Obstetrics and Gynaecology - Research Publications
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ItemKnock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.(Springer Science and Business Media LLC, 2022-12-30)BACKGROUND: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. METHODS: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. RESULTS: Approximately 70-90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. CONCLUSIONS: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.
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ItemPlatinum-resistance in epithelial ovarian cancer: an interplay of epithelial-mesenchymal transition interlinked with reprogrammed metabolism(BMC, 2022-12-03)BACKGROUND: Epithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases. METHODS: We explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial-mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan-Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME). RESULTS: Our proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME. CONCLUSIONS: Our research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.
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ItemPrecision Medicine: An Optimal Approach to Patient Care in Renal Cell Carcinoma.(Frontiers Media SA, 2022)Renal cell cancer (RCC) is a heterogeneous tumor that shows both intra- and inter-heterogeneity. Heterogeneity is displayed not only in different patients but also among RCC cells in the same tumor, which makes treatment difficult because of varying degrees of responses generated in RCC heterogeneous tumor cells even with targeted treatment. In that context, precision medicine (PM), in terms of individualized treatment catered for a specific patient or groups of patients, can shift the paradigm of treatment in the clinical management of RCC. Recent progress in the biochemical, molecular, and histological characteristics of RCC has thrown light on many deregulated pathways involved in the pathogenesis of RCC. As PM-based therapies are rapidly evolving and few are already in current clinical practice in oncology, one can expect that PM will expand its way toward the robust treatment of patients with RCC. This article provides a comprehensive background on recent strategies and breakthroughs of PM in oncology and provides an overview of the potential applicability of PM in RCC. The article also highlights the drawbacks of PM and provides a holistic approach that goes beyond the involvement of clinicians and encompasses appropriate legislative and administrative care imparted by the healthcare system and insurance providers. It is anticipated that combined efforts from all sectors involved will make PM accessible to RCC and other patients with cancer, making a tremendous positive leap on individualized treatment strategies. This will subsequently enhance the quality of life of patients.
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ItemExpression of TIMPs and MMPs in Ovarian Tumors, Ascites, Ascites-Derived Cells, and Cancer Cell Lines: Characteristic Modulatory Response Before and After Chemotherapy Treatment(FRONTIERS MEDIA SA, 2022-01-03)BACKGROUND: The tissue inhibitors of metalloproteinase (TIMPs) and their associated metalloproteinase (MMPs) are essential regulators of tissue homeostasis and are essential for cancer progression. This study analyzed the expression of TIMP-1,-2,-3 and the associated MMPs (MMP-2,-9,-11,-14) in different Stages, Grades and World Health Organization (WHO) classifications of serous ovarian tumors, ascites, ascites-derived cells from chemo-naïve (CN) and relapsed (CR) patients, and in ovarian cancer cell lines. The status of TIMPs and associated MMPs in response to chemotherapy treatment was assessed in cancer cell lines; TCGA data was interrogated to gauge TIMPs and associated MMPs as prognostic and platinum-response indicators. METHODS: The levels of TIMP-1, -2 and -3 were assessed by immunohistochemistry. The mRNA expression of TIMPs and MMPs was quantified by real time PCR (qRT-PCR). The chemosensitivity (IC50 values) to Cisplatin or Paclitaxel in cell lines was evaluated by MTT assay. The levels of TIMPs in ascites and cell lysates were analyzed by an ELISA assay. RESULTS: The expression of TIMP-2 was significantly upregulated in Type 2 compared to Type 1 tumors and normal/benign ovarian tissues. TIMP-3 expression was significantly enhanced in Stage III, Grade 3 and Type 2 tumors compared to normal/benign ovarian tissues. The mRNA expression of MMP-9,-11 and -14 was significantly upregulated in Stage IV compared to normal/benign ovarian tissues. The expression of TIMP-1 was highest, followed by TIMP-2 and then TIMP-3 in CN ascites. At the cellular level, TIMP-2 mRNA expression was significantly higher in CN compared to CR epithelial cells in patients. The expression of TIMP-1 and -2, MMPs and cancer stem cells (CSCs) were upregulated in response to chemotherapy treatments in cancer cell lines. Interrogation of the TCGA dataset suggests shifts in platinum responses in patients consistent with genetic alterations in TIMP-2, -3 and MMP-2, -11 genes in tumors; and decreased overall survival (OS) and progression-free survival (PFS) in patients with altered MMP-14 genes. CONCLUSIONS: TIMPs and related MMPs are differentially expressed in serous ovarian tumors, ascites, ascites-derived cells and ovarian cancer cell lines. Chemotherapy treatment modulates expression of TIMPs and MMPs in association with increased expression of genes related to cancer stem cells.
