Chromatin Immunoprecipitation Assays Research Articles

GR/P300 Regulates MKP1 Signaling Pathway and Mediates Depression-like Behavior in Prenatally Stressed Offspring.

Accumulating evidence suggests that prenatal stress (PNS) increases offspring susceptibility to depression, but the underlying mechanisms remain unclear. We constructed a mouse model of prenatal stress by spatially restraining pregnant mice from 09:00-11:00 daily on Days 5-20 of gestation. In this study, western blot analysis, quantitative real-time PCR (qRT‒PCR), immunofluorescence, immunoprecipitation, chromatin immunoprecipitation (ChIP), and mifepristone rescue assays were used to investigate alterations in the GR/P300-MKP1 and downstream ERK/CREB/TRKB pathways in the brains of prenatally stressed offspring to determine the pathogenesis of the reduced neurogenesis and depression-like behaviors in offspring induced by PNS. We found that prenatal stress leads to reduced hippocampal neurogenesis and depression-like behavior in offspring. Prenatal stress causes high levels of glucocorticoids to enter the fetus and activate the hypothalamic‒pituitary‒adrenal (HPA) axis, resulting in decreased hippocampal glucocorticoid receptor (GR) levels in offspring. Furthermore, the nuclear translocation of GR and P300 (an acetylation modifying enzyme) complex in the hippocampus of PNS offspring increased significantly. This GR/P300 complex upregulates MKP1, which is a negative regulator of the ERK/CREB/TRKB signaling pathway associated with depression. Interestingly, treatment with a GR antagonist (mifepristone, RU486) increased hippocampal GR levels and decreased MKP1 expression, thereby ameliorating abnormal neurogenesis and depression-like behavior in PNS offspring. In conclusion, our study suggested that the regulation of the MKP1 signaling pathway by GR/P300 is involved in depression-like behavior in prenatal stress-exposed offspring and provides new insights and ideas for the fetal hypothesis of mental health.

CCT6A facilitates lung adenocarcinoma progression and glycolysis via STAT1/HK2 axis

BackgroundChaperonin Containing TCP1 Subunit 6 A (CCT6A) is a prominent protein involved in the folding and stabilization of newly synthesized proteins. However, its roles and underlying mechanisms in lung adenocarcinoma (LUAD), one of the most aggressive cancers, remain elusive.MethodsOur study utilized in vitro cell phenotype experiments to assess CCT6A’s impact on the proliferation and invasion capabilities of LUAD cell lines. To delve into CCT6A’s intrinsic mechanisms affecting glycolysis and proliferation in lung adenocarcinoma, we employed transcriptomic sequencing and liquid chromatography-mass spectrometry analysis. Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) assays were also conducted to substantiate the mechanism.ResultsCCT6A was found to be significantly overexpressed in LUAD and associated with a poorer prognosis. The silencing of CCT6A inhibited the proliferation and migration of LUAD cells and elevated apoptosis rates. Mechanistically, CCT6A interacted with STAT1 protein, forming a complex that enhances the stability of STAT1 by protecting it from ubiquitin-mediated degradation. This, in turn, facilitated the transcription of hexokinase 2 (HK2), a critical enzyme in aerobic glycolysis, thereby stimulating LUAD’s aerobic glycolysis and progression.ConclusionOur findings reveal that the CCT6A/STAT1/HK2 axis orchestrated a reprogramming of glucose metabolism and thus promoted LUAD progression. These insights position CCT6A as a promising candidate for therapeutic intervention in LUAD treatment.

Brevilin A Inhibits Prostate Cancer Progression by Decreasing PAX5-Activated SOX4.

