Role of Extracellular Vesicles in Epithelial Ovarian Cancer: A Systematic Review

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions. EVs mediate the cellular crosstalk, thus contributing to defining the tumor microenvironment, including in epithelial ovarian cancer (EOC). The available literature investigating the role of EVs in EOC has been reviewed following PRISMA guidelines, focusing on the role of EVs in early disease diagnosis, metastatic spread, and the development of chemoresistance in EOC. Data were identified from searches of Medline, Current Contents, PubMed, and from references in relevant articles from 2010 to 1 April 2020. The research yielded 194 results. Of these, a total of 36 papers, 9 reviews, and 27 original types of research were retained and analyzed. The literature findings demonstrate that a panel of EV-derived circulating miRNAs may be useful for early diagnosis of EOC. Furthermore, it appears clear that EVs are involved in mediating two crucial processes for metastatic and chemoresistance development: the epithelial–mesenchymal transition, and tumor escape from the immune system response. Further studies, more focused on in vivo evidence, are urgently needed to clarify the role of EV assessment in the clinical management of EOC patients.     

Suppression of Ovarian Cancer Cell Growth by AT-MSC Microvesicles

Transport of bioactive cargo of microvesicles (MVs) into target cells can affect their fate and behavior and change their microenvironment. We assessed the effect of MVs derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) on the biological activity of the ovarian cancer cell lines ES-2 (clear cell carcinoma) and OAW-42 (cystadenocarcinoma). The HATMSC2-MVs were characterized using dynamic light scattering (DLS), transmission electron microscopy, and flow cytometry. The anti-tumor properties of HATMSC2-MVs were assessed using MTT for metabolic activity and flow cytometry for cell survival, cell cycle progression, and phenotype. The secretion profile of ovarian cancer cells was evaluated with a protein antibody array. Both cell lines internalized HATMSC2-MVs, which was associated with a decreased metabolic activity of cancer cells. HATMSC2-MVs exerted a pro-apoptotic and/or necrotic effect on ES-2 and OAW-42 cells and increased the expression of anti-tumor factors in both cell lines compared to control. In conclusion, we confirmed an effective transfer of HATMSC2-MVs into ovarian cancer cells that resulted in the inhibition of cell proliferation via different pathways, apoptosis and/or necrosis, which, with high likelihood, is related to the presence of different anti-tumor factors secreted by the ES-2 and OAW-42 cells.     

The Behavior of Matrix Metalloproteinases and Their Inhibitors in Colorectal Cancer

Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix components crucial for tumor growth, invasion and metastasis. MMPs are controlled by natural inhibitors called tissue inhibitors of metalloproteinases (TIMPs). We and others have demonstrated that MMPs and TIMPs are especially important in the process of tumor invasion, progression and the metastasis of colorectal cancer (CRC). It has been proposed that MMPs and TIMPs might play a part not only in tumor invasion and initiation of metastasis but also in carcinogenesis from colorectal adenomas. Several recent studies demonstrated that high preoperative serum or plasma MMP-2, MMP-9 and TIMP-1 antigen levels are strong predictive factors for poor prognosis in patients with CRC and their determination might be useful for identification of patients with higher risk for cancer recurrence. MMP-9 and TIMP-1 have significant potential tumor marker impact in CRC. Their diagnostic sensitivity is consistently higher than those of conventional biomarkers. The pharmacological targeting of CRC by the development of a new generation of selective inhibitors of MMPs, that is highly specific for certain MMPs, is a promising and challenging area for the future.     

The Proteolytic Landscape of Ovarian Cancer: Applications in Nanomedicine

Ovarian cancer (OvCa) is one of the leading causes of mortality globally with an overall 5-year survival of 47%. The predominant subtype of OvCa is epithelial carcinoma, which can be highly aggressive. This review launches with a summary of the clinical features of OvCa, including staging and current techniques for diagnosis and therapy. Further, the important role of proteases in OvCa progression and dissemination is described. Proteases contribute to tumor angiogenesis, remodeling of extracellular matrix, migration and invasion, major processes in OvCa pathology. Multiple proteases, such as metalloproteinases, trypsin, cathepsin and others, are overexpressed in the tumor tissue. Presence of these catabolic enzymes in OvCa tissue can be exploited for improving early diagnosis and therapeutic options in advanced cases. Nanomedicine, being on the interface of molecular and cellular scales, can be designed to be activated by proteases in the OvCa microenvironment. Various types of protease-enabled nanomedicines are described and the studies that focus on their diagnostic, therapeutic and theranostic potential are reviewed.     

