HIF-1α Stabilization Research Articles

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

The role of hypoxia in chronic kidney disease: a nuanced perspective.

This review critically examines the role of hypoxia in chronic kidney disease (CKD). While traditionally viewed as detrimental, recent insights suggest a more nuanced understanding of hypoxia's role during renal disease. Emerging evidence challenges the traditional view that hypoxia is universally harmful in CKD context. We review here the recent evidence about hypoxia and HIF activation in CKD. We also discuss the effect of hypoxia on the renal tissue, and the relative inhibition of different HIF isoforms. Recent advancements in therapies, such as HIF prolyl hydroxylase inhibitors (HIF-PHIs) and sodium-glucose cotransporter 2 (SGLT2) inhibitors seem to target the HIF pathway. These drugs impact anemia associated with CKDbut also renoprotection, hinting at a more complex interplay between hypoxia, HIF activation, and renal health. A certain level of hypoxia and specific HIF pathway activation, especially HIF-α, can be beneficial in CKD progression. Therapeutic strategies targeting HIF stabilization, such as with HIF-PHIs and SGLT2 inhibitors, offer promising avenues for enhancing renal protection. Future investigations should aim at better understanding the precise effects on HIF pathway and optimize their clinical application to improve outcomes for CKD patients.

The MUC1–HIF-1α signaling axis regulates pancreatic cancer pathogenesis through polyamine metabolism remodeling

Dysregulation of polyamine metabolism has been implicated in cancer initiation and progression; however, the mechanism of polyamine dysregulation in cancer is not fully understood. In this study, we investigated the role of MUC1, a mucin protein overexpressed in pancreatic cancer, in regulating polyamine metabolism. Utilizing pancreatic cancer patient data, we noted a positive correlation between MUC1 expression and the expression of key polyamine metabolism pathway genes. Functional studies revealed that knockdown of spermidine/spermine N1-acetyltransferase 1 (SAT1), a key enzyme involved in polyamine catabolism, attenuated the oncogenic functions of MUC1, including cell survival and proliferation. We further identified a regulatory axis whereby MUC1 stabilized hypoxia-inducible factor (HIF-1α), leading to increased SAT1 expression, which in turn induced carbon flux into the tricarboxylic acid cycle. MUC1-mediated stabilization of HIF-1α enhanced the promoter occupancy of the latter on SAT1 promoter and corresponding transcriptional activation of SAT1, which could be abrogated by pharmacological inhibition of HIF-1α or CRISPR/Cas9-mediated knockout of HIF1A. MUC1 knockdown caused a significant reduction in the levels of SAT1-generated metabolites, N1-acetylspermidine and N8-acetylspermidine. Given the known role of MUC1 in therapy resistance, we also investigated whether inhibiting SAT1 would enhance the efficacy of FOLFIRINOX chemotherapy. By utilizing organoid and orthotopic pancreatic cancer mouse models, we observed that targeting SAT1 with pentamidine improved the efficacy of FOLFIRINOX, suggesting that the combination may represent a promising therapeutic strategy against pancreatic cancer. This study provides insights into the interplay between MUC1 and polyamine metabolism, offering potential avenues for the development of treatments against pancreatic cancer.

Characterization of cell cycle, inflammation, and oxidative stress signaling role in non-communicable diseases: Insights into genetic variants, microRNAs and pathways