Brevilin A possesses inhibitory effects on the development of prostate cancer (PCa); however, the underlying mechanism remains unclear. The present work aims to analyze how Brevilin A regulates PCa cell malignancy. RNA expression of paired box 5 (PAX5) and SRY-box transcription factor 4 (SOX4) was analyzed by quantitative real-time polymerase chain reaction. Protein expression of PAX5, SOX4, and nuclear proliferation marker (Ki67) was detected by western blotting or immunohistochemistry assay. The viability, proliferation, apoptosis, and migratory and invasive abilities of PCa cells were investigated by cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays, respectively. The association between PAX5 and SOX4 was identified by dual-luciferase reporter assay and chromatin immunoprecipitation assay. Xenograft mouse model assay was used to reveal the effect of Brevilin A on tumor tumorigenesis in vivo. PAX5 and SOX4 expression were upregulated in PCa tissues and cells relative to normal prostate tissues and human prostate epithelial cells. Brevilin A treatment inhibited PAX5 protein expression in PCa cells. Additionally, Brevilin A inhibited proliferation, migration and invasion and induced apoptosis of PCa cells, whereas these effects were attenuated after PAX5 overexpression. SOX4 was transcriptionally activated by PAX5, and its introduction partially relieved the inhibitory effects of PAX5 knockdown on PCa cell malignancy. Moreover, Brevilin A delayed tumor formation in vivo. Brevilin A inhibited PCa progression by regulating SOX4 expression in a PAX5-dependent manner, providing a promising anti-tumor drug for PCa.

Curcumin Inhibits the Progression of Non-small Cell Lung Cancer by Regulating DMRT3/SLC7A11 Axis.

Non-small cell lung cancer (NSCLC) is a fatal malignancy all over the world. Emerging studies have shownthat curcumin might repress NSCLC progression by regulating ferroptosis, but the underlying mechanism remains unclear. 16HBE, LK-2, and H1650 cell viability was detected using Cell Counting Kit-8 assay. LK-2 and H1650 cell proliferation, apoptosis, and angiopoiesis were measured using 5-ethynyl-2'-deoxyuridine, flow cytometry, and tube formation assay. Superoxide dismutase, Malondialdehyde, Glutathione, and lactate dehydrogenase levels in LK-2 and H1650 cells were examined using special assay kits. Fe+ level was assessed using an iron assay kit. Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) and solutecarrierfamily7member11(SLC7A11) protein levels were detected using western in NSCLC tissues, adjacent matched normal tissues, 16HBE cells, LK-2 cells, H1650 cells, and xenograft tumor tissues. Glutathione peroxidase 4, Acyl-CoA Synthetase Long Chain Family Member 4, and transferrinreceptor1 protein levels in LK-2 and H1650 cells were examined by western blot assay. DMRT3 and SLC7A11 levels were determined using real-time quantitative polymerase chain reaction. After JASPAR prediction, binding between DMRT3 and SLC7A11 promoter was verified using Chromatin immunoprecipitation and dual-luciferase reporter assays in LK-2 and H1650 cells. Role of curcumin on NSCLC tumor growth was assessed using the xenograft tumor model in vivo. Curcumin blocked NSCLC cell proliferation and angiopoiesis, and induced apoptosis and ferroptosis. DMRT3 or SLC7A11 upregulation partly abolished the suppressive role of curcumin on NSCLC development. In mechanism, DMRT3 was a transcription factor of SLC7A11 and increased the transcription of SLC7A11 via binding to its promoter region. Curcumin inhibited NSCLC growth in vivo by modulating DMRT3. Curcumin might constrain NSCLC cell malignant phenotypes partly through the DMRT3/SLC7A11 axis, providing a promising therapeutic strategy for NSCLC.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma

BackgroundSorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC.MethodsWe investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells.ResultsOur study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition.ConclusionsFerroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

MSC Promotes the Secretion of Exosomal lncRNA KLF3-AS1 to Regulate Sphk1 Through YY1-Musashi-1 Axis and Improve Cerebral Ischemia-Reperfusion Injury.

