HIF-1 Target Research Articles

Unveiling the proteome of the fasting heart: Insights into HIF-1 pathway regulation.

Fasting is a common dietary intervention known for its protective effects against metabolic and cardiovascular diseases. While its effects are mostly systemic, understanding tissue-specific changes in the heart is crucial for the identification of the mechanisms underlying fasting-induced cardioprotection. In this study, we performed a proteomic analysis of the fasting heart and attempted to clarify the molecular basis of fasting-induced cardioprotection. Our investigation identified a total of 4,652 proteins, with 127 exhibiting downregulation and 118 showing upregulation after fasting. Annotation analysis highlighted significant changes in processes such as lipid metabolism, the peroxisome pathway, and reactive oxygen species metabolism. Notably, the HIF-1 signaling pathway emerged as one of the focal points, with various HIF-1 targets exhibiting differential responses to fasting. Further experiments demonstrated downregulation of HIF-1α at both transcript and protein levels. Intriguingly, while gene expression of Egln3 decreased, its protein product PHD3 remained unaffected by fasting. The unchanged levels of pro-inflammatory cytokines indicated that the observed reduction in Hif1a expression did not stem from a decrease in basal inflammation. These findings underscore the complex regulation of the well-established cardioprotective HIF-1 signaling within the heart during 3-day fasting.

Abstract B085: The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma

Abstract Ferroptosis is an iron-dependent form of cell death converging on lipid peroxidation, first identified by examining compounds with enhanced lethality to KRAS mutant cells. Two compounds widely used as ferroptosis inducers are erastin, which acts as an inhibitor of cystine import and thereby depletes cellular glutathione, and RSL-3, which inhibits glutathione peroxidase 4. Despite over 90% of pancreatic adenocarcinoma (PDAC) tumors harboring KRAS mutations, PDAC exhibits relative resistance to ferroptosis compared to other tumor types, and the mechanisms behind this resistance remain unclear. The pancreatic tumor environment is marked by severe hypoxia, due to poor vascularity and a dense fibrotic stroma, and so we hypothesized that hypoxia might mediate resistance to ferroptosis. Here we report that across a panel of human and murine PDAC cell lines, hypoxia induces resistance to both ferroptosis inducing agents, erastin and RSL-3. The effect of hypoxia was synergistic with exposure to pancreatic cancer tumor interstitial fluid, which induces a hypoxic-like gene expression profile even under normoxia and enhances hypoxic gene expression changes when combined with hypoxia. Our findings reveal that hypoxia-mediated resistance to ferroptosis is orchestrated by the hypoxia-inducible transcription factor HIF-2, as evidenced by its complete abolition in HIF-2 knockout cell lines, while the protective effect of hypoxia was maintained in HIF-1 knockout lines. RNA-seq analyses and chromatin immunoprecipitation studies (chIP) identified several HIF-2 target genes responsible for coordinating ferroptosis resistance mechanisms. Cysteine is a precursor for glutathione, and HIF-2 increased cystine import through upregulation of both components of the cystine-glutamate antiporter system Xc (SLC7A11 and SLC3A2) and increased cysteine biosynthesis via upregulation of transsulfuration pathway enzymes (CBS and CTH). In addition, HIF-2 upregulated Parkin, which we identify as a novel regulator of ferroptosis, thereby increasing mitophagy and decreasing reactive oxygen species. In vivo studies using human and murine PDAC xenograft models confirmed that HIF-2 knockout impaired tumor growth and increased susceptibility to the pro-ferroptotic agent sulfasalazine. Collectively, these data identify a mechanism by which PDAC evades ferroptosis and identifies a possible therapeutic strategy of combining HIF-2 inhibitors with pro-ferroptotic agents in pancreatic cancer. Citation Format: Maimon E Hubbi, Catherine Wang, Erin Hollander, Yasir Suhail, Kshitiz, Alexander Muir, Ben Z Stanger, Chi V Dang. The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B085.

