Decoding Epigenetic Regulators in Cancer: Acetylation of HIF2A by Histone Acetyltransferase 1 (HAT1) is essential for executing hypoxia response in glioma

Abstract

Gliomas are tumors of the central nervous system arising from glial cells. Based on the origin of the cell type, it can be astrocytoma, oligodendroglioma, and ependymoma. Astrocytoma is the most common type of glioma, further classified as Grades I, II, III, and IV. Grade IV tumor is also known as Glioblastoma (GBM) and is the most lethal and highly malignant brain tumor. Despite several medical interventions, the overall survival is very dismal, and half of the patients die within 15 months of diagnosis. The dismal picture of survival is further aggravated by the grim quality of life of GBM patients. Advancement in high-throughput technologies coupled with bioinformatics tools has revolutionized cancer research. The vast implication of next-generation sequencing techniques has empowered us to explore the molecular pathways leading to the pathogenesis and therapy resistance of GBM. Screening based on the whole genome, whole-exome, and transcriptome sequencing has identified major signaling pathways responsible for gliomagenesis and has played a broader role in diagnosis and treatment regimens. The genetic and epigenetic screens have stratified GBM into molecular features like G-CIMP+/-, IDH mutant and wild type, and MGMT promoter methylated and unmethylated, and these features have proven to be exceptional prognostic markers. These integrated genetic and epigenetic screenings have also revealed the molecular heterogeneity present in GBM and identified genes responsible for the resistance and phenotypic plasticity of GBM cells. In the recent decade, further augmentation in sequencing techniques like ChiP-Seq, ATAC-Seq, FAIRE-Seq, and global methylation assay has enabled researchers to understand the epigenome of cancer cells. E.g., In GBM, promoter methylation of MGMT makes the cells sensitive to chemotherapeutic drugs. In addition to this, IDH mutation leads to a change in the global methylation pattern of GBM cells compared to non-IDH mutant samples. Besides these, the growing knowledge about the role of long noncoding RNAs in fine-tuning gene regulation has propelled the field of epigenetics in cancer development and progression. Advancements in proteomic technology have recently identified protein deregulation in glioma. It has also helped us understand the role of post-translational regulation in cancer initiation and progression. These shreds of epigenetic evidence also indicate that the players performing these epigenetic functions may get deregulated or non-functional in cancer. All this information intrigued us to explore the status of epigenetic regulators in glioma. Our studies illustrate the various aspects of epigenetic regulators in GBM, where our focus ranges from surfacing the epigenetic regulator's landscape in GBM, expanding regulation of a particular epigenetic regulator, HAT1, and the role of this protein towards glioma pathogenesis. This thesis is divided into two work-related chapters: Chapter 3 and chapter 4. In chapter 3, we performed extensive bioinformatic analyses to understand altered epigenetic regulators and the probable causes of their misregulation. In chapter 4, we identify HAT1 (histone Acetyltransferase 1) as one of the epigenetic regulators dysregulated in GBM that promotes gliomagenesis, proliferation, and migration via HIF2A. Chapter 3: Integrative multi-omics analysis illustrates the genetic and epigenetic alterations of Epigenetic Regulators (ERs) in Glioblastoma. Epigenetic regulators are the proteins that participate in nucleotide modifications, histone modifications, and chromatin remodeling. Recent studies have illustrated the importance of these proteins in maintaining the epigenome of a cell and their deregulation leading to various cancer. However, the molecular mechanism by which this particular class of proteins gets regulated remains elusive. In this study, we have carried out an integrated bioinformatic analysis of epigenetic regulators (ERs) in various datasets to identify several genetic and epigenetic altered events among them in GBM. Alterations in the ERs include mutations, copy number variations (amplifications and deletions), and expression changes both at RNA and protein levels, and the