Stroke remains the 3rd leading cause of long-term disability globally. Over the past decade, mesenchymal stem cell (MSC) transplantation has been proven as an effective therapy for ischemic stroke. However, the mechanism of MSC-derived exosomal lncRNAs during cerebral ischemia/reperfusion (I/R) remains ambiguous. The oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) rat model were generated. MSCs were isolated and characterized by flow cytometry and histochemical staining, and MSC exosomes were purified and characterized by transmission electron microscopy, flow cytometry and Western blot. Western blot, RT-qPCR and ELISA assay were employed to examine the expression or secretion of key molecules. CCK-8 and TUNEL assays were used to assess cell viability and apoptosis. RNA immunoprecipitation and RNA pull-down were used to investigate the direct association between krüppel-like factor 3 antisense RNA 1 (KLF3-AS1) and musashi-1(MSI1). Yin Yang 1 (YY1)-mediated transcriptional regulation was assessed by chromatin immunoprecipitation and luciferase assays. The histological changes and immunoreactivity of key molecules in brain tissues were examined by H&E and immunohistochemistry. MSCs were successfully isolated and exhibited directionally differential potentials. MSC exosomal KLF3-AS1 alleviated OGD/R-induced inflammation in SK-N-SH and SH-SY5Y cells via modulating Sphk1. Mechanistical studies showed that MSI1 positively regulated KLF3-AS1 expression through its direct binding to KLF3-AS1. YY1 was identified as a transcription activator of MSI1 in MSCs. Functionally, YY1/MSI1 axis regulated the release of MSC exosomal KLF3-AS1 to modulate sphingosine kinase 1 (Sphk1)/NF-κB pathway, thereby ameliorating OGD/R- or cerebral I/R-induced injury. MSCs promote the release of exosomal KLF3-AS1 to regulate Sphk1 through YY1/MSI axis and improve cerebral I/R injury.

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing invivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3

BackgroundHelicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis.MethodsThe expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry.ResultsAfter H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased.ConclusionsIn summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

BackgroundDysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC.MethodsThe clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells.ResultsHCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells.ConclusionsIL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21

BackgroundSingle nuclear polymorphisms (SNPs) have been published to be correlated with multiple diseases. Transcription Factor 21 (TCF21) is a critical transcription factor involved in various types of cancers. However, the association of TCF21 genetic polymorphisms with gastric cancer (GC) susceptibility and prognosis remains unclear.MethodsA case-control study comprising 890 patients diagnosed with GC and an equal number of cancer-free controls was conducted. After rigorous statistical analysis, molecular experiments were carried out to elucidate the functional significance of the SNPs in the context of GC.ResultsTCF21 rs2327430 (OR = 0.78, P = 0.026) provides protection against GC, while rs4896011 (OR = 1.39, P = 0.005) exhibit significant associations with GC risk. Furthermore, patients with the (TC + CC) genotype of rs2327430 demonstrate a relatively favorable prognosis (OR = 0.47, P = 0.012). Mechanistically, chromatin immunoprecipitation assay and luciferase reporter assay revealed that the C allele of rs2327430 disrupts the binding of Transcription Factor AP-2 Alpha (TFAP2A) to the promoter region of TCF21, resulting in increased expression of TCF21 and inhibition of malignant behaviors in GC cells.ConclusionOur findings highlight the significant role of TCF21 SNPs in both the risk and prognosis of GC and provide valuable insights into the underlying molecular mechanisms. Specifically, the disruptive effect of rs2327430 on TCF21 expression and its ability to modulate malignant cell behaviors suggest that rs2327430 may serve as a potential predictive marker for GC risk and prognosis.

KIF22 promotes multiple myeloma progression by regulating the CDC25C/CDK1/cyclinB1 pathway

