Ovarian Cancer Cell Line SKOV3 Research Articles

Cell-cycle related centromere protein F deficiency suppresses ovarian cancer cells growth by inducing ferroptosis.

This study aimed to investigate the involvement of the cell cycle-related protein centriole protein F (CENPF) in the development of ovarian cancer (OC) and explored its relationship with ferroptosis. The databases were analyzed to identify differential expression of cell cycle-related proteins between individuals with ovarian cancer and normal individuals. Immunohistochemistry and statistical analysis were conducted on ovarian tissues obtained from 40 patients with epithelial ovarian cancer and 20 normal individuals. In vitro experiments were performed using SKOV3 and HEY epithelial ovarian cancer cell lines. The mRNA microarray data set, consisting of GSE14001, GSE54388, GSE40595, and GSE14407, was downloaded from the Gene Expression Omnibus (GEO) database to investigate the genes associated with cell cycle regulation in ovarian cancer cells. CENPF was selected as the subject of study through differential analysis. Assessed the expression of CENPF in both ovarian cancer (OC) patients and normal ovarian tissues using immunohistochemistry. Lentivirus infection was employed to downregulate CENPF expression, and subsequent experiments including Cell Counting Kit-8 (CCK-8) assay, cell cycle analysis, transwell assay, and wound-healing assay were conducted to investigate the effects of CENPF on proliferation, invasion, migration, and cell cycle regulation in OC cells. The Reactive oxygen species (ROS) and the malondialdehyde (MDA) assays were performed to assess the involvement of CENPF in cellular redox reactions. Western blot analysis was conducted to examine the expression levels of ferroptosis-related proteins (GPX4, SLC7A11, DMT1, and P53). By querying and integrating cell cycle-related genes from the GEO database, in silico analyses using The Cancer Genome Atlas (TCGA) database combined with immunohistochemical studies, we discovered that CENPF is upregulated in ovarian cancer tissues and is related to survival. Downregulation of CENPF inhibited biological function of OC cells, increased intracellular ROS and MDA levels, and downregulated the GPX4 protein and the SLC7A11/xCT protein, but upregulated the DMT1 protein and the tumor protein 53(P53) protein expression to induce ferroptosis. This study did not investigate ferroptosis-related studies following CENPF overexpression, and the findings have not been validated in animal studies. Our findings demonstrated that the deficiency of CENPF played a crucial anti-oncogenic role in the progression of OC through the mechanism of ferroptosis.

Intact cell mass spectrometry coupled with machine learning reveals minute changes induced by single gene silencing

Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.

Circ-RNF111 Promotes Proliferation of Ovarian Cancer Cell SKOV-3 by Targeting the MiR-556-5p/CCND1 Axis.

The aim of this study was to investigate the role and mechanism of circ-RNF111 in the human ovarian cancer cell line SKOV-3. First, qRT-PCR was used to detect circ-RNF111 and miR-556-5p expression levels in human normal ovarian epithelial cells IOSE80 and human ovarian cancer cells SKOV-3. CCK-8 and colony formation assays were adopted to determine the proliferation rate and cell viability of SKOV-3 cells, respectively. Additionally, in an attempt to reveal the mechanism of circ-RNF111, we predicted the targeting relationship between miR-556-5p and circ-RNF111 as well as miR-556-5p and CCND1 using the circinteractome and TargetScan databases, respectively, and validated their relationship by dual-luciferase reporter assay. The protein expression levels of CCND1 in SKOV-3 cells were detected by Western blot. Based on the above experiments, the expression of circ-RNF111 was found to be up-regulated in SKOV-3, and the knockdown of circ-RNF111 significantly inhibited the proliferation and viability of SKOV-3 cells. Then we confirmed that circ-RNF111 sponged miR-556-5p in SKOV-3 cells to up-regulate CCND1 expression. In addition, simultaneous inhibition of miR-556-5p or overexpression of CCND1 in SKOV-3 cells with knockdown of circ-RNF111 reversed the inhibitory effect of knockdown of circ-RNF111 on the protein expression level of CCND1, cell proliferation rate, and cell viability. In summary, circ-RNF111 promotes the proliferation of SKOV-3 cells by targeting the miR-556-5p/CCND1 axis. Circ-RNF111 may serve as a potential target for ovarian cancer therapy.

