Transcription Factor Hypoxia-inducible Factor-1 Research Articles

Hypoxia-induced Factor-1α and its Role in Endometrial Cancer.

The transcription factor hypoxia inducible factor-1 (HIF-1) is one of the main factors in the cell's response to a lack of oxygen. Hypoxia is a typical feature of a growing cancerous tumor. Increased activity of HIF-1 is observed in many cancers, including endometrial cancer. HIF-1 functions as a heterodimer consisting of three subunits HIF-1α, HIF-2α, and HIF-3α and one subunit β. HIF-1α is a subunit that is sensitive to oxygen concentration and is constitutively expressed. The HIF-1α gene is highly polymorphic. Literature data suggest that single nucleotide polymorphisms (SNPs) of the HIF-1α gene may be risk factors for endometrial cancer. A better understanding of the molecular mechanisms of cancer development, progression and prognosis, including the role of SNPs, could lead to the development of new anti-cancer therapies.

Oscillatory hypoxia induced gene expression predicts low survival in human breast cancer patients.

Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factorHypoxia-Inducible Factor 1(HIF-1), which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated noncanonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influences cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using the Cancer Genome Atlas RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticates low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding responsein cells resulting in gene expression predicting reduced survival.

Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed β subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1β. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and β subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.

Hypoxia and the Hypoxia-Inducible Factors in Lymphocyte Differentiation and Function.

Molecular oxygen doubles as a biomolecular building block and an element required for energy generation and metabolism in aerobic organisms. A variety of systems in mammalian cells sense the concentration of oxygen to which they are exposed and are tuned to the range present in our blood and tissues. The ability to respond to insufficient O2 in tissues is central to regulation of erythroid lineage cells, but challenges also are posed for immune cells by a need to adjust to very different oxygen concentrations. Hypoxia-inducible factors (HIFs) provide a major means of making such adjustments. For adaptive immunity, lymphoid lineages are initially defined in bone marrow niches; T lineage cells arise in the thymus, and B cells complete maturation in the spleen. Lymphocytes move from these first stops into microenvironments (bloodstream, lymphatics, and tissues) with distinct oxygenation in each. Herein, evidence pertaining to functions of the HIF transcription factors (TFs) in lymphocyte differentiation and function is reviewed. For the CD4+ and CD8+ subsets of T cells, the case is very strong that hypoxia and HIFs regulate important differentiation events and functions after the naïve lymphocytes emerge from the thymus. In the B lineage, the data indicate that HIF1 contributes to a balanced regulation of B-cell fates after antigen (Ag) activation during immunity. A model synthesized from the aggregate literature is that HIF in lymphocytes generally serves to modulate function in a manner dependent on the molecular context framed by other TFs and signals.

Translational mRNA Profiling Analysis of Pseudopalisading Cells in Bevacizumab Resistance of GBM

AbstractGlioblastoma (GBM) is a malignant brain tumor with a poor prognosis that easily recurs. The antiangiogenic agent bevacizumab is used to treat recurrent GBM. However, GBM treated with bevacizumab easily rebounds due to evasive mechanisms that induce resistance to treatment, such as hypoxia‐inducible factor (HIF) associated pathways. The transcription factor HIFs are oxygen‐dependent and are degraded via the ubiquitin‐dependent proteasomal pathway. Therefore, it is necessary to study bevacizumab resistance with HIF‐associated pathways through translational mRNA profiling. This work applies a hypoxia response element (HRE) promoter to the ribosome affinity purification (TRAP) system for hypoxia‐specific translational mRNA profiling analysis. Here, this work analyzes the translatome of pseudopalisading cells in the brain samples of orthotopic mouse models using hypoxia‐responsive 5× HRE‐TRAP system to investigate the molecular mechanism of drug resistance on pseudopalisading cells around the necrotic area induced by bevacizumab resistance. The translatomic analysis result shows that pseudopalisading cells exhibit notable enrichment of gene sets associated with neurodegenerative diseases, such as Alzheimer's disease. The outcomes of this study enhance clinical relevance, meaning that the translatomic analysis result of pseudopalisading cells during bevacizumab treatment‐induced resistance provides new insight into a promising approach for developing targeted therapeutics.

Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis

Abstract Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and the most common form of the disease is cutaneous leishmaniasis (CL). A fundamental question in CL is what regulates the development of severe disease, which is critical to develop therapies to ameliorate pathology. In a series of studies, we demonstrated CD8 T cell-dependent cytotoxicity as the main inducer of immunopathology in CL. This result was unexpected since IFN-γ production by CD8 T cells plays a protective role by promoting pathogen elimination. To resolve this paradox, we studied the CD8 T cells in different anatomic sites and found that the effector function of CD8 T cells in CL depends on their location: while CD8 T cells are cytotoxic (GzmB+) and produce little IFN-γ in the skin lesions, CD8 T cells in the draining lymph nodes (dLN) have the opposite profile. Importantly, GzmB-CD8 T cells from dLN quickly upregulate GzmB after injection into CL lesions. By transcriptional profiling, we found that CD8 T cells in lesions but not dLN have a hypoxic signature. In vivo, we observed that CL lesions are hypoxic using the Oxyphor G4 oxygen probe and pimonidazole staining. In vitro, we found that induction of hypoxia was sufficient to convert GzmB- into GzmB+ CD8 T cells, and significantly decreased CD8 T cell production of IFN-γ. In vivo, blocking hypoxia-inducible factor (HIF), a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Mice with CD8 T cell deficient in HIF-1α produced significantly less GzmB in the skin lesions compared to wildtype mice. Together, our results suggest that the hypoxic signaling through the transcription factor HIF in CL lesions is necessary to convert CD8 T cells into pathogenic CD8 T cells in the skin lesion. Supported by grants from NIH (1R01AI162711)

Selective virtual screening of hypoxia inducible factor agonists and antagonists using artificial intelligence and linear response theory.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor that mediates a signaling cascade under low oxygen concentration and prevents cell death. Under normoxia, enhanced HIF activity promotes tumorigenesis, and it is considered as a key oncogenic driver in renal cell carcinoma. Enhancing HIF activity is a therapeutic approach to treat renal anaemia, thus making both HIF activators (agonists) and inhibitors (antagonists) a potential therapeutic target under different disease conditions. While known HIF agonists and antagonists have opposing effect on the HIF activity, both binds to same cavity in the C-terminal domain of HIF (PASB domain). We identify the atomic level mechanism behind differential effect of agonists/antagonists on HIF activity using molecular dynamics simulations and linear response theory. Agonists and antagonists trigger different allosteric pathway from the PASB domain to the DNA-binding domain of HIF transcription factor. The ligand induced external forces on the protein residues explains the observed allosteric effect and used as a descriptor to predict the activation/inhibition potential of a given ligand. A large-scale structure-based virtual screening of 700 million molecules from ZINC database is performed to identify the potential new ligands of HIF. Binding energies of 10 million ligands is predicted using Autodock Vina and used to train a directed-message passing neural network (D-MPNN) to predict binding energies of the remaining molecules, hence enabling the screening of the larger portion of molecules at a reasonable computational cost. The top candidate molecules are further classified as potential inhibitors based on the forces exerted by the ligands on protein residues.

Evaluation of HIF-1 Involvement in the BDNF and ProBDNF Signaling Pathways among Obstructive Sleep Apnea Patients.

Obstructive Sleep Apnea (OSA) is a chronic condition characterized by intermittent hypoxia associated with multiple comorbidities, including psychiatric disorders, such as depression, insomnia, and cognitive impairment. The brain-derived neurotrophic factor (BDNF) and proBDNF singling pathways have been shown to be involved in this group of diseases. Furthermore, their expression might be affected by hypoxia-inducible factor 1 (HIF-1), which is an oxygen sensitive transcription factor due to its alpha subunit. Therefore, this study aimed to evaluate the association between HIF-1α, BDNF, and proBDNF protein levels among OSA patients. This study included 40 individuals who underwent polysomnography (PSG) and were divided into the OSA group (n = 20; AHI ≥ 30) and healthy control (n = 20; AHI < 5) based on the apnea−hypopnea index (AHI). All participants had their peripheral blood collected in the evening before and the morning after the PSG. BDNF, proBDNF, and HIF-1α protein concertation measurements were performed using ELISA. No differences were found in BDNF, proBDNF, and HIF-1α protein levels between OSA and the control group, both in the evening and in the morning. In the OSA group, i.e., the linear regression model, the morning BDNF protein level was predicted by age (ß = −0.389, p = 0.023) and the mean SpO2 of desaturations during sleep (ß = −0.577, p = 0.002). This model accounted for 63.3% of the variability in the morning BDNF protein level (F = 14.639, p < 0.001). The morning proBDNF protein level was predicted by age (ß = −0.395, p = 0.033) and HIF-1α morning protein level (ß = −3.192, p = 0.005). This model accounted for 52.4% of the variability in the morning BDNF protein level (F = 9.355, p = 0.002). The obtained results suggest that the HIF-1 transcription factor might be involved in the pathway activated by proBDNF, which may have protective properties from hypoxia in OSA patients.

