Dual influence of TNFα on diverse in vitro models of ovarian cancer subtypes.

Ovarian cancer is the most lethal gynecological cancer. Numerous subtypes exist, each with distinct risk factors and prognosis. What underlies these subtypes and their progression is not clear, although inflammation through NFκB may play a key role. We performed a study on a series of well-characterized in vitro ovarian cancer models including TOV21G, TOV112D and OV90 originally derived from clear cell, endometrioid and high grade serous carcinoma respectively. Cells were treated with 0-100 ng/ml TNFα over 6-72 h. The NFκB pathway was inhibited by a series of NFκB pathway inhibitors, 100µM PDTC, 1µM PS-1145 and 200nM TPCA and the influence on cellular viability and inflammation was measured via an MTS assay and qPCR respectively. TNFα stimulation of NFκB was confirmed via Western blot. We found TNFα facilitated continued growth of TOV21G and TOV112D cells in an NFκB independent method. In contrast, TNFα inhibited OV90 cell growth in an NFκB dependent manner. TNFα stimulated production of IL-6, IL-8, MCP-1 and RANTES on all three cells lines, but only IL-6 and IL-8 were via NFκB mediated mechanisms. These results indicate TNFα may have diverse effects mediated through both NFκB and non-NFκB pathways on ovarian cancer cells. Understanding the role for TNFα in each subtype may have significant implications for charting disease progression and designing personalized treatments.

Early-stage endometrial cancer, CTNNB1 mutations, and the relation between lymphovascular space invasion and recurrence.

INTRODUCTION: Women diagnosed with early-stage (FIGO 1) endometrial cancer, grade 1 and 2 can have a good prognosis. Most of these women can be treated successfully with a hysterectomy and bilateral salpingo-oophorectomy and without the additional adjuvant treatment that is accompanied by more risks for complications. However, when recurrence does occur, the consequences can be dire. Accurate decisions must therefore be made by surgeons to avoid either under- or over-treatment. Risk and patient stratification for tailoring treatment still need further improvement. Both histopathology and genetic variants could be integrated into the decision process if relevant factors were identified. MATERIAL AND METHODS: Morphological features and the presence of selected genetic mutations in isolated malignant endometrial epithelial cells from these tumors were analyzed in a strictly defined cohort of FIGO 1, grade 1 and 2 low-risk endometrial cancer. Their presence in this cohort, their relation to recurrence, and the association between histopathological features and mutations were determined. This analysis was performed using archival formalin-fixed paraffin-embedded tissue, complete re-evaluation of histopathological features, laser capture microdissection of epithelial cells, and a polymerase chain reaction-based mutational screening assay. RESULTS: Twenty-one women with recurrence, after initial identification as low-risk endometrial cancer, were compared with 20 matched control women. The histological marker of lymphovascular invasion was significantly associated with recurrence. There was also a very high prevalence of mutations in CTNNB1 gene, occurring in 50% of this cohort. PTEN mutations were also observed in 27.8% of cases and PIK3CA mutations in 22.2%; none of these mutations were significantly related to recurrence. CONCLUSIONS: This study supports the importance of lymphovascular space invasion to identify women with significant risk for recurrence in initially low-risk, early-stage endometrial cancer. It also identifies CTNNB1 as a significant mutation in early-stage disease, and although it may not represent a marker for recurrence its high prevalence in early stage disease could have relevance for both pathogenesis and early treatment.

TNFα-induced IKKß complex activation influences epithelial, but not stromal cell survival in endometriosis.

