Aims: Brown adipose tissue (BAT) relies heavily on mitochondrial activity and reactive oxygen species homeostasis to regulate thermogenesis and metabolic balance. However, the specific role of glutathione peroxidase 4 (GPX4), a critical antioxidant enzyme and central regulator of ferroptosis, in BAT remains unclear. This study aims to investigate the necessity of GPX4 for the functional integrity and thermogenic capacity of BAT. Methods: Initially, we employed pharmacological inhibition of GPX4 in vitro using differentiated brown adipocytes. To investigate its role in vivo, we generated a BAT-specific Gpx4 knockout mouse model. The physiological and metabolic impacts of GPX4 deficiency were evaluated across three different conditions: cold exposure, high-fat diet, and vitamin E-deficient diet. Comprehensive evaluations were conducted using metabolic, histological, ultrastructural, and transcriptomic (RNA-seq) analyses. Results: In vitro, pharmacological inhibition of GPX4 induced ferroptosis in differentiated brown adipocytes, suggesting its potential regulatory role. Strikingly, in vivo histological, ultrastructural, and metabolic analyses indicated that the genetic deletion of GPX4 does not impair BAT morphology or thermogenic function under any of the tested conditions. Consistent with these physiological findings, RNA-seq revealed that GPX4 deficiency did not significantly alter the expression of genes associated with ferroptosis or thermogenic pathways. Conclusion: Although pharmacological inhibition of GPX4 triggers ferroptosis in brown adipocytes in vitro, GPX4 is not essential for maintaining the morphological integrity and thermogenic capacity of BAT in vivo under the specific experimental conditions tested.

ABSTRACT:The complex pathogenesis of myocardial ischemia-reperfusion (I/R) injury is a major factor influencing clinical prognosis. It has been confirmed that microRNAs are involved in myocardial I/R injury, and that pyroptosis is closely associated with its underlying mechanisms. However, the specific mechanism by which miR-193b-3p inhibits cell death and alleviates myocardial I/R injury remains unclear. This study aimed to investigate whether miR-193b-3p can inhibit pyroptosis and protect injured myocardium by targeting the Gasdermin-D (GSDMD)/Nucleotide-binding oligomerization domain-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling axis, thereby offering a potential therapeutic strategy for myocardial I/R injury. Through bioinformatics analysis, pyroptosis-related signaling pathways and key genes involved in myocardial I/R injury were identified. A myocardial I/R injury model was established, and pathological changes in myocardial tissue were evaluated using hematoxylin and eosin staining. A dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-193b-3p and GSDMD. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were employed to detect mRNA and protein expression levels of miR-193b-3p, GSDMD, and NLRP3. The role of miR-193b-3p in myocardial I/R injury was comprehensively evaluated based on cardiac troponin I levels and the rate of myocardial pyroptosis. The findings confirmed that miR-193b-3p inhibited GSDMD expression, attenuated pathological changes in rat myocardium, downregulated NLRP3 and other pyroptosis-related proteins, and reduced both myocardial pyroptosis and serum cardiac troponin I levels.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Abstract Trop2 and Nectin4 are transmembrane proteins involved in cancer pathogenesis. The therapeutic potential of targeting Trop2 or Nectin4 has been demonstrated through the approval of Trodelvy™ (Sacituzumab govitecan; SG) and Padcev™ (Enfortumab Vedotin; EV). However, the clinical use of these agents is accompanied by safety concerns. Trodelvy carries Boxed Warnings for severe or life-threatening neutropenia and severe diarrhea, while Padcev is associated with serious skin reactions. These safety issues underscore the importance of improving the drugs' safety profiles. It should be noted that the co-expression of Trop2 and Nectin4 is high in solid tumors while limited or moderate in normal tissues, presents an opportunity for more tumor-specific targeting and supports the bispecific design that could potentially address these safety issues. VBC103 is a bispecific antibody-drug conjugate (ADC) that selectively targets both Trop2 and Nectin4, which are highly co-expressed in UC, TNBC and other tumors. It delivers a TOPOi payload with a strong bystander effect to tumor cells. Our unique design maximizes efficacy while minimizing safety concerns. VBC103 has the following features to differentiate itself from other drugs targeting Trop2 or Nectin4 separately in clinical development and on the market. 1. Modulated affinity and valency. VBC103 incorporates a moderate affinity arm that targets Trop2 and a moderate affinity but high avidity arm that targets Nectin4, allowing for enhanced binding avidity, internalization, and cytotoxicity compared to its parental molecules (anti-Nectin4 parental antibody) or approved drug like EV. Additionally, the moderate affinity of both Trop2 and Nectin4 in VBC103 helps to reduce target-driven toxicities in normal tissues. 2. Innovative format of VHH-Fc fusion protein (MW 90kDa) demonstrated the superior tumor penetration, accumulation (MFI ratio in tumor: 280 VS 174) and distribution (MFI ratio in liver: 10 VS 18) compared to the IgG1 mAb- enfortumab (MW 150kDa). 3. Stronger bystander cell killing effect of the TOPOi payload vs. that of the payload used in EV. 4. Superior in vivo efficacy. In the UC CDX model, TGI 95% [VBC103, 5 mpk] vs 77% [EV, 8 mpk] vs 70% [EV+SG, 4+4 mpk] (Q3W X 3; ADC molecular equivalence) at Day 42 post injection. In the TNBC CDX model, VBC103 demonstrated continued superior efficacy compared to EV (maximum tolerated dose) and SG when administered as a single dose treatment at Day 21 post injection. 5. Wide therapeutic widow (TW). Preliminary toxicity studies in cynomolgus monkeys demonstrated good tolerability of repeated doses (18 and 36 mpk) of VBC103, which is molecular equivalent to 30 and 60 mpk of IgG1 ADC. These findings could establish a TW of >10 (HNSTD/MED). 6. Good developability. VBC103 also exhibited excellent developability based on plasma stability and other stress test data, which suggests it a good candidate in future CMC development. In summary, VBC103, with its unique bispecific design and differentiated features in affinity, valency, linker-payload, has shown promising potential as a first-in-class ADC candidate. Discovery studies have revealed favorable efficacy and toxicity profiles, supporting advancing VBC103 into clinical trials. Citation Format: Wei Wang, Jing Li, Lingyu Guan, Man Xu, Qun Yin. VBC103: An innovative Trop2/Nectin4 targeted bispecific antibody drug conjugate (ADC) in bladder urothelial carcinoma (UC), triple-negative breast cancer (TNBC) and beyond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB448.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKβ-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKβ/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

