CDKN1A Expression Research Articles

Identification of immune-related ferroptosis prognostic marker and in-depth bioinformatics exploration of multi-omics mechanisms in thyroid cancer.

Background: Factors such as variations in thyroid carcinoma (THCA) gene characteristics could influence the clinical outcome. Ferroptosis and immunity have been verified to play an essential role in various cancers, and could affect the cancer patients’ prognosis. However, their relationship to the progression and prognosis of many types of THCA remains unclear. Methods: First, we extracted prognosis-related immune-related genes and ferroptosis-related genes from 2 databases for co-expression analysis to obtain prognosis-related differentially expressed immune-related ferroptosis genes (PR-DE-IRFeGs), and screened BID and CDKN2A for building a prognostic model. Subsequently, multiple validation methods were used to test the model’s performance and compare its performance with other 4 external models. Then, we explored the mechanism of immunity and ferroptosis in the occurrence, development and prognosis of THCA from the perspectives of anti-tumor immunity, CDKN2A-related competitive endogenous RNA regulatory, copy number variations and high frequency gene mutation. Finally, we evaluated this model’s clinical practice value. Results: BID and CDKN2A were identified as prognostic risk and protective factors, respectively. External data and qRT-PCR experiment also validated their differential expression. The model’s excellent performance has been repeatedly verified and outperformed other models. Risk scores were significantly associated with most immune cells/functions. Risk score/2 PR-DE-IRFeGs expression was strongly associated with BRAF/NRAS/HRAS mutation. Single copy number deletion of CDKN2A is associated with upregulation of CDKN2A expression and worse prognosis. The predicted regulatory network consisting of CYTOR, hsa-miRNA-873-5p and CDKN2A was shown to significantly affect prognosis. The model and corresponding nomogram have been shown to have excellent clinical practice value. Conclusion: The model can effectively predict the THCA patients’ prognosis and guide clinical treatment. Ferroptosis and immunity may be involved in the THCA’s progression through antitumor immunity and BRAF/NRAS/HRAS mutation. CYTOR-hsa-miRNA-873-5p-CDKN2A regulatory networks and single copy number deletion of CDKN2A may also affect THCA′ progression and prognosis.

Regulation of male germline transmission patterns by the Trp53-Cdkn1a pathway

SummaryA small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT+ differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern.

Profiling of gene expression in the brain associated with anxiety-related behaviors in the chronic phase following cranial irradiation

Although the brain is exposed to cranial irradiation in many clinical contexts, including malignant brain tumor therapy, such exposure can cause delayed neuropsychiatric disorders in the chronic phase. However, how specific molecular mechanisms are associated with irradiation-induced behavioral dysfunction, especially anxiety-like behaviors, is unclear. In the present study, we evaluated anxiety-like behaviors in adult C57BL/6 mice using the open-field (OF) and elevated plus maze (EPM) tests 3 months following single cranial irradiation (10 Gy). Additionally, by using RNA sequencing (RNA-seq), we analyzed gene expression profiles in the cortex and hippocampus of the adult brain to demonstrate the molecular mechanisms of radiation-induced brain dysfunction. In the OF and EPM tests, mice treated with radiation exhibited increased anxiety-like behaviors in the chronic phase. Gene expression analysis by RNA-seq revealed 89 and 106 differentially expressed genes in the cortex and hippocampus, respectively, following cranial irradiation. Subsequently, ClueGO and STRING analyses clustered these genes in pathways related to protein kinase activity, circadian behavior, and cell differentiation. Based on our expression analysis, we suggest that behavioral dysfunction following cranial irradiation is associated with altered expression of Cdkn1a, Ciart, Fos, Hspa5, Hspb1 and Klf10. These novel findings may provide potential genetic targets to investigate for the development of radioprotective agents.

Identification and Validation of Cuproptosis-Related Prognostic Signature and Associated Regulatory Axis in Uterine Corpus Endometrial Carcinoma.

