microRNAs have emerged as powerful regulators of many biological processes, and their expression in many cancer tissues has been shown to correlate with clinical parameters such as cancer type and ...prognosis. Present in a variety of biological fluids, microRNAs have been described as a 'gold mine' of potential noninvasive biomarkers. Release of microRNA content of blood cells upon hemolysis dramatically alters the microRNA profile in blood, potentially affecting levels of a significant number of proposed biomarker microRNAs and, consequently, accuracy of serum or plasma-based tests. Several methods to detect low levels of hemolysis have been proposed; however, a direct comparison assessing their sensitivities is currently lacking. In this study, we evaluated the sensitivities of four methods to detect hemolysis in serum (listed in the order of sensitivity): measurement of hemoglobin using a Coulter® AcT diff™ Analyzer, visual inspection, the absorbance of hemoglobin measured by spectrophotometry at 414 nm and the ratio of red blood cell-enriched miR-451a to the reference microRNA miR-23a-3p. The miR ratio detected hemolysis down to approximately 0.001%, whereas the Coulter® AcT diff™ Analyzer was unable to detect hemolysis lower than 1%. The spectrophotometric method could detect down to 0.004% hemolysis, and correlated with the miR ratio. Analysis of hemolysis in a cohort of 86 serum samples from cancer patients and healthy controls showed that 31 of 86 (36%) were predicted by the miR ratio to be hemolyzed, whereas only 8 of these samples (9%) showed visible pink discoloration. Using receiver operator characteristic (ROC) analyses, we identified absorbance cutoffs of 0.072 and 0.3 that could identify samples with low and high levels of hemolysis, respectively. Overall, this study will assist researchers in the selection of appropriate methodologies to test for hemolysis in serum samples prior to quantifying expression of microRNAs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key ...cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20–RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy.
The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function ...of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as 'Low', 'Intermediate' or 'High' for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer, including epithelial ovarian cancer (EOC), and its expression has been found to correlate with advanced stage and ...poor prognosis. The EGFR ligand amphiregulin (AREG) has been investigated as a target for human cancer therapy and is known to have an autocrine role in many cancers. A cytokine array identified AREG as one of several cytokines upregulated by EGF in a phosphatidylinositol 3-kinase (PI3–K) dependent manner in EOC cells. To investigate the functional role of AREG in EOC, its effect on cellular migration and proliferation was assessed in two EOC cells lines, OV167 and SKOV3. AREG increased both migration and proliferation of EOC cell line models through activation of PI3–K signaling, but independent of mitogen activated protein kinase (MAPK) signaling. Through an AREG autocrine loop mediated via PI3–K, upregulation of AREG led to increased levels of both AREG transcript and secreted AREG, while downregulation of endogenous AREG decreased the ability of exogenous AREG to induce cell migration and proliferation. Further, inhibition of endogenous AREG activity or metalloproteinase activity decreased EGF-induced EOC migration and proliferation, indicating a role for soluble endogenous AREG in mediating the functional effects of EGFR in inducing migration and proliferation in EOC.
•AREG is a potential therapeutic target in EGFR-driven epithelial ovarian cancer (EOC).•AREG-stimulated migration, proliferation of EOC cells is dependent on activated PI3–K.•AREG-stimulated migration, proliferation of EOC cells is independent of MAPK signaling.•Levels of AREG are regulated through an autocrine loop mediated by PI3–K.•Endogenous AREG mediates EGFR-related migration and proliferation in EOC.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
There is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of ...malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers.
To identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification.
microRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784).
We identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Computational models are becoming an increasingly valuable tool in biomedical research. Their accuracy and effectiveness, however, rely on the identification of suitable parameters and on appropriate ...validation of the in-silico framework. Both these steps are highly dependent on the experimental model used as a reference to acquire the data. Selecting the most appropriate experimental framework thus becomes key, together with the analysis of the effect of combining results from different experimental models, a common practice often necessary due to limited data availability. In this work, the same in-silico model of ovarian cancer cell growth and metastasis, was calibrated with datasets acquired from traditional 2D monolayers, 3D cell culture models or a combination of the two. The comparison between the parameters sets obtained in the different conditions, together with the corresponding simulated behaviours, is presented. It provides a framework for the study of the effect of the different experimental models on the development of computational systems. This work also provides a set of general guidelines for the comparative testing and selection of experimental models and protocols to be used for parameter optimization in computational models.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Chromatin remodelling is a major mechanism by which cells control fundamental processes including gene expression, the DNA damage response (DDR) and ensuring the genomic plasticity required by stem ...cells to enable differentiation. The post-translational modification of histone H2B resulting in addition of a single ubiquitin, in humans at lysine 120 (K120; H2Bub1) and in yeast at K123, has key roles in transcriptional elongation associated with the RNA polymerase II-associated factor 1 complex (PAF1C) and in the DDR. H2Bub1 itself has been described as having tumour suppressive roles and a number of cancer-related proteins and/or complexes are recognised as part of the H2Bub1 interactome. These include the RING finger E3 ubiquitin ligases RNF20, RNF40 and BRCA1, the guardian of the genome p53, the PAF1C member CDC73, subunits of the switch/sucrose non-fermenting (SWI/SNF) chromatin remodelling complex and histone methyltransferase complexes DOT1L and COMPASS, as well as multiple deubiquitinases including USP22 and USP44. While globally depleted in many primary human malignancies, including breast, lung and colorectal cancer, H2Bub1 is selectively enriched at the coding region of certain highly expressed genes, including at p53 target genes in response to DNA damage, functioning to exercise transcriptional control of these loci. This review draws together extensive literature to cement a significant role for H2Bub1 in a range of human malignancies and discusses the interplay between key cancer-related proteins and H2Bub1-associated chromatin remodelling.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
There is growing evidence highlighting the importance of monoubiquitination as part of the histone code. Monoubiquitination, the covalent attachment of a single ubiquitin molecule at specific lysines ...of histone tails, has been associated with transcriptional elongation and the DNA damage response. Sites function as scaffolds or docking platforms for proteins involved in transcription or DNA repair; however, not all sites are equal, with some sites resulting in actively transcribed chromatin and others associated with gene silencing. All events are written by E3 ubiquitin ligases, predominantly of the RING (really interesting new gene) finger type. One of the most well-studied events is monoubiquitination of histone H2B at lysine 120 (H2Bub1), written predominantly by the RING finger complex RNF20-RNF40 and generally associated with active transcription. Monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) is also well-studied, its E3 ubiquitin ligase constituting part of thePolycomb Repressor Complex 1 (PRC1), RING1B-BMI1, associated with transcriptional silencing. Both modifications are activated as part of the DNA damage response. Histone monoubiquitination is a key epigenomic event shaping the chromatin landscape of malignancy and influencing how cells respond to DNA damage. This review discusses a number of these sites and the E3 RING finger ubiquitin ligases that write them.
Abstract Purpose More women diagnosed with breast cancer (BC) are living with oncology treatment–induced hot flushes (HFs). This Australian-based survey explores why some women experience more severe ...or ongoing HF and whether specific population characteristics are predictive of HF occurrence, frequency, and/or severity. Methods A non-probabilistic anonymous survey distributed online (Register4) and two Australian hospitals collected demographic and clinical information. Eligibility was consenting Australian-based women, 18 years and over, with a primary BC diagnosis. Analysis included linear and logistic regression models. Results A total of 324 survey responses were analyzed. Chemotherapy and hormone therapy were each associated with HF occurrence (aOR = 2.92, 95% CI 1.27, 6.70, p = 0.01; and aOR = 7.50, 95% CI 3.02, 18.62, p < 0.001) and in combination (aOR = 5.98, 95% CI 2.61, 13.69, p < 0.001). Increased self-reported anxiety at BC diagnosis was significantly associated with HF frequency and severity scores (aCO = 0.71, 95% CI 0.31, 1.12, p = 0.001; and aCO = 0.44, 95% CI 0.33, 0.55, p < 0.001). Postmenopausal women had significantly lower HF severity and frequency scores than premenopausal women (aCO = −0.93, 95% CI −1.62, −0.25, p = 0.008; and aCO = −2.62, 95% CI −5.14, −0.11, p = 0.041). Conclusions Women with BC receiving chemotherapy and/or hormone therapy and premenopausal or experiencing elevated anxiety and/or stress will likely experience more severe oncology treatment–related HFs. Implications for Cancer Survivors HFs continue across the BC treatment trajectory with women >5-year survivorship still reporting life impacts, with premenopausal women at the time of BC diagnosis at higher risk of experiencing severe and more frequent oncology treatment-induced HFs than postmenopausal women. Women at high risk require information on methods to moderate HF potential life impacts and maintain treatment compliance.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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