bims-kishpe 2024-11-03 papers

bims-kishpe

Biomed News

on HSP70 role in hypoxia and metabolism in ECs

Issue of 2024–11–03
twenty-six papers selected by
Alia Ablieh, Universität Heidelberg

Characteristic disease defects in circulating endothelial cells isolated from patients with pulmonary arterial hypertension.
Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular cells.
The impact of ERP29 on the progression of pharyngeal squamous cell carcinoma.
Proteomics Analysis on the Effects of Oxidative Stress and Antioxidants on Proteins Involved in Sterol Transport and Metabolism in Human Telomerase Transcriptase-Overexpressing-Retinal Pigment Epithelium Cells.
Ischemia-induced endogenous Nrf2/HO-1 axis activation modulates microglial polarization and restrains ischemic brain injury.
Exploring the Role of miR-132 in Rat Bladders and Human Urothelial Cells during Wound Healing.
METTL14-mediated m6A methylation regulates pathological retinal neovascularization by targeting autophagy.
Single-cell transcriptomics reveal diverging pathobiology and opportunities for precision targeting in scleroderma-associated versus idiopathic pulmonary arterial hypertension.
NADPH Oxidase 4: Crucial for Endothelial Function under Hypoxia-Complementing Prostacyclin.
cAMP driven UCP1 induction in human adipocytes requires ATGL-catalyzed lipolysis.
VASN promotes the aggressive phenotype in ARID1A-deficient lung adenocarcinoma.
Ion Channel Piezo1 Induces Ferroptosis of Trabecular Meshwork Cells: A Novel Observation in the Pathogenesis in Primary Open Angle Glaucoma.
Transcriptional regulation by MYC: an emerging new model.
Conservation of the cooling agent binding pocket within the TRPM subfamily.
Hypoxia-inducible factor-1 as targets for neuroprotection : from ferroptosis to Parkinson's disease.
Regulation of Airway Epithelial-Derived Alarmins in Asthma: Perspectives for Therapeutic Targets.
HIF-1α regulates mitochondrial function in bone marrow-derived macrophages, but not in tissue-resident alveolar macrophages.
Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
Hypoxia-dependent recruitment of error-prone DNA polymerases to genome replication.
NAT10-mediated RNA ac4C acetylation contributes to the myocardial infarction-induced cardiac fibrosis.
Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.
The post-translational modification O-GlcNAc is a sensor and regulator of metabolism.
Comprehensive pan-cancer analysis reveals ENC1 as a promising prognostic biomarker for tumor microenvironment and therapeutic responses.
Elevated expression levels of the protein kinase DYRK1B induce mesenchymal features in A549 lung cancer cells.
Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.
RAB4A is a master regulator of cancer cell stemness upstream of NUMB-NOTCH signaling.

PLoS One. 2024 ;19(10): e0312535

Characteristic disease defects in circulating endothelial cells isolated from patients with pulmonary arterial hypertension.

Kulwant S Aulak, Lori Mavarakis, Liping Tian, Deborah Paul, Suzy A Comhair, Raed A Dweik, Adriano R Tonelli.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary arterial pressures that can lead to right heart failure and death. No cure exists for this disease, but therapeutic advancements have extended its median survival from 2 to 7 years. Mechanistic research in PAH has been limited by factors including that a) animal models do not fully recapitulate the disease or provide insights into its pathogenesis, and b) cellular material from PAH patients is primarily obtained from donor lungs during autopsy or transplantation, which reflect end-stage disease. Therefore, there is a need to identify tools that can elucidate the specific mechanisms of human disease in individual patients, a critical step to guide treatment decisions based on specific pathway abnormalities. Here we demonstrate a simple method to isolate and culture circulating endothelial cells (CECs) obtained at the time of right heart catheterization in PAH patients. We tested these CECs using transcriptomics and found that they have typical traits of PAH, including those involving key treatment pathways, i.e. nitric oxide, endothelin, prostacyclin and BMP/activin pathways. CECs show important gene expression changes in other central PAH disease pathways. In summary, we present a new cellular model for the ex-vivo mechanistic evaluation of critical PAH pathways that participate in the pathogenesis of the disease and may help personalized therapeutic decisions.

DOI: https://doi.org/10.1371/journal.pone.0312535
Nat Metab. 2024 Oct 29.

Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular cells.

Wusheng Xiao, Nishith Shrimali, Niv Vigder, William M Oldham, Clary B Clish, Huamei He, Samantha J Wong, Bradley M Wertheim, Elena Arons, Marcia C Haigis, Jane A Leopold, Joseph Loscalzo.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of biological processes in hypoxia. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in normal (primary) cells, remain elusive. Here we show that HIF1α signalling is activated in several human primary vascular cells in normoxia and in vascular smooth muscle cells of normal human lungs. Mechanistically, aerobic HIF1α activation is mediated by paracrine secretion of three branched-chain α-ketoacids (BCKAs), which suppress PHD2 activity via direct inhibition and via LDHA-mediated generation of L-2-hydroxyglutarate. BCKA-mediated HIF1α signalling activation stimulated glycolytic activity and governed a phenotypic switch of pulmonary artery smooth muscle cells, which correlated with BCKA metabolic dysregulation and pathophenotypic changes in pulmonary arterial hypertension patients and male rat models. We thus identify BCKAs as previously unrecognized signalling metabolites that aerobically activate HIF1α and that the BCKA-HIF1α pathway modulates vascular smooth muscle cell function, an effect that may be relevant to pulmonary vascular pathobiology.

DOI: https://doi.org/10.1038/s42255-024-01150-4
Sci Rep. 2024 10 27. 14(1): 25681

The impact of ERP29 on the progression of pharyngeal squamous cell carcinoma.