PurposeMultiple myeloma (MM) is an incurable hematological malignancy characterized by clonal proliferation of malignant plasma B cells in bone marrow, and its pathogenesis remains unknown. The aim of this study was to determine the role of kinesin family member 22 (KIF22) in MM and elucidate its molecular mechanism.MethodsThe expression of KIF22 was detected in MM patients based upon the public datasets and clinical samples. Then, in vitro assays were performed to investigate the biological function of KIF22 in MM cell lines, and subcutaneous xenograft models in nude mice were conducted in vivo. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay were used to determine the mechanism of KIF22-mediated regulation.ResultsThe results demonstrated that the expression of KIF22 in MM patients was associated with several clinical features, including gender (P = 0.016), LDH (P < 0.001), β2-MG (P = 0.003), percentage of tumor cells (BM) (P = 0.002) and poor prognosis (P < 0.0001). Furthermore, changing the expression of KIF22 mainly influenced the cell proliferation in vitro and tumor growth in vivo, and caused G2/M phase cell cycle dysfunction. Mechanically, KIF22 directly transcriptionally regulated cell division cycle 25C (CDC25C) by binding its promoter and indirectly influenced CDC25C expression by regulating the ERK pathway. KIF22 also regulated CDC25C/CDK1/cyclinB1 pathway.ConclusionKIF22 could promote cell proliferation and cell cycle progression by transcriptionally regulating CDC25C and its downstream CDC25C/CDK1/cyclinB1 pathway to facilitate MM progression, which might be a potential therapeutic target in MM.

Pan-cancer analysis of super-enhancer-induced LINC00862 and validation as a SIRT1-promoting factor in cervical cancer and gastric cancer

Immune checkpoints inhibitors are effective but it needs more precise biomarkers for patient selection. We explored the biological significance of LINC00862 in pan-cancer by bioinformatics. And we studied its regulatory mechanisms using chromatin immunoprecipitation and RNA immunoprecipitation assays etc. TCGA and single-cell sequencing data analysis indicated that LINC00862 was overexpressed in the majority of tumor and stromal cells, which was related with poor prognosis. LINC00862 expression was related with immune cell infiltration and immune checkpoints expression, and had a high predictive value for immunotherapy efficacy. Mechanistically, LINC00862 competitively bound to miR-29c-3p to unleash SIRT1’s tumor-promoting function. SIRT1 inhibitor-EX527 were screened by virtual screening and verified by in vitro and vivo assays. Notably, acetyltransferase P300-mediated super-enhancer activity stimulated LINC00862 transcription. Collectively, LINC00862 could be a diagnostic and prognostic biomarker. LINC00862 could also be a predictive biomarker for immunotherapy efficacy. Super-enhancer activity is the driver for LINC00862 overexpression in cervical cancer and gastric cancer.

PITX2 functions as a transcription factor for GPX4 and protects pancreatic cancer cells from ferroptosis

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.

HIF-1α participates in the regulation of S100A16-HRD1-GSK3β/CK1α pathway in renal hypoxia injury

S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-β1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and β-catenin while decreasing GSK-3β. HIF-1α inhibition restored HRD1 and β-catenin upregulation and GSK-3β downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α‘s transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3β/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.

Abstract PO5-24-08: Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53

Abstract Background: Cancer cells are constantly exposed to harsh conditions like nutrient deprivation, hypoxia, and calcium imbalance leading to the induction of UPR. In its early phase, UPR aims to restore homeostasis while prolonged stress leads to induction of apoptosis. Thus, it is not surprising to observe that expression of the UPR components has been reported to both promote as well as suppress tumorigenesis. Studies have shown that Cation transport regulator homolog 1 (CHAC1) plays a dual role in cancer progression. The objective of the present study is to investigate the p53 mediated regulation of CHAC1 in breast cancer. Methods: We initially subjected a breast cancer tissue microarray (TMA) to immunohistochemistry (IHC) with p53 and CHAC1 specific antibody. We further did in vitro experiments like western blotting, qRT-PCR to see the expression of CHAC1 when wild type (WT) p53 was either induced using camptothecin (CPT) and Nutlin-3 in WT p53 expressing breast cancer cells or by the ectopic WT and mutant p53 transfection in null p53 breast cancer cells (MDA-MB-157). The Chromatin immunoprecipitation (ChIP) assay was done to find a potential binding site of p53 on CHAC1 promoter. Further, in vitro study was done to elucidate a potential role of CHAC1 in cell proliferation. Results: The IHC staining analysis revealed that there was no significant difference in the CHAC1 expression between high and low p53 staining tissue cores as high CHAC1 staining was observed in both cases. Further, in vitro analysis reinforced the findings of CHAC1 in TMAs as cell line studies showed that both forms of p53 had enhancing effect on CHAC1 levels. Upon induction of WT p53 and/or ectopic transfection of WT and mutant p53, we observed that CHAC1 levels were upregulated in a dose dependent manner. ChIP assay revealed that while WT p53 occupies the promoter region of the CHAC1 gene to induce its expression, mutant p53 may be inducing CHAC1 expression through an unknown mediator. These observations provide the basis that CHAC1 may be a novel transcriptional target of p53. Ectopic transfection of WT and mutant 53 showed opposite effect on cellular proliferation as evident from BrdU cellular proliferation assay in MDA-MB-157 cells. Further, we showed that CHAC1 enhanced breast cancer cell proliferation possibly via upregulation of PI3K/Akt/mTOR pathway and may likely be a player in angiogenesis as shown by in vitro angiogenesis assay. Conclusion: Taken together, the data from present study suggests that the effect of CHAC1 on cellular proliferation was associated with the status of p53 in breast cancer cells. The results suggest a plausible explanation for the differential behavior of CHAC1 in tumorigenesis. Citation Format: Vikrant Mehta, Harish Chander. Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-08.

NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis

Breast cancer (BC) poses a great threat to women’s health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

GLYR1 transcriptionally regulates PER3 expression to promote the proliferation and migration of multiple myeloma

Period circadian regulator 3 (PER3) functions as a tumor suppressor in various cancers. However, the role of PER3 in multiple myeloma (MM) has not been reported yet. Through this study, we aimed to investigate the potential role of PER3 in MM and the underlying mechanisms. RT-qPCR and western blotting were used to determine the mRNA and protein expression levels of PER3. Glyoxylate reductase 1 homolog (GLYR1) was predicted to be a transcription factor of PER3. The binding sites of GLYR1 on the promoter region of PER3 were analyzed using UCSC and confirmed using luciferase and chromatin immunoprecipitation assays. Viability, apoptosis, and metathesis were determined using CCK-8, colony formation, TUNEL, and transwell assays. We found that PER3 expression decreased in MM. Low PER3 levels may predict poor survival rates; PER3 overexpression suppresses the viability and migration of MM cells and promotes apoptosis. Moreover, GLYR1 transcriptionally activates PER3, and the knockdown of PER3 alleviates the effects of GLYR1 and induces its malignant behavior in MM cells. To conclude, GLYR1 upregulates PER3 and suppresses the aggressive behavior of MM cells, suggesting that GLYR1/PER3 signaling may be a potential therapeutic target for MM.

YAP prevents senescence of dermal fibroblast and inhibits melanogenesis via paracrine effect of DKK1.

Senile skin hyperpigmentation displays remarkable histopathological features of dermal aging. The crosstalk between melanocytes and dermal fibroblasts plays crucial roles in aging-related pigmentation. While senescent fibroblasts can upregulate pro-melanogenic factors, the role of anti-melanogenic factors, such as dickkopf1 (DKK1), and the upstream regulatory mechanism during aging remain obscure. This study investigated the roles of yes-associated protein (YAP) and DKK1 in the regulation of dermal fibroblast senescence and melanogenesis. Our findings demonstrated decreased YAP activity and DKK1 levels in intrinsic and extrinsic senescent fibroblasts. YAP depletion induced fibroblast senescence and downregulated the expression and secretion of DKK1, whereas YAP overexpression partially reversed the effect. The transcriptional regulation of DKK1 by YAP was supported by dual-luciferase reporter and chromatin immunoprecipitation assays. Moreover, YAP depletion in fibroblasts upregulated Wnt/β-catenin in melanocytes and stimulated melanogenesis, which was partially rescued by the re-supplementation of DKK1. Conversely, overexpression of YAP in senescent fibroblasts decreased Wnt/β-catenin levels in melanocytes and inhibited melanogenesis. Additionally, reduced levels of YAP and DKK1 were verified in the dermis of solar lentigines. These findings suggest that, during skin aging, epidermal pigmentation may be influenced by YAP in the dermal microenvironment via the paracrine effect of DKK1.