Environmental physical factors produce mitotic aberration and multicellularity in the ovarian cancer cell line SKOV3

The spread of ovarian cancer (OC) to the coelomic cavity triggers the secretion and accumulation of ascitic fluid (AF). Although its biochemical composition has been well studied, less is known about the implications of physical factors such as the pH and the mechanical properties of the AF for the malignancy of tumor cells. In this work, we investigated the effect of pH and the mechanical properties of AF on cell proliferation and mitotic morphology. We employed biopsies from patients with OC and the SKOV3 cell line as an in vitro model of OC with HeLa cells as controls. Sections of each tumor were stained with HE, analyzed, and related to clinical data. AF from patients with OC exhibited an alkaline pH (ranging from 7.3 to 7.8). Compared to control conditions, the 3 AFs significantly enhanced the proliferation of SKOV3 and HeLa cells. These effects were more pronounced at a more alkaline pH. In addition, we found that AFs have different densities that correlated with a significant increase in multinucleated tumor cells and severe morphological defects in cells undergoing mitosis. In agreement with these data, we found that higher concentrations of soft agar provoked significantly higher numbers of multinucleated and morphologically abnormal SKOV3 cells with no effect on HeLa cells.We conclude that an alkaline pH and greater rigidity could enhance the metastatic potential of OC cells. We propose that these two physical factors could be parameters of clinical importance as predictors of malignancy.

Pb-214/Bi-214-TCMC-Trastuzumab inhibited growth of ovarian cancer in preclinical mouse models

Introduction: Better treatments for ovarian cancer are needed to eliminate residual peritoneal disease after initial debulking surgery. The present study evaluated Trastuzumab to deliver Pb-214/Bi-214 for targeted alpha therapy (TAT) for HER2-positive ovarian cancer in mouse models of residual disease. This study is the first report of TAT using a novel Radon-222 generator to produce short-lived Lead-214 (Pb-214, t1/2 = 26.8 min) in equilibrium with its daughter Bismuth-214 (Bi-214, t1/2 = 19.7 min); referred to as Pb-214/Bi-214. In this study, Pb-214/Bi-214-TCMC-Trastuzumab was tested.Methods: Trastuzumab and control IgG antibody were conjugated with TCMC chelator and radiolabeled with Pb-214/Bi-214 to yield Pb-214/Bi-214-TCMC-Trastuzumab and Pb-214/Bi-214-TCMC-IgG1. The decay of Pb-214/Bi-214 yielded α-particles for TAT. SKOV3 and OVAR3 human ovarian cancer cell lines were tested for HER2 levels. The effects of Pb-214/Bi-214-TCMC-Trastuzumab and appropriate controls were compared using clonogenic assays and in mice bearing peritoneal SKOV3 or OVCAR3 tumors. Mice control groups included untreated, Pb-214/Bi-214-TCMC-IgG1, and Trastuzumab only.Results and discussion: SKOV3 cells had 590,000 ± 5,500 HER2 receptors/cell compared with OVCAR3 cells at 7,900 ± 770. In vitro clonogenic assays with SKOV3 cells showed significantly reduced colony formation after Pb-214/Bi-214-TCMC-Trastuzumab treatment compared with controls. Nude mice bearing luciferase-positive SKOV3 or OVCAR3 tumors were treated with Pb-214/Bi-214-TCMC-Trastuzumab or appropriate controls. Two 0.74 MBq doses of Pb-214/Bi-214-TCMC-Trastuzumab significantly suppressed the growth of SKOV3 tumors for 60 days, without toxicity, compared with three control groups (untreated, Pb-214/Bi-214-TCMC-IgG1, or Trastuzumab only). Mice-bearing OVCAR3 tumors had effective therapy without toxicity with two 0.74 MBq doses of Pb-214/Bi-214-TCMC-trastuzumab or Pb-214/Bi-214-TCMC-IgG1. Together, these data indicated that Pb-214/Bi-214 from a Rn-222 generator system was successfully applied for TAT. Pb-214/Bi-214-TCMC-Trastuzumab was effective to treat mouse xenograft models. Advantages of Pb-214/Bi-214 from the novel generator systems include high purity, short half-life for fractioned therapy, and hourly availability from the Rn-222 generator system. This platform technology can be applied for a variety of cancer treatment strategies.

