Carmela Ricciardelli

Proteomics analysis of serum extracellular vesicle identifies UCHL1 as a potential therapeutic target for high grade serous ovarian cancer

This study characterised the proteins from EVs in the serum from high-grade serous ovarian cancer (HGSOC) compared to healthy controls. Serum EVs were isolated, followed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins. We validated the expression of 4 EV proteins increased in cancer serum (KRT4, MARCKS, SPP1/OPN, and UCHL1) in HGSOC tissues and normal ovarian tissues using online databases and independent HGSOC patient tissue cohorts. We additionally investigated the effects of the UCHL1 inhibitor, LDN-57444, on HGSOC cell metabolic activity, motility, invasion, and apoptosis in HGSOC tissues using patient-derived explant assays. Proteomics analysis identified 28 EV proteins that were upregulated in HGSOC compared to healthy controls. We confirmed that UCHL1 protein levels were increased in HGSOC tissues compared to normal (OSE and FT) and benign epithelium. High stromal UCHL1 levels were associated with reduced progression-free survival in HGSOC. The UCHL1 inhibitor, LDN-57444, reduced the cell metabolic activity of ovarian cancer cell lines and primary ovarian cancer cells with high UCHL1 levels. LDN-57444 blocked the motility and invasion of OVCAR3 cells and promoted apoptosis in the HGSOC patient explant tissue assay. UCHL1 has the potential to be used as a novel prognostic and therapeutic target for HGSOC.

Protease-Activated Receptor F2R Is a Potential Target for New Diagnostic/Prognostic and Treatment Applications for Patients with Ovarian Cancer

Effective treatment of ovarian cancer is limited by late-stage detection and chemotherapy resistance. There is a clinical need for the discovery of novel molecular targets to enable the development of innovative theranostic approaches. We investigated the coagulation factor II receptor/protease-activated receptor 1 (F2R/PAR1) as a potential diagnostic/prognostic biomarker and therapeutic target for ovarian cancer treatment. Public RNA sequence and DNA microarray data were used to analyze F2R gene expression in ovarian cancers, with protein expression confirmed in tumor samples by flow cytometry, immunofluorescence, and immunohistochemistry (IHC). Functional assays were conducted to study effects of F2R suppression on tumor progression. Our analysis confirmed elevated F2R mRNA and protein expression in ovarian cancers, notably in patients with metastatic and chemotherapy-resistant disease. Kaplan–Meier survival analysis demonstrated an association between high F2R protein detection and reduced progression-free survival. F2R suppression in ovarian cancer cell lines reduced tumor cell motility, invasion, spheroid formation, and metabolism and enhanced carboplatin sensitivity. F2R is a compelling diagnostic/prognostic and therapeutic target that could be used to treat chemotherapy-resistant and metastatic disease. The evaluation of novel F2R targeting strategies, using antibody-conjugated drugs or F2R ligand-decorated drug carriers, could lead to the development of effective therapeutics for patients with ovarian cancer.

Identification of Proteins Associated with Ovarian Cancer Chemotherapy Resistance Using MALDI-MSI

Ovarian cancer is the most lethal gynecological cancer. Up to 75% of cases are high-grade serous ovarian cancer (HGSOC) that have high chemosensitivity to first-line platinum-based therapies. However, 75% of patients will become chemoresistant following relapse. The underlying mechanism for developing resistance to chemotherapy in HGSOC is poorly understood. In this study, we employed Matrix-Assisted Laser Desorption/Ionization–Mass Spectrometry Imaging (MALDI-MSI) on matching formalin-fixed paraffin-embedded (FFPE) HGSOC tissues at the time of diagnosis and following relapse with chemotherapy-resistant disease (n = 4). We identified m/z values that were differentially abundant in the matching diagnosis and relapse HGSOC tissues. These were matched to proteins using nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified upregulated proteins in the HGSOC relapse tissues, including COL12A1, FUBP1, PLEC, SLC4A1, and TKT. These proteins were validated by immunohistochemistry (IHC) and gene expression using online databases. IHC showed COL12A1, FUBP1, PLEC, SLC4A1, and TKT protein abundance were significantly elevated in HGSOC relapse tissues compared to matching tissues at diagnosis. COL12A1, FUBP1, PLEC, and TKT mRNA expression levels were significantly increased in HGSOC compared to normal ovary and associated with poor prognosis in HGSOC. We confirmed that higher protein abundance of both COL12A1 and PLEC correlated with reduced progression-free survival in HGSOC patients. Furthermore, both COL12A1 and PLEC mRNA and protein levels were significantly associated with chemotherapy resistance. In summary, using MALDI-MSI, we have identified proteins, including COL12A1 and PLEC, associated with chemotherapy resistance to be further evaluated as HGSOC biomarkers and/or therapeutic targets.

Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer

AbstractAlthough the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.

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.