Exploring the pharmacological potential of Lepionurus sylvestris blume: from folklore medicinal usage to modern drug development strategies using in vitro and in silico analyses

BackgroundLepionurus sylvestris Blume has a long history of folklore medicinal usage against various ailments. However, studies on these plants were neglected particularly their pharmacological potential.MethodsThe crude extract was identified using LC-MS analysis. In vitro assays were carried out to determine the properties of antioxidant, anti-microbial, and anti-cancer. Further, network pharmacology was proposed to evaluate the potential targets of the compounds against breast cancer and type II diabetes. Molecular docking and molecular dynamic simulation were used to determine the potential compounds for the drug formulation of diabetes.ResultsVarious bioactive compounds were identfied using LC-MS and Galiposin, Fujikinetin, Boeravinone B, 4-Deoxybryaquinone, and Norbaeocystin were described for the first time from the plant. Determination of antioxidant potential showed that the IC50 value of ABTS, DPPH, and phosphomolybdate was 24.33 µg/ml, 37.81 µg/ml, 60.35 µg/ml, and reducing power assays 1.185. The antibacterial activity against Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was determined, and the minimum inhibition concentration (MIC) was found to be 5.3 mg/ml, 3.47 mg/ml, 3.33 mg/ml, and 2.7 mg/ml respectively, revealing the extracts as effective antibacterial agents. The IC50 values for the plant extract were determined to be 26 µg/ml, 30.52 µg/ml, and 24.39 µg/ml for HeLa, MCF-7, and K-562 cells, respectively, and the increasing concentration of the plant extract increased LDH release. Furthermore, the in silico network pharmacology, molecular docking which had the highest docking score for GAPDH and HIF-1 target proteins of -9.3 kcal/mol, and − 11.3 kcal/mol binding affinities, and molecular dynamic simulation analysis revealed the bioactive compound Boeravinone B present in the plant was significant for the treatment of various ailments.ConclusionBased on our findings, plant extracts could be a promising option for developing new drug formulations.

The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes

BackgroundProstate cancer (PCa) ranks as the second most prevalent cancer in men, with advanced stages posing significant treatment challenges. Given its solid tumor nature, PCa is highly susceptible to hypoxia, a condition associated with resistance to radiation and chemotherapy, metastasis, and unfavorable patient outcomes. Hypoxia-inducible factors (HIFs) play a pivotal role in cancer cell adaptation to hypoxic environments, contributing to treatment resistance. Consequently, inhibitors targeting HIFs hold promise for cancer therapy.MethodsIn this study, we aimed to characterize novel HIF-1α inhibitors including Sodwanones A (1), B (2), C (3), G (4) and Yardenone 2 (5) isolated from marine sponges belonging to the Axinella genus. Our investigation evaluated the impact of these compounds on various aspects of HIF-1α regulation, including stabilization, nuclear localization, expression of HIF-1 target genes (while sparing HIF-2 target genes), cellular metabolism, as well as cell proliferation and viability in prostate cells under hypoxic conditions.ResultsOur findings revealed that among the compounds tested, Yardenone 2 exhibited notable effects in hypoxia: it destabilized HIF-1α at the protein level, decreased its nuclear localization, selectively altered the expression of HIF-1 target genes, and restrained cell proliferation in aggressive PC3 prostate cancer cells as well as in an MSK-PCa3 patient-derived organoid line. Moreover, it affected the morphology of these organoid. Yardenone 2 was also compared to Docetaxel, a specific microtubule inhibitor and a drug used in the treatment of prostate cancer. The comparison between the two compounds revealed notable differences, such as a lack of specificity to hypoxic cells of Docetaxel.ConclusionThese results mark the first demonstration that Yardenone 2 functions as a cytostatic-like inhibitor impacting microtubules, specifically targeting hypoxic cancer cells. This discovery suggests a promising avenue for novel therapeutic interventions in prostate cancer.

Roxadustat Attenuates Adverse Remodeling Following Myocardial Infarction in Mice.

We evaluated roxadustat's impact on HIF-1 stimulation, cardiac remodeling, and function after MI. Therefore, we analyzed nuclear HIF-1 expression, the mRNA and protein expression of key HIF-1 target genes (RT-PCR, Western blot), inflammatory cell infiltration (immunohistochemistry), and apoptosis (TUNEL staining) 7 days after MI. Additionally, we performed echocardiography in male and female C57BL/6 mice 28 days post-MI. We found a substantial increase in nuclear HIF-1, associated with an upregulation of HIF-1α target genes like CXCL12/CXCR4/ACKR3 at the mRNA and protein levels. Roxadustat increased the proportion of myocardial reparative M2 CD206+ cells, suggesting beneficial alterations in immune cell migration and a trend towards reduced apoptosis. Echocardiography showed that roxadustat treatment significantly preserved ejection fraction and attenuated subsequent ventricular dilatation, thereby reducing adverse remodeling. Our findings suggest that roxadustat is a promising clinically approved treatment option to preserve myocardial function by attenuating adverse remodeling.

