastasis-related genes, oxidative stress, and DNA damage in HPV-positive cervical cancer cells.MethodsHeLa and Ca Ski cell lines were transfected with ATF3-expressing vectors. Western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to confirm ATF3 overexpression following transfection. ROS assays and Comet assays assessed the impact of ATF3 on oxidative stress and DNA damage, while RT-qPCR was used to evaluate changes in HPV E6/E7, SHARP1, and MMP1 gene expression.ResultsATF3 overexpression led to elevated ROS levels (p < 0.02), resulting in oxidative DNA damage. These results demonstrate ATF3’s cytotoxic impact on cervical cancer cells through oxidative stress and DNA damage. Additionally, ATF3 overexpression significantly decreased MMP1 expression (p < 0.03), indicating a potential anti-metastatic effect, while SHARP1 and HPV E6/E7 expression levels were not significantly altered, indicating selective gene modulation by ATF3.ConclusionThese findings reveal that ATF3 contributes to tumor suppression in cervical cancer by modulating oxidative stress and DNA damage, selectively targeting genes involved in metastasis. These findings supports ATF3’s role in regulating key pathways in HPV-positive cervical cancer cells, providing a basis for further exploration of ATF3 as a target in therapeutic strategies aimed at improving outcomes in cervical cancer.

E2F1-Dependent CDCA5 overexpression drives cervical cancer progression and correlates with poor prognosis.

Cervical cancer (CC) remains a leading cause of cancer-related mortality in women worldwide, highlighting the urgent need for novel therapeutic strategies. This study investigates the molecular mechanisms and clinical significance of Cell Division Cycle Associated 5 (CDCA5) in cervical cancer progression. We performed comprehensive analyses of CDCA5 expression in cervical cancer and normal tissues, correlating expression levels with clinicopathological features and patient outcomes. Functional studies using CC cell lines (SiHa, HeLa, and CaSki) examined the effects of CDCA5 manipulation on tumor cell behavior. We identified E2F1 as a key transcriptional regulator of CDCA5 and validated our findings using in vivo xenograft models. CDCA5 was significantly upregulated in CC tissues and correlated with advanced disease stages and poor survival outcomes. Mechanistically, CDCA5 depletion in SiHa and HeLa cells suppressed proliferation, migration, and invasion, while its overexpression in CaSki cells enhanced these malignant properties. We identified E2F1 as a transcriptional activator of CDCA5. Importantly, CDCA5 knockdown significantly inhibited tumor growth in nude mouse models. Our findings establish CDCA5 as a critical E2F1-regulated oncogenic factor in cervical cancer progression. The strong correlation between CDCA5 expression and poor clinical outcomes suggests its potential as both a prognostic biomarker and therapeutic target in cervical cancer treatment.

Addition of PARP1-inhibition enhances chemoradiotherapy and thermoradiotherapy when treating cervical cancer in an in vivo mouse model

Background: Efficacy of current treatment options for cervical cancer require improvement. Previous in vitro studies have shown the enhancing effects of the addition of PARP1-inhibitors to chemoradiotherapy and thermoradiotherapy. The aim of our present study was to test efficacy of different combinations of treatment modalities radiotherapy, cisplatin, hyperthermia and PARP1-inhibitors using in vitro tumor models, ex vivo treated patient samples and in vivo tumor models. Materials and Methods: In vitro clonogenic survival curves (0–6 Gy) show that PARP1-i (4–5 M Olaparib) enhances both chemoradiotherapy (0.3–0.5 µM cisplatin) and thermoradiotherapy (42 °C for 1 h) in SiHa, CaSki and HeLa cells. A cervical cancer mouse model and freshly obtained in-house developed patient-derived organoids were used to examine the effects of different treatment combinations. For the in vivo study, human cervical cancer (SiHa) cells were injected in the right hind leg of athymic nude mice. In vivo mouse experiments show that PARP1-i enhances thermoradiotherapy or chemoradiotherapy by reduction of tumor volumes. Five cycles of treatment were applied with the following doses per cycle: irradiation 3 Gy, hyperthermia 1 h at 42 °C, cisplatin at 2 mg/kg, and twice PARP1-i at 50 mg/kg. Results: Quadruple treatment, combining radiotherapy, hyperthermia, cisplatin and PARP1-i, was very effective but also lead to severe side effects causing severe weight loss and death. In contrast, thermoradiotherapy or chemoradiotherapy with addition of PARP1-i, were effective without serious side effects. Conclusion: The triple combinations are promising options for potentially more effective treatment of locally advanced cervical cancer without more toxicity.

Combination of Vitex pseudo-negundo methanolic-extract with cisplatin can induce antioxidant activity and apoptosis in HeLa and Caski cells.

Cisplatin-based chemotherapy as a common therapeutic regimen for cervical cancer patients, is becoming more and more ineffective due to high resistance. This urges the need for introducing novel metabolics such as botanical drugs with the capacity to increase the cisplatin effectiveness. In that regard, here we investigated the anticancer effects of the Cisplatin-Vitex pseudo-Negundo combination in cervical cancer cell lines. V. pseudo-Negundo fruits were dried and extracted methanolic fraction. The MTT assay was performed to evaluate cytotoxicity of both drugs in CaSki and HeLa cells. Then, apoptosis, ROS production, and cell cycling were assessed by flow cytometry assay in cells treated with V. pseudo-Negundo and Cisplatin and their combination. Also, the rate of cell migration and colony formation were measured, using wound healing and colony formation assay, respectively. Also, the expression level of related genes (CD133, BAX, BCL2, Casp-3/8/9, MMP-3) was evaluated using the RT-PCR method. The obtained results established that the V. pseudo-Negundo plant has medicinal properties to induce apoptotic and antioxidant signals. The combination treatment of methanol extraction and Cisplatin had a cytotoxic effect on cervical cancer cell lines (HeLa and CaSki) compared to monotherapy. Also, combination therapy resulted in an increased apoptosis rate and diminished ROS production in both CaSki and HeLa cell lines. Furthermore, V. pseudo-Negundo and Cisplatin combination therapy leads to cell cycle arrest in the G2-M and G0-G1 phase in HeLa and CaSki cell lines, respectively. Moreover, combination therapy decreased the colony formation and cell motility in both cell lines and upregulated caspases gene expression. The combination of V. pseudo-Negundo with Cisplatin therapy results in a significant anti-cancer and antioxidant effect compared to cisplatin, representing a promising candidate for future clinical investigations.

Therapeutic efficacy of CRISPR RNA nanoparticles on cervical cancer disease using ultrasound elastography imaging

Therapeutic efficacy of CRISPR RNA nanoparticles on cervical cancer disease using ultrasound elastography imaging