Pan-cancer analysis of tissue and single-cell HIF-pathway activation using a conserved gene signature.

Activation of cellular hypoxia pathways, orchestrated by HIF (hypoxia-inducible factor) transcription factors, is a common feature of multiple tumor types, resulting from microenvironment factors and oncogenic mutation. Although they help drive many of the "hallmarks" of cancer and are associated with poor outcome and resistance to therapy, the transcriptional targets of HIF vary considerably depending on the cell type. By integrating 72 genome-wide assays of HIF binding and transcriptional regulation from multiple cancer types, we define a consensus set of 48 HIF target genes that is highly conserved across cancer types and cell lineages. These genes provide an effective marker of HIF activation in bulk and single-cell transcriptomic analyses across a wide range of cancer types and in malignant and stromal cell types. This allows the tissue-orchestrated responses to the hypoxic tumor microenvironment and to oncogenic HIF activation to be deconvoluted at the tumor and single-cell level.

FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.

The FAM57A (family with sequence similarity 57 member A) gene is controversially discussed to possess pro- or anti-tumorigenic potential. Here, we analyze the regulation of cellular FAM57A protein levels and study the functional role of FAM57A in HPV-positive cervical cancer cells. We find that FAM57A protein expression strongly depends on cell density, with FAM57A being readily detectable at low cell density, but undetectable at high cell density. This regulation occurs post-transcriptionally and is not mirrored by corresponding changes at the RNA level. We further show that FAM57A protein levels are highly increased in cervical cancer cells cultivated at hypoxia compared to normoxia and provide evidence that FAM57A is a hypoxia-responsive gene under control of the α-subunit of the HIF-1 (hypoxia-inducible factor-1) transcription factor. Yet, the strong relative increase of FAM57A protein levels in hypoxic cells is predominantly cell-density-dependent and occurs post-transcriptionally. Other anti-proliferative effectors besides hypoxia, such as silencing of HPV E6/E7 oncogene expression in cervical cancer cells, also result in an increase of FAM57A levels compared to untreated cells. Functional analyses reveal that FAM57A repression leads to pronounced anti-proliferative as well as anti-migratory effects in cervical cancer cells. Taken together, these results provide insights into the regulation of FAM57A protein levels and reveal that they underlie a tight cell-density-dependent control. Moreover, they identify FAM57A as a critical determinant for the phenotype of cervical cancer cells, which promotes their proliferation and migration capacities.

P12.03.B Ascorbate alters the hypoxic pathway in glioblastoma cellsin vitro and associates with improved patient survival