Juliana Carron, Lilian de Oliveira Coser, Carmen Silvia Passos Lima, Gustavo Jacob Lourenço.

ERP29 gene encodes a chaperone protein critical for protein folding and secretion. Previous study linked ERP29 inhibition to an elevated risk of pharynx squamous cell carcinoma (PSCC) and reduced patients' survival. However, ERP29 role in PSCC progression remains unknown. Here, we investigated ERP29 impact on PSCC progression in cisplatin (CDDP)-sensitive (FaDu and LAU-2063), CDDP-treated (FaDu-CDDP), and CDDP-resistant (FaDu-R) cells. ERP29 silencing decreased necrosis and increased migration in CDDP-sensitive, treated, and resistant cells; and reduced E-cadherin and increased vimentin immunoexpression in CDDP-sensitive 3D-spheroids. During CDDP treatment, ERP29 silencing enhanced proliferation. In CDDP-sensitive cells, ERP29 silencing upregulated genes associated with WNT, MAPK, and PI3K/AKT signaling pathways while downregulating CASP9 expression. During CDDP treatment, ERP29 silencing downregulated MDM2 and CASP9 expression. In CDDP-resistant cells, ERP29 silencing upregulated SOS1, MAPK1, AKT1, ITGAV, and CCNE1, while downregulating KRAS, JUN, MDM2, and CASP9 expression. In addition, inhibition of microRNA miR-4421 increased ERP29 expression and decreased MAPK1, AKT1, and JUN expression in CDDP-sensitive cells, as well as SOS1, MAPK1, AKT1, and ITGAV in CDDP-resistant cells. Lower ERP29 and higher miR-4421 expressions were predictive of poor survival, suggesting a potential therapeutic use for miR-4421 inhibitors. Upon validation, these findings may contribute to targeted therapies for PSCC based on ensuring ERP29 expression.

DOI: https://doi.org/10.1038/s41598-024-76210-6
Int J Mol Sci. 2024 Oct 10. pii: 10893. [Epub ahead of print]25(20):

Proteomics Analysis on the Effects of Oxidative Stress and Antioxidants on Proteins Involved in Sterol Transport and Metabolism in Human Telomerase Transcriptase-Overexpressing-Retinal Pigment Epithelium Cells.

R Scott Duncan, Andrew Keightley, Adam A Lopez, Conner W Hall, Peter Koulen.

  Age-related macular degeneration (AMD) is the most prevalent ocular disease in the elderly, resulting in blindness. Oxidative stress plays a role in retinal pigment epithelium (RPE) pathology observed in AMD. Tocopherols are potent antioxidants that prevent cellular oxidative damage and have been shown to upregulate the expression of cellular antioxidant proteins. Here, we determined whether oxidative stress and tocopherols, using either normal cellular conditions or conditions of sublethal cellular oxidative stress, alter the expression of proteins mediating sterol uptake, transport, and metabolism. Human telomerase transcriptase-overexpressing RPE cells (hTERT-RPE) were used to identify differential expression of proteins resulting from treatments. We utilized a proteomics strategy to identify protein expression changes in treated cells. After the identification and organization of data, we divided the identified proteins into groups related to biological function: cellular sterol uptake, sterol transport and sterol metabolism. Exposure of cells to conditions of oxidative stress and exposure to tocopherols led to similar protein expression changes within these three groups, suggesting that α-tocopherol (αT) and γ-tocopherol (γT) can regulate the expression of sterol uptake, transport and metabolic proteins in RPE cells. These data suggest that proteins involved in sterol transport and metabolism may be important for RPE adaptation to oxidative stress, and these proteins represent potential therapeutic targets.

Keywords:  age-relate macular degeneration (AMD); antioxidant; oxidative stress; proteomics; retinal pigment epithelium (RPE); sterol; tocopherol; vitamin E

DOI:  https://doi.org/10.3390/ijms252010893
Front Immunol. 2024 ;15 1440592

Ischemia-induced endogenous Nrf2/HO-1 axis activation modulates microglial polarization and restrains ischemic brain injury.

Ping-Chang Kuo, Wen-Tsan Weng, Barbara A Scofield, Hallel C Paraiso, I-Chen Ivorine Yu, Jui-Hung Jimmy Yen.

  Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in the ischemic brain induce oxidative stress and inflammatory responses. Nrf2 is a transcription factor responsible for regulating cellular redox balance through the induction of protective antioxidant and phase II detoxification responses. Although the induction of endogenous Nrf2/HO-1 axis activation has been observed in the ischemic brain, whether ischemia-induced endogenous Nrf2/HO-1 axis activation plays a role in modulating microglia (MG) phenotypes and restraining ischemic brain injury is not characterized and requires further exploration. To investigate that, we generated mice with Nrf2 knockdown specifically in MG to rigorously assess the role of endogenous Nrf2 activation in ischemic brain injury after stroke. Our results showed that MG-specific Nrf2 knockdown exacerbated ischemic brain injury after stroke. We found that Nrf2 knockdown altered MG phenotypes after stroke, in which increased frequency of inflammatory MG and decreased frequency of anti-inflammatory MG were detected in the ischemic brain. Moreover, we identified attenuated Nrf2/HO-1 axis activation led to increased CD68/IL-1β and suppressed CD206 expression in MG, resulting in aggravated inflammatory MG in MG-specific Nrf2 knockdown mice after stroke. Intriguingly, using type II diabetic preclinical models, we revealed that diabetic mice exhibited attenuated Nrf2/HO-1 axis activation in MG and exacerbated ischemic brain injury after stroke that phenocopy mice with MG-specific Nrf2 knockdown. Finally, the induction of exogenous Nrf2/HO-1 axis activation in MG through pharmacological approaches ameliorated ischemic brain injury in diabetic mice. In conclusion, our findings provide cellular and molecular insights demonstrating ischemia-induced endogenous Nrf2/HO-1 axis activation modulates MG phenotypes and restrains ischemic brain injury. These results further strengthen the therapeutic potential of targeting Nrf2/HO-1 axis in MG for the treatment of ischemic stroke and diabetic stroke.