Background: Uterine corpus endometrial carcinoma (UCEC) is a common gynecological malignancy globally with high recurrence and mortality rates. Cuproptosis is a new type of programmed cell death involved in tumor cell proliferation and growth, angiogenesis, and metastasis. Methods: The difference in cuproptosis-related genes (CRGs) between UCEC tissues and normal tissues deposited in The Cancer Genome Atlas database was calculated using the “limma” R package. LASSO Cox regression analysis was conducted to construct a prognostic cuproptosis–related signature. Kaplan–Meier analysis was conducted to compare the survival of UCEC patients. A ceRNA network was constructed to identify the lncRNA–miRNA–mRNA regulatory axis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to verify CRG expression in UCEC. Results: The expression of FDX1, LIAS, DLAT, and CDKN2A were upregulated, whereas the expression of LIPT1, DLD, PDHB, MTF1, and GLS were downregulated in UCEC versus normal tissues. The genetic mutation landscape of CRGs in UCEC was also summarized. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these CRGs were enriched in the tricarboxylic acid (TCA) cycle, glycolysis, and HIF-1 signaling pathway. LASSO Cox regression analysis was performed and identified a cuproptosis-related prognostic signature including these three prognostic biomarkers (CDKN2A, GLS, and LIPT1). UCEC patients with high risk scores had a poor prognosis with an area under the curve of 0.782 and 0.764 on 3- and 5-year receiver operating characteristic curves. Further analysis demonstrated a significant correlation between CDKN2A and pTNM stage, tumor grade, immune cell infiltration, drug sensitivity, tumor mutational burden (TMB) score, and microsatellite instable (MSI) score. The data validation of qRT-PCR further demonstrated the upregulation of CDKN2A and the downregulation of LIPT1 and GLS in UCEC versus normal tissues. The ceRNA network also identified lncRNA XIST/miR-125a-5p/CDKN2A regulatory axis for UCEC. Conclusion: The current study identified a cuproptosis-related prognostic signature including these three prognostic biomarkers (CDKN2A, GLS, and LIPT1) for UCEC. The ceRNA network also identified that lncRNA XIST/miR-125a-5p/CDKN2A regulatory axis may be involved in the progression of UCEC. Further in vivo and in vitro studies should be conducted to verify these results.

Clinicopathological and histological analysis of secondary malignant giant cell tumors of bone without radiotherapy.

Giant cell tumor of bone (GCTB) is an intermediate bone tumor that rarely undergoes malignant transformation. Secondary malignant GCTB (SMGCTB) is defined as a lesion in which high-grade sarcoma occurs at the site of previously treated GCTB. The present study retrospectively reviewed the medical records of patients with GCTB treated at Okayama University Hospital between April 1986 and April 2020. The clinicopathological and histological features of patients with SMGCTB without prior radiotherapy were investigated. A total of three patients (4%) with SMGCTB were detected, and the tumor sites were the distal ulna, distal femur and sacrum. Two of the patients had been treated with curettage and bone graft, and one had been treated with denosumab. In all cases, the lesions were made up of two components, the conventional GCTB component and the malignant component. The Ki67 labeling index was higher in the malignant components of SMGCTB and metastatic lesions compared with that in primary and recurrent conventional GCTB, or the conventional GCTB component of SMGCTB. Moreover, p53 expression was higher in these same components in patients who underwent curettage and bone grafting; however, there was no difference in the patient that received denosumab treatment. In this patient, clinical cancer genomic profiling revealed loss of CDKN2A, CDKN2B and MTAP expression. All three patients developed distant metastasis. The patients with SMGCTB in the ulna and femur died 13 and 54 months after detection of malignant transformation, respectively. The patient with SMGCTB in the sacrum received carbon-ion radiotherapy to the sacrum and pazopanib; the treatment was effective and the patient was alive at the last follow-up 3 years later. In conclusion, p53 may be associated with malignant transformation in GCTB. Future studies should investigate the association of between denosumab treatment and malignant transformation, as well as molecular targeted therapy to improve the clinical outcomes of SMGCTB.

Multi-platform-based characterization of ferroptosis in human colorectal cancer

SummaryFerroptosis is a type of programmed cell death potentially playing an important role in colorectal cancer (CRC) development. However, comprehensive investigations toward ferroptosis in human CRC are lacking. Here, we performed multiple investigations on cancer and para-cancer tissues. We demonstrated that the changes of structural variation and chromatin accessibility in CRC were more associated with the altered mRNA expression of ferroptosis-related genes (FRGs), and the expression of CDKN2A, GPX4, ALOXE3, and LINC00336 was related to the overall survival rates. Subsequently, we revealed that CYBB and YAP1 were potentially the hub genes, and that HSF1 and STAT2 were potentially FRGs’ upstream transcription factors. Finally, we depicted the crosstalk between ferroptosis and necrosis, autophagy, and apoptosis. Based on multi-dimensional analyses, we characterized ferroptosis, probable core genes, and the upstream regulators in human CRC. The findings here may improve our understanding of ferroptosis in CRC and provide new opportunities for clinical diagnosis and treatment.

INK4 cyclin-dependent kinase inhibitors as potential prognostic biomarkers and therapeutic targets in hepatocellular carcinoma.

