This article reviews the mechanisms of gene silencing in cancer and clinical applications of this phenomenon. The silencing of genes, especially tumor-suppressor genes, is a key event in the development of cancer. The silencing can be effected by a disabling mutation or by a shutting down of the promoter region, the site at which transcription of the gene begins.

Gene Silencing in Cancer in Association with Promoter Hypermethylation

Elevated rates of reactive oxygen species (ROS) have been detected in almost all cancers, where they promote many aspects of tumour development and progression. However, tumour cells also express increased levels of antioxidant proteins to detoxify from ROS, suggesting that a delicate balance of intracellular ROS levels is required for cancer cell function. Further, the radical generated, the location of its generation, as well as the local concentration is important for the cellular functions of ROS in cancer. A challenge for novel therapeutic strategies will be the fine tuning of intracellular ROS signalling to effectively deprive cells from ROS-induced tumour promoting events, towards tipping the balance to ROS-induced apoptotic signalling. Alternatively, therapeutic antioxidants may prevent early events in tumour development, where ROS are important. However, to effectively target cancer cells specific ROS-sensing signalling pathways that mediate the diverse stress-regulated cellular functions need to be identified. This review discusses the generation of ROS within tumour cells, their detoxification, their cellular effects, as well as the major signalling cascades they utilize, but also provides an outlook on their modulation in therapeutics.

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Reactive oxygen species in cancer

Inflammasomes are key signalling platforms that detect pathogenic microorganisms and sterile stressors, and that activate the highly pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. In this Review, we discuss the complex regulatory mechanisms that facilitate a balanced but effective inflammasome-mediated immune response, and we highlight the similarities to another molecular signalling platform - the apoptosome - that monitors cellular health. Extracellular regulatory mechanisms are discussed, as well as the intracellular control of inflammasome assembly, for example, via ion fluxes, free radicals and autophagy.

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Activation and regulation of the inflammasomes

The past few years have seen an explosion of interest in the epigenetics of cancer. This has been a consequence of both the exciting coalescence of the chromatin and DNA methylation fields, and the realization that DNA methylation changes are involved in human malignancies. The ubiquity of DNA methylation changes has opened the way to a host of innovative diagnostic and therapeutic strategies. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic.

The power and the promise of DNA methylation markers

Hypoxia-inducible factors (HIFs) are broadly expressed in human cancers, and HIF1α and HIF2α were previously suspected to promote tumour progression through largely overlapping functions. However, this relatively simple model has now been challenged in light of recent data from various approaches that reveal unique and sometimes opposing activities of these HIFα isoforms in both normal physiology and disease. These effects are mediated in part through the regulation of unique target genes, as well as through direct and indirect interactions with important oncoproteins and tumour suppressors, including MYC and p53. As HIF inhibitors are currently undergoing clinical evaluation as cancer therapeutics, a more thorough understanding of the unique roles performed by HIF1α and HIF2α in human neoplasia is warranted.

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HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression

The physiological function of Sentrin/SUMO-specific proteases (SENPs) remains largely unexplored, and little is known about the regulation of SENPs themselves. Here, we show that a modest increase of reactive oxygen species (ROS) regulates SENP3 stability and localization. We found that SENP3 is continuously degraded through the ubiquitin-proteasome pathway under basal condition and that ROS inhibit this degradation. Furthermore, ROS causes SENP3 to redistribute from the nucleoli to the nucleoplasm, allowing it to regulate nuclear events. The stabilization and redistribution of SENP3 correlate with an increase in the transcriptional activity of the hypoxia-inducing factor-1 (HIF-1) under mild oxidative stress. ROS-enhanced HIF-1 transactivation is blocked by SENP3 knockdown. The de-SUMOylating activity of SENP3 is required for ROS-induced increase of HIF-1 transactivation, but the true substrate of SENP3 is the co-activator of HIF-1α, p300, rather than HIF-1α itself. Removing SUMO2/3 from p300 enhances its binding to HIF-1α. In vivo nude mouse xenografts overexpressing SENP3 are more angiogenic. Taken together, our results identify SENP3 as a redox sensor that regulates HIF-1 transcriptional activity under oxidative stress through the de-SUMOylation of p300.

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SENP3 is responsible for HIF‐1 transactivation under mild oxidative stress via p300 de‐SUMOylation

Purpose : This study was designed to detect aberrant p16 promoter methylation in the serum of patients with colorectal cancer (CRC) and to explore the possibility of using this assay in early detection or as a prognostic marker of CRC patients. Experimental Design : Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and matching serum samples and control serum samples from 34 patients with adenomatous polyps and 10 healthy individuals. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was then analyzed. Results : P16 hypermethylation was found in 20 of 52 (38%) CRCs. Among the 20 cases with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) cases. No methylated p16 sequences were detected in the peripheral serum of the other 32 CRC cases without these changes in the tumor, in 34 patients with adenomatous polyps, or in 10 healthy control subjects. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes’ stage ( P = 0.03). Conclusions : This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.

https://clincancerres.aacrjournals.org/content/clincanres/8/1/188.full.pdf

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

OBJECTIVES To detect aberrant p16 promoter methylation in serum of patients with colorectal cancer (CRC), and to explore the possibility of using this assay in early detection or as a prognostic marker. METHODS Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and corresponding serum samples. Serum samples from 34 patients with adenomatous polyps and 10 healthy individuals were used as controls. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was analyzed. RESULTS p16 methylation was found in 38% (20 of 52) of CRC tissues. Among the 20 patients with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) patients. No methylated p16 sequences were detected in the peripheral serum of the 32 CRC patients without these changes in the tumor, in 34 paitents with adenomatous polyps, or in 10 healthy controls. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (chi(2) = 5.7, P = 0.03). CONCLUSION This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.

The molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone/ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non-stress condition due to Hsp90-independent CHIP-mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP-mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non-stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.

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Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H(2)O(2)-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

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Hui Cang

Papers

SENP3 is responsible for HIF‐1 transactivation under mild oxidative stress via p300 de‐SUMOylation

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1.