Abstract GOLPH3 is a peripheral membrane protein that is significantly associated with the development of cancer. However, the signaling pathways and mechanisms of GOLPH3 mediated cancer are still unclear. Here, our objective is to identify the essential molecules and signaling pathways by analyzing the RNA-seq data. The GSE196875 was produced by the Illumina NovaSeq 6000 (Homo sapiens). The KEGG and GO analyses indicated that axon guidance, signaling pathways regulating pluripotency of stem cells, and circadian entrainment are the major affected processes during the development of prostate cancer with GOLPH3 overexpression. Moreover, the ten interacting molecules were figured out including BMP2, RAC2, CACNA1C, PAX6, COL4A1, SPARC, GRIN1, CAV3, COL5A1, COL4A2. Thus, our study provides novel knowledge on the GOLPH3 affected cancer cells.

High altitudes or exposure to hypoxia leads to female reproductive disorders. Circadian clocks are intrinsic time-tracking systems that enable organisms to adapt to the Earth’s 24-h light/dark cycle, which can be entrained by other environmental stimuli to regulate physiological and pathological responses. In this study, we focused on whether ovarian circadian clock proteins were involved in regulating female reproductive dysfunction under hypoxic conditions. Hypobaric hypoxia was found to induce a significantly prolonged estrous cycle in female mice, accompanied by follicular atresia, pituitary/ovarian hormone synthesis disorder, and decreased LHCGR expression in the ovaries. Under the same conditions, the levels of the ovarian circadian clock proteins, CLOCK and BMAL1, were suppressed, whereas E4BP4 levels were upregulated. Results from granulosa cells (GCs) further demonstrated that CLOCK: BMAL1 and E4BP4 function as transcriptional activators and repressors of LHCGR in ovarian GCs, respectively, whose responses were mediated by HIF1ɑ-dependent (E4BP4 upregulation) and ɑ-independent (CLOCK and BMAL1 downregulation) manners. The LHCGR agonist was shown to efficiently recover the impairment of ovulation-related gene ( EREG and PGR ) expression in GCs induced by hypoxia. We conclude that hypoxia exposure causes dysregulation of ovarian circadian clock protein (CLOCK, BMAL1, and E4BP4) expression, which mediates female reproductive dysfunction by impairing LHCGR-dependent signaling events. Adjusting the timing system or recovering the LHCGR level in the ovaries may be helpful in overcoming female reproductive disorders occurring in the highlands.