The INK4 family is an important family of cyclin-dependent kinase inhibitors (CDKIs) and consists of CDKN2A, CDKN2B, CDKN2, and CDKN2D. Abnormal expression of CDKN2A has been reported in hepatocellular carcinoma (HCC) and is associated with the prognosis of patients and infiltration of immune cells. However, there is a lack of systematic research on the roles of the other INK4 family members in the diagnosis, prognosis, and immune regulation of HCC. Using online public databases and clinical samples, we comprehensively analyzed the INK4 family in HCC. All four INK4 proteins were overexpressed in HCC and correlated with advanced cancer stage and poor prognosis. INK4 expression accurately distinguished tumor from normal tissue, particularly CDKN2A and CDKN2C. The INK4 family participated in cell-cycle regulation and the DNA damage repair pathway, which inhibited genotoxic-induced apoptosis in tumorigenesis. INK4 proteins were positively correlated with the infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) and immune checkpoints (CTLA-4, PD1, and PD-L1). CDKN2D had the highest correlation (correlation coefficient >0.3) with all the above-mentioned infiltrating immune cells and immune checkpoints, indicating that it may be useful as an immunotherapy target. The INK4 family was valuable for diagnosis and predicting the prognosis of HCC and participated in the occurrence, progression, and immune regulation of HCC, demonstrating its potential as a diagnostic and prognostic biomarker and therapeutic target in HCC.

Bexarotene-induced cell death in ovarian cancer cells through Caspase-4-gasdermin E mediated pyroptosis

Bexarotene selectively activates retinoid X receptor, which is a commonly used anticancer agent for cutaneous T-cell lymphoma. In this study, we aimed to investigate the anticancer effect of bexarotene and its underlying mechanism in ovarian cancer in vitro. The ES2 and NIH:OVACAR3 ovarian cancer cell lines were treated with 0, 5, 10, or 20 µM of bexarotene. After 24 h, cell number measurement and lactate dehydrogenase (LDH) cytotoxicity assay were performed. The effect of bexarotene on CDKN1A expression, cell cycle-related protein, cell cycle, pyroptosis, and apoptosis was evaluated. Bexarotene reduced cell proliferation in all concentrations in both the cells. At concentrations of > 10 µM, extracellular LDH activity increased with cell rupture. Treatment using 10 µM of bexarotene increased CDKN1A mRNA levels, decreased cell cycle-related protein expression, and increased the sub-G1 cell population in both cells. In ES2 cells, caspase-4 and GSDME were activated, whereas caspase-3 was not, indicating that bexarotene-induced cell death might be pyroptosis. A clinical setting concentration of bexarotene induced cell death through caspase-4–mediated pyroptosis in ovarian cancer cell lines. Thus, bexarotene may serve as a novel therapeutic agent for ovarian cancer.

A malignant prognostic indicator of Uterine Corpus Endometrial Carcinoma: CDKN2A

Mounting evidence have proved that the expression level of CHKN2A has a certain influence on the immune infiltration of tumor and the prognosis of cancer patients. Nevertheless, the association between CDKN2A and Uterine Corpus Endometrial Carcinoma (UCEC) still remains unclear. Therefore, the aim of this study is to investigate the effects of CDKN2A on the prognosis of UCEC patients and its relationship with immune infiltration. ONCOMINE, GEPIA 2, Kaplan-Meier Plotter, TIMER2.0, Metascape, STRING and cBioPortal were used in this study to explore the influence of CDKN2A expression on UCEC. Compared to adjacent normal tissue, we demonstrated that high CDKN2A expression was associated with poor survival of UCEC patients (OS:HR=2.9, Logrank p=0.0055; DFS:HR=2.3, Log rank p=0.016). Meanwhile, significant correlation between CDKN2A expression and immune infiltration was observed in UCEC tumor tissue, such as CD8+ T cells (R=-0.431, P=2.72E-05) and Monocyte (R=0.295, P=5.20E-03) etc. In addition, the enrichment analysis results showed that CDKN2A and its co-expressed genes were involved in the pathways of RNA metabolism, positive regulation of cell cycle process and TP53 activity. The main gene alteration of CDKN2A in UCEC patients was amplification, and the mutation rate was only 3%. In conclusion, CDKN2A is expected to become a novel prognostic indicator and a potential drug target for UCEC.

