Cell Biology and Toxicology

The pivotal role of ZNF384: driving the malignant behavior of serous ovarian cancer cells via the LIN28B/UBD axis

Zinc finger protein 384 (ZNF384) is a highly conserved transcribed gene associated with the development of multiple tumors, however, its role and mechanism in serous ovarian cancer (SOC) are unknown. We first confirmed that ZNF384 was abnormally highly expressed in SOC tissues by bioinformatics analysis and immunohistochemistry. We further used lentivirus packaging and transfection techniques to construct ZNF384 overexpression or knockdown cell lines, and through a series of cell function experiments, gradually verified that ZNF384 promoted a series of malignant behaviors of SOC cell proliferation, migration, and invasion. By establishing a xenotransplantation model in nude mice, it was confirmed that ZNF384 promoted the progress of SOC in vivo. Mechanistically, Overexpression of ZNF384 enhanced the transcriptional activity of Lin-28 homolog B (LIN28B), which promoted the malignant behavior of SOC cells. In addition, LIN28B could regulate the expression of the downstream factor ubiquitin D (UBD) in SOC cells, further promoting the development of SOC. This study shows that ZNF384 aggravates the malignant behavior of SOC cells through the LIN28B/UBD axis, which may be used as a diagnostic biomarker for patients with SOC.

The microRNA Let-7 and its exosomal form: Epigenetic regulators of gynecological cancers

AbstractMany types of gynecological cancer (GC) are often silent until they reach an advanced stage, and are therefore often diagnosed too late for effective treatment. Hence, there is a real need for more efficient diagnosis and treatment for patients with GC. During recent years, researchers have increasingly studied the impact of microRNAs cancer development, leading to a number of applications in detection and treatment. MicroRNAs are a particular group of tiny RNA molecules that regulate regular gene expression by affecting the translation process. The downregulation of numerous miRNAs has been observed in human malignancies. Let-7 is an example of a miRNA that controls cellular processes as well as signaling cascades to affect post-transcriptional gene expression. Recent research supports the hypothesis that enhancing let-7 expression in those cancers where it is downregulated may be a potential treatment option. Exosomes are tiny vesicles that move through body fluids and can include components like miRNAs (including let-7) that are important for communication between cells. Studies proved that exosomes are able to enhance tumor growth, angiogenesis, chemoresistance, metastasis, and immune evasion, thus suggesting their importance in GC management. Graphical Abstract Impact of let-7 on female malignancies and diseases of the female reproductive tract. Let-7 expression is dysregulated in a variety of gynaecological and obstetric disorders.

Methylsulfonylmethane sensitizes endometrial cancer cells to doxorubicin

Abstract Background Methylsulfonylmethane (MSM) is a commonly used diet supplement believed to decrease the inflammation in joints and fastens recovery in osteoarthritis, gastric mucosal injury, or obesity-related disorders. It was also suggested that MSM might play a beneficial role in cancer treatment. Purpose So far, the MSM might have a potentially beneficial effect in endometrial cancer (EC) treatment. Study design This study evaluated the effect and usefulness of MSM in combinatory therapy with known drug doxorubicin (DOX). Methods The effect of combinational treatment of MSM and DOX on the induction of apoptosis was evaluated in EC cell lines (ISHIKAWA, MFE-296, MFE-280). Results We observed that MSM itself induces apoptosis in EC cell lines, and pre-treatment with MSM for 24 h increases the sensitivity of EC cells to DOX-induced apoptosis and DNA damage and that effect might be regulated by p42/44 (Erk1/2) MAPK and Akt (protein kinase B). Conclusion These results for the first time show that MSM might act as a sensitizer of EC cells to known drugs, for which EC cells quickly acquire resistance.