probable causes of deregulation were assessed in our analysis. Whole exome sequencing data from the TCGA was analyzed to identify mutated ERs in GBM. We identified 13 ERs mutated in more than 2% of GBM samples. Integrated analysis of the transcriptome, copy number variation data, RPPA, Methylome, and miRNAome was carried out to identify ERs transcript differentially regulated in GBM compared to control samples and the putative mechanism behind this differential regulation. There were 236 ERs differentially regulated at the transcript level. Additional analysis of these 236 differentially regulated ERs disclosed that CyclinB-CDK1-FOXM1axis transcriptionally regulates the most significantly upregulated ERs. Besides this, around 50% of ERs are regulated by miRNAs, nearly 15% of ERs are deregulated because of promoter methylation, and around 10% by copy number variation. Analysis of proteome data from CPTAC identified differentially regulated proteins in GBM compared to control samples. We also found the concordance between the RNA and protein data, revealing the genes regulated at the protein level through post-translational modifications. To obtain insights into transformation and aggressiveness-related ERs in GBM, we analyzed the transcriptome data for ERs deregulation in the lower grade and GBM. 110 ERs were dysregulated in the lower grade and GBM and could be responsible for initial transformation and gliomagenesis. While 66 ERs were specifically deregulated in GBM, that might be responsible for aggressiveness and progression from lower to higher grade glioma. We also recognized 45 ERs uniquely expressed in recurrent GBM compared to the control and primary GBM samples. These differentially expressed ERs might give an extra edge to tumor cells in chemoresistance and radioresistance and could be implicated in tumor recurrence prediction. These gene-specific targeting could be used in combination with the standard therapy of GBM to prevent a recurrence. Chemoresistance and radioresistance of GBM, eventually leading to its recurrence, is also attributed to Glioma stem-like cells (GSCs). Hence, we compared the transcriptomic data of neural stem cells, Glioma stem-like cells (GSCs), and differentiated glioma cells to identify GSCs-specific ERs. We found one ER specifically upregulated and seven downregulated in GSCs compared to NSCs and DGCs. The upregulated ER, USP46, is a deubiquitinating enzyme and its silencing led to a reduction in sphere formation of MGG8 and 1035 GSCs cells in neural stem cell media. Upon qPCR analysis, it was revealed that ablation of USP46 leads to a reduction in the level of GSCs-specific reprogramming factors SOX2 and POU3F2. In this study, we also sought mutant ERs association with the survival of the patients. We carried out a univariate cox regression analysis for the ERs mutated in more than two percent of GBM samples; however, we did not find any significant association between the mutation and survival of GBM patients. In a similar analysis with the differentially regulated ERs in GBM, we identified 15 ERs expressions to be significantly associated with the survival of GBM patients. To further strengthen this observation, we carried out a multivariate cox regression analysis for these 15 ERs, and out of 15, one ER, i.e., PRDM14, showed significant association with overall survival of GBM patients and was found to be an excellent prognostic marker that was able to predict survival independent of other GBM prognostic markers. Chapter 4: HAT1-dependent acetylation of HIF2A at Lysine 512 and Lysine 596 promotes its stability and confers oncogenic function. From the previous study, we found that HAT1 was one of the top upregulated ER. It was found to be upregulated in various other GBM datasets analyzed. These findings led us to speculate whether upregulation has any pathophysiologic function in GBM and, if yes, how it is advantageous to GBM cells. Keeping these objectives in mind, we tried to explore the physiological role of HAT1 in GBM biology. Our analysis revealed that expression of HAT1 is significantly associated with tumor aggressiveness and overall survival in both GBM and lower-grade astrocytoma. In vitro experiments after silencing of HAT1 indicated its oncogenic function in GBM. HAT1 silencing induced apoptosis and reduced proliferative and migratory capacity of GBM cells