Histone Deacetylase Inhibitors: The Epigenetic Therapeutics That Repress Hypoxia-Inducible Factors

Histone deacetylase inhibitors (HDACIs) have been actively explored as a new generation of chemotherapeutics for cancers, generally known as epigenetic therapeutics. Recent findings indicate that several types of HDACIs repress angiogenesis, a process essential for tumor metabolism and progression. Accumulating evidence supports that this repression is mediated by disrupting the function of hypoxia-inducible factors (HIF-1, HIF-2, and collectively, HIF), which are the master regulators of angiogenesis and cellular adaptation to hypoxia. Since HIF also regulate glucose metabolism, cell survival, microenvironment remodeling, and other alterations commonly required for tumor progression, they are considered as novel targets for cancer chemotherapy. Though the precise biochemical mechanism underlying the HDACI-triggered repression of HIF function remains unclear, potential cellular factors that may link the inhibition of deacetylase activity to the repression of HIF function have been proposed. Here we review published data that inhibitors of type I/II HDACs repress HIF function by either reducing functional HIF-1α levels, or repressing HIF-α transactivation activity. In addition, underlying mechanisms and potential proteins involved in the repression will be discussed. A thorough understanding of HDACI-induced repression of HIF function may facilitate the development of future therapies to either repress or promote angiogenesis for cancer or chronic ischemic disorders, respectively

Interaction between HIF-1α (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1α

AbstractHypoxia inducible factor-1α (HIF-1α) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1α and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1α and thereby enhances HIF-1α ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1α. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1α. However, we find that hARD1 specifically binds HIF-1α, suggesting a putative, still unclear, connection between these proteins

Effects of Thermally Induced Configuration Changes on rAAV Genome’s Enzymatic Accessibility

Physical titers for recombinant adeno-associated viral (rAAV) vectors are measured by quantifying viral genomes. It is generally perceived that AAV virions disassemble and release DNA upon thermal treatment. Here, we present data on enzymatic accessibility of rAAV genomes when AAV virions were subjected to thermal treatment. For rAAV vectors with a normal genome size (≤4.7 kb), thermal treatment at 75°C–99°C allowed only ∼10% of genomes to be detectable by quantitative real-time PCR. In contrast, greater than 70% of AAV genomes can be detected under similar conditions for AAV vectors with an oversized genome (≥5.0 kb). The permeability of virions, as measured by ethidium bromide (EB) staining, was enhanced by thermal stimulation. These results suggest that in rAAV virions with standard-sized genomes, the capsid and DNA are close enough in proximity for heat-induced “crosslinking,” which results in inaccessibility of vector DNA to enzymatic reactions. In contrast, rAAV vectors with oversized genomes release their DNA readily upon thermal treatment. These findings suggested that the spatial arrangement of capsid protein and DNA in AAV virions is genome-size dependent. These results provide a foundation for future improvement of vector assays, design, and applications

VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer

Background The function of the non-coding portion of the human genome remains one of the most important questions of our time. Its vast complexity is exemplified by the recent identification of an unusual and notable component of the transcriptome - very long intergenic non-coding RNAs, termed vlincRNAs. Results Here we identify 2,147 vlincRNAs covering 10 percent of our genome. We show they are present not only in cancerous cells, but also in primary cells and normal human tissues, and are controlled by canonical promoters. Furthermore, vlincRNA promoters frequently originate from within endogenous retroviral sequences. Strikingly, the number of vlincRNAs expressed from endogenous retroviral promoters strongly correlates with pluripotency or the degree of malignant transformation. These results suggest a previously unknown connection between the pluripotent state and cancer via retroviral repeat-driven expression of vlincRNAs. Finally, we show that vlincRNAs can be syntenically conserved in humans and mouse and their depletion using RNAi can cause apoptosis in cancerous cells. Conclusions These intriguing observations suggest that vlincRNAs could create a framework that combines many existing short ESTs and lincRNAs into a landscape of very long transcripts functioning in the regulation of gene expression in the nucleus. Certain types of vlincRNAs participate at specific stages of normal development and, based on analysis of a limited set of cancerous and primary cell lines, they appear to be co-opted by cancer-associated transcriptional programs. This provides additional understanding of transcriptome regulation during the malignant state, and could lead to additional targets and options for its reversal

Characterization of the transcriptional regulatory functions of the transforming region of the adenovirus E1A oncoprotein

By interacting with proteins of host cells, the adenoviral E1A protein promotes DNA synthesis and cell cycle progression to provide an environment for viral replication, to immortalize host cells, and in coorboration with Ras, to transform rodent cells. The extreme N-terminus and CR1 of E1A are not only required for these activities, but also involved in transcriptional regulation of host gene expression. In this dissertation, evidence is provided that the N-terminus of 243R E1A has transactivation activity in yeast. Recruitment to a specific promoter is essential. Mutagenesis studies correlated the transactivation function with the extreme N-terminus and CR1. Cotransfection assays in rodent cells confirmed that two overlapping domains, aa1-65 and aa37-80, transactivated independently. When recruited to the PCNA promoter, either domain was sufficient to transactivate. Deletion of either the N-terminus or the carboxyl-terminus of E1A resulted in failure to induce PCNA expression. VP16 was able to restore the ability of N-terminal deletion mutants of E1A to induce the PCNA promoter while the lack of carboxyl-terminus was compensated by fusion with a DNA binding domain. Moreover, E1A repressed DBD.VP16-, DBD.E1A- and p53-mediated transactivation in an N-terminus-dependent manner. Repression of p53 transactivation can be released by overexpression of p300, hTBP, or TFIIB. Overexpression of the pocket proteins has no effect in either case. When p53 was cotransfected with E1A, similar inhibition was found in Saos-2 cells that lack endogenous pRb and p53 activity. E1A mutant defective in binding to pocket proteins remains repression activity. In vitro assays showed that the N-terminus of E1A was involved in interaction with TBP and a new set of cellular proteins with apparent molecular weights of 200, 90, 45, 30 and 28 kDa in addition to p300. These associations do not depend on E1A\u27s association with p300 or pRb. Therefore, the N-terminal transforming region of E1A contains two overlapping but distinct transcriptional regulatory domains that regulate cellular gene expression in evolutionarily distant species, and the N-terminus of E1A may possesses both p300-dependent and independent functions

Histone Deacetylase Inhibitors Induce VHL and Ubiquitin-Independent Proteasomal Degradation of Hypoxia-Inducible Factor 1α

Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxia-inducible factor 1α (HIF-1α) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1α expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1α expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1α levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1α by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1α with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6