IL-6 regulates epithelial ovarian cancer EMT, invasion, and metastasis by modulating Let-7c and miR-200c through the STAT3/HIF-1α pathway.

Interleukin-6 (IL-6) and hypoxia-inducible factor-1α (HIF-1α) play important roles in epithelial-mesenchymal transformation (EMT) and tumor development. Previous studies have demonstrated that IL-6 promotes EMT, invasion, and metastasis in epithelial ovarian cancer (EOC) cells by activating the STAT3/HIF-1α pathway. MicroRNA (miRNA) is non-coding small RNAs that also play an important role in tumor development. Notably, Let-7 and miR-200 families are prominently altered in EOC. However, whether IL-6 regulates the expression of Let-7 and miR-200 families through the STAT3/HIF-1α signaling to induce EMT in EOC remains poorly understood. In this study, we conducted in vitro and in vivo investigations using two EOC cell lines, SKOV3, and OVCAR3 cells. Our findings demonstrate that IL-6 down-regulates the mRNA levels of Let-7c and miR-200c while up-regulating their target genes HMGA2 and ZEB1 through the STAT3/HIF-1α signaling in EOC cells and in vivo. Additionally, to explore the regulatory role of HIF-1α on miRNAs, both exogenous HIF blockers YC-1 and endogenous high expression or inhibition of HIF-1α can be utilized. Both approaches can confirm that the downstream molecule HIF-1α inhibits the expression and function of Let-7c and miR-200c. Further mechanistic research revealed that the overexpression of Let-7c or miR-200c can reverse the malignant evolution of EOC cells induced by IL-6, including EMT, invasion, and metastasis. Consequently, our results suggest that IL-6 regulates the expression of Let-7c and miR-200c through the STAT3/HIF-1α pathway, thereby promoting EMT, invasion, and metastasis in EOC cells.

Spotlight on New Hallmarks of Drug-Resistance towards Personalized Care for Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy worldwide. Despite the latest advances, a major clinical issue in EOC is the disappointing prognosis related to chemoresistance in almost one-third of cases. Drug resistance relies on heterogeneous cancer stem cells (CSCs), endowed with tumor-initiating potential, leading to relapse. No biomarkers of chemoresistance have been validated yet. Recently, major signaling pathways, micro ribonucleic acids (miRNAs), and circulating tumor cells (CTCs) have been advocated as putative biomarkers and potential therapeutic targets for drug resistance. However, further investigation is mandatory before their routine implementation. In accordance with the increasing rate of therapeutic efforts in EOC, the need for biomarker-driven personalized therapies is growing. This review aims to discuss the emerging hallmarks of drug resistance with an in-depth insight into the underlying molecular mechanisms lacking so far. Finally, a glimpse of novel therapeutic avenues and future challenges will be provided.

Deregulated circRNAs in Epithelial Ovarian Cancer With Activity in Preclinical In Vivo Models: Identification of Targets and New Modalities for Therapeutic Intervention.

Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.

ZSWIM4 inhibition improves chemosensitivity in epithelial ovarian cancer cells by suppressing intracellular glycine biosynthesis.

BACKGROUND: Zinc finger SWIM-type containing 4 (ZSWIM4) induces drug resistance in breast cancer cells. However, its role in epithelial ovarian cancer (EOC) remains unknown. In this study, we aimed to investigate the clinical significance of ZSWIM4 expression in EOC and develop new clinical therapeutic strategies for EOC. METHODS: ZSWIM4 expression in control and EOC tumor tissues was examined using immunohistochemistry. Lentiviral transduction, Cell Counting Kit-8 assay, tumorsphere formation assay, flow cytometry, western blotting, and animal xenograft model were used to assess the role of ZSWIM4 in chemotherapy. Cleavage Under Targets and Tagmentation (CUT&Tag) assays, chromatin immunoprecipitation assays, and luciferase reporter assays were used to confirm FOXK1-mediated upregulation of ZSWIM4 expression. The mechanism by which ZSWIM4 inhibition improves chemosensitivity was evaluated using RNA-sequencing. A ZSWIM4-targeting inhibitor was explored by virtual screening and surface plasmon resonance analysis. Patient-derived organoid (PDO) models were constructed from EOC tumor tissues with ZSWIM4 expression. RESULTS: ZSWIM4 was overexpressed in EOC tumor tissues and impaired patient prognoses. Its expression correlated positively with EOC recurrence. ZSWIM4 expression was upregulated following carboplatin treatment, which, in turn, contributed to chemoresistance. Silencing ZSWIM4 expression sensitized EOC cells to carboplatin treatment in vitro and in vivo. FOXK1 could bind to the GTAAACA sequence of the ZSWIM4 promoter region to upregulate ZSWIM4 transcriptional activity and FOXK1 expression increased following carboplatin treatment, leading to an increase in ZSWIM4 expression. Mechanistically, ZSWIM4 knockdown downregulated the expression of several rate-limiting enzymes involved in glycine synthesis, causing a decrease in intracellular glycine levels, thus enhancing intracellular reactive oxygen species production induced by carboplatin treatment. Compound IPN60090 directly bound to ZSWIM4 protein and exerted a significant chemosensitizing effect in both EOC cells and PDO models. CONCLUSIONS: ZSWIM4 inhibition enhanced EOC cell chemosensitivity by ameliorating intracellular glycine metabolism reprogramming, thus providing a new potential therapeutic strategy for EOC.