UCHL1-dependent control of hypoxia-inducible factor transcriptional activity during liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies.

Novel Approaches with HIF-2α Targeted Therapies in Metastatic Renal Cell Carcinoma.

Germline inactivation of the Von Hippel-Lindau (VHL) tumor suppressor is the defining hallmark in hereditary VHL disease and VHL-associated renal cell carcinoma (RCC). However, somatic VHL mutations are also observed in patients with sporadic RCC. Loss of function VHL mutations result in constitutive activation of hypoxia-inducible factor-2 alpha (HIF-2α), which leads to increased expression of HIF target genes that promote angiogenesis and tumor growth. As of 2023, belzutifan is currently the only approved HIF-2α inhibitor for both VHL-associated and sporadic metastatic RCC (mRCC). However, there is potential for resistance with HIF-2α inhibitors which warrants novel HIF-2α-targeting strategies. In this review, we discuss the potential resistance mechanisms with belzutifan and current clinical trials evaluating novel combinations of belzutifan with other targeted therapies and immune checkpoint inhibitors which may enhance the efficacy of HIF-2α targeting. Lastly, we also discuss newer generation HIF-2α inhibitors that are currently under early investigation and outline future directions and challenges with HIF-2α inhibitors for mRCC.

PROTEOLYSIS TARGETING CHIMERAS (PROTACS) AS ATTRACTIVE THERAPEUTICS IN IBD THROUGH THE REGULATION OF HYPOXIA-INDUCIBLE FACTORS

Abstract Inflammatory bowel disease (IBD) represents a group of chronic mucosal inflammatory disorders, including Crohn’s disease (CD) and ulcerative colitis (UC). Many IBD patients have limited therapeutic options. Accumulating literature has implicated a prominent role for O2 metabolism and hypoxia in innate immunity of healthy intestinal tissue (“physiologic hypoxia”) and shifts associated with active inflammation (“inflammatory hypoxia”). Reduction of oxygen levels triggers the temporal stabilization of the transcription factors hypoxia-inducible factors HIF1a and HIF2a, which are essential in intestinal homeostasis. HIF-1/2α are regulated by a family of prolyl hydroxylases (PHDs). Inhibition of PHDs results in transient stabilization of HIF-1/2α, and thus, PHD inhibitors (PHDi) have become attractive targets for the therapeutic treatment of IBD and are reported as protective in mouse models of colitis. We have pursued the development of PHDi using proteolysis targeting chimeras (PROTACs), heterobifunctional compounds that contain a protein of interest ligand, a linker, and a E3 ubiquitin ligase ligand. PROTACs hold advantages over small-molecule inhibitors, including enhanced specificity, higher potency, and sustained pathway inhibition. In ongoing studies, we have synthesized and tested a panel of PHD targeting PROTACs. In cultured intestinal epithelial cells, we have observed PROTAC activity at the nanomolar concentrations in vitro, representing a 2-3 log increase in potency compared to PHDi small molecules. For these purposes, we monitor PHD protein degradation and HIF1/2α stabilization through immunoblotting. We have confirmed HIF transcriptional activity in intestinal epithelial cells exposed to these PROTACs through the induction of various HIF target genes. In parallel, we have established a stably transduced GFP-PHD cell line as a high-throughput assay to monitor PHD degradation in real time. Thus, PHD-targeted PROTACs hold promise as viable alternatives to small molecules for the stabilization of HIF in intestinal epithelial cells.

Pharmacological modulation of HIF-1 in the treatment of neuropsychiatric disorders.

Hypoxia-inducible factor 1 has been identified as an important therapeutic target in psychiatric illnesses. Hypoxia is a condition in which tissues do not receive enough oxygen, resulting in less oxidative energy production. HIF-1, the master regulator of molecular response to hypoxia, is destabilized when oxygen levels fall. HIF-1, when activated, increases the gene transcription factors that promote adaptive response and longevity in hypoxia. HIF-regulated genes encode proteins involved in cell survival, energy metabolism, angiogenesis, erythropoiesis, and vasomotor control. Multiple genetic and environmental variables contribute to the pathophysiology of psychiatric disease. This review focuses on the most recent findings indicating the role of oxygen deprivation in CNS damage, with strong attention on HIF-mediated pathways. Several pieces of evidence suggested that, in the case of hypoxia, induction and maintenance of HIF-1 target genes may help reduce nerve damage. Major new insights into the molecular mechanisms that control HIF's sensitivity to oxygen are used to make drugs that can change the way HIF works as a therapeutic target for some CNS diseases.

Potential anti-cancer activity of Moringa oleifera derived bio-active compounds targeting hypoxia-inducible factor-1 alpha in breast cancer.

Breast cancer (BC) will become a highly detected malignancy in females worldwide in 2023, with over 2 million new cases. Studies have established the role of hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls cellular response to hypoxic stress, and is essential for BC spread. HIF-1 is implicated in nearly every critical stage of the metastatic progression, including invasion, EMT, intravasation, extravasation, angiogenesis, and the formation of metastatic niches. HIF-1 overexpression has been associated with poor prognosis and increased mortality in BC patients. This is accomplished by controlling the expression of HIF-1 target genes involved in cell survival, angiogenesis, metabolism, and treatment resistance. Studies have indicated that inhibiting HIF-1 has an anti-cancer effect on its own and that inhibiting HIF-1-mediated signaling improves the efficacy of anti-cancer therapy. Approximately 74 % of recognized anti-cancer drugs are sourced from plantspecies. Studies on anti-cancer characteristics of phytochemicals derived from Moringa oleifera (MO), also known as the 'Tree of Life', have revealed a high therapeutic potential for BC. In this review, we have highlighted the various mechanisms through which bioactive compounds present in MO may modulate HIF and its regulatory genes/pathways, toprove their efficacy in treating and preventing BC.

Remote ischemic preconditioning and hypoxia-induced biomarkers in acute myocardial infarction: study on a porcine model

Objectives. Remote ischemic preconditioning (RIPC) mitigates acute myocardial infarction (AMI). We hypothesized that RIPC reduces the size and severity of AMI and explored molecular mechanisms behind this phenomenon. Design. In two series of experiments, piglets underwent 60 min of the circumflex coronary artery occlusion, resulting in AMI. Piglets were randomly assigned into the RIPC groups (n = 7 + 7) and the control groups (n = 7 + 7). The RIPC groups underwent four 5-min hind limb ischemia-reperfusion cycles before AMI. In series I, the protective efficacy of RIPC was investigated by using biomarkers and echocardiography with a follow-up of 24 h. In series II, the heart of each piglet was harvested for TTC-staining to measure infarct size. Muscle biopsies were collected from the hind limb to explore molecular mechanisms of RIPC using qPCR and Western blot analysis. Results. The levels of CK-MBm (p = 0.032) and TnI (p = 0.007) were lower in the RIPC group. Left ventricular ejection fraction in the RIPC group was greater at the end of the follow-up. The myocardial infarct size in the RIPC group was smaller (p = 0.033). Western blot indicated HIF1α stabilization in the skeletal muscle of the RIPC group. PCR analyses showed upregulation of the HIF target mRNAs for glucose transporter (GLUT1), glucose transporter 4 (GLUT4), phosphofructokinase 1 (PFK1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase 1 (ENO1), lactate dehydrogenase (LDHA) and endothelial nitric oxidate synthase (eNOS). Conclusions. Biochemical, physiologic, and histologic evidence confirms that RIPC decreases the size of AMI. The HIF pathway is likely involved in the mechanism of the RIPC.

No benefit of HIF prolyl hydroxylase inhibition for hypertensive renal damage in renovascular hypertensive rats.

Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

BrUOG 387: Phase Ib investigator-initiated trial of a heat shock protein 90 inhibitor (HSP90i) combined with a CDK4/6i in advanced breast cancer progressing on CDK4/6i and in solid tumors with retinoblastoma (Rb)-deficiency (IND163592).