Extracellular Matrix Biomarkers in Colorectal Cancer

The cellular microenvironment composition and changes therein play an extremely important role in cancer development. Changes in the extracellular matrix (ECM), which constitutes a majority of the tumor stroma, significantly contribute to the development of the tumor microenvironment. These alterations within the ECM and formation of the tumor microenvironment ultimately lead to tumor development, invasion, and metastasis. The ECM is composed of various molecules such as collagen, elastin, laminin, fibronectin, and the MMPs that cleave these protein fibers and play a central role in tissue remodeling. When healthy cells undergo an insult like DNA damage and become cancerous, if the ECM does not support these neoplastic cells, further development, invasion, and metastasis fail to occur. Therefore, ECM-related cancer research is indispensable, and ECM components can be useful biomarkers as well as therapeutic targets. Colorectal cancer specifically, is also affected by the ECM and many studies have been conducted to unravel the complex association between the two. Here we summarize the importance of several ECM components in colorectal cancer as well as their potential roles as biomarkers.     

Therapeutic Strategies for Targeting Ovarian Cancer Stem Cells

Ovarian cancer is a fatal gynecological malignancy. Although first-line chemotherapy and surgical operation are effective treatments for ovarian cancer, its clinical management remains a challenge owing to intrinsic or acquired drug resistance and relapse at local or distal lesions. Cancer stem cells (CSCs) are a small subpopulation of cells inside tumor tissues, and they can self-renew and differentiate. CSCs are responsible for the cancer malignancy involved in relapses as well as resistance to chemotherapy and radiation. These malignant properties of CSCs are regulated by cell surface receptors and intracellular pluripotency-associated factors triggered by internal or external stimuli from the tumor microenvironment. The malignancy of CSCs can be attenuated by individual or combined restraining of cell surface receptors and intracellular pluripotency-associated factors. Therefore, targeted therapy against CSCs is a feasible therapeutic tool against ovarian cancer. In this paper, we review the prominent roles of cell surface receptors and intracellular pluripotency-associated factors in mediating the stemness and malignancy of ovarian CSCs.     

Piperine Targets Different Drug Resistance Mechanisms in Human Ovarian Cancer Cell Lines Leading to Increased Sensitivity to Cytotoxic Drugs

Our goal was to examine the anticancer effects of piperine against the resistant human ovarian cancer cells and to explore the molecular mechanisms responsible for its anticancer effects. Our study used drug-sensitive ovarian cancer cell line W1 and its sublines resistant to paclitaxel (PAC) and topotecan (TOP). We analyzed the cytotoxic effect of piperine and cytostatic drugs using an MTT assay. The impact of piperine on protein expression was determined by immunofluorescence and Western blot. We also examined its effect on cell proliferation and migration. We noticed a different level of piperine resistance between cell lines. Piperine increases the cytotoxic effect of PAC and TOP in drug-resistant cells. We observed an increase in PTPRK expression correlated with decreased pTYR level after piperine treatment and downregulation of P-gp and BCRP expression. We also noted a decrease in COL3A1 and TGFBI expression in investigated cell lines and increased COL3A1 expression in media from W1PR2 cells. The expression of Ki67 protein and cell proliferation rate decreased after piperine treatment. Piperine markedly inhibited W1TR cell migration. Piperine can be considered a potential anticancer agent that can increase chemotherapy effectiveness in cancer patients.     

Cancer Stem Cells in Ovarian Cancer—A Source of Tumor Success and a Challenging Target for Novel Therapies