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5ß1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling.

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

EGFR, HLA-G, CD70, c-MET, and NY-ESO1 as potential biomarkers in high grade epithelial ovarian carcinoma.

High grade epithelial ovarian carcinoma is an aggressive tumor. Treatment includes platinum therapy, however it recurs in most patients due to therapy resistance. In this project, we study the immunohistochemical (IHC) expression of five potential biomarkers/prognostic markers in high grade epithelial ovarian carcinoma: EGFR, HLA-G, CD70, c-MET, and NY-ESO1. A cohort of 274 patients is used. We compare the IHC expression with age, stage, ascites status, family history of cancer, disease free survival (DFS) and overall survival (OS). EGFR expression is significantly correlated with family history and worse OS. HLA-G is associated with worse OS. To confirm the results of EGFR and HLA-G, a second separated cohort of 248 patients is used. Positive EGFR expression again shows worse OS, while HLA-G expression has worse prognostic trend. CD70 has a worse OS trend. C-MET and NY-ESO1 do not have any clinical correlations. EGFR can potentially serve as target in future clinical immune therapy trials.

CCT2 prevented ß-catenin proteasomal degradation to sustain cancer stem cell traits and promote tumor progression in epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is featured by rapid progression and dismal outcomes clinically. Chaperonin Containing TCP1 Subunit 2 (CCT2) was identified as a crucial regulator for tumor progression, however, its exact role in EOC remained largely unknown. METHODS: CCT2 expression and prognostic value in EOC samples were assessed according to TCGA dataset. Proliferation and mobility potentials were assessed by CCK8, colony-formation, wound healing, and Transwell assays. Cancer stem cell (CSC) traits were evaluated by RT-PCR, WB assays, sphere-forming assay and chemoresistance analysis. Bioinformatic analysis, co-IP assays and ubiquitin assays were performed to explore the mechanisms of CCT2 on EOC cells. RESULTS: CCT2 highly expressed in EOC tissues and predicted poor prognosis of EOC patients by TCGA analysis. Silencing CCT2 significantly restrained cell proliferation, migration, and invasion. Moreover, CCT2 could effectively trigger epithelial-mesenchymal transition to confer extensive invasion potentials to EOC cells, Importantly, CCT2 positively correlated with CSC markers in EOC, and CCT2 knockdown impaired CSC traits and sensitize EOC cells to conventional chemotherapy regimens. Contrarily, overexpressing CCT2 achieved opposite results. Mechanistically, CCT2 exerted its pro-oncogene function by triggering Wnt/ß-catenin signaling. Specifically, CCT2 could recruit HSP105-PP2A complex, a well-established dephosphorylation complex, to ß-catenin via direct physical interaction to prevent phosphorylation-induced proteasomal degradation of ß-catenin, resulting in intracellular accumulation of active ß-catenin and increased signaling activity. CONCLUSIONS: CCT2 was a novel promotor for EOC progression and a crucial sustainer for CSC traits mainly by preventing ß-catenin degradation. Targeting CCT2 may represent a promising therapeutic strategy for EOC.