TPS3167 Background: Cyclin-dependent kinases 4/6 (CDK4/6) promote cell-cycle progression and cancer growth. CDK4/6 inhibitors (CDK4/6i), in combination with hormonal therapy, increase progression-free and overall survival (PFS and OS) in breast cancer but resistance to CDK4/6i is associated with genomic abnormalities including Rb-deficiency. Combined CDK4/6i/HSP90i has been shown to inhibit HIF1-alpha including in tumor cells with Rb-deficiency. Synergy between multiple CDK4/6i’s and HSP90i’s has suggested a drug class effect for combination. In CDK4/6i-treated cells, E3-ubiquitin ligase Smurf2 associates with and targets degradation of HIF1-alpha independent of VHL or hypoxia. BrUOG 387 investigates whether dual targeting of CDK4/6 and HSP90, through HIF1-alpha in patients with ER/PR+ Her2- advanced breast cancer progressing on CDK4/6i and patients with refractory Rb-deficient tumors is safe and reasonably tolerated. Methods: A phase Ib open-label single-arm dose de-escalation study is evaluating safety, tolerability as primary outcomes, recommended doses for cohort expansion and preliminary efficacy of CDK4/6i and HSP90i (IND163592; NCT05655598; the only HSP90i combination study in US). Patients with ER/PR+ Her2- advanced breast cancer progressing on CDK4/6i, and in cohort expansion, patients with Rb-deficient solid tumors (SCLC, soft tissue sarcoma, endometrial and bladder cancer) are included. CDK4/6i (palbociclib at dose previously tolerated up to 125 mg daily PO D1-21 of 28 day cycle) in combination with HSP90i (TAS-116/pimitespib starting at 120 mg 5 days on 2 days off, for D1-28 of cycle) is used in 3+3 dose-de-escalation design. The study includes PK analysis, and PD analysis will include HIF1/2, Smurf2, Rb, HIF targets VEGF, erythropoietin, Glut1, proliferation, cell death, cancer stem cells, NK, T-cells, RNA-seq and angiogenesis in pre- and post-treatment biopsies. Biostatistical design and analysis includes 3 dose levels of TAS-116 + palbociclib (level 0: TAS-116 120 mg, palbociclib 125 mg; level -1: TAS-116 80 mg, palbociclib 125 mg); level -2: TAS-116 40 mg, palbociclib 125 mg). Limiting Toxicities will include grade 4 neutropenia lasting > 7 days, neutropenic fever, grade 4 thrombocytopenia or Grade 3 non-hematologic toxicity not controlled with medical management. The study will enroll a total of approximately 15-27 patients (6-18 patients in dose de-escalation portion of the study and 9 additional patients in the cohort expansion phase). Secondary outcome analyses include response rate (CR, PR) measured by RECIST v.1.1 with exact two-sided 95% confidence intervals. OS, PFS, TTP, and DOR will be summarized using methods of Kaplan and Meier. Clinical trial information: NCT05655598 .

VBP1 negatively regulates CHIP and selectively inhibits the activity of hypoxia-inducible factor (HIF)-1α but not HIF-2α

Hypoxia-inducible factor-1 (HIF-1) is a critical transcription factor that regulates the expression of genes involved in cellular adaptation to low oxygen levels. Aberrant regulation of the HIF-1 signaling pathway is linked to various human diseases. Previous studies have established that HIF-1α is rapidly degraded in a von Hippel-Lindau protein (pVHL)-dependent manner under normoxic conditions. In this study, we find that pVHL binding protein 1 (VBP1) is a negative regulator of HIF-1α but not HIF-2α using zebrafish as an invivo model and invitro cell culture models. Deletion of vbp1 in zebrafish caused Hif-1α accumulation and upregulation of Hif target genes. Moreover, vbp1 was involved in the induction of hematopoietic stem cells (HSCs) under hypoxic conditions. However, VBP1 interacted with and promoted the degradation of HIF-1α in a pVHL-independent manner. Mechanistically, we identify the ubiquitin ligase CHIP and HSP70 as new VBP1 binding partners and demonstrate that VBP1 negatively regulated CHIP and facilitated CHIP-mediated degradation of HIF-1α. In patients with clear cell renal cell carcinoma (ccRCC), lower VBP1 expression was associated with worse survival outcomes. In conclusion, our results link VBP1 with CHIP stability and provide insights into underlying molecular mechanisms of HIF-1α-driven pathological processes.

The Role of the Estrogen-Related Receptor Alpha (ERRa) in Hypoxia and Its Implications for Cancer Metabolism.