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ItemDeterminants of resistance to VEGF-TKI and immune checkpoint inhibitors in metastatic renal cell carcinoma(BMC, 2021-06-07)Vascular endothelial growth factor tyrosine kinase inhibitors (VEGF-TKIs) have been the mainstay of treatment for patients with advanced renal cell carcinoma (RCC). Despite its early promising results in decreasing or delaying the progression of RCC in patients, VEGF-TKIs have provided modest benefits in terms of disease-free progression, as 70% of the patients who initially respond to the treatment later develop drug resistance, with 30% of the patients innately resistant to VEGF-TKIs. In the past decade, several molecular and genetic mechanisms of VEGF-TKI resistance have been reported. One of the mechanisms of VEGF-TKIs is inhibition of the classical angiogenesis pathway. However, recent studies have shown the restoration of an alternative angiogenesis pathway in modulating resistance. Further, in the last 5 years, immune checkpoint inhibitors (ICIs) have revolutionized RCC treatment. Although some patients exhibit potent responses, a non-negligible number of patients are innately resistant or develop resistance within a few months to ICI therapy. Hence, an understanding of the mechanisms of VEGF-TKI and ICI resistance will help in formulating useful knowledge about developing effective treatment strategies for patients with advanced RCC. In this article, we review recent findings on the emerging understanding of RCC pathology, VEGF-TKI and ICI resistance mechanisms, and potential avenues to overcome these resistance mechanisms through rationally designed combination therapies.
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ItemThe attributes of plakins in cancer and disease: perspectives on ovarian cancer progression, chemoresistance and recurrence(BMC, 2021-05-17)The plakin family of cytoskeletal proteins play an important role in cancer progression yet are under-studied in cancer, especially ovarian cancer. These large cytoskeletal proteins have primary roles in the maintenance of cytoskeletal integrity but are also associated with scaffolds of intermediate filaments and hemidesmosomal adhesion complexes mediating signalling pathways that regulate cellular growth, migration, invasion and differentiation as well as stress response. Abnormalities of plakins, and the closely related spectraplakins, result in diseases of the skin, striated muscle and nervous tissue. Their prevalence in epithelial cells suggests that plakins may play a role in epithelial ovarian cancer progression and recurrence. In this review article, we explore the roles of plakins, particularly plectin, periplakin and envoplakin in disease-states and cancers with emphasis on ovarian cancer. We discuss the potential role the plakin family of proteins play in regulating cancer cell growth, survival, migration, invasion and drug resistance. We highlight potential relationships between plakins, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) and discuss how interaction of these processes may affect ovarian cancer progression, chemoresistance and ultimately recurrence. We propose that molecular changes in the expression of plakins leads to the transition of benign ovarian tumours to carcinomas, as well as floating cellular aggregates (commonly known as spheroids) in the ascites microenvironment, which may contribute to the sustenance and progression of the disease. In this review, attempts have been made to understand the crucial changes in plakin expression in relation to progression and recurrence of ovarian cancer. Video Abstract.
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ItemShort-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden(BMC, 2013-03-27)Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.
