Epithelial-to-mesenchymal transition supports ovarian carcinosarcoma tumorigenesis and confers sensitivity to microtubule-targeting with eribulin

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumour type with limited treatment options. OCS is hypothesised to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analysed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumours. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts (PDX). Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a down-regulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate EMT plays a key role in OCS tumourigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes

Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158–66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections

Candidate and non-candidate based approaches for the detection of biomarkers of ovarian cancer

© 2010 Dr. Ratana LimOvarian cancer affects around 1,200 women in Australia each year.There is no reliable screening test for early stage ovarian cancer, with 75% of patients already at an advanced stage at diagnosis. This thesis endeavoured to discover new biomarkers of ovarian cancer by validating four candidate biomarkers, and discovering novel biomarkers using proteomic techniques. The four candidate biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), CD163, hCAP-18 and lactoferrin, were assessed by ELISA, Western blot and immunohistochemistry for detection of ovarian cancer. CD163 concentration was highest in grade 2 and 3 blood samples while hCAP-18 expression was highest in grade 1 tissue and benign and borderline blood samples. In particular, NGAL expression was found to be highest in low grade tumours and decreased with invasiveness of the tumour, suggesting a potential role of NGAL as an early stage marker. Using ovarian cancer cell lines, it was found that NGAL may have a role in epithelial-mesenchymal transition (EMT). Proteomic approaches to detect possible novel biomarkers included2-dimensional gel electrophoresis of normal and grade 3 ovarian tissues and identification of differentially expressed protein spots by mass spectrometry. A total of ten proteins were identified to be up-regulated in grade 3 serous ovarian cancer and nine proteins were found to be down-regulated in grade 3 cancer. The proteins identified include those of the heat shock family, and proteins involved in the cytoskeleton, apoptosis, host response and metabolism. In order to improve the outcome for ovarian cancer, the identification of biomarkers for early detection is of great importance. Validation of the proteins identified in this study would contribute to ovarian cancer research

Candidate and non-candidate based approaches for the detection of biomarkers of ovarian cancer

© 2010 Dr. Ratana LimOvarian cancer affects around 1,200 women in Australia each year.There is no reliable screening test for early stage ovarian cancer, with 75% of patients already at an advanced stage at diagnosis. This thesis endeavoured to discover new biomarkers of ovarian cancer by validating four candidate biomarkers, and discovering novel biomarkers using proteomic techniques. The four candidate biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), CD163, hCAP-18 and lactoferrin, were assessed by ELISA, Western blot and immunohistochemistry for detection of ovarian cancer. CD163 concentration was highest in grade 2 and 3 blood samples while hCAP-18 expression was highest in grade 1 tissue and benign and borderline blood samples. In particular, NGAL expression was found to be highest in low grade tumours and decreased with invasiveness of the tumour, suggesting a potential role of NGAL as an early stage marker. Using ovarian cancer cell lines, it was found that NGAL may have a role in epithelial-mesenchymal transition (EMT). Proteomic approaches to detect possible novel biomarkers included2-dimensional gel electrophoresis of normal and grade 3 ovarian tissues and identification of differentially expressed protein spots by mass spectrometry. A total of ten proteins were identified to be up-regulated in grade 3 serous ovarian cancer and nine proteins were found to be down-regulated in grade 3 cancer. The proteins identified include those of the heat shock family, and proteins involved in the cytoskeleton, apoptosis, host response and metabolism. In order to improve the outcome for ovarian cancer, the identification of biomarkers for early detection is of great importance. Validation of the proteins identified in this study would contribute to ovarian cancer research

Inhibition of GPR91 Reduces Inflammatory Mediators Involved in Active Labor in Myometrium

Problem. Some G-protein-coupled receptors (GPCRs) are regulators of inflammation, yet the role of the GPRC, GPR91, is unknown in human myometrium during the processes of human labor and delivery, a major inflammatory event. Method of Study. GPR91 mRNA expression was assessed using RT-qPCR in myometrium obtained from women at term Caesarean section in the absence of labor and during active spontaneous labor and in a mouse model of inflammation-induced preterm labor. Human primary myometrial cells were used to determine the effect of proinflammatory mediators on GPR91 and the effect of GPR91 siRNA on prolabor mediators. Statistical significance was ascribed to a P<0.05. Results. GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF2α, and in situ collagen gel contraction), and the transcription factor NF-κB. Conclusion. Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes

Increased chemerin concentrations in fetuses of obese mothers and correlation with maternal insulin sensitivity

Objective: The aim of this study was to determine the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating concentrations of chemerin in cord and maternal plasma, and (ii) gene expression and release of chemerin from human placenta and adipose tissue. Design: Chemerin concentrations were measured in maternal and cord plasma from 62 normal glucose tolerant women (NGT) and 69 women with GDM at the time of term elective Caesarean section. Placenta and adipose tissue expression and release of chemerin was measured from 22 NGT and 22 GDM women. Results: There was no effect of maternal obesity or GDM on maternal chemerin concentrations. Chemerin concentrations were significantly higher in cord plasma from women with maternal obesity. Cord chemerin concentrations in NGT women negatively correlated with the concentrations of maternal insulin sensitivity. There was no effect of GDM on maternal and cord chemerin concentrations, and on the release of chemerin from placenta and adipose tissue. Conclusions: At the time of term Caesarean section, preexisting maternal obesity, and its associated insulin resistance, is associated with higher cord plasma chemerin concentrations

Dietary Flavonoids as Therapeutics for Preterm Birth: Luteolin and Kaempferol Suppress Inflammation in Human Gestational Tissues In Vitro

Infection/inflammation is commonly associated with preterm birth (PTB), initiating uterine contractions and rupture of fetal membranes. Proinflammatory cytokines induce matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) and prostaglandins which initiate uterine contractions. Nuclear factor-κB (NF-κB) and activator-protein- (AP-)1 have key roles in the formation of these prolabour mediators. In nongestational tissues, dietary flavonoids such as luteolin and kaempferol inhibit NF-κB, AP-1, and their downstream targets. The aim of this study was to determine if luteolin and kaempferol reduce infection-induced prolabour mediators in human gestational tissues. Fetal membranes were incubated with LPS, and primary amnion cells and myometrial cells were incubated with IL-1β in the absence or presence of luteolin or kaempferol. Luteolin and kaempferol significantly reduced LPS-induced secretion of proinflammatory cytokines (IL-6 and IL-8) and prostaglandins (PGE2 and PGF2α) in fetal membranes, IL-1β-induced COX-2 gene expression and prostaglandin production in myometrium, and IL-1β-induced MMP-9 activity in amnion and myometrial cells. Luteolin and kaempferol decreased IL-1β-induced NF-κB p65 DNA binding activity and nuclear c-Jun expression. In conclusion, luteolin and kaempferol inhibit prolabour mediators in human gestational tissues. Given the central role of inflammation in provoking preterm labour, phytophenols may be a therapeutic approach to reduce the incidence of PTB

SIRT1 is a novel regulator of key pathways of human labor

Human sirtuin (SIRT) 1 and SIRT2, which possess nicotinamide adenosine dinucleotide (NAD+)-dependent deacetylase activity, exhibit anti-inflammatory actions. However, there are no data available on SIRT1 and SIRT2 expression and regulation in human intrauterine tissues. Thus, the aim of this study was to characterize the localization and expression of SIRT1 and SIRT2 in 1) placenta and fetal membranes before and after term spontaneous labor onset, 2) prelabor fetal membranes from the supracervical site (SCS) and a distal site (DS), and 3) in response to proinflammatory stimuli. Further, the effect of SIRT activation using resveratrol and SRT1720 on prolabor mediators was also assessed. SIRT1 and SIRT2 were localized in the syncytiotrophoblast layer and the cytotrophoblasts of the placenta, amnion epithelium, trophoblast layer of the chorion, and decidual cells. Additionally, SIRT2 was found within the endothelial walls of placental vessels. SIRT2, but not SIRT1, staining was significantly lower in amnion and chorion obtained from the SCS compared to a DS. On the other hand, SIRT1, but not SIRT2, gene and/or protein expression was significantly lower in placenta, amnion, and chorion obtained after labor compared to prelabor. SIRT1 expression, but not SIRT2, was down-regulated by lipopolysaccharide (LPS) and proinflammatory cytokines TNF and IL1B. The SIRT1 activators resveratrol and SRT1720 significantly decreased LPS-induced TNF, IL6, and IL8 gene expression and release and PTGS2 mRNA expression and resultant prostaglandin (PG) E and PGF release from human gestational tissues. In conclusion, SIRT1 possesses anti-inflammatory actions and thus may play a role in regulating pregnancy and parturition