RNA modification regulators as promising biomarkers in gynecological cancers

RNF144A suppresses ovarian cancer stem cell properties and tumor progression through regulation of LIN28B degradation via the ubiquitin-proteasome pathway

Cancer stem cells (CSCs) are the main driving force of tumorigenesis, metastasis, recurrence, and drug resistance in epithelial ovarian cancer (EOC). The current study aimed to explore the regulatory effects of ring finger protein 144A (RNF144A), an E3 ubiquitin ligase, in the maintenance of CSC properties and tumor development in EOC. The expressions of RNF144A in EOC tissue samples and cells were examined. The knockdown or overexpression of a target gene was achieved by transfecting EOC cells with short hairpin RNA or adenoviral vectors. A mouse xenograft model was constructed by inoculating nude mice with EOC cells. Co-immunoprecipitation was used to determine the interaction between RNF144A and LIN28B. Downregulated RNF144A expression was observed in ovarian tumor tissues and EOC cells. Low RNF144A expression was positively associated with poor survival of EOC patients. RNF144A knockdown significantly enhanced sphere formation and upregulated stem cell markers in EOC cells, while RNF144A overexpression prevented EOC cells from acquiring stem cell properties. Also, the upregulation of RNF144A inhibited ovarian tumor growth and aggressiveness in cell culture and mouse xenografts. Further analysis revealed that RNF144A induced LIN28B degradation through ubiquitination in EOC cells. LIN28B upregulation restored the expressions of stem cell pluripotency-associated transcription factors in EOC cells overexpressing RNF144A. Taken together, our findings highlight the therapeutic potential of restoring RNF144A expression and thereby suppressing LIN28B-associated oncogenic signaling for EOC treatment. • Ring finger protein 144A (RNF144A) is downregulated in epithelial ovarian cancer (EOC) tissues and cell lines. • The overexpression of RNF144A prevents EOC cells from acquiring stem cell properties and inhibits ovarian tumor growth. • RNF144A induces LIN28B degradation through ubiquitination in EOC cells. • LIN28B upregulation restores the expressions of stem cell pluripotency-associated transcription factors in EOC cells overexpressing RNF144A.

Caveolin-1-ACE2 axis modulates xenobiotic metabolism-linked chemoresistance in ovarian clear cell carcinoma

Among epithelial ovarian cancers, ovarian clear cell carcinoma (OCCC) remains markedly resistant to platinum-based chemotherapy, leading to poor clinical outcomes. In response to xenobiotic insults, caveolar platforms play crucial roles in modulating stress signaling responses in cancer cells. It has been hypothesized that caveolin-1 (Cav-1), a main component of the lipid raft, may regulate the response to platinum-based treatment in OCCC. The clinical transcriptomic evaluation demonstrated that high Cav-1 expression was positively associated with a favorable prognosis in patients with ovarian cancer. Cav-1 overexpression enhanced sensitivity to cisplatin (CDDP) treatment, whereas Cav-1 deficiency promoted chemoresistance in OCCC cells. Mechanistically, although Cav-1 counteracted angiotensin-converting enzyme 2 (ACE2) expression, ACE2 positively facilitated resistance to CDDP in OCCC cells. Furthermore, ACE2 restricted aryl hydrocarbon receptor expression and subsequent transcription of drug-metabolizing enzymes. Of note, ACE2 positively regulated the expression of the platinum-clearing enzyme CYP3A4. These findings suggest that the Cav-1-ACE2 axis modulates xenobiotic metabolism-linked chemoresistance in OCCC, predicting potential roles for the stress sentinel networks in oncogenic processes.

Long non-coding RNA CTSLP8 mediates ovarian cancer progression and chemotherapy resistance by modulating cellular glycolysis and regulating c-Myc expression through PKM2

