Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFAdriven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402-11. Ó2014 AACR.

Parkinson’s disease (PD) is one of the major neurodegenerative disorders. Mitochondrial malfunction is implicated in PD pathogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1), a serine/threonine kinase, plays an important role in the quality control of mitochondria and more than 70 PINK1 mutations have been identified to cause early-onset PD. However, the regulation of PINK1 gene expression remains elusive. In the present study, we identified the transcription start site (TSS) of the human PINK1 gene using switching mechanism at 5’end of RNA transcription (SMART RACE) assay. The TSS is located at 91 bp upstream of the translation start site ATG. The region with 104 bp was identified as the minimal promoter region by deletion analysis followed by dual luciferase assay. Four functional cis-acting nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-binding sites within the PINK1 promoter were identified. NFκB overexpression led to the up-regulation of PINK1 expression in both HEK293 cells and SH-SY5Y cells. Consistently, lipopolysaccharide (LPS), a strong activator of NFκB, significantly increased PINK1 expression in SH-SY5Y cells. Taken together, our results clearly suggested that PINK1 expression is tightly regulated at its transcription level and NFκB is a positive regulator for PINK1 expression.

The zinc finger transcription factor cKrox is crucial for CD4 T cell development. Mice lacking a functional cKrox gene lack CD4 T cells. Furthermore, in cKrox-deficient mice, developing T cells expressing a MHC class II-restricted TCR are redirected into the CD8 T cell lineage. We investigated the effect of the cKrox transgene on the development and function of two other types of T cells, namely self-specific CD8 T cells and CD4 T regulatory cells. In TCR transgenic mice that express a male-specific TCR (H-Y TCR), the only CD8 T cells that developed in male TCR transgenic mice are self-specific CD8 T cells. These H-Y TCR+ self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11, NK cell markers and proliferative response in response to IL-2 or IL-15. We found that the cKrox transgene converted these self-specific CD8 T cells into CD4 T cells. Furthermore, the converted CD4 T cells lost the characteristics of these self-specific CD8 T cells described above. Notably, the converted self-specific CD8 T cells express genes that are characteristic of CD4 T cells (GATA-3 and IL-2). The numbers of CD4 T regulatory cells were significantly higher in cKrox transgenic mice and these cells expressed higher suppressor activity compared to CD4 T regulatory cells from control mice. These studies indicate that the cKrox transgene affects the development and function of multiple T cell types. This work is supported by the Canadian Cancer Society.