Abstract 1831: 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR

Abstract Purpose: The study aims to determine the activity of the blood brain barrier permeable glycogen synthase kinase 3B (GSK3B) inhibitor, 9-ING-41, in pediatric brain tumors: embryonal tumor with multilayered rosettes (ETMR) and atypical rhabdoid tumor (ATRT). Methods: ETMR cell line, BT183, was grown in ultralow attachment flasks and ATRT cell lines: ATRT-7316-2187, ATRT-787197, and CHLA02 were grown in suspension flasks. Cells were treated with 9-ING-41 at dose range (0-20µM). After 24 and 48 hours of 9-ING-41 cells were analyzed by western blot. After 72 hours of 9-ING-41 cell viability was assessed with Cell Titer Glo 2.0 and IC50s were analyzed by GraphPad. RNA seq was performed on cells after 12, 24, and 48 hours of 9-ING-41 treatment. Total RNA was isolated with miRNeasy Mini Kit. RNA integrity was evaluated by an Agilent 2100 Bioanalyzer profile. rRNA was selectively depleted by employing RiboZero Globin. Next generation sequencing libraries were prepared using an Illumina TruSeq Stranded Total RNA kits. Read mapping was performed using STAR in BaseSpace. DESeq2 was used for normalization and differential expression analysis. Ingenuity Pathway Analysis was performed. Results: The IC50s of 9-ING-41 in the cell lines BT183, CHLA02-ATRT, ATRT-787197, and ATRT-2187 are 145.5nM, 481.2nM, 503.1nM, and 528.3nM, respectively. In BT183 cells treated with 150nM 9-ING-41 for 48 hours, XIAP decreased significantly by 55% and cleaved caspase 3 increased significantly by 29-fold, quantified by densitometry. In addition, western blot showed a decrease in LIN28A and an increase in TP53 protein levels. RNA-seq data show an increase in the p53 signaling pathway in the ETMR BT183 cell line with z-scores of 2.53 and 3 (p=3.73x10-13 and 2.35x10-14) after 12 and 24 hours of 150nM 9-ING-41, respectively. In ATRT-787197 cells p53 signaling increased with z-scores of 1.0 and 2.236 (p=2.62x10-8 and 2.89x10-5) after 24 and 48 hours of 400nM 9-ING-41, respectively. RNA seq data also show the ability of 9-ING-41 to significantly increase the expression of CDK inhibitor, CDKN1A, and pro-apoptotic genes BBC3, DRAM1, and BAX in ETMR and ATRT while significantly decreasing stemness associated genes: SMO and GLI1 in ETMR. Conclusions: 9-ING-41 decreases ETMR and ATRT cell viability at clinically relevant concentrations and these doses increase the p53 pathway and induce apoptosis while reducing the expression of stemness associated genes. Citation Format: Jason L. Haw, Elizabeth M. Moore, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Nury Steuerwald, Giselle S. Sholler. 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1831.

Abstract 3311: Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model

Abstract Androgen receptor (AR) inhibitors are standard of care for the treatment of advanced prostate cancer. Despite an initial response to treatment, patients eventually progress, and novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is an integral membrane glycoprotein that is highly expressed in prostate cancer but has limited expression in normal tissues. PSMA-TTC (227Th-pelgifatamab corixetan; BAY 2315497) consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody. PSMA-TTC delivers potent radiation to PSMA expressing cells. Here we evaluated the efficacy of PSMA-TTC in combination with darolutamide, a novel AR inhibitor in a hormone insensitive prostate cancer model and investigated the mode of action of the combination treatment. In vitro, darolutamide induced the expression of PSMA in the androgen-independent cell lines C4-2 and 22Rv1 more than 3-fold and 2-fold, respectively. Expression of the apoptosis marker CDKN1A was significantly induced in the PSMA-TTC alone and in the combination group compared to vehicle. Expression of the DNA repair genes BRCA1, XRCC2 and XRCC3 were significantly reduced in PSMA-TTC alone and in the combination group compared to vehicle. In vivo, a single i.v. injection of PSMA-TTC at 300 kBq/kg resulted in a tumor/control (T/C) ratio of 0.44 on day 19 in the 22Rv1 hormone-insensitive prostate cancer model while a twice daily oral treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.82. The combination of darolutamide and PSMA-TTC resulted in a higher efficacy with a T/C ratio of 0.27. Tumor area on day 19 after treatment was significantly reduced in the PSMA-TTC alone group and more so in the combination group, compared to vehicle. As expected, darolutamide alone was not efficacious in this hormone insensitive model. Nine out of 10 mice showed disease control (CR, PR or SD) in the combination group whereas no animals showed disease control in either of the monotherapy groups. This difference was significant by Fisher’s exact test analysis. Treatments were well tolerated with no significant changes in body weight in any of the treatment groups. Using the tumor dissociation kit Miltenyi individual tumor cells were isolated from darolutamide treated and untreated 22Rv1 xenograft tumors. FACS analysis showed a 50fold increase in PSMA expression in the darolutamide treated 22Rv1 tumors compared to vehicle treated animals. These data indicate that darolutamide induces PSMA expression in the hormone independent 22Rv1 model, which may have contributed to the stronger efficacy observed when combined with PSMA-TTC. Altogether these results support the further evaluation of darolutamide combination with PSMA radionuclides. Citation Format: Christoph Schatz, Urs Hagemann, Sabine Zitzmann-Kolbe, Bernard Haendler, Hartwig Hennekes, Stefanie Hammer, Arne Scholz. Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3311.