Abstract Purpose Long non-coding RNAs (lncRNAs) play vital roles in tumor progression and resistance. Ovarian cancer (OC), a common gynecological cancer, is associated with poor prognosis as it can progress to peritoneal metastasis and develop resistance to chemotherapy. This study aimed to examine the role of lncRNAs in the development of chemotherapy resistance in OC. Methods The clinical samples were divided into chemotherapy-sensitive and chemotherapy-resistant groups based on the chemotherapy response at follow-up. The glycolysis levels in the two groups were analyzed using positron emission tomography/computed tomography (PET/CT) scanning and immunohistochemistry. GEO dataset analysis revealed the expression of CTSLP8 in chemotherapy-resistant patients with OC. Two pairs of normal and diamminodichloroplatinum (DDP)-resistant cells were transfected with CTSLP8 overexpression and knockdown constructs to examine the functions of CTSLP8 in the OC cells and elucidate the underlying mechanisms. The in vivo effect of CTSLP8 overexpression and knockdown on the chemotherapy response of tumors was examined using a mouse subcutaneous tumor model. The tissue chips were subjected to fluorescence in situ hybridization and immunohistochemical (IHC) staining to examine the correlation among CTSLP8 expression, DDP resistance, and prognosis in OC. Results The dataset analysis demonstrated that CTSLP8 was upregulated in chemotherapy-resistant tumor tissues. CTSLP8 promoted the proliferation and development of DDP resistance in the OC cells. Moreover, CTSLP8 promoted c-Myc expression by facilitating the binding of PKM2 to the promoter region of c-Myc, thereby upregulating glycolysis. The analysis of tissue chips revealed that the upregulation of CTSLP8 was associated with the development of DDP resistance and poor prognosis in patients with OC. Conclusions These findings indicate that CTSLP8 forms a complex with PKM2 to regulate c-Myc, and this action results in the upregulation of cellular glycolysis, consequently promoting OC progression and development of chemotherapy resistance. Headlights 1. CTSLP8 was upregulated in the chemotherapy-resistant tumor tissues. 2. CTSLP8 promoted the proliferation and cisplatin resistance in the OC cells. 3. CTSLP8 promoted glycolysis by facilitating the binding of PKM2 to the promoter region of c-Myc. 4. Inhibition of CTSLP8 or the combination of c-Myc inhibitors with cisplatin were potential therapeutic strategies for chemotherapy-resistant of OC.

Therapeutic implications of endoplasmic reticulum stress gene CCL3 in cervical squamous cell carcinoma

This study investigated ERS-related gene expressions in CESC, identifying two molecular subtypes, P1 and P2, and constructing a precise prognostic model based on these subtypes. TCGA's whole-genome expression profiles were used to recognize these subtypes through the ConsensusClusterPlus method, further refining prognostic models with univariate and Lasso Cox regression analyses validated by the GSE39001 dataset. The study analyzed the expression distribution of ERS marker genes within T cell subgroups using scRNA-seq data (GSE168652), highlighting T cell diversity. The critical role of the CCL3 gene in prognostic models was examined explicitly in CD8 + T cells from healthy individuals and CESC patients. Elevated CCL3 levels were observed in patients' CD8 + T cells compared to healthy controls. Functional experiments involving CCL3 knockdown and overexpression in HeLa and SiHa CESC cell lines were conducted to investigate its impact on cell proliferation, migration, and invasion. These findings were subsequently validated in a nude mouse model. The results demonstrated that suppressing CCL3 inhibited cell proliferation, migration, and invasion significantly, while its overexpression promoted these processes. In the mouse model, CCL3 silencing reduced tumor growth and decreased Ki-67 labeling within the tumor tissues, indicating the therapeutic potential of targeting CCL3 in CESC treatment, possibly through CD8 + T cell regulation. This study contributes new prognostic assessment tools and personalized treatment options for CESC patients, paving the way for more targeted therapies in CESC by discovering the CCL3 gene, presenting significant clinical implications.

ETV4 promotes ovarian cancer growth by regulating mitochondrial function through Mfn2 ubiquitination mediated by the E3 ubiquitin ligase MARCH9

Mitochondrial dysfunction affects the development of ovarian cancer (OC). ETV4 is involved in mitochondrial fusion. The regulatory pathways of ETV4 in OC cells have not been further investigated. In this study, we aimed to explore the effects of ETV4 on OC development and analyze the downstream regulatory pathways of ETV4. The expression of ETV4 in OC cell lines (SK-OV-3, HEY, A2780, and OVCAR-3) was verified. After silencing ETV4, indicators related to mitochondrial function, including ATP level, mitochondrial membrane potential, mitochondrial DNA (mtDNA), and mitochondrial ROS (mtROS), were analyzed. The expression of mitochondrial fission/fusion-related markers (Mfn1, Mfn2, OPA1, DRP1, MFF, and FIS1) was detected. In vivo experiments were used to verify the effect of ETV4 on OC development. The TCGA-OV data indicated that ETV4 was highly expressed in OC. Silencing ETV4 inhibited the proliferation of OC cells. Mitochondrial membrane potential and ATP levels increased after ETV4 silencing, while mtDNA and mtROS levels decreased. ETV4 silencing promoted Mfn2 protein expression but did not affect Mfn2 mRNA level. Mfn2-associated E3 ubiquitin ligase MARCH9 was targeted and regulated by ETV4. MARCH9 overexpression alleviated the regulation of ETV4 silencing on mitochondrial function in OC cells. Lysosomal inhibitor CQ blocked the degradation of ubiquitinated Mfn2 protein. MARCH9 was found to mediate robust ubiquitination of Mfn2 via the K63-linked ubiquitination. ETV4 was highly expressed in OC and involved in the regulation of mitochondrial function. ETV4 regulated Mfn2 ubiquitination linked by K63 by regulating MARCH9.