Keywords:  Nrf2/HO-1 axis; diabetic stroke; ischemic stroke; microglia; neuroinflammation

DOI:  https://doi.org/10.3389/fimmu.2024.1440592
Int J Mol Sci. 2024 Oct 14. pii: 11039. [Epub ahead of print]25(20):

Exploring the Role of miR-132 in Rat Bladders and Human Urothelial Cells during Wound Healing.

Clara I Chamorro, Magdalena Fossum.

  Urinary bladder wound healing shares many features with skin healing, involving several molecular players, including microRNAs (miRs). This study investigated the role of miR-132 in urothelial cells. We analyzed miR-132 expression in rat bladder using in situ hybridization and conducted gain and loss of miR-132 function assays in primary human urothelial cells (HUCs). These assays included cell proliferation and migration studies. To explore the regulation of miR-132 expression, cells were treated with wound-healing-related factors such as interleukin 6 (IL-6), interleukin 10 (IL-10), and transforming growth factor beta-1 (TGF-β1). Predictive bioinformatics and a literature review identified potential miR-132 targets, which were validated through real-time polymerase chain reaction (RT-PCR) and Western blot analysis. miR-132 was found to promote cellular proliferation and migration during the early stages of urothelial wound repair. Its expression was modulated by key cytokines such as IL-6, IL-10, and TGF-β1. miR-132 played a crucial role in urothelial wound healing by enhancing cell proliferation and migration, regulated by cytokines, suggesting its action within a complex regulatory network. These findings highlight the therapeutic potential of targeting miR-132 in bladder injury repair, offering new insights into bladder repair mechanisms.

Keywords:  bladder wound healing; human; microRNA; urinary bladder; urothelium

DOI:  https://doi.org/10.3390/ijms252011039
Exp Cell Res. 2024 Oct 24. pii: S0014-4827(24)00382-3. [Epub ahead of print]443(1): 114291

METTL14-mediated m6A methylation regulates pathological retinal neovascularization by targeting autophagy.

Yang Yu, Huiling Nie, Xun Qin, Xi Chen, Xiumiao Li, Jin Yao.

  Pathological retinal neovascularization (RNV) is a prevalent characteristic of various ocular diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and retinal vein occlusion (RVO). While the importance of N6-methyladenosine (m6A) modification in diverse disease contexts is well-established, its functional role in pathological RNV remains unclear. Herein, we investigated the involvement of m6A modification and its core methyltransferase, METTL14, in a model of oxygen-induced retinopathy (OIR) to elucidate their contribution to retinal angiogenesis. In this study, we observed heightened levels of m6A modification and elevated expression of METTL14 in the OIR model, suggesting their potential implication in pathological RNV. Employing targeted knockdown of METTL14, we revealed that its depletion activated autophagy flux in human retinal vascular endothelial cells (HRVECs), consequently inhibiting the angiogenic capacity of endothelial cells. Mechanistically, we demonstrated that METTL14 exerts its regulatory influence on autophagy flux by modulating the stability of ATG7, a pivotal protein involved in autophagy. Specifically, METTL14 knockdown led to increased ATG7 expression at both mRNA and protein levels, accompanied by reduced m6A methylation of ATG7 mRNA and enhanced mRNA stability. Moreover, silencing of ATG7 counteracted the effects of METTL14 knockdown on endothelial cell functions, emphasizing ATG7 as a downstream target of METTL14-mediated autophagy in HRVECs. After all, our findings provide valuable insights into the pathogenesis of retinal pathological angiogenesis and potential therapeutic targets for the treatment of ocular neovascular diseases.

Keywords:  Angiogenesis; Autophagy; METTL14; N6-methyladenosine modification; Ocular neovascular diseases; Retinal neovascularization

DOI:  https://doi.org/10.1016/j.yexcr.2024.114291
bioRxiv. 2024 Oct 25. pii: 2024.10.25.620225. [Epub ahead of print]

Single-cell transcriptomics reveal diverging pathobiology and opportunities for precision targeting in scleroderma-associated versus idiopathic pulmonary arterial hypertension.

Tijana Tuhy, Julie C Coursen, Tammy Graves, Michael Patatanian, Christopher Cherry, Shannon E Niedermeyer, Sarah L Khan, Darin T Rosen, Michael P Croglio, Mohab Elnashar, Todd M Kolb, Stephen C Mathai, Rachel L Damico, Paul M Hassoun, Larissa A Shimoda, Karthik Suresh, Micheala A Aldred, Catherine E Simpson.