LN299 and U87. HAT1, a type B histone acetyltransferase, acetylates histone H4 at K5 and K12 positions and helps in replication-dependent chromatin assembly. It has also been shown to promote homologous recombination in DNA repair. Very few reports also implicate its role in tumorigenesis. In this study, we tried to explore the pathological role of HAT1 in GBM biology. We did transcriptome analysis in control and HAT1 silenced conditions. We found 1020 genes to be differentially regulated in HAT1 silenced conditions. Functional enrichment analysis identified hypoxia signaling as one of the most significantly depleted pathways in the HAT1 silenced condition. Further experiments revealed no change in the protein level expression of HIF1A in the HAT1-compromised condition. However, a drastic reduction in the HIF2A level indicated that HAT1 specifically regulates HIF2A protein stability and activity. Thus, we identified HIF2A as a novel target of HAT1. The present study demonstrates that HIF2A and HAT1 physically interact inside the cell. We also proved that HAT1 acetylates HIF2A, and loss of HAT1-dependent acetylation causes destabilization and degradation of HIF2A via proteasomal degradation. In addition to this, we also find that abrogation of HAT1 leads to a reduction in global acetylation of cells suggesting HAT1 may play a significant role in maintaining the acetylome pool of a cell. Through LC-MS/MS approach, we found several acetylated lysine residues of HIF2A. We zeroed in on three Lysine residues around the oxygen-dependent degradation domain (ODD) of HIF2A as putative targets of HAT1. These were K385, K512, and K596. We mutated lysine (K) to Glutamine (Q, an acetylation mimic) of all three residues individually by site-directed mutagenesis. Subsequent experiments revealed that K512Q and K596Q mutants did not show any change in the protein level in HAT1 silenced condition compared to shNT, thus making the HIF2A protein more stable. However, K385Q mutants failed to stabilize in the HAT1 condition, similar to HIF2A Wt. Therefore, for the first time, we show HIF2A as the target of HAT1, which leads to an effective hypoxia response in glioma. Though the oxygen-dependent regulation of HIF1A and HIF2A is similar, both proteins act on the same HIF response element, and both have overlapping targets like VEGF; however, they have selective target specificity based on the tissue and cell-type expression of HIF alpha subunits. For example, HIF1A primarily promotes glucose consumption and glycolysis, while HIF2A promotes the storage of fatty acids. Moreover, knockdown and overexpression studies of HIF1A and HIF2A in VHL deficient clear cell renal cell carcinoma cell line indicated that HIF2A but not HIF1A is responsible for tumor growth. HIF2A has also been found to promote the transcription of OCT4 and NANOG, implicating its role in pluripotency and stem cell maintenance. Moreover, HIF2A is expressed explicitly in the CD133 positive subpopulation of glioblastoma, and neuroblastoma cells, whereas HIF1A levels were similar in both tumorigenic and nontumorigenic cells implying the specific role of HIF2A in cancer stem cells maintenance. Existing literature information about HIF2A and the interaction of HAT1 with HIF2A compelled us to investigate the role of HAT1 in the reprogramming and maintenance of Glioma stem cells. Our results show that HAT1 is essential for DGC reprogramming as HAT1 silenced condition resulted in reduced neurosphere formation. Rescue experiments using acetylation mimic mutants (K512 and K596) of HIF2A enabled the DGCs to reprogram efficiently even in HAT1 silenced conditions implying HAT1 mediated acetylation of HIF2A promotes hypoxia response in GSCs. In addition to this, we also investigated the probable reason for the robust expression of HAT1 in GBM. Upon inhibitor screening of oncogenic signaling pathways, we identified Wnt/β-catenin as a transcriptional regulator of HAT1. This observation was further validated using HAT1 promoter luc experiments in FH535 (a Wnt signaling inhibitor), LiCl (Wnt signaling activator) treatment, or in the silenced and overexpressed condition of β-catenin. Thus, our study identifies that HAT1 modulates hypoxia response through HIF2A stabilization and is responsible for gliomagenesis and the promotion of glioma reprogramming and glioma stem-like cells maintenance.