Synthesis and biological evaluation of selective Pepstatin based trifluoromethylated inhibitors of Cathepsin D

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Suitable reference gene for silencing methods using microRNA encapsulated nanoparticles chitosan for the ovarian cancer cell line

MicroRNAs (miRNAs) are key regulators of gene expression, and their accurate quantification is essential for elucidating miRNA functional roles. Quantitative reverse transcription PCR (qRT-PCR) is widely utilized for miRNA expression analyses, although appropriate data normalization is critical for generating reliable results. The present study aimed to identify a suitable reference gene for normalizing miRNA qRT-PCR data in the ovarian cancer cell line SKOV-3. SKOV-3 cells were treated with miRNA-loaded chitosan nanoparticles or left untreated as a control. Total RNA, including small RNAs, was extracted from SKOV-3 cells and assessed for quality. The expression stability of 15 candidate reference genes was evaluated by GeNorm analysis. miR-584-5p was identified as the most stably expressed reference gene. To validate miRNA profiling, miR-584-5p was used to normalize expression levels, revealing distinct patterns of differential miRNA expression. Our findings provide critical insights into aberrant miRNA expression in ovarian cancer cells and underscore proper normalization as essential for accurate quantification of miRNAs by qRT-PCR.

Silibinin Induces Apoptosis and G2/M Cell Cycle Arrest in Human Ovarian Cancer SKOV-3 Cell Line

Background: Silibinin, an herbal polyphenolic flavonolignan, has antioxidant and anticancer properties. Objectives: This study aimed to evaluate some cellular and molecular effects of silibinin on the human ovarian cancer SKOV-3 cell line. Methods: For cytotoxicity investigations of silibinin, MTT assay was used at 24, 48, and 72 hours. Apoptosis and cell cycle were studied by flow cytometry. The effect of silibinin on mRNA expression of B-cell lymphoma 2 (Bcl-2), cyclin E, and S-phase kinase-associated protein 2 (SKP2) was determined by Quantitative reverse transcription polymerase chain reaction (QRT-PCR). Results: Silibinin administration in lower concentrations (12.5 and 25 µg/mL) did not lead to significant (P < 0.05) changes in cell viability and even slightly increased cell growth after 72 hours. However, silibinin in higher concentrations (≥ 50µg/mL) inhibited SKOV-3 cell proliferation in a dose- and time-dependent manner. The mode of cell growth inhibition was apoptosis induction and G2/M cell cycle arrest. Silibinin caused down-regulation of the anti-apoptotic gene, namely Bcl-2. Additionally, silibinin resulted in down-regulation of the major genes in the cell cycle, including cyclin E and SKP2. Conclusions: Overall, this study confirmed the ability of silibinin to suppress ovarian cancer progression through the induction of apoptosis and inhibition of G2/M phase transition. Silibinin may be considered an efficient and safe herbal medication for ovarian cancer.

Catecholamines Promote Ovarian Cancer Progression through Secretion of CXC-Chemokines.

Considerable evidence has accumulated in the last decade supporting the notion that chronic stress is closely related to the growth, metastasis, and angiogenesis of ovarian cancer. In this study, we analyzed the conditioned media in SKOV3 ovarian cancer cell lines treated with catecholamines to identify secreted proteins responding to chronic stress. Here, we observed that epinephrine and norepinephrine enhanced the secretion and mRNA expression of CXC-chemokines (CXCL1, 2, 3, and 8). Neutralizing antibodies to CXCL8 and CXCL8 receptor (CXCR2) inhibitors significantly reduced catecholamine-mediated invasion of SKOV3 cells. Finally, we found that the concentration of CXCL1 and CXCL8 in the plasma of ovarian cancer patients increased with stage progression. Taken together, these findings suggest that stress-related catecholamines may influence ovarian cancer progression through the secretion of CXC-chemokines.

Small Extracellular Vesicles from adipose-derived stem cells suppress cell proliferation by delivering the let-7 family of microRNAs in ovarian cancer

IntroductionOvarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines. Materials and methodsADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis. ResultsADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients. ConclusionADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs.