Introduction: Pulmonary arterial hypertension (PAH) involves progressive cellular and molecular change within the pulmonary vasculature, leading to increased vascular resistance. Current therapies targeting nitric oxide (NO), endothelin, and prostacyclin pathways yield variable treatment responses. Patients with systemic sclerosis-associated PAH (SSc-PAH) often experience worse outcomes than those with idiopathic PAH (IPAH).
Methods: Lung tissue samples from four SSc-PAH, four IPAH, and four failed donor specimens were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) lung tissue bank. Single-cell RNA sequencing (scRNAseq) was performed using the 10X Genomics Chromium Flex platform. Data normalization, clustering, and differential expression analysis were conducted using Seurat. Additional analyses included gene set enrichment analysis (GSEA), transcription factor activity analysis, and ligand-receptor signaling. Pharmacotranscriptomic screening was performed using the Connectivity Map.
Results: SSc-PAH samples showed a higher proportion of fibroblasts and dendritic cells/macrophages compared to IPAH and donor samples. GSEA revealed enriched pathways related to epithelial-to-mesenchymal transition (EMT), apoptosis, and vascular remodeling in SSc-PAH samples. There was pronounced differential gene expression across diverse pulmonary vascular cell types and in various epithelial cell types in both IPAH and SSc-PAH, with epithelial to endothelial cell signaling observed. Macrophage to endothelial cell signaling was particularly pronounced in SSc-PAH. Pharmacotranscriptomic screening identified TIE2, GSK-3, and PKC inhibitors, among other compounds, as potential drug candidates for reversing SSc-PAH gene expression signatures.
Discussion: Overlapping and distinct gene expression patterns exist in SSc-PAH versus IPAH, with significant molecular differences suggesting unique pathogenic mechanisms in SSc-PAH. These findings highlight the potential for precision-targeted therapies to improve SSc-PAH patient outcomes. Future studies should validate these targets clinically and explore their therapeutic efficacy.

DOI: https://doi.org/10.1101/2024.10.25.620225
Antioxidants (Basel). 2024 Sep 27. pii: 1178. [Epub ahead of print]13(10):

NADPH Oxidase 4: Crucial for Endothelial Function under Hypoxia-Complementing Prostacyclin.

Heike Brendel, Jennifer Mittag, Anja Hofmann, Helene Hempel, Sindy Giebe, Patrick Diaba-Nuhoho, Steffen Wolk, Christian Reeps, Henning Morawietz, Coy Brunssen.

  Aim: The primary endothelial NADPH oxidase isoform 4 (NOX4) is notably induced during hypoxia, with emerging evidence suggesting its vasoprotective role through H2O2 production. Therefore, we aimed to elucidate NOX4's significance in endothelial function under hypoxia. Methods: Human vessels, in addition to murine vessels from Nox4-/- mice, were explored. On a functional level, Mulvany myograph experiments were performed. To obtain mechanistical insights, human endothelial cells were cultured under hypoxia with inhibitors of hypoxia-inducible factors. Additionally, endothelial cells were cultured under combined hypoxia and laminar shear stress conditions. Results: In human occluded vessels, NOX4 expression strongly correlated with prostaglandin I2 synthase (PTGIS). Hypoxia significantly elevated NOX4 and PTGIS expression and activity in human endothelial cells. Inhibition of prolyl hydroxylase domain (PHD) enzymes, which stabilize hypoxia-inducible factors (HIFs), increased NOX4 and PTGIS expression even under normoxic conditions. NOX4 mRNA expression was reduced by HIF1a inhibition, while PTGIS mRNA expression was only affected by the inhibition of HIF2a under hypoxia. Endothelial function assessments revealed hypoxia-induced endothelial dysfunction in mesenteric arteries from wild-type mice. Mesenteric arteries from Nox4-/- mice exhibited an altered endothelial function under hypoxia, most prominent in the presence of cyclooxygenase inhibitor diclofenac to exclude the impact of prostacyclin. Restored protective laminar shear stress, as it might occur after thrombolysis, angioplasty, or stenting, attenuated the hypoxic response in endothelial cells, reducing HIF1a expression and its target NOX4 while enhancing eNOS expression. Conclusions: Hypoxia strongly induces NOX4 and PTGIS, with a close correlation between both factors in occluded, hypoxic human vessels. This relationship ensured endothelium-dependent vasodilation under hypoxic conditions. Protective laminar blood flow restores eNOS expression and mitigates the hypoxic response on NOX4 and PTGIS.

Keywords:  NADPH oxidase 4; PTGIS; endothelial function; human endothelial cells; hypoxia; laminar flow

DOI:  https://doi.org/10.3390/antiox13101178
Mol Metab. 2024 Oct 23. pii: S2212-8778(24)00182-0. [Epub ahead of print] 102051

cAMP driven UCP1 induction in human adipocytes requires ATGL-catalyzed lipolysis.

Anand Desai, Zinger Yang Loureiro, Tiffany DeSouza, Qin Yang, Javier Solivan-Rivera, Silvia Corvera.

   OBJECTIVE: The uncoupling protein 1 (UCP1) is induced in brown or "beige" adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPARγ agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously.
METHODS: Adipocytes generated from primary human progenitor cells were differentiated without exposure to PPARγ agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses.
RESULTS: UCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that Rsg's effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPARγ agonists.
CONCLUSION: UCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPARγ-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPARγ in response to cAMP may result from the production of natural PPARγ activating ligands through ATGL-mediated lipolysis.

Keywords:  beige adipose; brown adipose; catecholamine; hormone signaling; natural ligand; nuclear receptor; thermogenesis

DOI:  https://doi.org/10.1016/j.molmet.2024.102051
BMC Cancer. 2024 Oct 29. 24(1): 1327

VASN promotes the aggressive phenotype in ARID1A-deficient lung adenocarcinoma.

Dan-Ni Wu, Kang-Liang Zhang, Rui-Heng Chen, Wen-Sheng Ye, Chong Zheng, Yuan-Liang Zheng, Xiao-Dan Zhao, Ri-Sheng Huang.

