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 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

Inhibition of VEGF-A gene synthesis decreased ranibizumab and aflibercept mediated CCL2 CCL5, and CX3CL1 mRNA and protein levels in HCAECs.

<p>HCAECs were transfected with VEGF-A siRNA or VEGF-B siRNA. After 72h, cells were co-incubated with ranibizumab (0.1nM) or aflibercept (0.45nM), or PBS (control) for 16 h. Changes in CCL2, CCL5 and CXC3L1 (panels A, C and E) mRNA levels were quantified using real-time PCR. Protein levels (B, D, F) were quantified using ELISAs. Results are expressed as means +/- SEM. *P < 0.05 ranibizumab-treated non VEGF-A siRNA transfected cells vs. ranibizumab-treated VEGF-A siRNA transfected cells; **P< 0.05 aflibercept-treated non VEGF-A siRNA transfected cells vs. aflibercept-treated VEGF-A siRNA transfected cells.</p

Ranibizumab and aflibercept increase CCL2, CCL5, and CX3CL1 mRNA and protein levels in HCAECs.

<p>HCAECs were incubated with ranibizumab (R) (0.11nM) or aflibercept (A) (0.45nM), or PBS (C) for 16 h. Changes in CCL2, CCL-5 and CXC3L1 (panels A, C and E) mRNA levels were quantified using real-time PCR. Protein levels (B, D, F) were quantified using ELISAs. Results are expressed as means +/- SEM. *P < 0.05 ranibizumab treated cells vs. non-treated cells; **P< 0.05 aflibercept treated cells vs. non-treated cells; †P<0.05 ranibizumab treated cells vs aflibercept-treated cells.</p

Ranibizumab and aflibercept increased p65, ICAM-1 and VCAM-1 mRNA in HCAECs, with these effects attenuated by blocking VEGF-A gene synthesis, or VEGFR2/VEGF-A ligand binding or TLR4 receptor inhibition.

<p>HCAECs were incubated with ranibizumab (0.1nM) or aflibercept (0.45nM), or PBS (control). For VEGF-A knockout studies, HCAECs were transfected with VEGF-A siRNA 72h prior to incubation. For VEGFR2 inhibitor and TLR4 inhibitor experiments, these agents were added simultaneously with the drug. Changes in p65, ICAM-1 and VCAM-1 (panels A, B and C) mRNA levels were quantified using real-time PCR. Results are expressed as means +/- SEM. *P < 0.05 ranibizumab treated cells vs. non-treated cells, **P< 0.05 aflibercept-treated cells vs. non-treated cells, ∧P<0.05 ranibizumab-treated non VEGF-A siRNA transfected cells vs. ranibizumab-treated VEGF-A siRNA transfected cells, ∧∧P<0.05 afliberept-treated non VEGF-A siRNA transfected cells vs. abflibercept-treated VEGF-A siRNA transfected cells, # P < 0.05 ranibizumab-treated cells vs. ranibizumab-treated VEGFR2 inhibitor treated cells; ##P< 0.05 aflibercept-treated cells vs. aflibercept treated VEGFR2 inhibitor treated cells, ° P < 0.05 ranibizumab treated cells vs. ranibizumab-treated TLR4 inhibitor treated cells; °°P< 0.05 aflibercept-treated cells vs. aflibercept treated TLR4 inhibitor treated cells.</p

TLR4 receptor inhibition decreased ranibizumab and aflibercept mediated CCL2, CCL5, and CX3CL1 mRNA and protein levels in HCAECs.

<p>HCAECs were incubated with ranibizumab (0.1nM) or aflibercept (0.45nM), or PBS (control) and a TLR4 inhibitor for 16 h. Changes in CCL2, CCL5 and CXC3L1 (panels A, C and E) mRNA levels were quantified using real-time PCR. Protein levels (B, D, F) were quantified using ELISAs. Results are expressed as means +/- SEM. *P < 0.05 ranibizumab treated cells vs. ranibizumab-treated TLR4 inhibitor treated cells; **P< 0.05 aflibercept-treated cells vs. aflibercept treated TLR4 inhibitor treated cells.</p

Halogenated 2′-benzoylpyridine thiosemicarbazone (XBpT) chelators with potent and selective anti-neoplastic activity: Relationship to intracellular redox activity

Iron chelators of the 20-benzoylpyridine thiosemicarbazone (BpT) class show substantial potential as anticancer agents. To explore structure-activity relationships, new BpT analogues were designed that incorporated halogen substituents on the noncoordinating phenyl group (XBpTs). These XBpT ligands exhibited potent antiproliferative activity with some analogues exceeding that of the parent BpT compound. Importantly, there was an appreciable therapeutic index in vitro, as mortal cells were significantly less affected by these chelators relative to neoplastic cells. The addition of a halogen led to a halogen-specific increase in the redox potential of XBpT-Fe complexes. Probing for chelator-induced intracellular reactive oxygen species (ROS) with the fluorescent probe, 20,70-dichlorofluorescein, revealed a 1.5-4.7-fold increase in fluorescence upon incorporation of Cl, Br, or I to the parent analogues. Furthermore, an important structure-activity relationship was deduced where the addition of halogens led to a positive correlation between intracellular ROS generation and antiproliferative activity in the more hydrophilic BpT parent compounds