Abstract 2967: ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers (>800,000 new cases each year worldwide) and a leading cause of cancer mortality (~700,000 deaths per year globally), with an average 5-year survival rate of 20~30%. The prevalence of HCC and the unmet clinical needs emphasize the urge for novel regimens for these malignancies. In this study, we performed an epigenetics-focused CRISPR interference (CRISPRi) library screen in the HepG2-dCas9-Krab cells (a total of 3,669 sgRNAs targeting 728 chromatin regulator genes at their transcription start sites) and identified ACTR5 as a novel essential gene in HCC. CRISPR depletion of ACTR5 led to arrested cell cycle and attenuated HCC tumor progression in vivo. Transcriptomic analysis revealed a significantly increased CDKN2A expression in the sgACTR5 transduced HCC cells, which led to the activation of Rb and repression of CDK6 and E2F signaling. Meanwhile, histone modification profiling identified loss of H3K9me2 and Hek9me3 at the CDKN2A promoters upon ACTR5 depletion, indicating the involvement of the epigenetic silencing of tumor suppressors (including CDKN2A) underlying the ACTR5-driven HCC progression. Mechanistically, we found that CRISPR targeting the ACTR5’s interacting protein IES6 (also known as INO80C) recapitulated the sgACTR5 phenotypes, highlighting the collaborative roles between these two partners. We then conducted a high-density CRISPR gene tiling scan using a sgRNA library that targets every NGG protospacer adjacent motif (PAM) within the ACTR5 (284 sgRNAs) and IES6 (90 sgRNAs) coding exons. This saturating mutagenesis screen revealed the requirement of 2 regions (G502-S519 and I552-C569) within ACTR5’s 2nd Actin-fold domain for IES6 interaction. Reciprocally, our unbiased screen identified a previously uncharacterized domain (T163-V187) in IES6’s C-terminal region to mediate the ACTR5-IES6 complex formation. To investigate the clinical impact of ACTR5, we examined the response of HCC cells to various inhibitors upon ACTR5 depletion. While sgACTR5 did not affect the cellular response to sorafenib (an FDA-approved RAF and VEGF inhibitor for HCC patients), we found that suppression of ACTR5 sensitized the HCC cells to ribociclib (a CDK6 inhibitor) and UNC0642 (an H3K9 methyltransferase G9a and GLP inhibitor). Based on these observations, we rationalized a combination treatment of ribociclib/UNC0642 that synergistically suppresses the proliferation of the HCC cells. In summary, our study revealed a pivotal role of ACTR5 (and its complex member IES6) in supporting the CDK6-driven cell cycle progression via an H3K9me2 H3K9me3-mediated silencing of CDKN2A. These notions can be exploited to develop novel combinational therapies against the difficult to cure liver cancers. Citation Format: Xiaobao Xu, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Mingli Li, Qiao Liu, Nicole Mattson, Chun-wei Chen. ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2967.

Exploring and Verifying the Mechanism and Targets of Shenqi Pill in the Treatment of Nonalcoholic Steatohepatitis via Network Pharmacology and Experiments.

Shenqi pill (SQP), a famous traditional Chinese medicine (TCM) herbal formula derived from Jinguiyaolue (Synopsis of Prescriptions of the Golden Chamber), has long been used to treat kidney yang deficiency syndrome. According to the TCM treatment principle that the liver and kidney are homologies, the clinical use of SQP in the treatment of nonalcoholic steatohepatitis (NASH) has achieved a good effect. However, the active targeted genes and underlying mechanism remain unclear. In this study, we aimed to explore the treatment mechanism of SQP in NASH rats, which may further contribute to the in-depth exploration of SQP in clinical applications. Network pharmacology analysis was used to screen the target genes of SQP for NASH treatment based on public databases. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein–protein interaction (PPI) analysis were used to search for crucial target genes and mechanisms. UPLC–MS/MS was used to verify the active compounds of the SQP screened. The hepatic pathology and biochemical indicators of rats were used to judge the modeling results and the curative effect of SQP. Western blotting and qRT–PCR were used to verify the expression of crucial target genes at the protein and RNA levels, respectively. Network pharmacology analysis and bioinformatics analysis showed that PTGS2, JUN, MYC, and CDKN1A might be crucial target genes in the primary mechanism of SQP in treating NASH and improving the inflammatory response. The UPLC–MS/MS results confirmed that the hub active compound, quercetin, screened out through the TCMSP database, is indeed present in SQP. Hepatic injury and lipid metabolism indicators of NASH rats were significantly improved after SQP treatment. The results of WB and qRT–PCR showed that the expression of PTGS2, JUN, MYC, and CDKN1A was higher in NASH rats than in normal rats and decreased after SQP treatment. The expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) was reduced after SQP treatment, which confirmed that SQP could improve hepatic inflammation in rats. These results suggested that SQP could ameliorate NASH in rats, and that quercetin may be the critical active compound that exerts the therapeutic effect.

Cytosolic Nuclear Sensor Dhx9 Controls Medullary Thymic Epithelial Cell Differentiation by p53-Mediated Pathways.