  Loss of ARID1A has been reported to drive the progression of lung adenocarcinoma, yet the underlying mechanism remains elusive. In this study, we performed secretome analysis to identify the key secreted proteins regulating lung adenocarcinoma progression. We showed that the VASN level was significantly elevated in the conditioned medium from ARID1A-depleted A549 and H1299 cells. Restoration of ARID1A in ARID1A-depleted lung adenocarcinoma cells prevented the upregulation and secretion of VASN. Clinical analysis demonstrated a negative correlation between ARID1A and VASN expression in ARID1A-mutated lung adenocarcinomas. The patients with ARID1A-mutated lung adenocarcinoma had significantly higher concentrations of serum VASN than healthy controls. Moreover, serum VASN concentrations were associated with TNM stage, lymph node metastasis, and overall survival of the patients with ARID1A-mutated lung adenocarcinoma. Functional studies indicated that VASN overexpression potentiated the proliferation, invasion, and tumorigenesis of lung adenocarcinoma cells. Antibody neutralization of VASN suppressed the aggressiveness of ARID1A-depleted lung adenocarcinoma cells both in vitro and in vivo. Addition of recombinant VASN protein promoted the proliferation and invasion of lung adenocarcinoma cells. Additionally, knockdown of Notch1 blocked the aggressive phenotype induced by recombinant VASN protein. In conclusion, our data uncover the role of VASN in mediating the progression of ARID1A-depleted lung adenocarcinoma and highlight VASN as a promising therapeutic target for this disease.

Keywords:  ARID1A; Lung adenocarcinoma; Secretion; VASN

DOI:  https://doi.org/10.1186/s12885-024-13083-y
Am J Physiol Cell Physiol. 2024 Oct 28.

Ion Channel Piezo1 Induces Ferroptosis of Trabecular Meshwork Cells: A Novel Observation in the Pathogenesis in Primary Open Angle Glaucoma.

Kexin Liu, Jing Xu, Rufei Yang, Feng Wang, Ying Su.

   PURPOSE: This study aims to elucidate the role of Piezo1, a mechanosensitive molecule, in trabecular meshwork cells (TMCs) in the context of Primary Open Angle Glaucoma (POAG), a leading cause of irreversible visual impairment. Dysfunction of the trabecular meshwork (TM) is a key factor in the elevated intraocular pressure (IOP) observed in POAG, yet the specific mechanisms leading to TM dysfunction are not fully understood.
METHODS: We performed cell stretching on human trabecular meshwork cells (HTMCs) and pharmacologically activated HTMCs with Yoda1 to study the role of Piezo1 in HTMCs. We focused on assessing cell viability, mitochondrial changes, lipid peroxidation, and the expression of ferroptosis-related targets such as acyl-CoA synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4).
RESULTS: Cell stretching induces ferroptosis in HTMCs, and this phenomenon is reversed by Piezo1 knockdown. Additionally, pharmacological activation of Piezo1 also leads to ferroptosis in HTMCs. Furthermore, inhibiting the JNK/p38 signaling pathway was found to mitigate the ferroptotic response induced by Yoda1, thereby confirming that Piezo1 induces ferroptosis in TMCs through this pathway. Notably, our experiments suggest that Yoda1 may trigger ferroptosis in the TM of mouse eyes.
CONCLUSIONS: Our findings demonstrate that the Piezo1 pathway is a crucial mediator of ferroptosis in TMCs, providing new insights into the pathogenic mechanisms of glaucoma, particularly POAG. This study highlights the potential of targeting the Piezo1 pathway as a therapeutic approach for mitigating TM dysfunction and managing POAG.

Keywords:  Piezo1; ferroptosis; glaucoma; shear stress; trabecular meshwork

DOI:  https://doi.org/10.1152/ajpcell.00173.2024
Oncogene. 2024 Oct 28.

Transcriptional regulation by MYC: an emerging new model.

Simon T Jakobsen, Rasmus Siersbæk.

The transcription factor MYC has long been recognized for its pivotal role in transcriptional regulation of genes fundamental for cellular processes such as cell cycle, apoptosis, and metabolism. Dysregulation of MYC activity is implicated in various diseases, most notably cancer, where MYC drives uncontrolled cell proliferation and growth. Despite its significant role in cancer biology, targeting MYC for therapeutic purposes has been challenging due to its highly disordered protein structure. Hence, recent research efforts have focused on identifying the transcriptional mechanisms underlying MYC function to identify alternative strategies for intervention. This review summarizes recent advances in our understanding of how MYC orchestrates context-dependent and -independent gene-regulatory activities in cancer. Based on recent insights into the gene-regulatory function of MYC at enhancers, we propose an extension of the gene-specific affinity model. In this revised model, MYC enhancer activity drives context-specific gene programs that are distinct from the ubiquitously activated set of core MYC target genes driven by MYC promoter binding. The increased MYC enhancer activity in cancer and the distinct function of MYC at these regions compared to promoters may provide an opportunity for designing therapeutic approaches selectively targeting MYC enhancer activity in cancer cells.

DOI: https://doi.org/10.1038/s41388-024-03174-2
Elife. 2024 Nov 01. pii: RP99643. [Epub ahead of print]13

Conservation of the cooling agent binding pocket within the TRPM subfamily.

Kate Huffer, Matthew C S Denley, Elisabeth V Oskoui, Kenton J Swartz.

  Transient receptor potential (TRP) channels are a large and diverse family of tetrameric cation-selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca2+. Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here, we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4, and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of mouse TRPM4 to intracellular Ca2+, enhancing the sensitivity of the channel to Ca2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore.

Keywords:  TRPM channels; cool agents; intracellular calcium; molecular biophysics; mouse; structural biology; voltage

DOI:  https://doi.org/10.7554/eLife.99643
Neurol Sci. 2024 Oct 28.

Hypoxia-inducible factor-1 as targets for neuroprotection : from ferroptosis to Parkinson's disease.

Changyong Wang, Shanyu Lv, Hongyan Zhao, Guoguo He, Hongshuo Liang, Kemiao Chen, Minghai Qu, Yonghua He, Chaoyan Ou.

   BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by motor paralysis, tremor,and cognitive impairment. Risk factors such as brain hypoxia caused by aging and abnormal expression of HIF-1α areconsidered to be key to the development of PD, including α-synuclein accumulation and ferroptosis. However, therelationship between HIF-1α signaling and ferroptosis in PD has not been elucidated. The stable expression of HIF-1αinhibits the pathological development of PD. Aging aggravates PD pathology by promoting α-synuclein accumulationand oxidative stress.
METHODS: The literature on lipid peroxidation, oxidative stress, iron metabolism and other key factors in Parkinson'sdisease in recent years was reviewed through a variety of literature search channels, such as PubMed and Elsevier.
RESULTS: HIF-1α mediated ferroptosis through oxidative stress and GPX4-GSH system. HIF-1α mediates ferroptosisthrough Keap1-Nrf2-ARE, Grx3 and Grx4. HIF-1α mediates ferroptosis through iron metabolism.
CONCLUSION: This article reviews the oxygen-dependent regulatory mechanism of HIF-1α and its role in cerebralhypoxia homeostasis. Studies in the past decade have shown that Hif-1α mediated ferroptosis is important in PD.HIF-1α has a dual role, depending on the degree of cellular hypoxia and the environment. The equilibrium complexityneeds to be explained, and the role of ferroptosis needs to be investigated. The literature shows that the stabilizationof HIF-1α with PHD inhibitors and the combination of antioxidants and iron chelators are potential therapeuticdirections. In the future, the optimal use time and dose of inhibitors should be studied to improve the efficacy.

Keywords:  Ferroptosis; Hypoxia-inducible factor-1α; Iron metabolism; Oxidative stress levels; Parkinson’s disease

DOI:  https://doi.org/10.1007/s10072-024-07832-x
Biomedicines. 2024 Oct 11. pii: 2312. [Epub ahead of print]12(10):

Regulation of Airway Epithelial-Derived Alarmins in Asthma: Perspectives for Therapeutic Targets.

Ravneet K Hansi, Maral Ranjbar, Christiane E Whetstone, Gail M Gauvreau.

  Asthma is a chronic respiratory condition predominantly driven by a type 2 immune response. Epithelial-derived alarmins such as thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and IL-25 orchestrate the activation of downstream Th2 cells and group 2 innate lymphoid cells (ILC2s), along with other immune effector cells. While these alarmins are produced in response to inhaled triggers, such as allergens, respiratory pathogens or particulate matter, disproportionate alarmin production by airway epithelial cells can lead to asthma exacerbations. With alarmins produced upstream of the type 2 inflammatory cascade, understanding the pathways by which these alarmins are regulated and expressed is critical to further explore new therapeutics for the treatment of asthmatic patients. This review emphasizes the critical role of airway epithelium and epithelial-derived alarmins in asthma pathogenesis and highlights the potential of targeting alarmins as a promising therapeutic to improve outcomes for asthma patients.

Keywords:  airway epithelium; alarmin cytokines; allergies; asthma; biologic therapy; pollutants; respiratory infections

DOI:  https://doi.org/10.3390/biomedicines12102312
bioRxiv. 2024 Oct 14. pii: 2024.10.14.618294. [Epub ahead of print]

HIF-1α regulates mitochondrial function in bone marrow-derived macrophages, but not in tissue-resident alveolar macrophages.

Parker S Woods, Rengül Cetin-Atalay, Angelo Y Meliton, Kaitlyn A Sun, Obada R Shamaa, Kun Woo D Shin, Yufeng Tian, Benjamin Haugen, Robert B Hamanaka, Gökhan M Mutlu.

HIF-1α plays a critical role in shaping macrophage phenotype and effector function. We have previously shown that tissue-resident alveolar macrophages (TR-AMs) have extremely low glycolytic capacity at steady-state, but can shift toward glycolysis under hypoxic conditions. Here, using inducible HIF-1α knockout ( Hif1a -/- ) TR-AMs and bone marrow-derived macrophages (BMDMs) and show that TR-AM HIF-1α is required for the glycolytic shift under prolyl hydroxylase inhibition, but is dispensable at steady-state for inflammatory effector function. In contrast, HIF-1α deletion in BMDMs led to diminished glycolytic capacity at steady-state and reduced inflammatory capacity, but higher mitochondrial function. Gene set enrichment analysis revealed enhanced c-Myc transcriptional activity in Hif1a -/- BMDMs, and upregulation of gene pathways related to ribosomal biogenesis and cellular proliferation. The findings highlight the heterogeneity of HIF-1α function in distinct macrophage populations and provide new insight into how HIF-1α regulates gene expression, inflammation, and metabolism in macrophages.

DOI: https://doi.org/10.1101/2024.10.14.618294
EMBO Mol Med. 2024 Oct 28.

Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.

Louise Nuyttens, Jolien Vandewalle, Claude Libert.

  Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.

Keywords:  Lactate; Metabolism; Mitochondria; Pyruvate; Sepsis

DOI:  https://doi.org/10.1038/s44321-024-00155-6
Oncogene. 2024 Oct 28.

Hypoxia-dependent recruitment of error-prone DNA polymerases to genome replication.

Ran Yehuda, Ido Dromi, Yishai Levin, Thomas Carell, Nicholas Geacintov, Zvi Livneh.