Loss of LECT2 promotes ovarian cancer progression by inducing cancer invasiveness and facilitating an immunosuppressive environment.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that can bind to several receptors and mediate distinct molecular pathways in various cell settings. Changing levels of LECT2 have been implicated in multiple human disease states, including cancers. Here, we have demonstrated reduced serum levels of LECT2 in patients with epithelial ovarian cancer (EOC) and down-regulated circulating Lect2 as the disease progresses in a syngeneic mouse ID8 EOC model. Using the murine EOC model, we discovered that loss of Lect2 promotes EOC progression by modulating both tumor cells and the tumor microenvironment. Lect2 inhibited EOC cells' invasive phenotype and suppressed EOC's transcoelomic metastasis by targeting c-Met signaling. In addition, Lect2 downregulation induced the accumulation and activation of myeloid-derived suppressor cells (MDSCs). This fostered an immunosuppressive microenvironment in EOC by inhibiting T-cell activation and skewing macrophages toward an M2 phenotype. The therapeutic efficacy of programmed cell death-1 (PD-1)/PD-L1 pathway blockade for the ID8 model was significantly hindered. Overall, our data highlight multiple functions of Lect2 during EOC progression and reveal a rationale for synergistic immunotherapeutic strategies by targeting Lect2.

[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity. Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs). Results: Activated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature. Conclusion: These data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches.

Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer.

Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.

Patient-derived organoid culture in epithelial ovarian cancers-Techniques, applications, and future perspectives.

Epithelial ovarian cancer (EOC) is a heterogeneous disease composed of different cell types with different molecular aberrations. Traditional cell lines and mice models cannot recapitulate the human tumor biology and tumor microenvironment (TME). Patient-derived organoids (PDOs) are freshly derived from patients' tissues and are then cultured with extracellular matrix and conditioned medium. The high concordance of epigenetic, genomic, and proteomic landscapes between the parental tumors and PDOs suggests that PDOs can provide more reliable results in studying cancer biology, allowing high throughput drug screening, and identifying their associated signaling pathways and resistance mechanisms. However, despite having a heterogeneity of cells in PDOs, some cells in TME will be lost during the culture process. Next-generation organoids have been developed to circumvent some of the limitations. Genetically engineered organoids involving targeted gene editing can facilitate the understanding of tumorigenesis and drug response. Co-culture systems where PDOs are cultured with different cell components like immune cells can allow research using immunotherapy which is otherwise impossible in conventional cell lines. In this review, the limitations of the traditional in vitro and in vivo assays, the use of PDOs, the challenges including some tips and tricks of PDO generation in EOC, and the future perspectives, will be discussed.

LAMA5 promotes cell proliferation and migration in ovarian cancer by activating Notch signaling pathway.

LAMA5 (laminin α5) is a member of the laminin family. Despite the recent research implicating LAMA5 in cancer, the function of LAMA5 has remained uncertain in the progression of ovarian cancer (OC). Here, we investigated the functional influences of LAMA5 knockdown on OC in vitro and in vivo. In this study, we used immunohistochemistry (IHC) analysis to detect the relative expression of LAMA5 in OC and non-cancer tissues, and we analyzed its connection with the overall survival (OS) of OC patients. To prove the role of LAMA5 in cell proliferation, migration, and invasion, LAMA5 expression in OC cell lines was inhibited by lentivirus. Compared with normal fallopian tube tissue, epithelial ovarian cancer (EOC) tissue showed critically higher LAMA5 expression levels; additionally, high LAMA5 levels were a poor predictor of OS. We found that cell progression was restrained in LAMA5-knockdown OC cell lines in vivo and in vitro. Finally, LAMA5 might be a commanding inducer of the expression of epithelial-mesenchymal transition (EMT) and Notch signaling pathway-related markers. Together, our research indicates that LAMA5 is highly connected to OC progression as it may play a role in the EMT process through the Notch signaling pathway.

Efficacy and Safety of Glycosphingolipid SSEA-4 Targeting CAR-T Cells in an Ovarian Carcinoma Model.

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

Gut microbiota dysbiosis promotes the development of epithelial ovarian cancer via regulating Hedgehog signaling pathway.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer, which remains a threat to female health at all ages. Hypotheses for EOC development include the continuous presence of inflammation, in which microbiota and inflammatory cytokines participate in cancer-related signaling pathway activation. Hedgehog (Hh) signaling is prominent for EOC progression, and interacts with inflammation response related to gut microbiota (GM). However, the precise roles of GM during this process are unknown. Here, we showed that the GM from patients with EOC differed from that of healthy women and had GM dysbiosis. We found that EOC modeling may lead to GM changes in mice, and it restored after the administration of GM from healthy controls, while GM from patients with EOC further exacerbated GM dysbiosis. Furthermore, we found that GM from EOC markedly promoted tumor progression and activated Hh signaling; meanwhile, it increased the extent of inflammation and activated NF-κB signaling, but GM from healthy controls improved them. Our results demonstrate how GM dysbiosis promoted EOC progression by activating Hh signaling mediated by TLR4/NF-κB signaling. We anticipate our assay to be a new thought for exploring the role of GM in EOC development. Furthermore, improving GM dysbiosis is a novel therapeutic approach for delaying EOC development.