Thymic epithelial cells (TECs) critically participate in T cell maturation and selection for the establishment of immunity to foreign antigens and immune tolerance to self-antigens of T cells. It is well known that many intracellular and extracellular molecules elegantly have mastered the development of medullary TECs (mTECs) and cortical TECs (cTECs). However, the role played by NTP-dependent helicase proteins in TEC development is currently unclear. Herein, we created mice with a TEC-specific DExD/H-box helicase 9 (Dhx9) deletion (Dhx9 cKO) to study the involvement of Dhx9 in TEC differentiation and function. We found that a Dhx9 deficiency in TECs caused a significant decreased cell number of TECs, including mTECs and thymic tuft cells, accompanied by accelerated mTEC maturation but no detectable effect on cTECs. Dhx9-deleted mTECs transcriptionally expressed poor tissue-restricted antigen profiles compared with WT mTECs. Importantly, Dhx9 cKO mice displayed an impaired thymopoiesis, poor thymic T cell output, and they suffered from spontaneous autoimmune disorders. RNA-seq analysis showed that the Dhx9 deficiency caused an upregulated DNA damage response pathway and Gadd45, Cdkn1a, Cdc25, Wee1, and Myt1 expression to induce cell cycle arrest in mTECs. In contrast, the p53-dependent upregulated RANK-NF-κB pathway axis accelerated the maturation of mTECs. Our results collectively indicated that Dhx9, a cytosolic nuclear sensor recognizing viral DNA or RNA, played an important role in mTEC development and function in mice.

Phase II trial of palbociclib in advanced sarcoma overexpressing CDK4 gene excluding dedifferentiated liposarcoma (DD LPS): A study from the Spanish Group for Research on Sarcoma (GEIS).

11511 Background: CDK4/6 inhibitors showed a favorable progression-free survival (PFS) in DD LPS, a sarcoma bearing 12q 13-15 amplicon that implies CDK4 amplification. The median PFS was 4 and 7 months (m) for palbociclib and abemaciclib, respectively. Preclinical experiments in 10 sarcoma cell lines and 6 PDX models, including only one DD LPS, showed higher efficacy of anti-CDK4 in cases with high expression of CDK4 and low expression of p16. This rationale supported the design of a phase II trial exploring palbociclib in a wide range of sarcomas, excluding DD LPS. Methods: Progressing pretreated advanced soft tissue sarcoma, excluding DD LPS, or osteosarcoma adult patients (pts), whose tumors overexpressed CDK4 and underexpressed CDKN2A mRNA in a baseline mandatory biopsy, were enrolled. CDK4 and CDKN2A expression were assessed by qRT-PCR, using an external control as reference (Universal human reference RNA; Agilent Technologies). The primary endpoint was 6-m PFS rate. Minimax Simon’s two-stage with type 1 and 2 errors of 10%, and null and alternative hypothesis of H0 15%, H1 40%, 6-month PFS rates were specified. The study will warrant further investigation if 6 or more pts had a PFS > 6 m from 21 evaluable pts. Palbociclib was administered orally at 125 mg/ day 21 out of 28 days. Pre-screening intended to increase the probability of positive profile in the baseline biopsy. Results: A total of 214 pts with 236 CDK4/ CDKN2A determinations were assessed for enrolment; 141 for prescreening, in archive tumor sample, and 95 for screening, in a baseline biopsy. There were 38/141 (27%) and 28/95 (29%) pts with favorable mRNA profile from pre and screening, respectively. Twenty-two pts were enrolled with a median of previous systemic lines of 3 (1-5). There were 9 different sarcoma subtypes, including 2 osteosarcomas. With a median FU of 10 m (0.4-23.3), the median PFS was 4.2 m (95% CI 0.9-7.4), while the 6- and 12-m PFS rates were 30% (95% CI 9-51) and 18% (95% CI 12-48) respectively. From 19 evaluable pts (1 early death by COVID, 1 withdrew consent and for 1 it was too early to be assessed) 11 had stable disease (58%) and 8 progressed (42%) as the best response. Patients with CDK4 expression above the median value had significantly longer mPFS in the univariate analysis: 5.9 m (95% CI 1.4-10.4) vs 1.9 m (95% CI 0.6-3.2), p = 0.046; and longer OS: 15.5 m (95% CI 6.8-24.3) vs 10.6 m (95% CI 0-23.2), p = 0.047, respectively. The probability to find a positive profile in the screening was 29%, but this proportion increased up to 41% if in pre-screening had been positive. Conclusions: Palbociclib showed to be effective in a wide variety of sarcoma subtypes, other than DD LPS, selected by CDK4/CDKN2A biomarkers. Clinical trial information: NCT03242382.