Hypoxia is common in tumors and is associated with cancer progression and drug resistance, driven, at least in part, by genetic instability. Little is known on how hypoxia affects Translesion DNA Synthesis (TLS), in which error-prone DNA polymerases bypass lesions, thereby maintaining DNA continuity at the price of increased mutations. Here we show that under acute hypoxia, PCNA monoubiquitination, a key step in TLS, and expression of error-prone DNA polymerases increased under regulation of the HIF1α transcription factor. Knocking-down expression of DNA polymerase η, or using PCNA ubiquitination-resistant cells, inhibited genomic DNA replication specifically under hypoxia, and iPOND analysis revealed massive recruitment of TLS DNA polymerases to nascent DNA under hypoxia, uncovering a dramatic involvement of error-prone DNA polymerases in genomic replication. Of note, expression of TLS-polymerases correlates with VEGFA (primary HIF1α target) in a database of renal cell carcinoma, a cancer which accumulates HIF1α. Our results suggest that the tumor microenvironment can lead the cell to forgo, to some extent, the fast and accurate canonical DNA polymerases, for the more flexible and robust, but low-fidelity TLS DNA polymerases. This might endow cancer cells with resilience to overcome replication stress, and mutability to escape the immune system and chemotherapeutic drugs.

DOI: https://doi.org/10.1038/s41388-024-03192-0
J Cell Mol Med. 2024 Nov;28(21): e70141

NAT10-mediated RNA ac4C acetylation contributes to the myocardial infarction-induced cardiac fibrosis.

Jun Li, Feierkaiti Yushanjiang, Zhao Fang, Wan-Li Liu.

  Cardiac fibrosis is featured cardiac fibroblast activation and extracellular matrix accumulation. Ac4C acetylation is an important epigenetic regulation of RNAs that has been recently discovered, and it is solely carried out by NAT10, the exclusive enzyme used for the modification. However, the potential regulatory mechanisms of ac4C acetylation in myocardial fibrosis following myocardial infarction remain poorly understood. In our study, we activated fibroblasts in vitro using TGF-β1 (20 ng/mL), followed by establishing a myocardial infarction mouse model to evaluate the impact of NAT10 on collagen synthesis and cardiac fibroblast proliferation. We utilized a NAT10 inhibitor, Remodelin, to attenuate the acetylation capacity of NAT10. In the cardiac fibrosis tissues of chronic myocardial infarction mice and cultured cardiac fibroblasts (CFs) in response to TGF-β1 treatment, there was an elevation in the levels of NAT10 expression. This increase facilitated proliferation, the accumulation of collagens, as well as fibroblast-to-myofibroblast transition. Through the administration of Remodelin, we effectively reduced cardiac fibrosis in myocardial infarction mice by inhibiting NAT10's ability to acetylate mRNA. Inhibition of NAT10 resulted in changes in collagen-related gene expression and ac4C acetylation levels. Mechanistically, we found that NAT10 upregulates the acetylation modification of BCL-XL mRNA and enhances the stability of BCL-XL mRNA, thereby upregulating its protein expression, inhibiting the activation of Caspase3 and blocking the apoptosis of CFs. Therefore, the crucial involvement of NAT10-mediated ac4C acetylation is significant in the cardiac fibrosis progression, affording promising molecular targets for the treatment of fibrosis and relevant cardiac diseases.

Keywords:  NAT10; ac4C acetylation; apoptosis; cardiac fibrosis; myocardial infarction

DOI:  https://doi.org/10.1111/jcmm.70141
J Biol Chem. 2024 Oct 23. pii: S0021-9258(24)02432-3. [Epub ahead of print] 107930

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

Benjamin M Murter, Sean C Robinson, Hridesh Banerjee, Louis Lau, Uzodinma U Uche, Andrea L Szymczak-Workman, Lawrence P Kane.

  The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

Keywords:  ADAM; PIK3IP1; T-cell receptor (TCR); TrIP; T‐cell; cell signaling; cellular immune response; matrix metalloproteinase (MMP); phosphatidylinositide 3‐kinase (PI 3‐kinase)

DOI:  https://doi.org/10.1016/j.jbc.2024.107930
Open Biol. 2024 Oct;14(10): 240209

The post-translational modification O-GlcNAc is a sensor and regulator of metabolism.

Murielle M Morales, Matthew R Pratt.

  Cells must rapidly adapt to changes in nutrient conditions through responsive signalling cascades to maintain homeostasis. One of these adaptive pathways results in the post-translational modification of proteins by O-GlcNAc. O-GlcNAc modifies thousands of nuclear and cytoplasmic proteins in response to nutrient availability through the hexosamine biosynthetic pathway. O-GlcNAc is highly dynamic and can be added and removed from proteins multiple times throughout their life cycle, setting it up to be an ideal regulator of cellular processes in response to metabolic changes. Here, we describe the link between cellular metabolism and O-GlcNAc, and we explore O-GlcNAc's role in regulating cellular processes in response to nutrient levels. Specifically, we discuss the mechanisms of elevated O-GlcNAc levels in contributing to diabetes and cancer, as well as the role of decreased O-GlcNAc levels in neurodegeneration. These studies form a foundational understanding of aberrant O-GlcNAc in human disease and provide an opportunity to further improve disease identification and treatment.

Keywords:  O-GlcNAc; metabolism; modifications; post-translational; regulators; sensor

DOI:  https://doi.org/10.1098/rsob.240209
Sci Rep. 2024 10 25. 14(1): 25331

Comprehensive pan-cancer analysis reveals ENC1 as a promising prognostic biomarker for tumor microenvironment and therapeutic responses.

Zhenyu Cao, Jinfeng Zhu, Zicheng Wang, Yuhuai Peng, Liyun Zeng.

  Accumulating research showed that ENC1 plays a critical role in maintaining the physiological functions. However, little is known about its role in predicting prognosis and immunotherapy response across cancers. In our results, compared to normal tissues, most cancer tissues exhibit increased ENC1 expression. We found that the most common type of genetic variation was gene mutation. In addition, a positive correlation was found between CNV and ENC1 expression. Moreover, the overexpression of ENC1 was positively correlated with poor clinical outcomes. The GSEA results showed that ENC1 is closely correlated with tumor-promoting biological functions in most cancers. ENC1 is also closely negatively associated with the infiltration levels of T cells, activated NK cells, and B cells. Most immunomodulators are positively associated with ENC1. Further, we verified that inhibition of ENC1 expression suppressed the proliferation and migration of breast cancer, pancreatic cancer and glioma cells. In conclusion, our study demonstrated that ENC1 plays a protumorigenic role in most cancers. Additionally, ENC1 is closely correlated with tumor microenvironment features and immune checkpoint inhibitors expression. Overall, ENC1 could serve as a promising potential prognostic biomarker in various tumors.