Ovarian cancer is the most lethal neoplasm of the female genital organs. Despite indisputable progress in the treatment of ovarian cancer, the problems of chemo-resistance and recurrent disease are the main obstacles for successful therapy. One of the main reasons for this is the presence of a specific cell population of cancer stem cells. The aim of this review is to show the most contemporary knowledge concerning the biology of ovarian cancer stem cells (OCSCs) and their impact on chemo-resistance and prognosis in ovarian cancer patients, as well as to present the treatment options targeted exclusively on the OCSCs. The review presents data concerning the role of cancer stem cells in general and then concentrates on OCSCs. The surface and intracellular OCSCs markers and their meaning both for cancer biology and clinical prognosis, signaling pathways specifically activated in OCSCs, the genetic and epigenetic regulation of OCSCs function including the recent studies on the non-coding RNA regulation, cooperation between OCSCs and the tumor microenvironment (ovarian cancer niche) including very specific environment such as ascites fluid, the role of shear stress, autophagy and metabolic changes for the function of OCSCs, and finally mechanisms of OCSCs escape from immune surveillance, are described and discussed extensively. The possibilities of anti-OCSCs therapy both in experimental settings and in clinical trials are presented, including the recent II phase clinical trials and immunotherapy. OCSCs are a unique population of cancer cells showing a great plasticity, self-renewal potential and resistance against anti-cancer treatment. They are responsible for the progression and recurrence of the tumor. Several completed and ongoing clinical trials have tested different anti-OCSCs drugs which, however, have shown unsatisfactory efficacy in most cases. We propose a novel approach to ovarian cancer diagnosis and therapy.     

Immune Tumor Microenvironment in Ovarian Cancer Ascites

Ovarian cancer (OC) has a specific type of metastasis, via transcoelomic, and most of the patients are diagnosed at advanced stages with multiple tumors spread within the peritoneal cavity. The role of Malignant Ascites (MA) is to serve as a transporter of tumor cells from the primary location to the peritoneal wall or to the surface of the peritoneal organs. MA comprise cellular components with tumor and non-tumor cells and acellular components, creating a unique microenvironment capable of modifying the tumor behavior. These microenvironment factors influence tumor cell proliferation, progression, chemoresistance, and immune evasion, suggesting that MA play an active role in OC progression. Tumor cells induce a complex immune suppression that neutralizes antitumor immunity, leading to disease progression and treatment failure, provoking a tumor-promoting environment. In this review, we will focus on the High-Grade Serous Carcinoma (HGSC) microenvironment with special attention to the tumor microenvironment immunology.     

Androgen/Androgen Receptor Signaling in Ovarian Cancer: Molecular Regulation and Therapeutic Potentials

Ovarian cancer (OVCA) arises from three cellular origins, namely surface epithelial cells, germ cells, and stromal cells. More than 85% of OVCAs are EOCs (epithelial ovarian carcinomas), which are the most lethal gynecological malignancies. Cancer stem/progenitor cells (CSPCs) are considered to be cancer promoters due to their capacity for unlimited self-renewal and drug resistance. Androgen receptor (AR) belongs to the nuclear receptor superfamily and can be activated through binding to its ligand androgens. Studies have reported an association between AR expression and EOC carcinogenesis, and AR is suggested to be involved in proliferation, migration/invasion, and stemness. In addition, alternative AR activating signals, including both ligand-dependent and ligand-independent, are involved in OVCA progression. Although some clinical trials have previously been conducted to evaluate the effects of anti-androgens in EOC, no significant results have been reported. In contrast, experimental studies evaluating the effects of anti-androgen or anti-AR reagents in AR-expressing EOC models have demonstrated positive results for suppressing disease progression. Since AR is involved in complex signaling pathways and may be expressed at various levels in OVCA, the aim of this article was to provide an overview of current studies and perspectives regarding the relevance of androgen/AR roles in OVCA.     

Targeting Signaling Pathways in Epithelial Ovarian Cancer

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Response to platinum-based chemotherapy is poor in some patients and, thus, current research is focusing on new therapy options. The various histological types of OC are characterized by distinctive molecular genetic alterations that are relevant for ovarian tumorigenesis. The understanding of these molecular pathways is essential for the development of novel therapeutic strategies.

Purpose

We want to give an overview on the molecular genetic changes of the histopathological types of OC and their role as putative therapeutic targets.

In Depth Review of Existing Data

In 2012, the vascular endothelial growth factor (VEGF) inhibitor, bevacizumab, was approved for OC treatment. Bevacizumab has shown promising results as single agent and in combination with conventional chemotherapy, but its target is not distinctive when analyzed before treatment. At present, mammalian target of rapamycin (mTOR) inhibitors, poly-ADP-ribose polymerase (PARP) inhibitors and components of the EGFR pathway are in the focus of clinical research. Interestingly, some phytochemical substances show good synergistic effects when used in combination with chemotherapy.

Conclusion

Ongoing studies of targeted agents in conjunction with chemotherapy will show whether there are alternative options to bevacizumab available for OC patients. Novel targets which can be assessed before therapy to predict efficacy are needed. The assessment of therapeutic targets is continuously improved by molecular pathological analyses on tumor tissue. A careful selection of patients for personalized treatment will help to reduce putative side effects and toxicity.     