Potential Prognostic Markers for Relapsed/Refractory vs. Responsive Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is the most common blood cancer in the elderly, which progresses rapidly and is often fatal. The prognosis for AML remains poor in most older patients: only about 15% of patients over 60 years of age can recover. Our aim is to determine new potential AML clinical treatment prognosis markers. We analyzed certain genes, proteins and the epigenome profile in therapy-resistant and responsive AML patients at diagnosis stage and after clinical treatment. We determined that MYC, WT1, IDH1, CDKN1A, HDAC2, TET1, KAT6A and GATAD2A gene expression changes might characterize refractory AML. Therefore, these genes could have an impact for AML prognosis.Acute myeloid leukemia (AML) is a heterogeneous disease. A significant proportion of AML patients is refractory to clinical treatment or relapses. Our aim is to determine new potential AML clinical treatment prognosis markers. We investigated various cell fate and epigenetic regulation important gene level differences between refractory and responsive AML patient groups at diagnosis stage and after clinical treatment using RT-qPCR. We demonstrated that oncogenic MYC and WT1 and metabolic IDH1 gene expression was significantly higher and cell cycle inhibitor CDKN1A (p21) gene expression was significantly lower in refractory patients’ bone marrow cells compared to treatment responsive patients both at diagnosis and after clinical treatment. Moreover, we determined that, compared to clinical treatment responsive patients, refractory patients possess a significantly higher gene expression of histone deacetylase 2 (HDAC2) and epigenetic DNA modulator TET1 and a significantly lower gene expression of lysine acetyltransferase 6A (KAT6A) and nucleosome remodeling and deacetylase (NuRD) complex component GATAD2A. We suggest that MYC, WT1, IDH1, CDKN1A, HDAC2, TET1, KAT6A and GATAD2A gene expression changes might characterize refractory AML. Thus, they might be useful for AML prognosis. Additionally, we suggest that epigenetic modulation might be beneficial in combination with standard treatment.

Yaobishu Regulates Inflammatory, Metabolic, Autophagic, and Apoptosis Pathways to Attenuate Lumbar Disc Herniation.

Objective Here, we aimed to explore the main mechanism of Yaobishu (YBS) in lumbar disc herniation (LDH). Methods and Results Eighteen compounds that might act on LDH were obtained through a combination of network pharmacology prediction and identification by high-performance liquid chromatography-mass spectrometry. The key compounds were palmitic acid and trans-4-hydroxy-3-methoxycinnamate (cinnamate). KEGG analysis demonstrated that palmitic acid target genes mainly regulate the PPAR signaling pathway, Ras signaling pathway, and fatty acid metabolism. Cinnamate target genes were primarily involved in chemical carcinogenesis-receptor activation, lipid and atherosclerosis, the HIF-1 signaling pathway, and nitrogen metabolism. The rat LDH model was constructed using autologous nucleus pulposus tissue implantation. Differential expression gene (DEGs) related to metabolism (CDKN1A and UHRF1), inflammation (S100A9 and SOCS3), autophagy (DCN and LEPR), and apoptosis (CTSW and BCL2A1) in dorsal root ganglion (DRG) tissues of the control and LDH groups was evaluated by RNA-Seq. TNF-α stimulated DRG neuronal cells were used to establish an in vitro LDH model. YBS, palmitic acid, and cinnamate reduced the expression of substance P, CGRP, S100A9, CTSW, and cleaved caspase-3, while enhancing the expression of CDKN1A, UHRF1, PCNA, Ki67, SOCS3, DCN, LEPR, and BCL2A1, as well as telomerase activity. Pearson's correlation analysis confirmed that DCN was positively correlated with BCL2A1, indicating that autophagy might be negatively correlated with apoptosis in LDH. YBS, palmitic acid, and cinnamate reduced the Siegal neurological score and serum IL-1β and IL-18 levels, while increasing changes in the hind paw mechanical withdrawal threshold. The RNA-Seq results further showed that YBS downregulated S100A9 and CTSW expression, while upregulating SOCS3, CDKN1A, UHRF1, DCN, LEPR, and BCL2A1 expression. Conclusion YBS and its compounds, palmitic acid, and cinnamate, attenuated LDH by regulating the inflammatory, metabolic, autophagic, and apoptotic pathways. Our results might improve the theoretical and experimental basis for clinical applications of LDH disease treatment.

In Vitro Study on the Biological Characteristics of Adipose-Derived Stem Cells from Liposuction Area in Patients with Progressive Hemifacial Atrophy.