Non-Communicable Diseases (NCDs) significantly impact global health, contributing to over 70% of premature deaths, as reported by the World Health Organization (WHO). These diseases have complex and multifactorial origins, involving genetic, epigenetic, environmental and lifestyle factors. While Genome-Wide Association Study (GWAS) is widely recognized as a valuable tool for identifying variants associated with complex phenotypes; the multifactorial nature of NCDs necessitates a more comprehensive exploration, encompassing not only the genetic but also the epigenetic aspect. For this purpose, we employed a bioinformatics-multiomics approach to examine the genetic and epigenetic characteristics of NCDs (i.e. colorectal cancer, coronary atherosclerosis, squamous cell lung cancer, psoriasis, type 2 diabetes, and multiple sclerosis), aiming to identify novel biomarkers for diagnosis and prognosis. Leveraging GWAS summary statistics, we pinpointed Single Nucleotide Polymorphisms (SNPs) independently associated with each NCD. Subsequently, we identified genes linked to cell cycle, inflammation and oxidative stress mechanisms, revealing shared genes across multiple diseases, suggesting common functional pathways. From an epigenetic perspective, we identified microRNAs (miRNAs) with regulatory functions targeting these genes of interest. Our findings underscore critical genetic pathways implicated in these diseases. In colorectal cancer, the dysregulation of the “Cytokine Signaling in Immune System” pathway, involving LAMA5 and SMAD7, regulated by Hsa-miR-21-5p, Hsa-miR-103a-3p, and Hsa-miR-195-5p, emerged as pivotal. In coronary atherosclerosis, the pathway associated with “binding of TCF/LEF:CTNNB1 to target gene promoters” displayed noteworthy implications, with the MYC factor controlled by Hsa-miR-16-5p as a potential regulatory factor. Squamous cell lung carcinoma analysis revealed significant pathways such as “PTK6 promotes HIF1A stabilization,” regulated by Hsa-let-7b-5p. In psoriasis, the “Endosomal/Vacuolar pathway,” involving HLA-C and Hsa-miR-148a-3p and Hsa-miR-148b-3p, was identified as crucial. Type 2 Diabetes implicated the “Regulation of TP53 Expression” pathway, controlled by Hsa-miR-106a-5p and Hsa-miR-106b-5p. In conclusion, our study elucidates the genetic framework and molecular mechanisms underlying NCDs, offering crucial insights into potential genetic/epigenetic biomarkers for diagnosis and prognosis. The specificity of pathways and related miRNAs in different pathologies highlights promising candidates for further clinical validation, with the potential to advance personalized treatments and alleviate the global burden of NCDs.

Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells.

Nuclear factor of activated T cells 5 (NFAT5) and cyclooxygenase 2 (COX2; PTGS2) both participate in diverse pathologies including cancer progression. However, the biological role of the NFAT5-COX2 signaling pathway in human endometrial cancer has remained elusive. The present study explored whether NFAT5 is expressed in endometrial tumors and if NFAT5 participates in cancer progression. To gain insights into the underlying mechanisms, NFAT5 protein abundance in endometrial cancer tissue was visualized by immunohistochemistry and endometrial cancer cells (Ishikawa and HEC1a) were transfected with NFAT5 or with an empty plasmid. As a result, NFAT5 expression is more abundant in high-grade than in low-grade endometrial cancer tissue. RNA sequencing analysis of NFAT5 overexpression in Ishikawa cells upregulated 37 genes and downregulated 20 genes. Genes affected included cyclooxygenase 2 and hypoxia inducible factor 1α (HIF1A). NFAT5 transfection and/or treatment with HIF-1α stabilizer exerted a strong stimulating effect on HIF-1α promoter activity as well as COX2 expression level and prostaglandin E2 receptor (PGE2) levels. Our findings suggest that activation of NFAT5-HIF-1α-COX2 axis could promote endometrial cancer progression.