Exosomes derived from ovarian cancer cells regulate proliferation and migration of cancer-associated fibroblasts

Cancer-associated fibroblast (CAF) is an essential risk factor for ovarian cancer. Exosomes can mediate cellular communication in the tumour microenvironment, but the interaction of tumour cell exosomes with CAF is less studied in Ovarian cancer. This study identified H19/miR-29c-3p/LOXL2-COL1A1 as a ceRNA regulatory network involved in regulating tumour matrix-associated signaling pathways associated with CAF. Cellular assays demonstrated that exosomes from ovarian cancer cell line SKOV3 significantly promoted the proliferation and migration of CAF. The results of mixed transplantation tumour experiments in nude mice showed that exosomes of SKOV3 significantly promoted tumour growth. Ovarian cancer tumour-derived exosomes can regulate CAF proliferation and migration through H19/miR-29c-3p/LOXL2-COL1A1. This study reveals the regulatory role of tumour exosomes on CAF, which may provide a theoretical basis for the development of therapeutic regimens targeting fibroblasts in ovarian cancer.

Chromosome instability region analysis and identification of the driver genes of the epithelial ovarian cancer cell lines A2780 and SKOV3.

Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers worldwide. The molecular mechanisms of serous ovarian cancer (SOC) remain unclear and not well understood. SOC cases are primarily diagnosed at the late stage, resulting in a poor prognosis. Advances in molecular biology techniques allow us to obtain a better understanding of precise molecular mechanisms and to identify the chromosome instability region and key driver genes in the carcinogenesis and progression of SOC. Whole-exome sequencing was performed on the normal ovarian cell line IOSE80 and the EOC cell lines SKOV3 and A2780. The single-nucleotide variation burden, distribution, frequency and signature followed the known ovarian mutation profiles, without chromosomal bias. Recurrently mutated ovarian cancer driver genes, including LRP1B, KMT2A, ARID1A, KMT2C and ATRX were also found in two cell lines. The genome distribution of copy number alterations was found by copy number variation (CNV) analysis, including amplification of 17q12 and 4p16.1 and deletion of 10q23.33. The CNVs of MED1, GRB7 and MIEN1 located at 17q12 were found to be correlated with the overall survival of SOC patients (MED1: p = 0.028, GRB7: p = 0.0048, MIEN1: p = 0.0051), and the expression of the three driver genes in the ovarian cell line IOSE80 and EOC cell lines SKOV3 and A2780 was confirmed by western blot and cell immunohistochemistry.

Ginsenoside 20(S)-Rg3 reduces KIF20A expression and promotes CDC25A proteasomal degradation in epithelial ovarian cancer

BackgroundGinsenoside 20(S)-Rg3 shows promising tumor-suppressive effects in ovarian cancer via inhibiting NF-κB signaling. This study aimed to explore the downstream tumor suppressive mechanisms of ginsenoside Rg3 via this signaling pathway. Materials and methodsA systematical screening was applied to examine the expression profile of 41 kinesin family member genes in ovarian cancer. The regulatory effect of ginsenoside Rg3 on KIF20A expression was studied. In addition, we explored interacting proteins of KIF20A and their molecular regulations in ovarian cancer. RNA-seq data from The Cancer Genome Atlas (TCGA) was used for bioinformatic analysis. Epithelial ovarian cancer cell lines SKOV3 and A2780 were used as in vitro and in vivo cell models. Commercial human ovarian cancer tissue arrays were used for immunohistochemistry staining. ResultsKIF20A is a biomarker of poor prognosis among the kinesin genes. It promotes ovarian cancer cell growth in vitro and in vivo. Ginsenoside Rg3 can suppress the transcription of KIF20A. GST pull-down and co-immunoprecipitation (IP) assays confirmed that KIF20A physically interacts with BTRC (β-TrCP1), a substrate recognition subunit for SCFβ−TrCP E3 ubiquitin ligase. In vitro ubiquitination and cycloheximide (CHX) chase assays showed that via interacting with BTRC, KIF20A reduces BTRC-mediated CDC25A poly-ubiquitination and enhances its stability. Ginsenoside Rg3 treatment partly abrogates KIF20A overexpression-induced CDC25A upregulation. ConclusionThis study revealed a novel anti-tumor mechanism of ginsenoside Rg3. It can inhibit KIF20A transcription and promote CDC25A proteasomal degradation in epithelial ovarian cancer.