Under low oxygen conditions (hypoxia), cells activate survival mechanisms including metabolic changes and angiogenesis, which are regulated by HIF-1. The estrogen-related receptor alpha (ERRα) is a transcription factor with important roles in the regulation of cellular metabolism that is overexpressed in hypoxia, suggesting that it plays a role in cell survival in this condition. This review enumerates and analyses the recent evidence that points to the role of ERRα as a regulator of hypoxic genes, both in cooperation with HIF-1 and through HIF-1- independent mechanisms, in invertebrate and vertebrate models and in physiological and pathological scenarios. ERRα's functions during hypoxia include two mechanisms: (1) direct ERRα/HIF-1 interaction, which enhances HIF-1's transcriptional activity; and (2) transcriptional activation by ERRα of genes that are classical HIF-1 targets, such as VEGF or glycolytic enzymes. ERRα is thus gaining recognition for its prominent role in the hypoxia response, both in the presence and absence of HIF-1. In some models, ERRα prepares cells for hypoxia, with important clinical/therapeutic implications.

Abstract 494: BPI-452080: A potent and selective HIF-2α inhibitor for the treatment of clear cell renal cell carcinoma, von Hippel-Lindau disease, and other solid tumors

Abstract The most observed alteration in clear cell renal cell carcinoma (ccRCC) is the inactivation of the tumor suppressor von Hippel-Lindau (VHL) E3 ligase due to genetic predisposition, somatic mutations, or methylation. Loss of VHL can lead to hypoxia-inducible factor 2α (HIF-2α) accumulation and elevated expression of HIF target genes, such as VEGFA, many of which facilitate angiogenesis, proliferation, and metastasis of ccRCC. Therefore, inhibiting HIF-2α has become a new strategy for the treatment of ccRCC. Here, we present the discovery of BPI-452080, a selective small molecule HIF-2α inhibitor. In VHL defective ccRCC cell lines, BPI-452080 potently and selectively blocked the heterodimerization of HIF-2α with HIF-1β, but not that of HIF-1α with HIF-1β. It inhibited the downstream hypoxia-responsive gene transcription and VEGFA protein secretion. In multiple xenograft mouse models, oral administration of BPI-452080 significantly inhibited tumor growth in a dose-dependent manner. BPI-452080 demonstrated good PK/PD correlation in the 786-O xenograft model after a single dosing, as shown by a concentration-dependent inhibition of VEGFA secretion in plasma and a reduction of the mRNA levels of VEGFA, CXCR4, CCND1, and GLUT1 genes in tumor tissue. Moreover, BPI-452080 exhibited good bioavailability in multiple pre-clinical species, favorable ADME properties, and good tolerance in toxicology studies. In conclusion, BPI-452080 is a potent, selective, and orally bioavailable HIF-2α inhibitor which presents a novel therapeutic option for ccRCC, von Hippel-Lindau disease, and other solid tumors. BPI-452080 is planned to enter Phase I clinical trial in China in early 2023. Citation Format: Pingping Wang, Rongwen Yang, Yun Sun, Xuepeng Ju, Jiayu Zhao, Yanju Liu, Xiaoyun Liu, Zhengyao Zou, Jialin Ren, Min Wang, Haibo Chen, Xuegang Yi, Jian Zhang, Teng Ma, Gaoliang Cheng, Lijia Liu, Jing Guo, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-452080: A potent and selective HIF-2α inhibitor for the treatment of clear cell renal cell carcinoma, von Hippel-Lindau disease, and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 494.

PHF6 recruits BPTF to promote HIF-dependent pathway and progression in YAP-high breast cancer