In this study, we investigate the biological characteristics of ADSCs from the liposuction area in patients with hemifacial atrophy in vitro. ADSCs were respectively extracted from the donor site of patients with hemifacial atrophy and healthy ones. ADSCs of two groups were respectively tested for proliferation ability, phenotype, multipotency, migration ability, self-repair ability, apoptosis, and autophagy. Exosomes extracted from the supernatant of two groups were detected by NTA particle size, electron microscopy (TEM), and WB for CD63 and TSG10, respectively. CCK-8 showed a statistically less increase in cell proliferation in PHA-ADSCs after the sixth day. ADSCs in both groups had typical phenotypes and multidirectional abilities. PHA-ADSCs exhibited weaker droplet formation. The cell migration ability in PHA-ADSCs was weaker tested by Transwell assay. The live/dead proportion calculated by ImageJ following calcein-AM/PI double staining revealed live cells in PHA-ADSCs was 46.11% compared with 54.21% in NORM-ADSCs after OGD treatment. A significant down-regulation of ATG7 and ATG12 and a higher percentage of apoptosis were found in PHA-ADSCs. A significant up-regulation of BAX occurred in PHA-ADSCs.ARPC5 expression in the PHA group was extremely distinct down-regulated.CDKN1A and CDKN2A expression in the PHA group was significantly up-regulated.WB analyses confirmed that both groups' ADSCs-Exosomes surface markers CD63 and TSG101 were positively expressed but varied significantly. PHA-ADSCs exhibited a poorer proliferation ability, higher apoptosis percentage, weaker lipid droplets formation, weaker cell migration, poorer intolerance to OGD, aging earlier, and weaker self-renewal and repairability.PHA-ADSCs-Exosomes showed low expressions of CD63 and TSG101.This study provides strong evidence that the addition of exosomes with specific cytokines can improve the fat survival rate after fat filling in patients with hemifacial atrophy. This journal requires that authors 42 assign a level of evidence to each submission to which 43 Evidence-Based Medicine rankings are applicable. This 44 excludes Review Articles, Book Reviews, and manuscripts 45 that concern Basic Science, Animal Studies, Cadaver 46 Studies, and Experimental Studies. For a full description of 47 these Evidence-Based Medicine ratings, please refer to the 48 Table of Contents or the online Instructions to Authors 49 https://www.springer.com/00266 .

A Novel Cuproptosis-Related Prognostic Gene Signature and Validation of Differential Expression in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma, which is characterized by metabolic reprogramming. Cuproptosis, a novel form of cell death, is highly linked to mitochondrial metabolism and mediated by protein lipoylation. However, the clinical impacts of cuproptosis-related genes (CRGs) in ccRCC largely remain unclear. In the current study, we systematically evaluated the genetic alterations of cuproptosis-related genes in ccRCC. Our results revealed that CDKN2A, DLAT, DLD, FDX1, GLS, PDHA1 and PDHB exhibited differential expression between ccRCC and normal tissues (|log2(fold change)| > 2/3 and p < 0.05). Utilizing an iterative sure independence screening (SIS) method, we separately constructed the prognostic signature of CRGs for predicting the overall survival (OS) and progression-free survival (PFS) in ccRCC patients. The prognostic score of CRGs yielded an area under the curve (AUC) of 0.658 and 0.682 for the prediction of 5-year OS and PFS, respectively. In the Kaplan−Meier survival analysis of OS, a higher risk score of cuproptosis-related gene signature was significantly correlated with worse overall survival (HR = 2.72 (2.01–3.68), log-rank p = 1.76 × 10−7). Patients with a higher risk had a significantly shorter PFS (HR = 2.83 (2.08–3.85), log-rank p = 3.66 × 10−7). Two independent validation datasets (GSE40435 (N = 101), GSE53757 (N = 72)) were collected for meta-analysis, suggesting that CDKN2A (log2(fold change) = 1.46, 95%CI: 1.75–2.35) showed significantly higher expression in ccRCC tissues while DLAT (log2(fold change) = −0.54, 95%CI: −0.93–−0.15) and FDX1 (log2(fold change) = −1.01, 95%CI: −1.61–−0.42) were lowly expressed. The expression of CDKN2A and FDX1 in ccRCC was also significantly associated with immune infiltration levels and programmed cell death protein 1 (PD-1) expression (CDKN2A: r = 0.24, p = 2.14 × 10−8; FDX1: r = −0.17, p = 1.37 × 10−4). In conclusion, the cuproptosis-related gene signature could serve as a potential prognostic predictor for ccRCC patients and may offer novel insights into the cancer treatment.

Marine toxin (+)‐chaetocin‐induced apoptosis of lung large cell carcinoma cell lines through cell cycle arrest via CDKN1A expression and replicative stress

AbstractLung large cell carcinoma is a common type of lung cancer with poor prognosis. Although targeted drugs have achieved enormous success in treating non‐small‐cell lung carcinoma (NSCLC), new chemotherapy is needed since the emerged drug resistance always hinders curative effects. The fungal toxin (+)‐chaetocin demonstrated strong antineoplastic activities against the tested lung large cell carcinoma cell lines H460 and H661 at submicromolar concentrations. Further research demonstrated that (+)‐chaetocin effectively induced H460 apoptosis at 10–30 nM concentrations, while cell death occurred at 60 nM concentrations due to DNA duplication errors. Cell cycle and transcriptional analyses proved that cell cycle arrest via CDKN1A expression and the comprehensive replicative stress of (+)‐chaetocin are key factors in the (+)‐chaetocin working mechanisms.