Synthesis, characterization, and anticancer properties of iminopyridine platinum(II) complexes containing long aliphatic chains

A series of iminopyridine ligands were prepared from the condensation reaction of 6-methylpyridine-2-carboxaldehyde and long-chain aliphatic primary amines. Square planar platinum(II) complexes were synthesized from the reaction of [PtCl2(η2-coe)]2 (coe = cis-cyclooctene) with two equivalents of ligand. The synthesis of the platinum compounds appears to go through a five-coordinate trigonal pyramidal species where coe remains coordinated initially but eventually dissociates to afford the four-coordinate complexes. Ligands and platinum complexes were characterized using multi-nuclear NMR and FT-IR spectroscopies as well as an X-ray diffraction study for the platinum complex derived from n-hexylamine. The cytotoxic properties of the platinum complexes against the ovarian cancer cell line SKOV-3 using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method were examined. Relative cytotoxicities of the platinum complexes follow a trend of greater potency with increasing chain length against the SKOV-3 cell line, with the longest aliphatic hydrocarbon chain (C16) exhibiting anticancer effects comparable to that of cisplatin.

Domatinostat Targets the FOXM1-Survivin Axis to Reduce the Viability of Ovarian Cancer Cells Alone and in Combination with Chemotherapeutic Agents.

The deregulation of the FOXM1 transcription factor is a key molecular alteration in ovarian cancer, contributing to the development and progression of ovarian cancer via activation of the target genes. As such, FOXM1 is a highly attractive therapeutic target in the treatment of ovarian cancer, but there has been no clinically tested FOXM1 inhibitor to date. We investigated in this study the effects of domatinostat, a class I-selective HDAC inhibitor currently in the clinical stage of development as a cancer therapeutic, on the expression of FOXM1 and viability of ovarian cancer cells. Cell viability, as well as protein and mRNA expression of FOXM1 and its transcriptional target survivin, was examined after domatinostat treatment of TOV21G and SKOV3 ovarian cancer cell lines in the absence or presence of cisplatin and paclitaxel. The effect of FOXM1 knockdown on survivin expression and those of genetic and pharmacological inhibition of survivin alone or in combination with the chemotherapeutic agents on cell viability were also examined. Domatinostat reduced the protein and mRNA expression of FOXM1 and survivin and also the viability of ovarian cancer cells alone and in combination with cisplatin or paclitaxel at clinically relevant concentrations. Knockdown experiments showed survivin expression was dependent on FOXM1 in ovarian cancer cells. Survivin inhibition was sufficient to reduce the viability of ovarian cancer cells alone and in combination with the chemotherapeutic agents. Our findings suggest that domatinostat, which effectively targets the FOXM1-survivin axis required for the viability of ovarian cancer cells, is a promising option for the treatment of ovarian cancer.

Abstract 5556: Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer

Abstract The objective of these in vitro and in vivo studies was to validate Lemur Tyrosine Kinase 3 (LMTK3) as a specific target and predictor of clinical outcome in ovarian cancer. LMTK3 belongs to a family of regulated tyrosine kinases with three structurally related isoforms, LMTK1, LMTK2, and LMTK3. Both nuclear and cytoplasmic LMTK3 expressions correlated with tumor grade and patient survival in cancers such as breast and colorectal cancer. We tested the clinical significance of the LMTK3 gene by immunohistochemistry (IHC) using LMTK3 monoclonal antibody on formalin-fixed paraffin-embedded sections (FFPE) collected from 204 early-stage (stage I-II) ovarian cancer patients. Results from this IHC LMTK3 study revealed a higher cytoplasmic to nuclear localization of LMTK3 correlated with worse overall survival (P<0.01). Further investigation of LMTK3’s prognostic value by screening LMTK3 signaling in 270 stage III-IV ovarian cancer patients is ongoing. We utilized the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for testing the killing efficacy of targeting LMTK3 by a monoclonal antibody, siRNA, and specific LMTK3 binding peptides (LMTK3BP) in ovarian cancer cell lines SKOV3, MDAH-2774, A2780, and TOV-21G. Treatment with LMTK3 specific monoclonal antibody, siRNA, and LMTK3BP significantly induced killing of both chemosensitive and chemoresistant ovarian cancer cells without affecting normal cells in vitro. Moreover, we observed this killing as synergistic with both Cisplatin and Taxotere treatment in vitro. Lastly, we used an A2780 cell line derived orthotopic xenograft mouse model of ovarian cancer to test the efficacy of specific LMTK3BP in vivo. Strikingly, LMTK3BP 20 mg/kg IV dose given three times a week for three weeks showed a 35% tumor reduction. Furthermore, in vivo safety studies showed no signs of toxicity of the LMTK3BP, even at a very high dose of 40 mg/kg. Thus, with dose optimization in ongoing experiments, we could expect a higher efficacy. In conclusion, this study highlighted the importance of LMTK3 as a predictor of patient clinical outcome and potential target for treatment in ovarian cancer. Citation Format: Thea K. Kirsch-Mangu, Axel S. Tullberg, Anna Portela, Khalil Helou, Ghassan M. Saed. Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5556.