Abstract Background Glioblastomas are highly aggressive and hypoxic tumours. This environment activates the hypoxic pathway, driving glioma progression and treatment resistance. The hypoxic pathway is regulated by the hypoxia inducible factor (HIF) hydroxylases, which require oxygen as a substrate. Under normoxic conditions, the HIF hydroxylases are active, causing degradation and inhibition of HIF transcription factors. Under hypoxia, the activity of the hydroxylases reduces and HIF accumulates, activating the hypoxic response. HIF hydroxylases also require ascorbate as a cofactor for optimal function. The brain has one of the highest ascorbate levels in the human body, yet data on ascorbate levels in gliomas is scarce. Cellular ascorbate uptake occurs through solute carrier family 23 member 2 (SLC23A2). My aim is to understand the relationship between ascorbate, SLC23A2 and the hypoxic pathway in brain cancer using both in vitro cell culture and clinical samples. Material and Methods Ascorbate uptake was measured in human glioblastoma cell lines (T98G, U251MG, U87MG; ATCC) using reverse phase high performance liquid chromatography (HPLC-ECD). CRISPR-Cas was designed to knock-out SLC23A2. Clinical glioblastoma samples (n=37) and follow-up data were provided by the Cancer Society Tissue Bank and University of Otago Dunedin. Ethics and informed consent were obtained (H19/163, MEC/08/02/016). Ascorbate levels, measured by HPLC-ECD, and HIF-1α and downstream targets were measured using Western blotting or ELISA. A HIF score was calculated from HIF-1α and downstream target protein levels to estimate hypoxic pathway activity. Results In this study we have shown that T98G and U251 cells accumulate up to 15 nmol ascorbate/106 cells when exposed to 500 µM ascorbate for up to 24 hours, compared to U87MG cells with up to 3 nmol ascorbate/106 cells. Cancer Cell Line Encyclopaedia data shows that T98G and U251MG cells express higher levels of SLC23A2 compared to U87MG cells, aligning with our results. Clinical glioblastoma tissue contained a median of 7.6 µg ascorbate/100 mg tissue. Patients survival was significantly longer with above, vs below, median tumour ascorbate levels (Gehan-Breslow-Wilcoxon p = 0.027). The HIF score was negatively correlated with tumour ascorbate levels (Pearson r -0.327, p = 0.048). Patients with higher HIF-score had significantly shorter survival time compared to those with a lower HIF score (Gehan-Breslow-Wilcoxon p = 0.005). Conclusion Ascorbate uptake in glioblastoma cells varies between cell lines and appears reliant on the level of SLC23A2. Higher ascorbate content in clinical glioblastoma samples was associated with reduced hypoxic pathway activity and longer patient survival. Ongoing work, using SLC23A2 CRISPR-Cas knock-out cells, is investigating the effect of disrupting ascorbate uptake on hypoxic pathway signalling in glioblastoma cells.

Hypoxia Affects HIF-1/LDH-A Signaling Pathway by Methylation Modification and Transcriptional Regulation in Japanese Flounder (Paralichthys olivaceus).

Simple SummaryWith global climate change and increased aquaculture production, fishes in natural waters or aquaculture systems are easily subjected to hypoxic stress. However, our understanding about their responsive mechanisms to hypoxia is still limited. Japanese flounder (Paralichthys olivaceus) is a widely cultivated marine economical flatfish, whose hypoxic responsive mechanisms are not fully researched. In this study, responses to hypoxia were investigated at blood physiological, biochemical, hormonal, and molecular levels. Responsive mechanisms of the HIF-1/LDH-A signaling pathway in epigenetic modification and transcriptional regulation were also researched. These results are important for enriching the theory of environmental responsive mechanisms and guiding aquaculture.Japanese flounder (Paralichthys olivaceus) responsive mechanisms to hypoxia are still not fully understood. Therefore, we performed an acute hypoxic treatment (dissolved oxygen at 2.07 ± 0.08 mg/L) on Japanese flounder. It was confirmed that the hypoxic stress affected the physiological phenotype through changes in blood physiology (RBC, HGB, WBC), biochemistry (LDH, ALP, ALT, GLU, TC, TG, ALB), and hormone (cortisol) indicators. Hypoxia inducible factor-1 (HIF-1), an essential oxygen homeostasis mediator in organisms consisting of an inducible HIF-1α and a constitutive HIF-1β, and its target gene LDH-A were deeply studied. Results showed that HIF-1α and LDH-A genes were co-expressed and significantly affected by hypoxic stress. The dual-luciferase reporter assay confirmed that transcription factor HIF-1 transcriptionally regulated the LDH-A gene, and its transcription binding sequence was GGACGTGA located at −2343~−2336. The DNA methylation status of HIF-1α and LDH-A genes were detected to understand the mechanism of environmental stress on genes. It was found that hypoxia affected the HIF-1α gene and LDH-A gene methylation levels. The study uncovered HIF-1/LDH-A signaling pathway responsive mechanisms of Japanese flounder to hypoxia in epigenetic modification and transcriptional regulation. Our study is significant to further the understanding of environmental responsive mechanisms as well as providing a reference for aquaculture.