Metalloproteinases 1 and 3 as Potential Biomarkers in Breast Cancer Development

Breast cancer continues to be one of the main causes of morbidity and mortality globally and was the leading cause of cancer death in women in Spain in 2020. Early diagnosis is one of the most effective methods to lower the incidence and mortality rates of breast cancer. The human metalloproteinases (MMP) mainly function as proteolytic enzymes degrading the extracellular matrix and plays important roles in most steps of breast tumorigenesis. This retrospective cohort study shows the immunohistochemical expression levels of MMP-1, MMP-2, MMP-3, and MMP-9 in 154 women with breast cancer and 42 women without tumor disease. The samples of breast tissue are assessed using several tissue matrices (TMA). The percentages of staining (≤50%–>50%) and intensity levels of staining (weak, moderate, or intense) are considered. The immunohistochemical expression of the MMP-1-intensity (p = 0.043) and MMP-3 percentage (p = 0.018) and intensity, (p = 0.025) present statistically significant associations with the variable group (control–case); therefore, expression in the tumor tissue samples of these MMPs may be related to the development of breast cancer. The relationships between these MMPs and some clinicopathological factors in breast cancer are also evaluated but no correlation is found. These results suggest the use of MMP-1 and MMP-3 as potential biomarkers of breast cancer diagnosis.     

Effect of ALDH1A1 Gene Knockout on Drug Resistance in Paclitaxel and Topotecan Resistant Human Ovarian Cancer Cell Lines in 2D and 3D Model

Ovarian cancer is the most common cause of gynecological cancer death. Cancer Stem Cells (CSCs) characterized by drug transporters and extracellular matrix (ECM) molecules expression are responsible for drug resistance development. The goal of our study was to examine the role of aldehyde dehydrogenase 1A1 (ALDH1A1) expression in paclitaxel (PAC) and topotecan (TOP) resistant ovarian cancer cell lines. In both cell lines, we knocked out the ALDH1A1 gene using the CRISPR/Cas9 technique. Additionally, we derived an ALDH1A1 positive TOP-resistant cell line with ALDH1A1 expression in all cells via clonal selection. The effect of ALDH1A1 gene knockout or clonal selection on the expression of ALDH1A1, drug transporters (P-gp and BCRP), and ECM (COL3A1) was determined by Q-PCR, Western blot and immunofluorescence. Using MTT assay, we compared drug resistance in two-dimensional (2D) and three-dimensional (3D) cell culture conditions. We did not observe any effect of ALDH1A1 gene knockout on MDR1/P-gp expression and drug resistance in the PAC-resistant cell line. The knockout of ALDH1A1 in the TOP-resistant cell line resulted in a moderate decrease of BCRP and COL3A1 expression and weakened TOP resistance. The clonal selection of ALDH1A1 cells resulted in very strong downregulation of BCPR and COL3A1 expression and overexpression of MDR1/P-gp. This finally resulted in decreased resistance to TOP but increased resistance to PAC. All spheroids were more resistant than cells growing as monolayers, but the resistance mechanism differs. The spheroids’ resistance may result from the presence of cell zones with different proliferation paces, the density of the spheroid, ECM expression, and drug capacity to diffuse into the spheroid.     

Metastatic Voyage of Ovarian Cancer Cells in Ascites with the Assistance of Various Cellular Components

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and has a unique metastatic route using ascites, known as the transcoelomic root. However, studies on ascites and contained cellular components have not yet been sufficiently clarified. In this review, we focus on the significance of accumulating ascites, contained EOC cells in the form of spheroids, and interaction with non-malignant host cells. To become resistant against anoikis, EOC cells form spheroids in ascites, where epithelial-to-mesenchymal transition stimulated by transforming growth factor-β can be a key pathway. As spheroids form, EOC cells are also gaining the ability to attach and invade the peritoneum to induce intraperitoneal metastasis, as well as resistance to conventional chemotherapy. Recently, accumulating evidence suggests that EOC spheroids in ascites are composed of not only cancer cells, but also non-malignant cells existing with higher abundance than EOC cells in ascites, including macrophages, mesothelial cells, and lymphocytes. Moreover, hetero-cellular spheroids are demonstrated to form more aggregated spheroids and have higher adhesion ability for the mesothelial layer. To improve the poor prognosis, we need to elucidate the mechanisms of spheroid formation and interactions with non-malignant cells in ascites that are a unique tumor microenvironment for EOC.     