STUDY QUESTION: Can the activity of the IκB kinase (IKKß) complex in endometriotic cells contribute to endometriotic lesion survival? SUMMARY ANSWER: There is a constitutive activity of the IKKß catalytic complex in peritoneal and deeply infiltrating lesions that can influence epithelial, but not stromal cell viability. WHAT IS KNOWN ALREADY: Endometriotic lesions exist in an inflammatory microenvironment with higher local concentrations of cytokines, such as tumour necrosis factor α (TNFα). TNFα stimulates the activation of the IKKß complex, an important nodal point in multiple signalling pathways that influence gene transcription, proliferation and apoptosis. However, few data on the regulation of IKKß in endometriotic tissue are currently available. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of endometriotic tissue from peritoneal, ovarian and deeply infiltrating lesions from 37 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Basal and activated (phosphorylated) IKKß concentrations were analysed by western blotting and immunohistochemistry. The relationship between the expression and activation of these proteins and peritoneal fluid (TNFα) concentrations, measured via ELISA, was examined. A subsequent in vitro analysis of TNFα treatment on the activation of IKKß and the effect on epithelial and stromal cell viability by its inhibition with PS1145 was also performed. MAIN RESULTS AND ROLE OF CHANCE: Levels of the phosphorylated IKKß complex in endometriotic lesions had a significant positive correlation with peritoneal fluid TNFα concentrations. Phosphorylated IKKß complex was more prevalent in peritoneal and deeply infiltrating endometriosis lesions compared with ovarian lesions. IKKß was present in both epithelial and stromal cells in all lesions but active IKKß was limited to epithelial cells. TNFα stimulated an increased expression of phosphorylated IKKß and the inhibition of this kinase with PS1145 significantly influenced ectopic epithelial cells viability but not eutopic epithelial cells, or endometrial stromal cells. LIMITATIONS, REASONS FOR CAUTION: In vitro analysis on epithelial cells was performed with immortalized cell lines and not primary cell cultures and only low sample numbers were available for the study. WIDER IMPLICATIONS OF THE FINDINGS: The regulation of aberrant signalling pathways represents a promising yet relatively unexplored area of endometriosis progression. The IKKß complex is activated by inflammation and is critical nodal point of numerous downstream kinase-signalling pathways, including NFκB (nuclear factor κB), mTOR (mammalian target of rapamycin) and BAD (Bcl2-antagonist of cell death). This study shows a significant relationship between peritoneal fluid TNFα and IKKß activation in epithelial cells that will have significant consequences for the continued survival of these cells at ectopic locations through the regulation of downstream pathways. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Swiss National Science Foundation (Grant Number 320030_140774). The authors have no conflict of interest to declare.

Inflammation influences steroid hormone receptors targeted by progestins in endometrial stromal cells from women with endometriosis.

Endometriosis is an estrogen-dependent disease characterised by the growth of endometrial epithelial and stromal cells outside the uterus creating a chronic inflammatory environment that further contributes to disease progression. The first choice treatment for endometriosis is currently progestin mediated hormone modulation. In addition to their progestogenic activity however, progestins also have the potential to bind to other nuclear receptors influencing their local activity on endometriotic cells. This local activity will be dependent on the steroid hormone receptor expression that occurs in endometrial cells in a chronic inflammatory environment. We therefore aimed to quantify receptors targeted by progestins in endometrial stromal cells after exposure to inflammation. Using primary endometrial stromal cells isolated from women with endometriosis we examined the mRNA and protein expression of the progesterone receptors A and B, membrane progesterone receptors 1 and 2, androgen receptors, mineralocorticoid receptors and glucocorticoid receptors after exposure to the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1ß (IL-1ß). The results indicate that both cytokines reduced the expression of progesterone receptors and increased the expression of the glucocorticoid receptors in the endometrial stromal cells. The change in expression of progestin targets in endometrial stromal cells in an inflammatory environment could contribute to the progesterone resistance observed in endometriotic cells and ultimately influence the design of hormonal therapies aimed at treating this disease.

Kinase signalling pathways in endometriosis: potential targets for non-hormonal therapeutics.

BACKGROUND: Endometriosis, the growth of endometrial tissue outside the uterine cavity, is associated with chronic pelvic pain, subfertility and an increased risk of ovarian cancer. Current treatments include the surgical removal of the lesions or the induction of a hypoestrogenic state. However, a reappearance of the lesion after surgery is common and a hypoestrogenic state is less than optimal for women of reproductive age. Additional approaches are required. Endometriosis lesions exist in a unique microenvironment characterized by increased concentrations of hormones, inflammation, oxidative stress and iron. This environment influences cell survival through the binding of membrane receptors and a subsequent cascading activation of intracellular kinases that stimulate a cellular response. Many of these kinase signalling pathways are constitutively activated in endometriosis. These pathways are being investigated as therapeutic targets in other diseases and thus may also represent a target for endometriosis treatment. METHODS: To identify relevant English language studies published up to 2015 on kinase signalling pathways in endometriosis, we searched the Pubmed database using the following search terms in various combinations; 'endometriosis', 'inflammation', 'oxidative stress', 'iron', 'kinase', 'NF kappa', 'mTOR', 'MAPK' 'p38', 'JNK', 'ERK' 'estrogen' and progesterone'. Further citing references were identified using the Scopus database and finally current clinical trials were searched on the clinicaltrials.gov trial registry. RESULTS: The current literature on intracellular kinases activated by the endometriotic environment can be summarized into three main pathways that could be targeted for treatments: the canonical IKKß/NFκB pathway, the MAPK pathways (ERK1/2, p38 and JNK) and the PI3K/AKT/mTOR pathway. A number of pharmaceutical compounds that target these pathways have been successfully trialled in in vitro and animal models of endometriosis, although they have not yet proceeded to clinical trials. The current generation of kinase inhibitors carry a potential for adverse side effects. CONCLUSIONS: Kinase signalling pathways represent viable targets for endometriosis treatment. At present, however, further improvements in clinical efficacy and the profile of adverse effects are required before these compounds can be useful for long-term endometriosis treatment. A better understanding of the molecular activity of these kinases, including the specific extracellular compounds that lead to their activation in endometriotic cells specifically should facilitate their improvement and could potentially lead to new, non-hormonal treatments of endometriosis.

Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis.

Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 healthy women undergoing sterilization or patients with benign ovarian cysts (controls) using multiplexed double fluorescence-based immunometric assay platform and enzyme-linked immunosorbent assay. Significantly higher concentrations of glycodelin-A were shown in serum, and significantly higher levels of glycodelin-A, IL-6, and IL-8, and lower levels of leptin were measured in the peritoneal fluid of cases versus controls. In serum, the best performance was shown by models that included the ratio of leptin/glycodelin-A and the ratio of ficolin 2/glycodelin-A, whereas in the peritoneal fluid the best models included the ratio of biglycan/leptin, regulated on activation normal T-cell expressed and secreted/IL-6 and ficolin-2/glycodelin-A, and IL-8 per milligram of total protein, all in combination with age. The models using serum and peritoneal fluid distinguished between ovarian endometriosis patients and controls regardless of the menstrual cycle phase with relatively high sensitivity (72.5% to 84.2%), specificity (78.4% to 91.2%), and area under the curve (0.85 to 0.90).

Expression of AKR1B1, AKR1C3 and other genes of prostaglandin F2α biosynthesis and action in ovarian endometriosis tissue and in model cell lines.

Endometriosis is a frequent benign gynecological disease characterized by endometrial tissue outside the uterine cavity. The estimated prevalence in the general population is 6-10%, but this reaches 30-50% in women with infertility and/or pain. As ectopic tissue within the pelvic cavity provokes inflammation, endometriosis is also considered a chronic inflammatory disease, and is characterized by increased peritoneal fluid levels of prostaglandin (PG)E2 and PGF2α. The AKR1B1 and AKR1C3 enzymes act as PG synthases and catalyze reduction of PGH2 to PGF2α, and PGD2 to 9α,11ß-PGF2α, respectively. AKR1B1 and AKR1C3 may thus be associated with increased PGF2α production in endometriosis patients, as supported by our previous report of increased AKR1C1-AKR1C3 mRNA levels in endometriotic tissue, compared to control endometrium. Here, we initially evaluated PGF2α concentrations in peritoneal fluid from endometriosis patients and healthy women. We also examined expression of AKR1B1, AKR1C3 and other genes involved in PGF2α biosynthesis, metabolism, and action in ovarian endometriosis tissue versus healthy endometrium, and in peritoneal endometriosis and control endometrium model cell lines. Compared to controls, increased PGF2α concentrations in peritoneal fluid of patients were supported by endometriotic tissue showing increased AKR1B1 mRNA and protein levels, but unchanged AKR1C3 protein levels. Among genes involved in PGF2α biosynthesis, metabolism and action PLA2G2A, PTGS2/COX-2, ABCC4 and PTGFR were up-regulated, mRNA levels of SLCO2A, PTGDS and HPGDS were unchanged, and genes PLA2G4A and HPGD were down-regulated in diseased tissue. All of these PGF2α-associated genes were also expressed in control endometrial HIEEC epithelial and HIESC stromal cell lines, and in peritoneal endometriosis 12-Z epithelial and 22-B stromal cell lines. Higher expression of PLA2G2A, PTGS2, AKR1B1, AKR1C3 and ABCC4 was seen in 22-B endometriosis cells compared to HIESC control cells. These cell models characterized in this study will enable further investigations into the role of PGF2α in the pathophysiology of endometriosis and the involvement of AKR1B1 and AKR1C3.

Phospholipase A2 group IIA is elevated in endometriomas but not in peritoneal fluid and serum of ovarian endometriosis patients.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.

Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients.

In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD=220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p<0.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.

Elevated glycodelin-A concentrations in serum and peritoneal fluid of women with ovarian endometriosis.

The aim of this study was to evaluate serum and peritoneal fluid (PF) glycodelin-A concentrations in women with ovarian endometriosis. Ninety-nine matched pairs of serum and PF samples were included in our study. The case group comprised 57 women with ovarian endometriosis and the control group 42 healthy women undergoing sterilization or patients with benign ovarian cysts. Glycodelin-A concentrations were measured using ELISA. Endometriosis patients had significantly higher serum and PF glycodelin-A concentrations compared to controls, and this increase was observed in both proliferative and secretory cycle phases. Glycodelin-A concentrations were more than 10-fold higher in PF than in serum and correlated with each other. Intensity and frequency of menstrual pain positively correlated with glycodelin-A concentrations. Sensitivity and specificity of glycodelin-A as a biomarker for ovarian endometriosis were 82.1% and 78.4% in serum, and 79.7% and 77.5% in PF, respectively. These results indicate that Glycodelin-A has a potential role as a biomarker to be used in combination with other, independent marker molecules.