Abstract 677: Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer

Abstract Hypoxia is a key feature of the solid tumour microenvironment, in which it is associated with poor prognosis and resistance to therapy. Hypoxia-inducible factor (HIF) is the central mediator of cellular responses to hypoxia and drives a transcriptional program with central roles in tumorigenesis. Clear cell renal cell cancers (ccRCC) exhibit the most direct and profound upregulation of the HIF pathway - through bi-allelic inactivation of the VHL tumour suppressor, a ubiquitin ligase that targets HIF-alpha subunits for degradation under normal oxygenated conditions. HIF-alpha proteins are members of the bHLH-PAS family and interact with HIF-1beta (also known as ARNT) to form functional heterodimers binding chromatin. However, the aryl hydrocarbon receptor (AHR), another bHLH-PAS protein that dimerises with HIF-1beta, is also up-regulated in ccRCC, raising the possibility of crosstalk between these two transcriptional pathways. Previous studies support an antagonistic relationship between HIF and AHR, with evidence of a context-dependent and gene-specific crosstalk. Here, we investigate the pan-genomic and precise relationship between the HIF and AHR transcription factors in ccRCC. Using immunoprecipitation, we show direct competition for HIF-1beta binding between the HIF-alpha and AHR proteins in renal cancer cell lines. Pan-genomic analysis of HIF-1alpha, HIF-2alpha, HIF-1beta and AHR chromatin binding by ChIP-seq analysis reveals a widespread competitive interaction between HIF and AHR. In RCC4 cells, this interaction is bidirectional, with induction of HIF leading to a reduction in AHR binding, while induction of AHR reduces binding of both HIF-1alpha and HIF-2alpha to chromatin. However, this competition is diminished at sites bound by both HIF and AHR, indicating a cooperative interaction between the two transcription factors in cis. Thus, while the HIF-alpha and AHR proteins compete for the common binding partner HIF-1beta, they appear to form a cooperative interaction when bound to neighbouring sites on chromatin. Linking these findings to changes in gene expression by RNA-seq analysis, we show the global downstream effects of the competition between HIF and AHR, but also reveal a degree of overlap and cooperativity between their transcriptional programs. Furthermore, we provide evidence of this antagonistic relationship in ccRCC tumours (TCGA database), in which we observe a negative correlation between HIF and AHR target gene expression. These findings shed light on the complex crosstalk between the HIF and AHR transcriptional pathways, which encompasses both a widespread inhibitory interaction and specific cooperative events and highlight a role for AHR in modifying the HIF transcriptional output in ccRCC. Citation Format: Véronique N. Lafleur, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 677.

Abstract 3766: Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia

Abstract Low oxygen availability (hypoxia) is a common feature of many solid tumors and is associated with poor prognosis and resistance to therapy. The main regulator of cellular responses to hypoxia is the transcription factor HIF (hypoxia-inducible factor). In addition, several histone lysine demethylases (KDMs) are 2-OG dependent dioxygenases and require oxygen for their activity. When oxygen availability is limited, the activity of these KDMs is inhibited leading to global histone hypermethylation, potentially altering gene expression. However, some KDMs are also known to be direct transcriptional targets of HIF, increasing their expression in hypoxia and counteracting this histone hypermethylation. Furthermore, HIF can facilitate recruitment of histone methyltransferase activity to chromatin in cis, leading to local increases in methylation status. Histone methylation can be associated with both gene activation and gene repression, and it remains unclear how the balance of these widespread changes relates to locus-specific regulation of gene expression. Furthermore, previous attempts to analyze regulation of histone methylation by ChIP-seq in hypoxia have not employed normalization methods that take into account global changes in histone modification. We have performed ChIP-seq analysis of histone trimethylation (H3K4me3, H3K9me3, H3K27me3 and H3K36me3) in PC3 prostate cancer cells incubated in normoxia or (16hrs, 1%) hypoxia. We have then examined the effects of the HIF-transcriptional pathway on these marks using the same cell line following deletion of HIF-1b to block the HIF response. We employ an integrated spike-in with drosophila chromatin to provide a normalization control. We then relate our findings to changes in gene expression using RNA-seq analysis, again normalized to a spike-in control. We observe global increases in all 4 histone trimethylations across the genome in hypoxia. In particular, we observe hypoxic increases in histone trimethylation at all gene loci. However, while basal levels of histone trimethylation correlated with basal transcript levels as previously reported, hypoxic increases in promoter-associated histone H3K4me3 trimethylation bore no relation to either the direction or magnitude of gene regulation in hypoxia. Although, more subtle increases were observed across the gene body at gene loci induced by hypoxia. These findings question the currently accepted model that changes in H3K4me3 instruct changes in transcription in hypoxia and suggest that the overall effect of histone trimethylation is a more subtle balance of competing effects. Citation Format: Jessica D. Kindrick, Peter J. Ratcliffe, WIlliam Doug Figg, David R. Mole. Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3766.