SPOCK1 Overexpression Suggests Poor Prognosis of Ovarian Cancer

Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been found in a variety of malignant tumors and is associated with a poor prognosis. We aimed to explore the role of SPOCK1 in ovarian cancer. Ovarian cancer cell lines SKOV3 and SW626 were transfected with SPOCK1 overexpressing or empty vector using electroporation. Cells were studied by immunostaining and an automated Western blotting system. BrdU uptake and wound healing assays assessed cell proliferation and migration. SPOCK1 expression in human ovarian cancer tissues and in blood samples were studied by immunostaining and ELISA. Survival of patients with tumors exhibiting low and high SPOCK1 expression was analyzed using online tools. Both transfected cell lines synthesized different SPOCK1 variants; SKOV3 cells also secreted the proteoglycan. SPOCK1 overexpression stimulated DNA synthesis and cell migration involving p21CIP1. Ovarian cancer patients had increased SPOCK1 serum levels compared to healthy controls. Tumor cells of tissues also displayed abundant SPOCK1. Moreover, SPOCK1 levels were higher in untreated ovarian cancer serum and tissue samples and lower in recipients of chemotherapy. According to in silico analyses, high SPOCK1 expression was correlated with shorter survival. Our findings suggest SPOCK1 may be a viable anti-tumor therapeutic target and could be used for monitoring ovarian cancer.

Design, Synthesis and Antitumor Activity of Novel pyran-functionalized Uracil Derivatives: Docking Studies

Aim: Synthesis of novel pyran-based uracils that may have potent antitumor activity against hepatocellular carcinoma HepG2 and ovarian cancer SKOV3 cell lines. Materials & methods: Novel pyran-based uracils were synthesized and their anticancer activity was assessed using methyl thiazolyl tetrazolium and wound-healing assays to detect their cytotoxicity and their antiproliferative and antimigratory activities. Results: Compounds 3, 5, 6, 7, 8, 9, 10, 11 and 13 significantly inhibited cell proliferation of the HepG2 cell line. Compounds 7, 8, 9 and 13 significantly inhibited the proliferation of SKOV3 cells, which was also proven through docking studies with topoisomerase I. Conclusion: The molecular docking analysis revealed that 7 and 9 are two major compounds found to possess higher degrees of interaction with DNA gyrase.

SAMHD1 expression modulates innate immune activation and correlates with ovarian cancer prognosis.

SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase which has been proposed as a putative prognostic factor in haematological cancers and certain solid tumours, although with controversial data. Here, we evaluate SAMHD1 function in ovarian cancer, both in vitro and in ovarian cancer patients. SAMHD1 expression was downregulated in ovarian cancer cell lines OVCAR3 and SKOV3 by RNA interference. Gene and protein expression changes in immune signalling pathways were assessed. SAMHD1 expression in ovarian cancer patients was evaluated by immunohistochemistry and survival analysis was performed according to SAMHD1 expression. SAMHD1 knockdown induced a significant upregulation of proinflammatory cytokines concomitant to increased expression of the main RNA-sensors, MDA5 and RIG-I, and interferon-stimulated genes, supporting the idea that the absence of SAMHD1 promotes innate immune activation in vitro. To assess the contribution of SAMHD1 in ovarian cancer patients, tumours were stratified in SAMHD1-low and SAMHD1-high expressing tumours, resulting in significantly shorter progression free survival (PFS) and overall survival (OS) in SAMHD1-high expression subgroup (p=0.01 and 0.04, respectively). SAMHD1 depletion correlates with increased innate immune cell signalling in ovarian cancer cells. In clinical samples, SAMHD1-low expressing tumors showed increased progression free survival and overall survival irrespective of BRCA mutation status. These results point towards SAMHD1 modulation as a new therapeutic strategy, able to enhance innate immune activation directly in tumour cells, leading to improved prognosis in ovarian cancer.