Keywords:  ENC1; Immunomodulators; Immunotherapy; Pan-cancer; Prognostic biomarker; Tumor immune infiltration

DOI:  https://doi.org/10.1038/s41598-024-76798-9
BMC Cancer. 2024 Oct 31. 24(1): 1341

Elevated expression levels of the protein kinase DYRK1B induce mesenchymal features in A549 lung cancer cells.

Soraya Sester, Gerrit Wilms, Joana Ahlburg, Aaron Babendreyer, Walter Becker.

   BACKGROUND: The protein kinase DYRK1B is a negative regulator of cell proliferation but has been found to be overexpressed in diverse human solid cancers. While DYRK1B is recognized to promote cell survival and adaption to stressful conditions, the consequences of elevated DYRK1B levels in cancer cells are largely uncharted.
METHODS: To elucidate the role of DYRK1B in cancer cells, we established a A549 lung adenocarcinoma cell model featuring conditional overexpression of DYRK1B. This system was used to characterize the impact of heightened DYRK1B levels on gene expression and to monitor phenotypic and functional changes.
RESULTS: A549 cells with induced overexpression of wild type DYRK1B acquired a mesenchymal cell morphology with diminished cell-cell contacts and a reorganization of the pericellular actin cytoskeleton into stress fibers. This transition was not observed in cells overexpressing a catalytically impaired DYRK1B variant. The phenotypic changes were associated with increased expression of the transcription factors SNAIL and SLUG, which are core regulators of epithelial mesenchymal transition (EMT). Further profiling of DYRK1B-overexpressing cells revealed transcriptional changes that are characteristic for the mesenchymal conversion of epithelial cells, including the upregulation of genes that are related to cancer cell invasion and metastasis. Functionally, DYRK1B overexpression enhanced the migratory capacity of A549 cells in a wound healing assay.
CONCLUSIONS: The present data identify DYRK1B as a regulator of phenotypic plasticity in A549 cells. Increased expression of DYRK1B induces mesenchymal traits in A549 lung adenocarcinoma cells.

Keywords:  A549; Cancer cell migration; DYRK1B; Lung adenocarcinoma; Phenotypic plasticity; Protein kinase; Stress fibers

DOI:  https://doi.org/10.1186/s12885-024-13057-0
Mol Cell. 2024 Oct 24. pii: S1097-2765(24)00826-8. [Epub ahead of print]

Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.

Matthew J Payea, Showkat A Dar, Carlos Anerillas, Jennifer L Martindale, Cedric Belair, Rachel Munk, Sulochan Malla, Jinshui Fan, Yulan Piao, Xiaoling Yang, Abid Rehman, Nirad Banskota, Kotb Abdelmohsen, Myriam Gorospe, Manolis Maragkakis.

  Senescence is a state of indefinite cell-cycle arrest associated with aging, cancer, and age-related diseases. Here, we find that translational deregulation, together with a corresponding maladaptive integrated stress response (ISR), is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response activating transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames (uORFs). Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. We also find that stress augments the senescence-associated secretory phenotype with sustained remodeling of inflammatory factors expression that is suppressed by non-uORF carrying ATF4 mRNA expression. Our results thus show that senescent cells possess a unique response to stress, which entails an increase in their inflammatory profile.

Keywords:  ATF4; ER stress; ISR; SASP; integrated stress response; nanopore direct RNA sequencing; proteomics; ribosome sequencing; senescence; senescence-associated secretory phenotype; translation

DOI:  https://doi.org/10.1016/j.molcel.2024.10.003
Cell Death Dis. 2024 Oct 27. 15(10): 778

RAB4A is a master regulator of cancer cell stemness upstream of NUMB-NOTCH signaling.

Subbulakshmi Karthikeyan, Patrick J Casey, Mei Wang.

Cancer stem cells (CSCs) are a group of specially programmed tumor cells that possess the characteristics of perpetual cell renewal, increased invasiveness, and often, drug resistance. Hence, eliminating CSCs is a major challenge for cancer treatment. Understanding the cellular programs that maintain CSCs, and identifying the critical regulators for such programs, are major undertakings in both basic and translational cancer research. Recently, we have reported that RAB4A is a major regulator of epithelial-to-mesenchymal transition (EMT) and it does so mainly through regulating the activation of RAC1 GTPase. In the current study, we have delineated a new signaling circuitry through which RAB4A transmits its control of cancer stemness. Using in vitro and in vivo studies, we show that RAB4A, as the upstream regulator, relays signal stepwise to NUMB, NOTCH1, RAC1, and then SOX2 to control the self-renewal property of multiple cancer cells of diverse tissue origins. Knockdown of NUMB, or overexpression of NICD (the active fragment NOTCH1) or SOX2, rescued the in vitro sphere-forming and in vivo tumor-forming abilities that were lost upon RAB4A knockdown. Furthermore, we discovered that the chain of control is mostly through transcriptional regulation at every step of the pathway. The discovery of the novel signaling axis of RAB4A-NUMB-NOTCH-SOX2 opens the path for further expansion of the signaling chain and for the identification of new regulators and interacting proteins important for CSC functions, which can be explored to develop new and effective therapies.

DOI: https://doi.org/10.1038/s41419-024-07172-w