Abstract 1587: Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors

Abstract Kidney cancer is the seventh most common malignancy in the Western world and 80% of these are clear cell renal cell cancers (ccRCC). ccRCC arises following inactivation of the VHL gene, which negatively regulates HIF (hypoxia-inducible factor) transcription factors. Therefore, in ccRCC, HIF is constitutively active and drives a gene expression program that is central to tumorigenesis. Whilst this is necessary for ccRCC formation, it is not sufficient, and other mutations must also occur in combination with VHL inactivation to drive ccRCC tumorigenesis. The mechanisms underlying their synergy with VHL inactivation remain poorly understood. These secondary driver mutations are often subclonal in nature, suggesting the presence of phenotypically distinct malignant cells within an individual tumor. This intratumor heterogeneity renders it difficult to study the functional impact of driver mutations in ccRCC by conventional ‘bulk’ analyses such as RNA-seq. Consequently, the molecular mechanisms underlying ccRCC initiation are not known. Furthermore, non-malignant stromal cells (including the vasculature and immune response) can support tumor growth, and little is known as to how they interact with malignant cells in ccRCC. This is particularly important since current therapeutic strategies in kidney cancer target the stromal compartment. Therefore, there is an unmet need to better-understand transcriptional heterogeneity and cellular diversity in ccRCC. To that end, we have sampled multiple regions from ccRCC tumors (and tumor-adjacent kidney tissue) and performed single cell RNA-seq (10x Genomics) on approximately 150,000 individual cells. These encompass malignant cells as well as non-malignant cells (including fibroblasts, endothelial cells, pericytes and immune cells), both of which exhibit pronounced transcriptional heterogeneity within and between tumors. Analysis of these transcriptomes has identified evolutionary gene expression programs underlying cancer cell dedifferentiation. In addition, we observe concurrent heterogeneity in angiogenic signaling between malignant cells and non-malignant blood vessel cells within these tumors. Lastly, we have explored the effect of microenvironmental hypoxia across different ccRCC tumor cell types. In summary, we present a single cell atlas derived from multi-region heterogenous ccRCC samples. This sheds light on the mechanisms underlying tumor evolution and progression, as well as angiogenic heterogeneity (which may contribute to the variable response to receptor tyrosine kinase inhibition) and the effect of hypoxia on ccRCC biology. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Peter J. Ratcliffe, David R. Mole. Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1587.

The Protective Role of pVHL in Imiquimod-Induced Psoriasis-like Skin Inflammation.

Psoriasis is a chronic inflammatory disease distinguished by an excessive proliferation and abnormal differentiation of keratinocytes. Immune cells, such as T lymphocytes and neutrophils, and inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and interleukin 17 (IL-17), are essential for maintaining psoriatic lesions. Additionally, a hypoxic milieu present in the skin promotes the expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α). This protein regulates the expression of angiogenic and glycolytic factors, such as vascular endothelial grown factor and lactate dehydrogenase (LDH), both relevant in chronic inflammation. The von Hippel–Lindau protein (pVHL) is a negative regulator of HIF-1α. Previously, we found that pVHL was almost absent in the lesions of psoriasis patients; therefore, we investigated the impact of rescue pVHL expression in lesional skin. We used the imiquimod-induced psoriasis-like mouse model as an adenoviral vector that allowed us to express pVHL in the skin. Our data show that, in lesional skin, pVHL expression was reduced, whereas HIF-1α was increased. Remarkably, the retrieval of pVHL prevented psoriatic lesions, diminishing erythema, scale, and epidermal and vascular thickness. Furthermore, pVHL expression was capable of reducing HIF-1α, LDH, TNF-α and immune cell infiltration (mainly IL-17+ neutrophils). In conclusion, our results demonstrate that pVHL has a protective role to play in the pathophysiology of psoriasis.

EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1α is repressed by SET7-catalyzed lysine methylation

Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 α under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor–mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 α. Notably, we demonstrate that the methylation mimic mutant of EGLN1 loses the capability to suppress the hypoxia signaling pathway, leading to the enhancement of cell proliferation and the oxygen consumption rate. Collectively, our data identify a novel modification of EGLN1 that is critical for inhibiting its enzymatic activity and which may benefit cellular adaptation to conditions of hypoxia.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 μg ascorbate/100 mg tissue, range 0.8 – 20.4 μg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.

Ethiology and Pathophysiology of Whart Hpv Infection: A Review Article

Genital Human Papillomavirus (HPV) infection is a self-limiting and often asymptomatic disease. Genital HPV infection is divided into high-risk HPV (HPV 16 and 18) and low-risk HPV (HPV 6 and 11). Low-risk or non-oncogenic HPV produces a clinical picture of anogenital warts, which may be condylomatous, papules, or keratotic. The subclinical form of genital HPV infection can present with an "aceto-white" lesion found on the cervix that will appear when examined using an acetic acid solution evaluated by colposcopy. Based on the association with cervical cancer and precursor lesions, HPV can also be classified as high-risk (HR-HPV) and low-risk (LR-HPV) oncogenic types. LR-HPV types, such as HPV 6 and 11, can cause generalized genital warts or benign hyperproliferative lesions with a very limited predisposition to malignant progression, while infection with HR-HPV types, HPV 16 and 18, is associated with the development of pre-malignant and cervical lesions. Discussion: Human Papillomavirus is a small DNA virus (50-55nm) belonging to the family Papillomaviridae and genus Papillomavirus. Human papillomavirus (HPV) is a relatively small non-enveloped virus containing a double-stranded circular DNA genome associated with histone-like proteins and protected by a capsid formed by two final proteins, L1 and L2. Each capsid consists of 72 capsomeres, The pathogenesis of HPV begins with infection of stem cells in the basal layer of the epithelium. Once inside the cell, the virus requires expression of the E1 and E2 genes to maintain a low genome copy number. These proteins bind to the origin of replication and the virus secretes cellular DNA polymerases and other proteins required for DNA replication. In the suprabasal layer, the expression of genes E1, E2, E5, E6 and E7 contributes to the maintenance of the viral genome and induces cell proliferation, increasing the number of HPV-infected cells in the epithelium, resulting in a higher number of cells that will eventually produce infectious virions. Conclusion: In benign HPV lesions, cell proliferation increases leading to increased nutrition, resulting in competition for nutrients and oxygen. Both HR-HPV and LR-HPV E7 proteins increase the level of the transcription factor Hypoxia-inducible factor-1 (HIF-1), and induce increased expression of HIF-1 target genes under hypoxic conditions. Increased HIF-1 activity results in increased transcription of a subset of genes that support angiogenesis, and the induction of this angiogenesis is critical for the persistence and growth of HPV lesions such as genital warts.

Sima, a Drosophila homolog of HIF-1α, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression.

Limited oxygen availability impairs normal body growth, although the underlying mechanisms are not fully understood. In Drosophila, hypoxic responses in the larval fat body (FB) disturb the secretion of insulin-like peptides from the brain, inhibiting body growth. However, the cell-autonomous effects of hypoxia on the insulin-signaling pathway in larval FB have been underexplored. In this study, we aimed to examine the effects of overexpression of Sima, a Drosophila hypoxia-inducible factor-1 (HIF-1) α homolog and a key component of HIF-1 transcription factor essential for hypoxic adaptation, on the insulin-signaling pathway in larval FB. Forced expression of Sima in FB reduced the larval body growth with reduced Akt phosphorylation levels in FB cells and increased hemolymph sugar levels. Sima-mediated growth inhibition was reversed by overexpression of TOR or suppression of FOXO. After Sima overexpression, larvae showed higher expression levels of Tribbles, a negative regulator of Akt activity, and a simultaneous knockdown of Tribbles completely abolished the effects of Sima on larval body growth. Furthermore, a reporter analysis revealed Tribbles as a direct target gene of Sima. These results suggest that Sima in FB evokes Tribbles-mediated insulin resistance and consequently protects against aberrant insulin-dependent larval body growth under hypoxia.