Estrogen promotes Epithelial ovarian cancer cells proliferation via down-regulating expression and activating phosphorylation of PTEN.

Epithelial ovarian cancer (EOC) is the most common of cancer death among malignant tumors in women, its occurrence and development are strongly linked to estrogen. Having identified the phosphatase and tensin homologue (PTEN) is a potent tumor suppressor regulating cell proliferation, migration, and survival. Meanwhile, there is a correlation between PTEN protein expression and estrogen receptor expression in EOC. However, no study has amplified on the molecular regulatory mechanism and function between estrogen and PTEN in the development of EOC. In this research, we found that PTEN shows a low expression level in EOC tissues and estrogen decreased PTEN expression via the estrogen receptor 1 (ESR1) in EOC cells. Knockdown of PTEN enhanced the proliferation and migration level of EOC cells driven by estrogen. Moreover, PTEN was also phosphorylated by G protein-coupled receptor 30 (GPR30)-Protein kinase C (PKC) signaling pathway upon estrogen stimulation. Inhibiting the phosphorylation of PTEN weakened the proliferation and migration of estrogen induced-EOC cells estrogen and decreased the phosphorylation of Protein kinase B (AKT) and Mammalian target of rapamycin (mTOR). These results indicated that estrogen decreased PTEN expression level via the ESR1 genomic pathway and phosphorylated PTEN via the GPR30-PKC non-genomic pathway to activate the PI3K/AKT/mTOR signaling pathway, thereby determining the fate of EOC cells.

Immune profile of primary and recurrent epithelial ovarian cancer cases indicates immune suppression, a major cause of progression and relapse of ovarian cancer.

BACKGROUND: Ovarian cancer is the third most prevalent cancer in Indian women. Relative frequency of High grade serous epithelial ovarian cancer (HGSOC) and its associated deaths are highest in India which suggests the importance of understanding their immune profiles for better treatment modality. Hence, the present study investigated the NK cell receptor expression, their cognate ligands, serum cytokines, and soluble ligands in primary and recurrent HGSOC patients. We have used multicolor flow cytometry for immunophenotyping of tumor infiltrated and circulatory lymphocytes. Procartaplex, and ELISA were used to measure soluble ligands and cytokines of HGSOC patients. RESULTS: Among the enrolled 51 EOC patients, 33 were primary high grade serous epithelial ovarian cancer (pEOC) and 18 were recurrent epithelial ovarian cancer (rEOC) patients. Blood samples from 46 age matched healthy controls (HC) were used for comparative analysis. Results revealed, frequency of circulatory CD56Bright NK, CD56Dim NK, NKT-like, and T cells was reduced with activating receptors while alterations in immune subsets with inhibitory receptors were observed in both groups. Study also highlights differential immune profile of primary and recurrent ovarian cancer patients. We have found increased soluble MICA which might have acted as "decoy" molecule and could be a reason of decrease in NKG2D positive subsets in both groups of patients. Furthermore, elevated level of serum cytokines IL-2, IL-5, IL-6, IL-10, and TNF-α in ovarian cancer patients, might be associated with ovarian cancer progression. Profiling of tumor infiltrated immune cells revealed the reduced level of DNAM-1 positive NK and T cells in both groups than their circulatory counterpart, which might have led to decrease in NK cell's ability of synapse formation. CONCLUSIONS: The study brings out differential receptor expression profile on CD56BrightNK, CD56DimNK, NKT-like, and T cells, cytokines levels and soluble ligands which may be exploited to develop alternate therapeutic approaches for HGSOC patients. Further, few differences in the circulatory immune profiles between pEOC and rEOC cases, indicates the immune signature of pEOC undergoes some changes in circulation that might facilitated the disease relapse. They also maintains some common immune signatures such as reduced expression of NKG2D, high level of MICA as well as IL-6, IL10 and TNF-α, which indicates irreversible immune suppression of ovarian cancer patients. It is also emphasized that a restoration of cytokines level, NKG2D and DNAM-1on tumor infiltrated immune cells may be targeted to develop specific therapeutic approaches for high-grade serous epithelial ovarian cancer.

HCK Promotes High-Grade Serous Ovarian Cancer Tumorigenesis through CD44 and NOTCH3 Signaling.

High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse and succumb to their disease. Despite significant advances in our understanding of this disease, the mechanisms that govern the distinctions between HGSOC with good and poor prognosis remain unclear. In this study, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumor samples to identify molecular pathways that distinguish HGSOC tumors relative to clinical outcome. Our analyses identify significant upregulation of hematopoietic cell kinase (HCK) expression and signaling in poor prognostic HGSOC patient samples. Analyses of independent gene expression datasets and IHC of patient samples confirmed increased HCK signaling in tumors relative to normal fallopian or ovarian samples and demonstrated aberrant expression in tumor epithelial cells. Consistent with the association between HCK expression and tumor aggressiveness in patient samples, in vitro phenotypic studies showed that HCK can, in part, promote cell proliferation, colony formation, and invasive capacity of cell lines. Mechanistically, HCK mediates these phenotypes, partly through CD44 and NOTCH3-dependent signaling, and inhibiting CD44 or NOTCH3 activity, either genetically or through gamma-secretase inhibitors, can revert HCK-driven phenotypes. IMPLICATIONS: Collectively, these studies establish that HCK acts as an oncogenic driver of HGSOC through aberrant activation of CD44 and NOTCH3 signaling and identifies this network as a potential therapeutic opportunity in a subset of patients with aggressive and recurrent HGSOC.

LRG1 Is Involved in the Progression of Ovarian Cancer via Modulating FAK/AKT Signaling Pathway.

BACKGROUND: Rapid progression and early metastasis remain the main cause of high mortality in epithelial ovarian cancer (EOC) patients. The objective of this study was to explore the mechanisms of EOC progression and detect the function of leucine-rich alpha-2-glycoprotein 1 (LRG1) in modulating the pathologic process. METHODS: Ultracentrifugation was initially performed to extract exosomes from the urine samples of EOC patients and healthy female subjects. Mass spectrometry (MS) was employed to analyze differentially expressed proteins. Survival analysis was performed to examine the association between LRG1 levels and the prognosis of EOC patients. LRG1 silencing ovarian cancer cell lines were built and cell migration was further evaluated via wound healing and transwell assays. Immunoblot, immunofluorescence and immunohistochemistry analyses were performed. A subcutaneous tumor model was established to study the function of LRG1 in vivo. RESULTS: Exosomal LRG1 was specifically expressed in urine samples of EOC patients and high LRG1 levels were significantly associated with poor prognosis. Function analyses showed that LRG1 was associated with ovarian cancer migration and progression. Mechanistically, LRG1 was significantly related to the focal adhesion kinase/protein kinase B (FAK/AKT) signaling pathway. CONCLUSIONS: LRG1 participated in progression and metastasis of ovarian cancer via activation of the FAK/AKT pathway probably.

KLF5-mediated CDCA5 expression promotes tumor development and progression of epithelial ovarian carcinoma.

Cell division cycle associated 5 (CDCA5) is correlated with the development and progression of many malignant tumors. However, little is known about its role in epithelial ovarian cancer (EOC) progression. In this study, the clinical value, biological function and underlying mechanisms of CDCA5 in EOC were evaluated. CDCA5 mRNA and protein levels were substantially upregulated in EOC and had a significant positive correlation with adverse clinicopathological characteristics and a poor prognosis. CDCA5 facilitated proliferation, invasion, and metastasis and disrupted mitochondrial-mediated endogenous apoptosis by activating the cell cycle pathway and inhibiting the P53 pathway in EOC cells. Conversely, knockdown of CDCA5 expression blocked the malignant activities of EOC cells and suppressed the growth of xenograft tumors in vivo. Mechanistically, the transcription factor KLF5 bound to a specific site in the CDCA5 promoter and promoted CDCA5 expression. Moreover, KLF5 overexpression rescued the negative regulation of inhibited CDCA5 expression on EOC cell proliferation. In conclusion, our findings revealed that CDCA5 promoted tumor progression of EOC via the KLF5/CDCA5/cell cycle and P53 axes, which might provide new insights into the roles of CDCA5 in EOC.