The FGFR Family Inhibitor AZD4547 Exerts an Antitumor Effect in Ovarian Cancer Cells

The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.     

Reduced Gonadotrophin Receptor Expression Is Associated with a More Aggressive Ovarian Cancer Phenotype

Follicle-stimulating hormone (FSH) and luteinising hormone (LH) play important roles in regulating cell growth and proliferation in the ovary. However, few studies have explored the expression of FSH and LH receptors (FSHR and LHCGR) in ovarian cancer, and their functional roles in cancer progression remain inconclusive. This study investigated the potential impact of both mRNA (FSHR, LHCGR) and protein (FSHR, LHCGR) expression on ovarian cancer progression using publicly available online databases, qRT-PCR (high grade serous ovarian cancers, HGSOC, n = 29 and benign ovarian tumors, n = 17) and immunohistochemistry (HGSOC, n = 144). In addition, we investigated the effect of FSHR and LHCGR siRNA knockdown on the pro-metastatic behavior of serous ovarian cancer cells in vitro. High FSHR or high LHCGR expression in patients with all subtypes of high-grade ovarian cancer was significantly associated with longer progression-free survival (PFS) and overall survival (OS). High FSHR protein expression was associated with increased PFS (p = 0.050) and OS (p = 0.025). HGSOC patients with both high FSHR and high LHCGR protein levels had the best survival outcome, whilst both low FSHR and low LHCGR expression was associated with poorest survival (p = 0.019). Knockdown of FSHR significantly increased the invasion of serous ovarian cancer cells (OVCAR3 and COV362) in vitro. LHCGR knockdown also promoted invasion of COV362 cells. This study highlights that lower FSHR and LHCGR expression is associated with a more aggressive epithelial ovarian cancer phenotype and promotes pro-metastatic behaviour.     

Expression of the MT1 Melatonin Receptor in Ovarian Cancer Cells

Ovarian cancer (OC) is the leading cause of death among women with genital tract disorders. Melatonin exhibits oncostatic properties which it may effect through binding to its membrane receptor, MT1. The aim of this study was to determine the expression of MT1 in OC cells and to correlate this with clinical and pathological data. Immunohistochemistry was performed on 84 cases of OC. Normal ovarian epithelial (IOSE 364) and OC (SK-OV-3, OVCAR-3) cell lines were used to examine the MT1 expression at protein level using the western blot and immunofluorescence technique. The expression of MT1 was observed as cytoplasmic-membrane (MT1_CM) and membrane (MT1_M) reactions. A positive correlation between MT1_CM and MT1_M was found in all the studied cases. There were no significant differences between the expression of MT1_CM, MT1_M, and histological type, staging, grading, presence of residual disease, or overall survival time. Immunofluorescence showed both MT1_M and MT1_CM expression in all the tested cell lines. Western blot illustrated the highest protein level of MT1 in IOSE 364 and the lowest in the OVCAR-3. The results indicate the limited prognostic significance of MT1 in OC cells.     

Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.     

Akt-Activated Endothelium Increases Cancer Cell Proliferation and Resistance to Treatment in Ovarian Cancer Cell Organoids

Ovarian cancer (OC) is a heterogeneous disease characterized by its late diagnosis (FIGO stages III and IV) and the importance of abdominal metastases often observed at diagnosis. Detached ovarian cancer cells (OCCs) float in ascites and form multicellular spheroids. Here, we developed endothelial cell (EC)-based 3D spheroids to better represent in vivo conditions. When co-cultured in 3D conditions, ECs and OCCs formed organized tumor angiospheres with a core of ECs surrounded by proliferating OCCs. We established that Akt and Notch3/Jagged1 pathways played a role in angiosphere formation and peritoneum invasion. In patients’ ascites we found angiosphere-like structures and demonstrated in patients’ specimens that tumoral EC displayed Akt activation, which supports the importance of Akt activation in ECs in OC. Additionally, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCC–EC crosstalk relating to OCC proliferation and during peritoneal invasion. Our results support the use of multicellular spheroids to better model tumoral and stromal interaction. Such models could help decipher the complex pathways playing critical roles in metastasis spread and predict tumor response to chemotherapy or anti-angiogenic treatment.