Multiple myeloma (MM) is a plasma cell malignancy that is characterized by the overabundance of monoclonal paraprotein. Aurora kinase A (AURKA) was upregulated in patients with high-risk MM. AURKA inhibitors were used to inhibit MM cell proliferation by inducing cell apoptosis and injury. In our study, we aim to identify biological processes and pathways of MM cells under the knockout of AURKA (AURKA KO) by using a bioinformatics method to elucidate their potential pathogenesis. The gene expression profiles of the GSE163589 dataset were originally produced by using the high-throughput BGISEQ-500 (Homo sapiens). The biological categories and pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactom enrichment. KEGG and GO results indicated the biological pathways related to the immune responses and cancer activities were mostly affected in the development of MM with AURKA KO. Moreover, we identified several genes including GNG5, UBE2D1, and BUB1B were involved in the regulation of cancer genesis. We further predicted novel regulators that had the ability to affect the progression of MM with AURKA KO based on the L1000fwd analysis. Therefore, this study provides further insights into the mechanism of MM under AURKA inhibitor treatments.

Abstract The COVID-19 pandemic caused by the infection with SARS-CoV-2 has overwhelmed many health systems globally. Our study is to identify differentially expressed genes (DEGs) and the associated biological pathways of COVID-19 to elucidate the potential pathogenesis and metabolism. The gene expression profile of the GSE155363 dataset was originally produced using the high-throughput Illumina HiSeq 4000 (Macaca mulatta). Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed to discover their functional categories and biochemical pathways. The results suggested that four biological pathways: Fatty acid elongation, Biosynthesis of unsaturated fatty acids, Fatty acid metabolism, and Ribosome were mostly involved in the macaques with COVID-19. Thus, our study provides novel insights into the underlying pathogenesis of COVID-19.

Disturbed sleep is closely associated with an increased risk of metabolic diseases. However, the underlying mechanisms of circadian clock genes linking sleep and lipid profile abnormalities have not been fully elucidated. This study aimed to explore the important role of the circadian clock in regulating impaired cholesterol metabolism at an early stage of sleep deprivation (SD). Sleep disturbance was conducted using an SD instrument. Our results showed that SD increased the serum cholesterol levels. Concentrations of serum leptin and resistin were much lower after SD, but other metabolic hormone concentrations (adiponectin, glucagon, insulin, thyroxine, norepinephrine, and epinephrine) were unchanged before and after SD. Warning signs of cardiovascular diseases [decreased high density lipoprotein (HDL)-cholesterol and increased corticosterone and 8-hydroxyguanosine levels] and hepatic cholestasis (elevated total bile acids and bilirubin levels) were observed after SD. Cholesterol accumulation was also observed in the liver after SD. The expression levels of HMGCR, the critical enzyme for cholesterol synthesis, remained unchanged in the liver. However, the expression levels of liver CYP7A1, the enzyme responsible for the conversion of cholesterol into bile acids, significantly reduced after SD. Furthermore, expression of NR1D1, a circadian oscillator and transcriptional regulator of CYP7A1, strikingly decreased after SD. Moreover, NR1D1 deficiency decreased liver CYP7A1 levels, and SD could exacerbate the reduction of CYP7A1 expression in NR1D1−/− mouse livers. Additionally, NR1D1 deficiency could further increase serum cholesterol levels under SD. These results suggest that sleep disturbance can induce increased serum cholesterol levels and liver cholesterol accumulation by NR1D1 mediated CYP7A1 inhibition.