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ItemAttributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary(BMC, 2012-11-21)Epithelial ovarian cancer (EOC) remains the most lethal of all the gynaecological malignancies with drug resistance and recurrence remaining the major therapeutic barrier in the management of the disease. Although several studies have been undertaken to understand the mechanisms responsible for chemoresistance and subsequent recurrence in EOC, the exact mechanisms associated with chemoresistance/recurrence continue to remain elusive. Recent studies have shown that the parallel characteristics commonly seen between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) are also shared by a relatively rare population of cells within tumors that display stem cell-like features. These cells, termed 'cancer initiating cells' or 'cancer stem cells (CSCs)' have been shown not only to display increased self renewal and pluripotent abilities as seen in ESCs and iPSCs, but are also highly tumorigenic in in vivo mouse models. Additionally, these CSCs have been implicated in tumor recurrence and chemoresistance, and when isolated have consistently shown to express the master pluripotency and embryonic stem cell regulating gene Oct4. This article reviews the involvement of Oct4 in cancer progression and chemoresistance, with emphasis on ovarian cancer. Overall, we highlight why ovarian cancer patients, who initially respond to conventional chemotherapy subsequently relapse with recurrent chemoresistant disease that is essentially incurable.
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ItemNeuronal transcription factor Brn-3a(l) is over expressed in high-grade ovarian carcinomas and tumor cells from ascites of patients with advanced-stage ovarian cancer(BMC, 2010-07-29)OBJECTIVES: In view of the recent association of Brn-3 transcription factors with neuroblastomas, cervical, breast, and prostate cancers we examined the expression of Brn-3a(l) in normal ovaries and in different histological grades of ovarian tumors. The expression of Brn-3a(l) was also evaluated in normal ovarian and cancer cell lines and tumor cells isolated from the ascites of advanced-stage ovarian cancer patients. METHODS: Normal ovaries, benign, borderline, grades 1, 2 and 3 ovarian tumors were analyzed by immunohistochemistry for Brn-3a(l) expression. A total of 46 ovarian specimens were included in the study. Immunofluorescence was used to investigate the expression of Brn-3a in normal ovarian and cancer cell lines. Brn-3a(l) expression was also evaluated by Western blot in tumor cells isolated from ascites of advanced-stage ovarian cancer patients and also in ovarian cancer cell lines. RESULTS: Nearly 12% of normal and benign ovarian tissues and 57% of borderline ovarian tumors were positive for epithelial Brn-3a(l) expression. Stromal staining was higher and it constituted 40% of normal non-cancerous ovaries compared to 50 and 86% in benign and borderline tumors. On the other hand, 85-100% of grades 1, 2 & 3 ovarian tumors demonstrated nuclear and cytoplasmic Brn-3a(l) staining in the epithelium. Stromal staining in grades1, 2 and 3 tumors constituted 71-88% of total staining. Overall, immunoreactive Brn-3a was present in all grades of ovarian tumors. The extent of epithelial and stromal Brn-3a staining was significantly different between the normal and histological grades of tumors (epithelial-chi2 = 41.01, df = 20, P = 0.004, stromal-chi2 = 24.66. df = 15, P = 0.05). The extent of epithelial staining was significantly higher in grades 1 and 2 ovarian tumors compared to normal ovaries and benign ovarian tumors (p < 0.05). In parallel, stromal staining was significantly higher in grade 3 tumors compared to normal ovaries (p < 0.05). In addition, cytoplasmic and nuclear Brn-3a expression was evident in ovarian cancer cell lines while no such expression was observed in SV40 antigen immortalized normal ovarian cell lines. CONCLUSION: These data suggest that like other cancers, Brn-3a(l) expression is enhanced in ovarian tumors and its expression is consistent with its known role in inhibiting apoptosis and enhancing tumorigenesis. Specific targeting of Brn-3a may provide a useful strategy for regulating multiple tumor related genes involved with ovarian carcinomas.
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ItemIsolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors(PUBLIC LIBRARY SCIENCE, 2012-10-08)Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.