Abstract 433: BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization

Abstract Hepatocellular carcinoma (HCC) is a highly lethal primary liver malignancy characterized by hypoxia, a common feature of solid tumors, particularly in HCC. Hypoxia-inducible factors (HIFs) play a pivotal role in mediating various cellular processes, such as metabolic adaptation, to support the survival of hypoxic cells. The cytosolic form of branched-chain amino transferase (BCAT1) is responsible for the reversible transamination reaction between branched-chain keto-amino acids (BCKAs) and branched-chain amino acids (BCAAs), facilitating the interconversion between α-ketoglutarate (α-KG) and glutamate. Interestingly, we found that in the context of HCC, BCAT1 consumes α-KG, thereby favoring the stabilization of HIF-1α through suppressing the α-KG-dependent prolyl hydroxylase (PHD). Knockdown (KD) or knockout (KO) of BCAT or BCAT inhibitor (ERG245) suppressed HIF-1 stability, as well as HIF-1 dependent transcriptomic and metabolic changes. Mass spectrometry (MS)-based isotopic carbon tracing showed that KD/KO or ERG 245 suppressed hypoxia/HIF-1-induced glycolytic flux. Interestingly, ERG245 effectively inhibited the proliferative output of hypoxic HCC cells and suppressed HCC growth. ERG245 also profoundly inhibited HCC growth in vitro and in vivo together with Sorafenib, a tyrosine kinase inhibitor (TKI) used in HCC by increasing the apoptotic cell death. Furthermore, we confirmed that the expressions of BCAT1 and the HIF-1α-targeted metabolic genes are positively correlated in human HCCs. Our findings unequivocally demonstrate that BCAT1 confers a growth advantage to HCC cells by stabilizing HIF-1α and orchestrating HIF-1α-mediated metabolic reprogramming. Consequently, targeting BCAT1 emerges as a promising therapeutic strategy for HCC patients. Funding Support: This work was supported by the Centre for Oncology and Immunology under the Health@InnoHK initiative funded by the Innovation and Technology Commission, The Government of Hong Kong SAR, China and National Natural Science Foundation of China (NSFC) (82173123, C.C.-L.W.) Citation Format: Misty Shuo Zhang, Kenneth Kin-Leung Kwan, Weixing Li, Adonia E. Papathanassiu, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 433.

Abstract 5413: In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions

Abstract Hypoxia is a common condition in the tumor microenvironment that contributes to tumor cell migration, invasion, and metastasis, partly through inducing epithelial-mesenchymal transition (EMT). One of the key proteins in this process is hypoxia-inducible factor 1 (HIF-1), which regulates the expression of various genes as transcription factor and is vital for the cellular response in tissue hypoxia. Several studies have investigated the causation between hypoxic conditions and increased in vitro tumor cell motility and the role of potential regulatory signaling pathways. Understanding the precise molecular mechanisms underlying hypoxia-induced responses is crucial for better targeting tumor progression. However, establishing sound and predictive models is urgently needed to achieve this.In this study, we investigated the migration ability of four non-small cell lung cancer cell lines carrying different mutations of the growth factor pathway (H1975: EGFR T790M, PF901: BRAF V600E, PF139: KRAS G12C mutant, and H838: triple WT) under normoxic, hypoxic, and hypoxia-mimicking CoCl2-treated conditions. The motility of the cells was investigated by 48 h video microscopy (single cell random migration) and 72 h scratch assay. We analyzed the expression of crucial genes and proteins, including HIF-1α, as well as molecules associated with epithelial-mesenchymal transition (vimentin, n-cadherin, and e-cadherin), proliferation (PCNA), and apoptosis (PARP), using Western blot and qPCR techniques. Our work showed no change in cellular motility between any of the cell lines following treatment with CoCl2 compared to normoxic conditions. However, PF139 and PF901 cell lines showed a significant decrease in single-cell motility upon hypoxia. Interestingly, immunoblot analysis revealed stabilization of HIF-1α in response to both CoCl2 and hypoxia in the meantime. Additionally, we observed cell line-dependent changes in EMT marker expression upon CoCl2 treatment and hypoxia. Our work complements previous results published by our group and others on the tumor-promoting role of hypoxia, demonstrating that short-term exposure to hypoxia may act as an acute stressor for tumor cell lines. Our work suggests that the tumor progression-promoting effects are tissue-specific and should be investigated using long-term hypoxia models. Acknowledgments: This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Sára Eszter Surguta is a grantee of the ITM-NKFIA funded Cooperative Doctoral Program (1018567). Citation Format: Sára Eszter Surguta, Marcell Baranyi, Laura Svajda, Ivan Ranđelović, Mihály Cserepes, József Tóvári. In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5413.

LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

BackgroundCancer stem-like cell is a key barrier for therapeutic resistance and metastasis in various cancers, including breast cancer, yet the underlying mechanisms are still elusive. Through a genome-wide lncRNA expression profiling, we identified that LINC00115 is robustly upregulated in chemoresistant breast cancer stem-like cells (BCSCs).MethodsLncRNA microarray assay was performed to document abundance changes of lncRNAs in paclitaxel (PTX)-resistant MDA-MB-231 BCSC (ALDH+) and non-BCSC (ALDH−). RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to determine the binding proteins of LINC00115. The clinical significance of the LINC00115 pathway was examined in TNBC metastatic lymph node tissues. The biological function of LINC00115 was investigated through gain- and loss-of-function studies. The molecular mechanism was explored through RNA sequencing, mass spectrometry, and the CRISPR/Cas9-knockout system. The therapeutic potential of LINC00115 was examined through xenograft animal models.ResultsLINC00115 functions as a scaffold lncRNA to link SETDB1 and PLK3, leading to enhanced SETDB1 methylation of PLK3 at both K106 and K200 in drug-resistant BCSC. PLK3 methylation decreases PLK3 phosphorylation of HIF1α and thereby increases HIF1α stability. HIF1α, in turn, upregulates ALKBH5 to reduce m6A modification of LINC00115, resulting in attenuated degradation of YTHDF2-dependent m6A-modified RNA and enhanced LINC00115 stability. Thus, this positive feedback loop provokes BCSC phenotypes and enhances chemoresistance and metastasis in triple-negative breast cancer. SETDB1 inhibitor TTD-IN with LINC00115 ASO sensitizes PTX-resistant cell response to chemotherapy in a xenograft animal model. Correlative expression of LINC00115, methylation PLK3, SETDB1, and HIF1α are prognostic for clinical triple-negative breast cancers.ConclusionsOur findings uncover LINC00115 as a critical regulator of BCSC and highlight targeting LINC00115 and SETDB1 as a potential therapeutic strategy for chemotherapeutic resistant breast cancer.

Hypoxia-induced miR-210-3p expression in lung adenocarcinoma potentiates tumor development by regulating CCL2 mediated monocyte infiltration.

In most cancers, tumor hypoxia downregulates the expression of C-C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is not yet clear. We compared noncancerous and lung-adenocarcinoma human samples for hypoxia-inducible factor 1-alpha (HIF-1A), microRNA-210-3p (mir-210-3p), and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia-induced miR-210-3p in the regulation of CCL2 expression and macrophage polarization. HIF-1Α stabilization increases miR-210-3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR-210-3p directly binds to the 3'untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR-210-3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR-210-3p mimic-transfected HIF-1A silenced cancer cells. In contrast, inhibition of miR-210-3p in HIF-1A-overexpressed cells markedly restored monocyte migration, highlighting a direct link between the miR-210-3p level and tumor monocyte burden. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an antitumor M1 phenotype. Anti-hsa-miR-210-3p-locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR-210-3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Abstract 5538: Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer

Abstract Metastatic prostate cancer (PCA) exhibits a predilection for bone metastasis, initiating a complex interplay with bone cells, leading to dysregulated remodeling and mixed-pathological bone alterations. Human PCA primarily yields osteoblastic bone lesions, linked to bone pain and an adverse prognosis. In this study, we explored the activation of HIF-1α in myeloid phagocytic cells by apoptotic cancer cells in the context of bone metastatic PCA. Our previous findings demonstrated HIF-1α stabilization in bone macrophages engulfing apoptotic PCA cells in vitro. This study extends our understanding to an in vivo setting using mouse prostate cancer cells designed to express inducible caspase 9, triggering apoptosis (and subsequent efferocytosis) when exposed to the dimerizer molecule AP20187 (AP). Our assessment of PCA tumor growth in bone utilized a syngeneic mouse model, introducing a loss-of-function Hif1a mutation into myeloid lineage cells using LysM-Cre mice crossed with Hif1a-floxed mice (Hif1amut (LysM-Cre+/- Hif1aflox)), compared with the LysM-Cre-/- Hif1aflox (WT). In the first model, cancer cells were inoculated into the tibiae of these mice and apoptosis was induced with AP on days 3 and 5 post-inoculation (AP model). Results were compared to a model where apoptosis was not induced after inoculation (No-AP). In the AP model, fractional bone volume (BV/TV) in the medullae was significantly higher in tumor-inoculated tibiae than in non-inoculated (No-tumor) contralateral bone, highlighting the predominance of osteoblastic lesions. Conversely, in the No-AP model, increased BV/TV significance was achieved in WT mice (tumor vs. No-tumor), but not in mutant mice. Trabecular thickness (Tb.Th) was increased in both models in the presence of tumor, regardless of the Hif1a phenotype. Importantly, significant differences in BV/TV and Tb.Th between WT and Hif1amut tumor tibiae were exclusively noted in the context of induced apoptosis. Histological differences between tumors in the AP model were observed by immunofluorescence (IF) staining, which revealed a reduced fractional F4/80+ area in Hif1amut vs. WT tumors, with no differences in TRAP+ (osteoclasts) lining bone surfaces. RNAseq analysis of HIF1amut vs. WT macrophages efferocytosing apoptotic cancer cells showed downregulated processes related to vasculature development and blood vessel morphogenesis (p< 10-5). In correlation, IF analysis with endomucin (EMCN) demonstrated a reduction in stained area (EMCN+) relative to tumor area in Hif1amut tibiae vs. WT, suggesting a role of efferocytosis-induced HIF-1α in tumor vascularization. This study unveils intricacies of metastatic tumor growth in bone and proposes a connection between efferocytosis-induced HIF-1α in myeloid phagocytic cells and the promotion of tumor vascularization, alongside the enhancement of osteoblastic bone remodeling. Citation Format: Gabriel G. Kleer, Maria Molina Sanchez, Veronica Mendoza-Reinoso, Amy J. Koh, John Rubin, Laurie K. McCauley, Hernan Roca. Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5538.

Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression. Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup. Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes. Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs. Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2864.

Abstract 378: PHD3 dependent stabilization of β3-adrenoceptor induces HIF-1αactivation in Ewing sarcoma

Abstract Hypoxia is a condition of oxygen deficiency which occurs in most growing solid tumors and stimulates a cascade of cell signals through a family of transcription factors named as hypoxia inducible factors (HIFs) that transactivate several regulatory genes involved in tumor proliferation. β3-adrenoceptors (β3-ARs) are involved in several hypoxic scenarios and in pathological conditions where hypoxia leads to important steps for cancer progression. Studies showed that β-AR blockade of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. In this work we assumed that through PHD3 activity, β3-AR mediates hypoxia sensing needed for HIF-1α stabilization, thus we investigated a putative correlation between β3-AR/HIF-1α and PHD3. Data showed a similar outcome of β3-ARs and HIF-1α at short time of hypoxia, and that β3-AR antagonist, SR59230A reduced HIF-1α protein expression under hypoxia. β3-AR silencing under hypoxia revealed an evident reduction of HIF-1α protein expression that confirmed the β3-AR modulation of HIF-1α expression. SR59230A treatment strongly reduced the HIF-1α target genes as GLUT1, HK-2 and HIF-1α expression at long time treatment confirming the β3-AR modulation of HIF-1α transcriptional activity. Conversely, the HIF-1α-transcriptional inhibitor Topotecan did not affect β3-AR expression confirming that β3-AR mediates HIF-1α activation. Immunofluorescence and nuclear-cytoplasmic fragmentation showed that β3-AR co-localized with the nucleus under hypoxia, more than normoxia, and presence of both β3-AR and HIF-1α in the nucleus. Co-immunoprecipitation of β3-AR/HIF-1α revealed that under hypoxia, HIF-1α dissociates from β3-AR binding, but this process was abrogated by SR59230A. HIF-1α, released under hypoxia from the link with β3-AR, can play its transcriptional activity in the nucleus, while the binding with β3-AR blocks its transcriptional activity. Moreover, the β3-AR upregulation under hypoxia occurs trough an early inhibition of PHD3 activity that did not influence HIF-1α expression. Hypoxic PHD2 inhibition results after PHD3 leading to HIF-1α upregulation. Moreover, the blockade β3-AR by SR59230A, inhibited HIF-1α upregulation by increasing PHD2 activity. Citation Format: Amada Pasha, Laura Zocca, Francesco Carrozzo, Cristina Banella, Rachele Amato, Annalisa Tondo, Claudio Favre, Maura Calvani. PHD3 dependent stabilization of β3-adrenoceptor induces HIF-1αactivation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 378.

