Reverse Engineering the Neuroblastoma Regulatory Network Uncovers MAX as One of the Master Regulators of Tumor Progression

Neuroblastoma is the most common extracranial tumor and a major cause of infant cancer mortality worldwide. Despite its importance, little is known about its molecular mechanisms. A striking feature of this tumor is its clinical heterogeneity. Possible outcomes range from aggressive invasion to other tissues, causing patient death, to spontaneous disease regression or differentiation into benign ganglioneuromas. Several efforts have been made in order to find tumor progression markers. In this work, we have reconstructed the neuroblastoma regulatory network using an information-theoretic approach in order to find genes involved in tumor progression and that could be used as outcome predictors or as therapeutic targets. We have queried the reconstructed neuroblastoma regulatory network using an aggressive neuroblastoma metastasis gene signature in order to find its master regulators (MRs). MRs expression profiles were then investigated in other neuroblastoma datasets so as to detect possible clinical significance. Our analysis pointed MAX as one of the MRs of neuroblastoma progression. We have found that higher MAX expression correlated with favorable patient outcomes. We have also found that MAX expression and protein levels were increased during neuroblastoma SH-SY5Y cells differentiation. We propose that MAX is involved in neuroblastoma progression, possibly increasing cell differentiation by means of regulating the availability of MYC:MAX heterodimers. This mechanism is consistent with the results found in our SH-SY5Y differentiation protocol, suggesting that MAX has a more central role in these cells differentiation than previously reported. Overexpression of MAX has been identified as anti-tumorigenic in other works, but, to our knowledge, this is the first time that the link between the expression of this gene and malignancy was verified under physiological conditions.     

Sensitivity of Malignant Peripheral Nerve Sheath Tumor Cells to TRAIL Is Augmented by Loss of NF1 through Modulation of MYC/MAD and Is Potentiated by Curcumin through Induction of ROS

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.     

Combined analysis of murine and human microarrays and ChIP analysis reveals genes associated with the ability of MYC to maintain tumorigenesis

The MYC oncogene has been implicated in the regulation of up to thousands of genes involved in many cellular programs including proliferation, growth, differentiation, self-renewal, and apoptosis. MYC is thought to induce cancer through an exaggerated effect on these physiologic programs. Which of these genes are responsible for the ability of MYC to initiate and/or maintain tumorigenesis is not clear. Previously, we have shown that upon brief MYC inactivation, some tumors undergo sustained regression. Here we demonstrate that upon MYC inactivation there are global permanent changes in gene expression detected by microarray analysis. By applying StepMiner analysis, we identified genes whose expression most strongly correlated with the ability of MYC to induce a neoplastic state. Notably, genes were identified that exhibited permanent changes in mRNA expression upon MYC inactivation. Importantly, permanent changes in gene expression could be shown by chromatin immunoprecipitation (ChIP) to be associated with permanent changes in the ability of MYC to bind to the promoter regions. Our list of candidate genes associated with tumor maintenance was further refined by comparing our analysis with other published results to generate a gene signature associated with MYC-induced tumorigenesis in mice. To validate the role of gene signatures associated with MYC in human tumorigenesis, we examined the expression of human homologs in 273 published human lymphoma microarray datasets in Affymetrix U133A format. One large functional group of these genes included the ribosomal structural proteins. In addition, we identified a group of genes involved in a diverse array of cellular functions including: BZW2, H2AFY, SFRS3, NAP1L1, NOLA2, UBE2D2, CCNG1, LIFR, FABP3, and EDG1. Hence, through our analysis of gene expression in murine tumor models and human lymphomas, we have identified a novel gene signature correlated with the ability of MYC to maintain tumorigenesis.     

Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes.

We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from Colo 320 cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins.     

Hypoxia reduces MAX expression in endothelial cells by unproductive splicing

The MYC–MAX–MXD network is involved in the regulation of cell differentiation and proliferation. Hypoxia affects the expression levels of several members of this network, but changes specific to MAX expression have so far not been shown. We found that in endothelial cells, hypoxia induces alternative splicing of MAX, thereby increasing the expression of two MAX isoforms that differ from the wild type in their 3′ end. Isoform C is degraded by nonsense-mediated decay and isoform E encodes a highly unstable protein. The instability of isoform E is conferred by 36 isoform-specific amino acids, which have the capacity to destabilize heterologous proteins. Both splicing events are therefore unproductive and serve the purpose to downregulate the wild type protein.     

植物对非生物胁迫应答的转录因子及调控机制

植物对非生物胁迫的应答反应涉及到许多基因和生化分子机制,胁迫相关基因、蛋白质及代谢物构成了一个复杂的调控网络,其中转录控制具有举足轻重的作用。本文主要对近年来发现的几种在转录控制中起关键作用的转录因子CBF/DREB、bZIP、MYB/MYC和HSF及其调控机制进行介绍。这几种转录因子可以分别和胁迫应答顺式作用元件CRT/DRE、ABRE、MYB/MYC识别位点及HSE结合,在非生物胁迫条件下调控下游靶基因的表达,进而使一些胁迫保护物质如脯氨酸、可溶性糖类、自由基的清除剂、热休克蛋白和分子伴侣等的表达水平升高,最终增强植物对非生物胁迫的耐受能力。