BACH1 transcriptionally upregulates FOSL2 to induce M2 macrophages phenotype by activating TGFβ/SMAD signaling to promote the transformation of lung fibroblasts into myofibroblasts

Pulmonary fibrosis is a chronic and progressive lung disorder. The pro-fibrosis factors induced by M2 macrophage phenotype promote the differentiation of fibroblasts into myofibroblasts, which is essential for pulmonary fibrosis. We aimed to explore the role and mechanism of BTB domain and CNC homology 1 (BACH1) in pulmonary fibrosis. BACH1 was knocked down in THP‐1 polarized M2 macrophages with or without FOS-like antigen 2 (FOSL2) overexpression, the expression of M2 macrophage markers was detected. Cell viability, migration, invasion and extracellular matrix (ECM) accumulation were estimated by CCK-8, wound healing, transwell, western bot and immunofluorescence staining. Luciferase reporter and chromatin immunoprecipitation assays were used to verify the binding of BACH1 to FOSL2 promotor region. In vivo, a bleomycin (BLM)-induced pulmonary fibrosis mice model was established to evaluate the effect of BACH1 silencing on the histopathological changes, M2 macrophage phenotype and extracellular matrix (ECM) deposition. Expression of proteins was assessed with western blot. Results indicated that BACH1 expression was upregulated in M2 macrophages polarized from THP-1 cells. BACH1 deficiency inhibited the polarization of THP-1 to the M2 macrophage phenotype to promote the transformation of lung fibroblasts into myofibroblasts. Additionally, BACH1 could transcriptionally activate FOSL2 expression in THP-1-derived macrophages to upregulate TGFβ/SMAD signaling in HFL-1 cells. The animal experiments indicated that BACH1 knockdown alleviated BLM-induced pulmonary fibrosis, M2 macrophage polarization and inactivated FOSL2/TGFβ/SMAD signaling in mice lung tissues. Together, this finding suggests BACH1/FOSL2 may be useful therapeutic targets for the treatment of pulmonary fibrosis.

MIER2/PGC1A elicits sunitinib resistance via lipid metabolism in renal cell carcinoma

IntroductionRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system and accounts for more than 90 % of all renal tumors. Resistance to targeted therapy has emerged as a pivotal factor that contributes to the progressive deterioration of patients with advanced RCC. Metabolic reprogramming is a hallmark of tumorigenesis and progression, with an increasing body of evidence indicating that abnormal lipid metabolism plays a crucial role in the advancement of renal clear cell carcinoma. ObjectivesClarify the precise mechanisms underlying abnormal lipid metabolism and drug resistance. MethodsBioinformatics screening and analyses were performed to identify hub gene. qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assays, and other biological methods were used to explore and verify related pathways. Various cell line models and animal models were used to perform biological functional experiments. ResultsIn this study, we identified Mesoderm induction early response 2 (MIER2) as a novel biomarker for RCC, demonstrating its role in promoting malignancy and sunitinib resistance by influencing lipid metabolism in RCC. Mechanistically, MIER2 facilitated P53 deacetylation by binding to HDAC1. Acetylation modification augmented the stability and transcriptional function of P53, while deacetylation of P53 hindered the transcriptional process of PGC1A, leading to intracellular lipid accumulation in RCC. Furthermore, Trichostatin A (TSA), an inhibitor of HDAC1, was found to impede the MIER2/HDAC1/P53/PGC1A pathway, offering potential benefits for patients with sunitinib-resistant renal cell cancer. ConclusionOur findings highlight MIER2 as a key player in anchoring HDAC1 and inhibiting PGC1A expression through the deacetylation of P53, thereby inducing lipid accumulation in RCC and promoting drug resistance. Lipid-rich RCC cells compensate for energy production and sustain their own growth in a glycolysis-independent manner, evading the cytotoxic effects of targeted drugs and ultimately culminating in the development of drug resistance.