Expression of HIF1α in intestinal epithelium restricts arthritis inflammation by inhibiting RIPK3-induced cell death machinery

ObjectivesTo investigate the mechanism by which intestinal epithelial cell (IEC) death induces arthritis.MethodsIEC death was assessed by staining for necroptosis and apoptosis markers and fluorescence in situ hybridisation at different...

Lactiplantibacillus plantarum K8 lysates regulate hypoxia-induced gene expression

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.

Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

MiR-1224-5p reverses gefitinib resistance in non-small-cell lung cancer cells by modulating RFX5/YAP1/HIF1α axis

Purpose: To investigate the molecular pathways by which miR-1224-5p modulate RFX5/YAP1/HIF1α pathway, thereby promoting gefitinib tolerance within non-small-cell lung cancer (NSCLC). 
 Methods: To screen differentially expressed miR-1224-5p in NSCLC samples and predict its downstream target gene – RFX5 – by bioinformatics analysis, 60 NSCLC tissues and their corresponding paraneoplastic tissues were collected. Dual-luciferase assays were performed to verify the targeting relationship between miR-1224-5p and RFX5. Four NSCLC cell lines (A549, H1299, H2170, and H1975) and BEAS-2B normal lung epithelial cell lines were used for in vitro experiments. Co-immunoprecipitation (Co-IP) and Western blotting after cycloheximide (CHX) treatment were used to determine the regulatory interaction between YAP1 and HIF1α, with YAP1 modulating HIF1α protein stability. 
 Results: In NSCLC, downregulation of miR-1224-5p was observed, which resulted in the decrease of RFX5 levels. This reduction in miR-1224-5p levels leads to a decrease in RFX5 levels. Furthermore, restoring miR-1224-5p expression in NSCLC cells made them more sensitive to gefitinib. In vitro, RFX5 elevates YAP1, which in turn boosts the stability of HIF1α. However, miR-1224-5p disrupts this mechanism by influencing the RFX5/YAP1/HIF1α pathway, thus mitigating resistance to gefitinib. 
 Conclusion: The findings show that miR-1224-5p targets RFX5 to suppress YAP1 transcription, thereby diminishing HIF1α stability and overcoming gefitinib tolerance in NSCLC. These findings identify miR-1224-5p as a promising approach to address gefitinib tolerance in NSCLC.

Growing concerns about using hypoxia-inducible factor prolyl hydroxylase inhibitors for the treatment of renal anemia.

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) have emerged as a novel therapeutic class for treating anemia in patients with chronic kidney disease. Small molecule analogs of α-ketoglutarate (AKG), an essential substrate for 2-oxoglutarate-dependent dioxygenases (2-OGDDs), including prolyl hydroxylase domain proteins (PHDs), inhibit PHDs pharmacologically and thereby prevent HIF degradation. HIF stabilization alleviates anemia through several stimulatory effects on erythropoiesis, but it also affects the expression of many anemia-unrelated genes whose protein products exert important functions in vivo. Therefore, the pleiotropic effects of HIF stabilization under normoxic conditions deserve to be examined in more detail. Specifically, we believe that particular attention should be given to epigenetic modifications among the various AKG-based metabolic systems that may be altered by HIF-PHIs. It is noteworthy that AKG has been reported to exert health-protective actions. AKG-based metabolic systems include enzymes associated with the tricarboxylic acid cycle and amino acid metabolism, as well as 2-OGDD-mediated processes, which play important roles in many biological reactions. In this review, we examine the multifaceted effects of HIF-PHIs, encompassing not only their on-target effect of HIF stabilization but also their off-target inhibitory effects on various AKG-based metabolic systems. Furthermore, we examine its potential relevance to cardiovascular complications, based on clinical and animal studies suggesting its involvement in vascular calcification, thrombogenesis and heart failure. In conclusion, although HIF-PHIs offer a promising avenue for anemia treatment in CKD patients, their broader impact on multiple biological systems raises substantial concerns. The intricate interplay between HIF stabilization, AKG competition and cardiovascular complications warrants extensive, long-term investigations to ensure the safety and usefulness of HIF-PHIs in clinical practice.