Study of the female sex survival advantage in melanoma—a focus on x-linked epigenetic regulators and immune responses in two cohorts

Background: Survival from melanoma is strongly related to patient sex, with females having a survival rate almost twice that of males. Many explanations have been proposed but have not withstood critical scrutiny. Prior analysis of different cancers with a sex bias has identified six X-linked genes that escape X chromosome inactivation in females and are, therefore, potentially involved in sex differences in survival. Four of the genes are well-known epigenetic regulators that are known to influence the expression of hundreds of other genes and signaling pathways in cancer. Methods: Survival and interaction analysis were performed on the skin cutaneous melanoma (SKCM) cohort in The Cancer Genome Atlas (TCGA), comparing high vs. low expression of KDM6A, ATRX, KDM5C, and DDX3X. The Leeds melanoma cohort (LMC) on 678 patients with primary melanoma was used as a validation cohort. Results: Analysis of TCGA data revealed that two of these genes—KDM6A and ATRX—were associated with improved survival from melanoma. Tumoral KDM6A was expressed at higher levels in females and was associated with inferred lymphoid infiltration into melanoma. Gene set analysis of high KDM6A showed strong associations with immune responses and downregulation of genes associated with Myc and other oncogenic pathways. The LMC analysis confirmed the prognostic significance of KDM6A and its interaction with EZH2 but also revealed the expression of KDM5C and DDX3X to be prognostically significant. The analysis also confirmed a partial correlation of KDM6A with immune tumor infiltrates. Conclusion: When considered together, the results from these two series are consistent with the involvement of X-linked epigenetic regulators in the improved survival of females from melanoma. The identification of gene signatures associated with their expression presents insights into the development of new treatment initiatives but provides a basis for exploration in future studies

The thermodynamics of general anesthesia

It is known that the action of general anesthetics is proportional to their partition coefficient in lipid membranes (Meyer-Overton rule). This solubility is, however, directly related to the depression of the temperature of the melting transition found close to body temperature in biomembranes. We propose a thermodynamic extension of the Meyer-Overton rule which is based on free energy changes in the system and thus automatically incorporates the effects of melting point depression. This model provides a quantitative explanation of the pressure reversal of anesthesia. Further, it explains why inflammation and the addition of divalent cations reduce the effectiveness of anesthesia.Comment: 7 pages, 2 figure

Phase transitions in biological membranes

Native membranes of biological cells display melting transitions of their lipids at a temperature of 10-20 degrees below body temperature. Such transitions can be observed in various bacterial cells, in nerves, in cancer cells, but also in lung surfactant. It seems as if the presence of transitions slightly below physiological temperature is a generic property of most cells. They are important because they influence many physical properties of the membranes. At the transition temperature, membranes display a larger permeability that is accompanied by ion-channel-like phenomena even in the complete absence of proteins. Membranes are softer, which implies that phenomena such as endocytosis and exocytosis are facilitated. Mechanical signal propagation phenomena related to nerve pulses are strongly enhanced. The position of transitions can be affected by changes in temperature, pressure, pH and salt concentration or by the presence of anesthetics. Thus, even at physiological temperature, these transitions are of relevance. There position and thereby the physical properties of the membrane can be controlled by changes in the intensive thermodynamic variables. Here, we review some of the experimental findings and the thermodynamics that describes the control of the membrane function.Comment: 23 pages, 15 figure

Intratumoral acidosis fosters cancer-induced bone pain through the activation of the mesenchymal tumor-associated stroma in bone metastasis from breast carcinoma

Cancer-induced bone pain (CIBP) is common in patients with bone metastases (BM), significantly impairing quality of life. The current treatments for CIBP are limited since they are often ineffective. Local acidosis derived from glycolytic carcinoma and tumor-induced osteolysis is only barely explored cause of pain. We found that breast carcinoma cells that prefer bone as a metastatic site have very high extracellular proton efflux and expression of pumps/ion transporters associated with acid-base balance (MCT4, CA9, and V-ATPase). Further, the impairment of intratumoral acidification via V-ATPase targeting in xenografts with BM significantly reduced CIBP, as measured by incapacitance test. We hypothesize that in addition to the direct acid-induced stimulation of nociceptors in the bone, a novel mechanism mediated by the acid-induced and tumor-associated mesenchymal stroma might ultimately lead to nociceptor sensitization and hyperalgesia. Consistent with this, short-term exposure of cancer-associated fibroblasts, mesenchymal stem cells, and osteoblasts to pH 6.8 promotes the expression of inflammatory and nociceptive mediators (NGF, BDNF, IL6, IL8, IL1b and CCL5). This is also consistent with a significant correlation between breakthrough pain, measured by pain questionnaire, and combined high serum levels of BDNF and IL6 in patients with BM, and also by immunofluorescence staining showing IL8 expression that was more in mesenchymal stromal cells rather than in tumors cells, and close to LAMP-2 positive acidifying carcinoma cells in BM tissue sections.In summary, intratumoral acidification in BM might promote CIBP also by activating the tumor-associated stroma, offering a new target for palliative treatments in advanced cancer

Defining the functional roles of X-linked epigenetic regulator lysine demethylase 6A (KDM6A) in Melanoma.