BackgroundAberrant epigenetic remodeling events contribute to progression and metastasis of breast cancer (Bca). The specific mechanims that epigenetic factors rely on to mediate tumor aggressiveness remain unclear. We aimed to elucidate the roles of epigenetic protein PHF6 in breast tumorigenesis.MethodsPublished datasets and tissue samples with PHF6 staining were used to investigate the clinical relevance of PHF6 in Bca. CCK-8, clony formation assays were used to assess cell growth capacity. Cell migration and invasion abilities were measured by Transwell assay. The gene mRNA and protein levels were measured by quantitative real-time PCR and western blot. Chromatin immunoprecipitation (ChIP)-qPCR assays were used to investigate transcriptional relationships among genes. The Co-immunoprecipitation (Co-IP) assay was used to validate interactions between proteins. The CRISPR/Cas9 editing technology was used to construct double HIF knockout (HIF-DKO) cells. The subcutaneous xenograft model and orthotopic implantation tumor model were used to asess in vivo tumor growth.ResultsIn this study, we utilized MTT assay to screen that PHF6 is required for Bca growth. PHF6 promotes Bca proliferation and migration. By analyzing The Cancer Genome Atlas breast cancer (TCGA-Bca) cohort, we found that PHF6 was significantly higher in tumor versus normal tissues. Mechanistically, PHF6 physically interacts with HIF-1α and HIF-2α to potentiate HIF-driven transcriptional events to initiate breast tumorigenesis. HIF-DKO abolished PHF6-mediated breast tumor growth, and PHF6 deficiency in turn impaired HIF transcriptional effects. Besides, hypoxia could also rely on YAP activation, but not HIF, to sustain PHF6 expressions in Bca cells. In addition, PHF6 recuits BPTF to mediate epigenetic remodeling to augment HIF transcriptional activity. Targeting PHF6 or BPTF inhibitor (AU1) is effective in mice models. Lastly, PHF6 correlated with HIF target gene expression in human breast tumors, which is an independent prognostic regulator.ConclusionsCollectively, this study identified PHF6 as a prognostic epigenetic regulator for Bca, functioning as a HIF coactivator. The fundamental mechanisms underlying YAP/PHF6/HIF axis in breast tumors endowed novel epigenegtic targets for Bca treatment.

Molecular pharmacology of multitarget cyclin-dependent kinase inhibitors in human colorectal carcinoma cells

ABSTRACT Background Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. Objective Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. Method Protein expression was analyzed for key signaling proteins by western blotting. β-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. Results CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. Conclusion Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.

Association of mitochondrial metabolic activity and immune pathways with disease stage in renal cell carcinoma.

722 Background: Previous large scale genomic studies have described somatic alterations that affect tumor progression. However, metabolic and immune pathway changes that occur with advancing disease stage are not well characterized. Herein, we performed transcriptomic profiling of clear-cell renal carcinoma (ccRCC) tumors across clinical stages to define these changes. Methods: TCGA Firehose Legacy cohort was used to determine differential gene expression between stage 3 ccRCCs from patients who remained disease free (S3DF) vs. those who relapsed (S3RL) within two years of nephrectomy. Single cell RNA-seq (scRNAseq) analysis was performed on primary ccRCCs from nine patients (stage 1 n=4, stage 3 n=4, metastatic n=1). Seurat V4 package and module score was used to analyze patterns of gene expression associated with tumor progression at the single cell level. Results: Transcriptomic comparison of ccRCCs from TCGA Firehose Legacy cohort revealed induction of genes encoding mitochondrial respiratory complexes and mitochondrial ribosomal proteins (MRPs) and decrease in expression of immune genes in S3RL as compared to S3DF tumors. Using a set of 23 differentially regulated mitochondrial and immune genes we established a transcriptomic signature that stratified patients into those at risk of relapse vs. not. ScRNA-seq data analysis determined increased expression of mitochondrial respiratory complexes’ gene-sets, and decreased levels of glycolytic genes and HIF targets during tumor progression from stage-I to metastatic. This was supported by spatial transcriptomics of cancer cells, which revealed clusters of cells with high mitochondrial activity and decreased MHC class I and II gene expression. Conclusions: The data support a model of progressive metabolic reprogramming from glycolysis and HIF-regulated pathways to mitochondrial respiration in parallel with diminished immune expression of antigens on cancer cells with advancing disease stage in ccRCC. Identification of targetable mitochondrial vulnerability could be a therapeutic option in patients who progress on immune checkpoint inhibitors.

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia.

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia-inducible factors (HIFs). HIF-1 mediates extensive metabolic re-programming, and expression of HIF-1α, its oxygen-regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF-1 activity. PDP1 is a key hormone-regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl-CoA. Silencing of PDP1 down-regulated HIF transcriptional activity and the expression of HIF-dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF-1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP-1 decreased histone H3 acetylation of HIF-1 target gene promoters and inhibited binding of HIF-1 to the respective hypoxia-response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl-CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF-1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl-CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia-inducible genes.