Advances in hybrid hydrogel design for biomedical applications: innovations in drug delivery and tissue engineering for gynecological cancers

The mechanism of sevoflurane affecting ovarian cancer cell proliferation and migration by regulating RNA methylase TRDMT1 to activate the β-catenin pathway

Sevoflurane (Sevo), a commonly used inhalant anesthetic clinically, is associated with a worsened cancer prognosis, and we investigated its effect on RNA methylase tRNA aspartic acid methyltransferase 1 (TRDMT1) expression and ovarian cancer (OC) cell malignant phenotypes. Human OC cells (OVCAR3/SKOV3) were pretreated with 3.6% Sevo and cultured under normal conditions for 48 h, with their viability assessed. After 2-h Sevo treatment or interference plasmid transfections to down-regulate TRDMT1/adenomatous polyposis coli (APC), changes in TRDMT1, APC and β-catenin expression, cell proliferative activity, cycle, apoptosis, migration, invasion, and 5-methylcytosine (m5C) methylation potential modification sites were evaluated. Additionally, APC mRNA m5C methylation level and stability, the binding of APC mRNA with TRDMT1, the binding intensity of APC and β-catenin, and β-catenin nuclear translocation were detected Lastly, Cyclin D1, cellular-myelocytomatosis viral oncogene (C-myc) and β-catenin protein levels, and ki67-positive rate were assessed. Sevo treatment boosted cell cycle, proliferation, migration and invasion, suppressed apoptosis and APC expression, and up-regulated C-myc, β-catenin, TRDMT1 and Cyclin D1 levels. Silencing TRDMT1 or β-catenin partially averted Sevo-mediated promotion effects on cell malignant biological behaviors. Lowly-expressed APC annulled the effect of silencing TRDMT1 and promoted cell malignant behaviors. Sevo enhanced APC mRNA m5C modification and degradation and activated the APC/β-catenin pathway by increasing TRDMT1, thus encouraging OC growth in vivo. Sevo stimulated APC m5C modification and curbed its expression by up-regulating TRDMT1, which in turn activated the β-catenin pathway to stimulate OC cell cycle, invasion, proliferation, and migration and to suppress apoptosis.

Sevoflurane but not propofol enhances ovarian cancer cell biology through regulating cellular metabolic and signaling mechanisms

AbstractPerioperative risk factors, including the choice of anesthetics, may influence ovarian cancer recurrence after surgery. Inhalational anesthetic sevoflurane and intravenous agent propofol might affect cancer cell metabolism and signaling, which, in turn, may influence the malignancy of ovarian cancer cells. The different effects between sevoflurane and propofol on ovarian cancer cell biology and underlying mechanisms were studied. Cultured ovarian cancer cells were exposed to 2.5% sevoflurane, 4 μg/mL propofol, or sham condition as the control for 2 h followed by 24-h recovery. Glucose transporter 1 (GLUT1), mitochondrial pyruvate carrier 1 (MPC1), glutamate dehydrogenase 1 (GLUD1), pigment epithelium-derived factor (PEDF), p-Erk1/2, and hypoxia-inducible factor 1-alpha (HIF-1α) expressions were determined with immunostaining and/or Western blot. Cultured media were collected for 1H-NMR spectroscopy-based metabolomics analysis. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to analyze metabolomics data. Sevoflurane increased the GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α expressions but decreased the PEDF expression relative to the controls. In contrast to sevoflurane, propofol decreased GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α but increased PEDF expression. Sevoflurane increased metabolite isopropanol and decreased glucose and glutamine energy substrates in the media, but the opposite changes were found after propofol treatment. Our data indicated that, unlike the pro-tumor property of sevoflurane, propofol negatively modulated PEDF/Erk/HIF-1α cellular signaling pathway and inhibited ovarian cancer metabolic efficiency and survival, and hence decreased malignancy. The translational value of this work warrants further study. Graphical abstract • Sevoflurane promoted but propofol inhibited ovarian cancer cell biology. • Sevoflurane upregulated but propofol downregulated the GLUT1, MPC1, and GLUD1 expressions of ovarian cancer cells. • Sevoflurane enhanced but propofol inhibited ovarian cancer cellular glucose. metabolism and glutaminolysis. • Sevoflurane downregulated PEDF but upregulated the Erk pathway and HIF-1α, while propofol had the adverse effects on ovarian cancer cells.

Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence

AbstractSenescence induction and epithelial-mesenchymal transition (EMT) events are the opposite sides of the spectrum of cancer phenotypes. The key molecules involved in these processes may get influenced or altered by genetic and epigenetic changes during tumor progression. Double C2-like domain beta (DOC2B), an intracellular vesicle trafficking protein of the double C2 protein family, plays a critical role in exocytosis, neurotransmitter release, and intracellular vesicle trafficking. DOC2B is repressed by DNA promoter hypermethylation and functions as a tumor growth regulator in cervical cancer. To date, the molecular mechanisms of DOC2B in cervical cancer progression and metastasis is elusive. Herein, the biological functions and molecular mechanisms regulated by DOC2B and its impact on senescence and EMT are described. DOC2B inhibition promotes proliferation, growth, and migration by relieving G0/G1-S arrest, actin remodeling, and anoikis resistance in Cal27 cells. It enhanced tumor growth and liver metastasis in nude mice with the concomitant increase in metastasis-associated CD55 and CD61 expression. Inhibition of EMT and promotion of senescence by DOC2B is a calcium-dependent process and accompanied by calcium-mediated interaction between DOC2B and CDH1. In addition, we have identified several EMT and senescence regulators as targets of DOC2B. We show that DOC2B may act as a metastatic suppressor by inhibiting EMT through induction of senescence via DOC2B-calcium-EMT-senescence axis. Graphical abstract

ZNF471 modulates EMT and functions as methylation regulated tumor suppressor with diagnostic and prognostic significance in cervical cancer

AbstractCervical cancer (CC) is a leading cause of cancer-related death among women in developing countries. However, the underlying mechanisms and molecular targets for therapy remain to be fully understood. We investigated the epigenetic regulation, biological functions, and clinical utility of zinc-finger protein 471 (ZNF471) in CC. Analysis of cervical tissues and five independent public datasets of CC showed significant hypermethylation of the ZNF471 gene promoter. In CC cell lines, promoter DNA methylation was inversely correlated with ZNF471 expression. The sensitivity and specificity of the ZNF471 hypermethylation for squamous intraepithelial lesion (SIL) vs tumor and normal vs tumor was above 85% with AUC of 0.937. High methylation and low ZNF471 expression predicted poor overall and recurrence-free survival. We identified −686 to +114 bp as ZNF471 promoter, regulated by methylation using transient transfection and luciferase assays. The promoter CpG site methylation of ZNF471 was significantly different among cancer types and tumor grades. Gal4-based heterologous luciferase reporter gene assays revealed that ZNF471 acts as a transcriptional repressor. The retroviral mediated overexpression of ZNF471 in SiHa and CaSki cells inhibited growth, proliferation, cell migration, invasion; delayed cell cycle progression in vitro by increasing cell doubling time; and reduced tumor growth in vivo in nude mice. ZNF471 overexpression inhibited key members of epithelial-mesenchymal transition (EMT), Wnt, and PI3K-AKT signaling pathways. ZNF471 inhibited EMT by directly targeting vimentin as analyzed by bioinformatic analysis, ChIP-PCR, and western blotting. Thus, ZNF471 CpG specific promoter methylation may determine the prognosis of CC and could function as a potential tumor suppressor by targeting EMT signaling.

m6A mRNA methylation regulates the ERK/NF-κB/AKT signaling pathway through the PAPPA/IGFBP4 axis to promote proliferation and tumor formation in endometrial cancer

N6-methyladenosine (m