Melanoma is an aggressive form of skin cancer and Australia has one of highest incidences of melanoma in the world. Current treatments for metastatic melanoma are plagued by the resistance melanomas develop against immunotherapies and targeted therapies. Lysine demethylases (KDMs) are epigenetic enzymes that remove methyl groups from the amino acid lysine (K) on histone proteins, which effects gene expression. One of these KDMs is KDM6A (an X-linked gene also known as UTX) that removes methyl groups from histone 3, lysine number 27 (H3K27me3) inducing activation of gene expression. KDM6A has been reported to play roles in the progression of multiple cancers, however the role of KDM6A in melanoma is yet to be investigated. In this study, the effects of KDM6A expression on clinical parameters, including survival, and gene expression patterns were investigated in a cohort of 458 melanoma patients obtained from The Cancer Genome Atlas (TCGA). In addition, the effects of a KDM6 inhibitor GSK-J4 and KDM6A knockout using the CRISPR-Cas9 system in melanoma cells was investigated in vitro using a variety of molecular and cell biology assays. RNA sequencing was used to determine which genes and pathways were significantly upregulated and downregulated in drug treated and KDM6A knockout melanoma cells. Results showed that high KDM6A expression significantly correlated with gender in melanoma patients KDM6A expression was associated with better overall survival in melanoma patients, particularly in females but not in males. High KDM6A expression was associated with upregulation of interferon pathways and downregulation of pro-survival pathways which may prevent melanoma growth. High KDM6A expression was also associated with multiple immune cell infiltration in melanoma patient tumours, especially in females. In addition, KDM6A expression significantly correlated with COMPASS components, an important epigenetic complex in which KDM6A is an essential enzymatic component. In vitro studies showed that KDM6A knockout in melanoma cells significantly increased proliferation and colony formation, hence promoting melanoma cell growth and supporting the role of KDM6A in tumour suppressive function. RNA-seq analysis in KDM6A depleted cells showed significant upregulation of oncogenic pathways and downregulation of tumour suppressive pathways. Surprisingly, GSK-J4 treatment in melanoma cells showed the opposite effect to KDM6A knockdown with increased apoptosis and decreased viability, colony formation and 3D spheroid formation, but had no effect on cell cycle regardless of basal KDM6A expression. In addition, GSK-J4 also targeted other histone markers which include H3K4me3. RNA-seq analysis in drug treated melanoma cells showed a significant upregulation of pathways involved in DNA regulation and significant downregulation of cell metabolic pathways. These results suggest that KDM6A appears to have a protective effect in melanoma patients, especially in females, indicating a potential tumour suppressive role

Wnt-5a is up-regulated and associated with poor survival in ovarian cancer

Ovarian cancer is the ninth most common cancer diagnosed in Australian women, but the fifth most common cause of cancer related death. One pathway involved in ovarian cancer is Wnt, a key developmental pathway which is often dysregulated in human cancer and is thought to play a role in epithelial to mesenchymal transition (EMT) and metastasis.Expression of the Wnt ligand Wnt-5a was investigated in a large cohort of 721 benign, borderline and ovarian cancer patients. Wnt-5a expression was significantly higher in ovarian cancer patients compared to benign and borderline patients (p 0.05). Wnt-5a expression wasalso correlated with clinico-pathological parameters of ovarian cancer, namely the present tumour marker CA-125 (p < 0.05), patient age (p < 0.0001), ascites (p < 0.0001), residual disease (p < 0.0001) and advanced FIGO Stage (p < 0.0001). In addition, there was a significant negative correlation between Wnt-5a expression and SFRP4 an antagonist of the Wnt signalling pathway (p < 0.0001). This data shows that Wnt-5a is associated with more aggressive disease and is a predictor of poor prognosis in ovarian cancer patients.In vitro studies were conducted to investigate the functional effect of Wnt-5a modulation in the normal epithelial ovarian cell line HOSE 6.3 by using a recombinant protein form of Wnt-5a. Wnt-5a expression significantly decreased the expression of &#946;&#150;catenin dependent Wnt signalling targets CCDN1 (p < 0.01), Axin2 (p < 0.01) and MYC (p < 0.001), and significantly increased the expression of &#946;&#150;catenin independent Wnt signalling targets NFAT (p < 0.01), JNK (p < 0.01), PRKCA (p < 0.01) and MMP13 (p < 0.001). Wnt-5a expression in HOSE 6.3 induced EMT since CDH1 (E-cadherin) expression decreased (p < 0.05) and VIM (Vimentin) (p < 0.01), SNAI2 (Slug) (p < 0.01), and SNAI1 (Snail) (p < 0.001) expression increased. Wnt-5a expression increased cell proliferation (p < 0.05), increased cell migration (p < 0.001) anddecreased cell adhesion (p < 0.05)

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells.

Recent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.Human coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug's peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.Both drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.We demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo