Sheri Nixdorf

Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.

Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community.

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A(2) (PLA(2)) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA(2)-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA(2) enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA(2) enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA(2)s), particularly the best studied enzyme Group IIA sPLA(2) in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA(2) in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA(2). Group VI calcium-independent PLA(2) enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA(2) enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.

Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the “bisecting N-acetyl-glucosamine” type N-glycans, increased levels of α 2–6 sialylated N-glycans and “N,N′-diacetyl-lactosamine” type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique “bisecting GlcNAc” type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic programming of their associated synthetic enzymes in ovarian cancer could potentially be used for the development of novel anti-glycan drug targets and clinical diagnostic tools.

The Wnt gatekeeper SFRP4 modulates EMT, cell migration and downstream Wnt signalling in serous ovarian cancer cells

Aberrant Wnt signalling is implicated in numerous human cancers, and understanding the effects of modulation of pathway members may lead to the development of novel therapeutics. Expression of secreted frizzled related protein 4 (SFRP4), an extracellular modulator of the Wnt signalling pathway, is progressively lost in more aggressive ovarian cancer phenotypes. Here we show that recombinant SFRP4 (rSFRP4) treatment of a serous ovarian cancer cell line results in inhibition of β-catenin dependent Wnt signalling as measured by TOP/FOP Wnt reporter assay and decreased transcription of Wnt target genes, Axin2, CyclinD1 and Myc. In addition, rSFRP4 treatment significantly increased the ability of ovarian cancer cells to adhere to collagen and fibronectin, and decreased their ability to migrate across an inflicted wound. We conclude that these changes in cell behaviour may be mediated via mesenchymal to epithelial transition (MET), as rSFRP4 treatment also resulted in increased expression of the epithelial marker E-cadherin, and reduced expression of Vimentin and Twist. Combined, these results indicate that modulation of a single upstream gatekeeper of Wnt signalling can have effects on downstream Wnt signalling and ovarian cancer cell behaviour, as mediated through epithelial to mesenchymal plasticity (EMP). This raises the possibility that SFRP4 may be used both diagnostically and therapeutically in epithelial ovarian cancer.

Loss of secreted frizzled-related protein 4 correlates with an aggressive phenotype and predicts poor outcome in ovarian cancer patients.

Background Activation of the Wnt signaling pathway is implicated in aberrant cellular proliferation in various cancers. In 40% of endometrioid ovarian cancers, constitutive activation of the pathway is due to oncogenic mutations in β-catenin or other inactivating mutations in key negative regulators. Secreted frizzled-related protein 4 (SFRP4) has been proposed to have inhibitory activity through binding and sequestering Wnt ligands. Methodology/Principal Findings We performed RT-qPCR and Western-blotting in primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators activated β-catenin, β-catenin and GSK3β. SFRP4 was then examined by immunohistochemistry in a cohort of 721 patients and due to its proposed secretory function, in plasma, presenting the first ELISA for SFRP4. SFRP4 was most highly expressed in tubal epithelium and decreased with malignant transformation, both on RNA and on protein level, where it was even more profound in the membrane fraction (p<0.0001). SFRP4 was expressed on the protein level in all histotypes of ovarian cancer but was decreased from borderline tumors to cancers and with loss of cellular differentiation. Loss of membrane expression was an independent predictor of poor survival in ovarian cancer patients (p = 0.02 unadjusted; p = 0.089 adjusted), which increased the risk of a patient to die from this disease by the factor 1.8. Conclusions/Significance Our results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets.

The non-canonical Wnt ligand, Wnt5a, is upregulated and associated with epithelial to mesenchymal transition in epithelial ovarian cancer

OBJECTIVE Aberrant Wnt signalling has previously been associated with gynaecological cancers, and the aim of this study was to investigate the expression of Wnt5a in epithelial ovarian cancer, and clarify its role in activating or inhibiting β-catenin dependent and independent Wnt signalling pathways. METHOD Wnt5a expression was investigated in a large cohort of epithelial ovarian cancer patient samples using immunohistochemistry and correlated with clinicopathological variables. Wnt5a function was investigated in vitro in ovarian cell lines. RESULTS Wnt5a expression was found to be upregulated in all major subtypes (serous, endometrioid, clear cell and mucinous) of epithelial ovarian cancer compared to borderline tumours and benign controls. Treatment of ovarian surface epithelial cells with recombinant Wnt5a decreased cell adhesion and was associated with increased epithelial to mesenchymal transition (EMT). In addition, downstream targets of β-catenin dependent Wnt signalling were inhibited, and β-catenin independent targets increased following Wnt5a upregulation. Knockdown of Wnt5a in ovarian cancer cells was associated with a mesenchymal to epithelial transition (MET), but had no significant effect on cell migration or proliferation. CONCLUSION This study adds to the increasing evidence that Wnt signalling may play an important role in ovarian cancer development. Utilising an unparalleled large cohort of 623 patients, Wnt5a protein expression was shown to be significantly higher in ovarian cancer patients when compared to benign and borderline ovarian tumours and healthy control patients. In addition, we have utilised in vitro models to show for the first time in ovarian cancer that Wnt5a driven non-canonical pathways can alter epithelial to mesenchymal transition (EMT).

Reliable in vitro studies require appropriate ovarian cancer cell lines.

Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future.

Elevated levels of prostate‐specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis

The underlying basis for rising levels of prostate‐specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer–derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established. Expression of mutant p53 was demonstrated by Western blot analysis, inactivation of wtp53 function, and a loss of p53‐dependent responses to DNA damage induced by UV‐irradiation and cisplatin. Levels of PSA mRNA and secreted protein were determined by RT‐PCR and Western blotting, respectively. Serine protease activity was assessed using an esterase assay. In vivo effects of mutant p53 expression were examined after orthotopic implantation into prostates of nude mice. Expression of all p53 mutants was associated with elevated PSA mRNA and secreted PSA protein. In a representative line, mutant p53 was also associated with increased PSA protease‐like activity compared with a control line expressing wildtype p53. Overall PSA levels, and PSA levels in serum from mice bearing tumors derived from cells expressing mutant p53, were increased compared with levels in mice bearing tumors derived from control cells. In addition, the tumors derived from cells with mutant p53 had increased vascularization and induced lymph node metastases. These data provide in vitro and in vivo support for the notion that p53 mutations directly contribute to increased levels of serum PSA, and are associated with more aggressive tumors.

A clinicopathological review of 33 patients with vulvar melanoma identifies c-KIT as a prognostic marker

Vulvar melanoma is the second most common vulvar cancer. Patients with vulvar melanoma usually present with the disease at a late stage and have a poor prognosis. The prognostic predictors reported in the literature are not unequivocal and the role of lichen sclerosus and c-KIT mutations in the aetiology of vulvar melanoma is unclear. Breslow staging currently seems to be the most adequate predictor of prognosis. We thus performed a clinicopathological and literature review to identify suitable predictors of prognosis and survival and investigated the expression of c-KIT (by immunohistochemistry) in patients with vulvar melanoma (n=33) from the Gynaecological Cancer Centres of the Royal Hospital for Women (Sydney, Australia) and John Hunter Hospital (Newcastle, Australia). Our series of 33 patients fitted the expected clinical profile of older women: delayed presentation, high stage, limited response to treatment and poor prognosis. We identified 3 patients (9.1%) with lichen sclerosus associated with melanoma in situ, although no lichen sclerosus was found in the areas of invasive melanoma. No patient had vulvar nevi. We identified a) Breslow’s depth, b) an absence of any of the pathological risk factors, such as satellitosis, in-transit metastasis, lymphovascular space invasion (LVSI) and dermal mitosis, c) removal of inguino-femoral lymph nodes, d) lateral margin of >1 cm, and e) c-KIT expression as valuable prognostic predictors for disease-free survival. We conclude that c-KIT expression is, apart from Breslow’s depth, another valuable predictor of prognosis and survival. Lichen sclerosus may be associated with vulvar melanoma.

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells.

OBJECTIVE The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer.