CDKN1A Expression Research Articles

Crosstalk of cuproptosis-related prognostic signature and competing endogenous RNAs regulation in hepatocellular carcinoma.

Cuproptosis is a new type of programmed cell death involved in the regulation of neuroendocrine tumors, immune microenvironment, and substance metabolism. However, the role of cuproptosis-related genes (CRGs) in Hepatocellular carcinoma (HCC) remains unclear. Through multiple bioinformatics analysis, we constructed a prognostic gene model and competing endogenous RNA (ceRNA) network. The correlation between CRGs and prognosis, immune infiltration, immune checkpoints, microsatellite instability (MSI) and tumor mutational burden (TMB) was analyzed by Kaplan-Meier curve, univariate Cox, multivariate regression, and Spearman's analysis in HCC patients. Besides, the qRT-PCR and immunohistochemistry assays were used to determine prognostic CRGs mRNA and protein expression in HCC. We established a novel 3-gene signature related to CRGs for evaluating the prognosis of HCC patients. HCC patients with high risk scores had a poor prognosis with an area under the curve of 0.737, 0.646, and 0.634 on 1-year, 3-year, and 5-year receiver operating characteristic curves. Significant correlation was observed between prognostic CRGs and immune infiltration, immune checkpoints, MSI and TMB. We also developed five ceRNA networks to regulate the occurrence and progression of HCC. CDKN2A, DLAT, and PDHA1 protein expression was up-regulated in HCC versus normal tissues. Besides, the mRNA expression levels of CDKN2A, DLAT, GLS, and PDHA1 were elevated in the HCC cell lines compared to the normal liver cell lines. This novel prognostic CRGs signature could be accurately predict the prognosis of patients with HCC. The ceRNA regulatory network might be potential prognostic biomarkers and therapeutic targets for HCC patients.

Expression of the CDKN1A, MDM2, and ATM genes as a biomarker of the toxic effect of heavy metals (literature review)

The development of new approaches enabling differentiation of a wide range of toxic effects can significantly improve risk assessment. To understand the response mechanisms at the molecular level, it is important to study the expression of genes responsible for DNA repair, since this process is one of the early responses to toxic effects.
 The purpose of the study was to summarize available data on the expression of repair CDKN1A, MDM2, and ATM genes associated with toxic effects of exposure to heavy metals.
 A systematic search was carried out to identify studies on a given topic in the PubMed, Web of Science, eLIBRARY and Google Scholar electronic databases using the following keywords: heavy metals, CDKN1A, MDM2, ATM, toxicity, DNA repair, and gene expression. The initial search for scientific publications was carried out independently by three authors; then all sources found were checked and compared to filter out duplicate papers. This review covers 50 literature sources.
 The analysis of toxicogenome studies allowed us to identify several genes for assessing heavy metal toxicity among a large number of candidate biomarkers. The most commonly considered genes are the p21/CDKN1A gene, the MDM2 proto-oncogene, and the ATM gene.
 Limitations. The review is limited to considering changes in the expression of only a small number of genes responsible for DNA repair.
 Conclusion. The expression of the above biomarker genes provides a detailed picture of the response of a biological system to hazardous exposures and can be used as part of the assessment of toxic effects.

Comparative Transcriptome Analyses in Mantle Cell Lymphoma (MCL): Evidence of E2F1 As a Major Target in Aggressive Blastoid MCL Model

Background Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma characterized by the translocation t(11;14)(q13;q32) which triggers the dysregulation of the cyclin D1 by placing its coding gene CC1ND1 under the control of an immunoglobulin promoter. One of the major consequences of this molecular event is the disturbance of the cyclin D/Rb pathway leading to Rb inactivation and activation the elongation factor E2F1 mainly implicated in cell cycle progression. The mainstay of MCL therapies remains the chemo-immunotherapy followed by autologous transplantation but recently several novel therapies have been developed such as BTK inhibitors, Venetoclax, CDK4/6 inhibitors and more recently CAR-T therapies. However, patients with aggressive forms of MCL exhibit low response rates, requiring the identification of novel therapeutic targets. Materials and Methods We have previously generated an MCL cell line (UPN-1) from a patient with a blastoid variant of MCL presenting unique morphological and cytogenetics characteristics (M'kacher et al, Oncogene 2003). This cell line has been shown to harbor a TP53 mutation and a highly increased sensitivity to radiation despite the absence of functional p53. The molecular basis of this phenomenon has not been explored. In this work, we further characterized UPN-1 cell line using the Clarium S human microarray which has been compared to the gene expression profile of other existing MCL cell lines (GRANTA-519, JEKO-1, JVM-2, MAVEK-1, MINO, REC-1, SP-49, SP-53, Z-138). After mathematical correction, a supervised analysis was performed between triplicate samples of UPN-1 and a group constituted with all other MCL cell lines. We have then set up in vitro assays to determine the effect of a direct E2F1 inhibitor on the proliferative potential of UPN-1. Results The comparative transcriptome analyses revealed a list of 47 genes specifically upregulated in UPN-1. Unsupervised classification allowed to discriminate UPN-1 samples from other MCL cell lines based on the expression of these 47 genes. These data suggested a specific expression profile of UPN-1 as compared to other cell lines. Among the upregulated genes, E2F1 elongation factor was found to be highly upregulated in UPN-1 as compared to other MCL cell lines along with positive enrichment of E2F targets which are mainly implicated in cell cycle progression (Figure 1). Interestingly, UPN-1 has a low level of BCL-2 as compared to other MCL lines that could explain its radio sensitivity. However, other signaling pathways involved in the inhibition of apoptosis were found to be present in UPN-1 by geneset enrichment analysis. The expression of CDKN1A (p21), major cell cycle regulator and known as a marker of aggressive MCL, was found to be low in UPN-1. A repression of immune response pathway such as NFKB with low expression of NFKBIA was identified. Finally, the expression of PIK3CD which encodes for a therapeutic target in MCL (such as PI3KDelta inhibitor Idelalisib) was also significantly repressed in UPN-1 as compared to nine other MCL cell lines. To evaluate the effect of the direct inhibition of E2F1, we treated UPN-1 cells with a targeted E2F1 inhibitor 5‘-Deoxy-5‘-(methylthio)adenosine (MTA) which is a protein methylation inhibitor. MTT assays revealed a 50% reduction of proliferation at + 4 hours in comparison to the vehicle-treated conditions (P<0.0001).Annexin assays showed an increase in early apoptosis (36.6 %) at + 8 hours with subsequent rise in late apoptotic cells (64.5 %) observed after 24 hours of treatment. Interestingly, qRT-PCR analyses of MTA-treated cells exhibited a decrease in the expression of downstream factors associated with E2F1, such as RAD51, BRCA1, CDK2, CCNB2, RANBP1. Conclusions We report here for the first time the comparative transcriptome analysis of the unique aggressive blastoid MCL line UPN-1 as compared to other MCL lines described. This cell line exhibited a major upregulation of E2F1 expression and a down regulation of the cell cycle inhibitor CDKN1A(p21). The potential effects of the direct inhibition of E2F1 using the targeted inhibitor MTA demonstrated a reduction in cell proliferation and induction of apoptosis, suggesting the possibility of associating clinically acceptable E2F1 inhibition strategies to other current therapies.

The PP2A-B56α Heterocomplex Regulates Response to Venetoclax Plus Azacitidine Treatment in AML

Venetoclax (VEN), a selective inhibitor of BCL2, combined with azacitidine (Aza) has emerged as the new standard of care for patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. However, the long-term medical benefit is modest, and relapse appears to be unavoidable. We have recently reported that PP2A activators enhance the efficacy of VEN and VEN+Aza in primary samples and pre-clinical models of AML, and that the PP2A-B56α complex drives the synergistic pro-apoptotic activity observed. Our aim here is to further decipher the role of PP2A-B56α in VEN+Aza treatment response in AML. We used CRISPR-Cas9 to generate HL-60 sublines lacking the B56α or B55α PP2A regulatory subunits. Interestingly, while there were no significant differences in their baseline rate of proliferation, HL-60 B56α knockdown (KD) clones were significantly more resistant to VEN and VEN+Aza than wild type (WT) cells. In contrast, HL-60 B55α KD clones were sensitive to VEN+Aza, pointing out a specific role for specific PP2A heterotrimers, in this case the PP2A-B56α complex in the combined treatment response. Protein expression analysis of known substrates of PP2A-B56α demonstrated that pSer62-MYC and total MYC decreased after VEN+Aza treatment in WT cells but not in KD cells. It is known that the PP2A-B56α complex directly dephosphorylates MYC-Ser62, inducing MYC ubiquitin-mediated proteasomal degradation. Moreover, p21/CDKN1A, that is transcriptionally activated by p53 and inhibited by MYC, was up-regulated at both the mRNA and protein level after VEN+Aza treatment in WT cells but not in KD cells. HL-60 is a p53 null cell line; therefore, p21 up-regulation upon the combined treatment might be MYC-dependent. Of note, treatment of HL-60 B56α KD cells with the MYC inhibitor MYCi975 rescued the VEN+Aza synergy seen in these cells, confirming the relevance of MYC degradation to VEN+Aza response. Interestingly, VEN+MYCi975 had synergistic anti-leukemic effects in these same cell lines. Furthermore, CDKN1A expression was also rescued upon the triple therapy consisting of VEN+Aza+MYCi975. Combined, our results suggest that the PP2A-B56α complex is involved in the regulation of VEN+Aza response through changes in MYC expression. Interestingly, a novel small molecular glue (PMG) developed by our group, which specifically stabilizes the PP2A-B56α complex, enhanced VEN plus Aza response in vitro; however, this triple therapy did not work in HL-60 B56α KD cells and we observed a clearly decrease in MYC protein expression upon the triple therapy only in HL-60 B56α WT cells, supporting the rationale to translate these novel PMGs into the clinic. Altogether, our data suggest that PP2A-B56α might have an important role in VEN plus Aza treatment response through the regulation of MYC degradation.

Dnmt3a-Mutant Haematopoietic Stem and Progenitor Cells Induce Mesenchymal Stromal Cell Senescence through the Production of Alarmins S100A8/A9

Clonal haematopoiesis (CH) is an age-related condition occurring due to somatic genetic variation in haematopoietic stem and progenitor cells (HSPCs), most frequently observed in the gene DNA methyltransferase DNMT3A. CH can be a precursor condition for haematologic malignancy, but the mechanisms driving progression of CH to malignancy are incompletely understood. Given that malignant cells are understood to reprogram their bone marrow (BM) microenvironment to create a self-reinforcing niche, we hypothesized that HSPCs carrying a CH-associated mutation in DNMT3A have microenvironment-remodelling properties that promote their clonal advantage. Using single-cell RNA-seq, we comprehensively captured bone and BM cells of haematopoietic and non-haematopoietic lineages in Dnmt3a R878H/+ mice in which the Dnmt3a mutation is present in the haematopoietic but not the non-haematopoietic compartments. In the non-haematopoietic fractions, we identified enrichment of a cellular senescence program, including transcriptional signatures of cell cycle arrest and expression of the canonical senescence-associated gene Cdkn1a (p21), in BM mesenchymal stromal cells (MSCs). The MSC cellular senescence phenotype caused by Dnmt3a-mutant haematopoiesis was confirmed at the protein level by increased expression of senescence-associated beta galactosidase (SA-β-gal) and the anti-apoptotic proteins BCL-2 and BCL-xL. In ex vivo co-culture assays, primary wild-type MSCs cultured with Dnmt3a-mutant HSPCs versus control HSPCs had increased SA-β-gal, Cdkn1a (p21), and Cdkn2a (p16) expression. Moreover, transwell assays demonstrated that MSC senescence induced by Dnmt3a-mutant HSPCs is cell contact-independent. To undercover the mechanisms driving MSC senescence, we analyzed soluble factors produced by Dnmt3a-mutant HSPCs. Conditioned mediafrom Dnmt3a-mutant HSPCs had significantly elevated levels of S100A8/A9, IL-6 and TNFα, which we confirmed were also elevated in BM fluid of Dnmt3a-mutant mice. In our single cell RNA-seq data, S100a8 and S100a9 were the top significantly increased genes in Dnmt3a-mutant versus control HSCs, making S100A8/A9 our top candidates for functional studies. We tested the direct effect of recombinant S100A8/A9 on primary wild-type MSCs and found that this was sufficient to induce SA-β-gal. Ongoing studies using the S100A8/A9 inhibitor Tasquinimod are examining the dependency of Dnmt3a-mutant hematopoiesis on alarmin production ex vivo and in vivo. Given that S100A8/A9 are heavily involved in the pathogenesis of acute inflammation and myeloproliferative neoplasms, induction of senescence through the alarmin axis is a mechanistic link between aging, clonal hematopoiesis and haematologic malignancy.

An alternative spliced UPF2 transcript in pancreatic inflammatory myofibroblastic tumors

BackgroundInflammatory myofibroblastic tumors (IMTs) are characterized by myofibroblast proliferation and an inflammatory cell infiltrate. Our previous study on IMTs reveals that disrupt NMD pathway causes to lower the threshold for triggering the immune cell infiltration, thereby resulting in inappropriate immune activation. However, myofibroblast differentiation and proliferation is not yet known. MethodsRT-PCR, RT-qPCR, DNA sequence, western bolt, 5′race analysis and site-specific mutagenesis were used in this study. ResultsHere, an alternative spliced (ALS) UPF2 mRNA skipping exon 2 and 3 and corresponding to the truncated UPF2 protein were found in 2 pancreatic IMTs. We showed that the uORF present in the 5′UTR of UPF2 mRNA is responsible for the translation inhibition, whiles ALS UPF2 is more facilitated to be translated into the truncated UPF2 protein. Several mRNA targets of the NMD were upregulated in IMT samples, indicating that the truncated UPF2 function is strongly perturbed, resulted in disrupted NMD pathway in IMTs. These upregulated NMD targets included cdkn1a expression and the generation of high levels of p21 (waf1/cip1), which may contribute to triggering IMTs. ConclusionThe disrupt UPFs/NMD pathway may link to molecular alteration associated with differentiation and proliferation for IMTs.

Differential expression of BCL11b and CDKN2A in CD30-positive peripheral T cell lymphoma: Retrospective study.

Peripheral T-cell lymphoma is a disease that includes multiple T-cell lymphoma subtypes. It is still unclear whether CD30 can be used as a new target molecule and classification standard for PTCL. Differences in the molecular characteristics of CD30-positive PTCL and CD30-negative PTCL have rarely been reported. This study aimed to analyze the expression of BCL11b and CDKN2A in CD30-positive PTCL and CD30-negative PTCL, in order to guide the pathological classification, prognosis, and clinical treatment of PTCL. Immunohistochemical staining and quantitative reverse-transcription PCR (qRT-PCR) were performed on formalin-fixed paraffin-embedded tissue. Verification of BCL11b and CDKN2A expression in ALCL, PTCL-NOS, AITL and NK/TCL. Based on immunohistochemical analysis, the expression level of BCL11b in the lymph node reactive hyperplasia control group was high at 85.0%, which was higher than 68.8% in CD30-positive PTCL and 44.1% in CD30-negative PTCL (P < .05, respectively). CDKN2A showed expression rates of 70.0% in the control group, 79.2% in CD30-positive PTCL and 79.4% in CD30-negative PTCL. qRT-PCR showed that the relative BCL11b mRNA expression levels in patients with PTCL were lower than those in the control group (0.694 vs 1.832, P = .045). Univariate analysis showed that international prognostic index score, CD30 expression, and BCL11b expression were closely related to prognosis (P < .05, respectively). Multivariate Cox regression analysis revealed that high expression of BCL11b mRNA was an independent factor affecting prognosis (respectively, P < .05). Spearman correlation analysis indicated that BCL11b expression had a significant positive correlation with CD30 expression (P = .005). These results indicate that BCL11b may be involved in CD30 differentiation and PTCL prognosis. The detection and targeting of BCL11b and CD30 may provide new strategies for the treatment and classification of PTCL.

The Combination of Radiation with PARP Inhibition Enhances Senescence and Sensitivity to the Senolytic, Navitoclax, in Triple Negative Breast Tumor Cells.

Despite significant advances in the treatment of triple-negative breast cancer, this disease continues to pose a clinical challenge, with many patients ultimately suffering from relapse. Tumor cells that recover after entering into a state of senescence after chemotherapy or radiation have been shown to develop a more aggressive phenotype, and to contribute to disease recurrence. By combining the PARP inhibitor (PARPi), talazoparib, with radiation, senescence was enhanced in 4T1 and MDA-MB-231 triple-negative breast cancer cell lines (based on SA-β-gal upregulation, increased expression of CDKN1A and the senescence-associated secretory phenotype (SASP) marker, IL6). Subsequent treatment of the radiation- and talazoparib-induced senescent 4T1 and MDA-MB231 cells with navitoclax (ABT-263) resulted in significant apoptotic cell death. In immunocompetent tumor-bearing mice, navitoclax exerted a modest growth inhibitory effect when used alone, but dramatically interfered with the recovery of 4T1-derived tumors induced into senescence with ionizing radiation and talazoparib. These findings support the potential utility of a senolytic strategy in combination with the radiotherapy/PARPi combination to mitigate the risk of disease recurrence in triple-negative breast cancer.

High expression of CDKN2A is associated with poor prognosis in colorectal cancer and may guide PD-1-mediated immunotherapy

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Immunotherapy targeting the programmed death protein 1(PD-1) and its ligand (PD-L1), is a promising treatment option for many cancers, but has exhibited poor therapeutic efficacy in CRC. This study aimed to identify and validate the prognostic value of immune-related genes and PD-1-associated genes for immunotherapy treatment of CRC.MethodsAn extensive analysis of prognostic immune-related DEGs and PD-1-related genes has highlighted CDKN2A as a vital overlapping gene. To further explore its expression in CRC and its prognostic value, we conducted qRT-PCR, Western blot experiments, and consulted various databases. Subsequently, we conducted gene expression analysis, survival and prognostic analysis, enrichment analysis, immune infiltration assessment, and TIDE analysis to assess the significance of CDKN2A.ResultsIn CRC, CDKN2A was highly expressed compared to normal tissue. It was found that CDKN2A expression was related to clinicopathological features such as inflammation and tumor stage. Furthermore, a significant correlation was identified between CDKN2A and immune infiltration, specifically involving CD4 T cells, CD8 T cells, and macrophages. The analysis of the GSEA of CRC samples with high CDKN2A expression identified enrichment of genes involved in MYC target-v2 and metabolism pathways. Furthermore, UBE2I, CDK4, CDK6, TP53, and CCND1 were found to be significantly coexpressed with CDKN2A, suggesting a potential role that these gene play in CRC and immunotherapy.ConclusionsOur study revealed that high CDKN2A expression in CRC is a potentially valuable prognostic biomarker, which may guide PD-1-mediated immunotherapy.

Abstract 15509: “Root” Endothelial Cells: A Novel Population That Regulates Lung Capillary and Artery Maintenance and Repair

Introduction: Pulmonary capillary endothelial cells (ECs) are vital for various lung functions, such as maintaining the capillary network, promoting angiogenesis and facilitating gas and nutrient exchange. Recent advancements in single-cell transcriptomics have uncovered novel populations of general capillary ECs (gCaps) in the lungs. Understanding specialized EC types provides valuable insights into pulmonary vascular diseases. We aimed to challenge current understanding of the regenerative capabilities of lung resident ECs. Methods: Highly enriched ECs isolated from rat and mouse lungs underwent single-cell transcriptomics to explore EC population diversity. Results: UMAP annotation of ECs identified five novel general capillary cell populations (gCaps A-E). Lineage confirmation was achieved by Apelin receptor expression. Two distinct zonations (AV-zonation) were observed between pulmonary artery and vein ECs, with specific gCaps phenotypes (A: Clic4+, B: Flot1+, D: Scn7a+, E: Lingo2+), indicating a gradual phenotypic transition from arterial to vein ECs. Within the AV-zonation, a small population of gCapB (Flot1+) cells, resided at the interface of small artery ECs and gCap A and D, exhibiting characteristics of Root cells. RNA velocity analysis suggested Root cells as the origin of capillary and arterial cell lineages. PAGA analysis revealed high transcriptional similarity between gCapB and gCaps A, D, E and arterial cells, suggesting gCapB's role in capillary and arteriole formation and repair. Root cells exhibited upregulation of transcription factors Fos and Jun, promoting EC proliferation and angiogenesis, along with the Foxo and associated cell cycle regulation pathways. Root cells also displayed high G2M and S scores, indicating mitotic activity, supporting their role as the origin of capillary and arterial ECs. Elevated expression of CDKn1a and 1c, crucial for stem cell maintenance and differentiation further implied the repair potential of Root cells. Conclusions: Root Cells (gCapB) play a crucial role in regulating capillary and arteriole formation and repair by serving as a source of capillary and arterial ECs. These findings enhance our understanding of pulmonary vascular maintenance and repair mechanisms.

Loss of EFL1 Which Causes a Shwachman-Diamond Syndrome Reduces Cell Proliferation and Alters Transcriptional Profiling during Neutrophil Differentiation

Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myeloid malignancy. Almost all individuals with SDS harbor biallelic mutations in SBDS. The partner of SBDS is EFL1, a ribosomal protein with GTPase activity. More than a dozen patients have been found with SDS due to biallelic mutations in EFL1. EFL1 serves to release EIF6, facilitating the assembly of the 80S ribosome from 60S and 40S ribosome subunits. Our lab has generated both sbds and efl1 null zebrafish, which phenocopy much of the human syndrome. We recently reported that Sbds and Efl1 depletion leads to upregulation of tp53 and cdkn1a ( p21) and accumulation of Eif6 in SDS zebrafish models, which is hypothesized to cause bone marrow failure. However, the mechanism(s) of EFL1's loss-of-function leading to neutropenia is not known. Methods. We used CRISPR/Cas9 editing to generate mutations in exon 17 of Efl1 of 32Dcl3, an IL-3-dependent murine myeloid cell line that can be differentiated to mature neutrophils. We analyzed the effects of Efl1 mutations on cell proliferation, survival, and differentiation with G-CSF. Immunoblotting and qPCR-based gene expression analysis were performed to identify affected mechanistic pathways. Results. Two clones were isolated and were sequenced to verify biallelic deleterious mutations. A clone with compound heterozygous inframe deletion and frameshift mutations showed trace expression of EFL1 (EFL1-KD) and the other with compound heterozygous frameshift mutations showed no expression (EFL1-KO). They presented a myeloblast-like morphology indistinguishable from parental cells. They proliferated significantly less than parental cells, although there was no increased apoptosis. Both EFL1-KD and KO cells showed significantly increased protein expression of EIF6 and transcriptional expression of Trp53 and Cdkn1a. EFL1 was downregulated during differentiation of parental 32Dcl3 cells to neutrophils, whereas EIF6 levels did not change. Withdrawal and replenishment of IL-3 from culturing media did not alter EFL1 protein expression. When EFL1-KD and KO were cultured in differentiating media, they did not proliferate and underwent greater apoptosis, particularly after 3 days of differentiation compared to parental 32Dcl3. The surviving EFL1-KD and KO matured into neutrophils after 7 days. Gene profiling for neutrophil differentiation identified equivalent expression of key transcriptional factors ( Cebpa, Cebpe, and Spi1) of EFL1-KD and KO in the maintenance media, except for Gfi1 which was two-fold and three-fold high in EFL1-KD and KO compared to parental cells, respectively. G-CSF induced significantly higher expression of Cebpa and lower expression of Cebpe in EFL1-KO from those in parental cells, respectively. Based on the observation that somatic genetic rescue by EIF6 mutations occurs in human SDS patients and animal models, we transfected 32Dcl3 cells with siRNA against Eif6. In parental 32Dcl3 cells, reduction of EIF6 caused slow cell growth; however, transfection of siRNA to Eif6 partially rescued proliferation of EFL1-KD and KO in both maintenance and differentiating media. Conclusions. Our results suggested that imbalance of late maturing factors of ribosome associated with SDS may cause decreased proliferation in myeloid-committed cells, thereby leading to neutropenia in SDS. Newly established Efl1-deficient cells recapitulated key observation from SDS patients, with an increased proapoptotic profile in hematopoietic cells. Because EFL1 has only been recognized recently as a rare cause of SDS, we do not know if EFL1 deficiency serves as a leukemia predisposition syndrome. We speculate that the aberrant normal neutrophil differentiation may form the foundation for myeloid neoplasia in SDS.

Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway.

Long noncoding RNA plasmacytoma variant translocation1 is overexpressed in cutaneous squamouscell carcinoma and exon 2 is critical for itsoncogenicity.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long noncoding RNAs (lncRNAs) are a group of RNA molecules with essential regulatory functions in both physiological and pathological processes. To investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC. Quantitative reverse transcriptase polymerase chain reaction and single-molecule in situ hybridization were used to quantify the expression level of PVT1 in normal skin, premalignant skin lesions, actinic keratosis (AK) and primary and metastatic cSCCs. The function of PVT1 in cSCC was investigated both in vivo (tumour xenografts) and in vitro (competitive cell growth assay, 5-ethynyl-2'-deoxyuridine incorporation assay, colony formation assay and tumour spheroid formation assay) upon CRISPR-Cas9-mediated knockout of the entire PVT1 locus, the knockout of exon 2 of PVT1, and locked nucleic acid (LNA) gapmer-mediated PVT1 knockdown. RNA sequencing analysis was conducted to identify genes and processes regulated by PVT1. We identified PVT1 as a lncRNA upregulated in cSCC in situ and cSCC, associated with the malignant phenotype of cSCC. We showed that the expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9 deletion of the entire PVT1 locus and LNA gapmer-mediated knockdown of PVT1 transcript impaired the malignant behaviour of cSCC cells, suggesting that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro, demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we showed that PVT1 was localized in the cell nucleus and its deletion resulted in cellular senescence, increased cyclin-dependent kinase inhibitor 1 (p21/CDKN1A) expression and cell cycle arrest. Our study revealed a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence in cSCC. PVT1 may be a potential biomarker and therapeutic target in cSCC.

PSVI-14 Mitigating the Negative Effects of Heat Stress in Grow-Finish Pigs Using Water Delivered Oregano Essential Oil

Abstract Grow-finish pigs (n = 192) were used in a 28-day experiment to determine if adding oregano essential oil to the water would mitigate the detrimental effects of heat stress. Pigs (initial BW = 51.9 kg) were blocked by sex and BW and randomly allotted to a 2x2 factorial arrangement of water treatment (oregano essential oil vs. control) and environment (heat stress vs. thermoneutral). There were 6 pigs/pen (4 barrows and 2 gilts/pen) and 8 pens/treatment. Pigs were given 15 d to acclimate to pens and water treatment under thermoneutral conditions (21.1℃). Following acclimation, one-half the pigs were subjected to a 3-day diurnal heat stress (12 h at 33.3℃ and 12 h at 26.7℃). On d 22 of the experiment (3 d post-heat stress), one barrow/pen was euthanized; jejunal mucosa scrapings were collected, flash frozen in liquid nitrogen, and stored at -80oC for subsequent RNA isolation and cDNA creation. Gene expression of CDKN1A, TP53, GPX1, SOD1, SLC2A2, SLC2A5, and MUC2 were determined by qPCR using SYBR green, normalized using housekeeper genes and expressed relative to the control treatment using the 2-ΔΔCT method. Data were analyzed as RCB using GLM procedure in SAS 9.4 with fixed effects of water treatment, environment, and the interaction. Heat-stressed pigs had reduced ADG (P &amp;lt; 0.003) and Gain:Feed (P &amp;lt; 0.008) during the 3d heat stress compared with pigs housed in thermoneutral conditions. Post-heat stress, ADG (P &amp;lt; 0.001) and Gain:Feed (P &amp;lt; 0.001) increased in previously heat-stressed pigs compared with thermoneutral pigs. There was no effect of oregano supplementation before or during heat stress. However, in the post-heat stress period, ADG was increased (P &amp;lt; 0.05) by oregano supplementation in the water for both heat stressed and non-heat stressed pigs. Oregano supplementation did not affect ADFI before heat stress. An interaction (P &amp;lt; 0.005) of heat stress and water treatment was observed for ADFI during heat stress and after heat stress. During heat stress, oregano supplementation resulted in reduced feed intake. Post-heat stress ADFI was lowest for non-heat stressed pigs with no oregano supplementation. Oregano essential oil increased water consumption before (P &amp;lt; 0.02), during (P &amp;lt; 0.03), and after heat stress (P &amp;lt; 0.02), with an overall increase in water consumption of 35% compared with control pigs (P &amp;lt; 0.04). No interactions of heat stress and oregano supplementation were observed for gene expression. SLC2A2 expression tended (P &amp;lt; 0.08) to be downregulated when oregano essential oils were added to the water and upregulated following an acute heat stress. SLC2A5 expression was increased in pigs given oregano compared with control pigs (P &amp;lt; 0.02), but was unaffected by heat stress. Water delivered oregano essential oil increased water intake and had a slight effect on intestinal gene expression but did not mitigate the effects of a short-term heat stress.

Necroptosis-related genes are associated with prognostic features of kidney renal clear cell carcinoma

IntroductionRenal clear cell carcinoma is a common type of cancer in the adult urological system. It has a high mortality rate, with 30% of patients developing metastasis and 60% dying within 1–2 years of diagnosis. Recent advancements in tumor immunology and necroptosis have provided new insights into kidney cancer therapy. Therefore, it is crucial to identify potential targets for combining immunotherapy with necroptosis.Materials and methodsUsing the GSE168845 dataset and necroptosis-related genes, we identified genes that are differentially expressed in relation to necroptosis. We analyzed the prognostic value of these genes through differential expression analysis, prognostic analysis, and Cox regression analysis. The expression levels of the MYCN and CDKN2A genes were verified using the GSE53757 dataset. We also examined the association between the differentially expressed genes and clinicopathological features, as well as overall survival in our cohorts. In addition, we constructed a lasso Cox regression model to assess the correlation between these genes and immune score, ICP, and OCLR score. We conducted qRT-PCR to detect the expression of MYCN, CDKN2A, and ZBP1 in different samples of kidney renal clear cell carcinoma (KIRC). The expression levels of these genes were verified in a normal kidney cell line (HK-2 cells) and two KIRC cell lines (786-O, ACHN). The protein levels of MYCN and CDKN2A were detected using immunohistochemistry (IHC). SiRNA was used to silence the expression of MYCN and CDKN2A in the ACHN cell line, and wound healing assays were performed to measure cell migration.ResultsMYCN, CDKN2A, and ZBP1 were identified as necroptosis-related genes with independent prognostic value, leading to the development of a risk prognostic model. The expression of the CDKN2A gene was significantly higher in KIRC tissues compared to normal tissues, while the expression of the MYCN gene was significantly lower in KIRC tissues. The expression of MYCN and CDKN2A was associated with tumor stage, metastasis, and overall survival in our cohort. Furthermore, MYCN, CDKN2A, and ZBP1 were significantly correlated with immune score, ICP, and OCLR score. The expression levels of CDKN2A and ZBP1 were higher in KIRC cells compared to normal kidney cells, while the expression of MYCN was lower in KIRC cells. The protein expression of MYCN and CDKN2A was also higher in KIRC tissues, as confirmed by IHC. The results of the wound healing assay indicated that silencing CDKN2A inhibited cell migration, while silencing MYCN enhanced cell migration.ConclusionsMYCN and CDKN2A are potential targets and valuable prognostic biomarkers for combining immunotherapy with necroptosis in kidney renal clear cell carcinoma. CDKN2A promotes the migration of renal cancer cells, while MYCN inhibits their migration.

A candidate prognostic biomarker: TFEB inhibits tumor progression via elevating CDKN1A in bladder cancer

Bladder cancer(BC) is among the most prevalent malignancies in the world, with 549,393 new cases documented in 2018, and most BC patients have a poor prognosis. Transcription factor EB (TFEB) is considered a crucial controller of lysosomal-associated diseases, but a growing number of research in recent years have reported that TFEB plays other functions in tumors independent of lysosomal autophagy. In this study, we aimed to assess whether TFEB is a biomarker for BC and a molecular target for BC therapy. TFEB was lowly expressed in BC tissues relative to paracancerous tissues, and its elevated expression was strongly associated to a better prognosis for BC patients. TFEB overexpression markedly suppressed cell proliferation, limited cell migration, and accelerated apoptosis. Tumor growth in vivo was also suppressed. Mechanistically, we found that TFEB promoted CDKN1A expression by binding to the upstream progenitor of the CDKN1A promoter, which was also dependent on p53. Finally, Immune cell infiltration in BC tissues, PDL-1 expression, and Single-cell RNA sequencing data revealed immunotherapy may have a positive correlation with TFEB expression. Our study identifies that TFEB regulates CDKN1A in BC and has a positive prognostic value, while its expression is also positively correlated with immune cell infiltration. Therefore, TFEB may represent a recent therapeutic target for BC.

Compound AC1Q3QWB upregulates CDKN1A and SOX17 by interrupting the HOTAIR—EZH2 interaction and enhances the efficacy of tazemetostat in endometrial cancer

Endometrial cancer (EC) is a common malignancy of the female reproductive system, with an escalating incidence. Recurrent/metastatic EC presents a poor prognosis. The interaction between the long non-coding RNA (lncRNA) HOTAIR and the polycomb repressive complex 2 (PRC2) induces abnormal silencing of tumor suppressor genes, exerting a pivotal role in tumorigenesis. We have previously discovered AC1Q3QWB (AQB), a small-molecule compound targeting HOTAIR-EZH2 interaction. In the present study, we unveil that AQB selectively hampers the interaction between HOTAIR and EZH2 within EC cells, thus reversing the epigenetic suppression of tumor suppressor genes. Furthermore, our findings demonstrate AQB's synergistic effect with tazemetostat (TAZ), an EZH2 inhibitor, significantly boosting the expression of CDKN1A and SOX17. This, in turn, induces cell cycle arrest and impedes EC cell proliferation, migration, and invasion. In vivo experiments further validate AQB's potential by enhancing TAZ's anti-tumor efficacy at lower doses. Our results advocate AQB, a recently discovered small-molecule inhibitor, as a promising agent against EC cells. When combined with TAZ, it offers a novel therapeutic strategy for EC treatment.

Comprehensive analysis of cuproptosis genes and cuproptosis-related genes as prognosis factors in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common invasive and pernicious cancer with a low five-year survival rate. To identify potential therapeutic targets, we first investigated the characteristics of cuproptosis genes (CUGs) in ESCC. The expression patterns of 10 CUGs (FDX1, LIPT1, LIAS, DLAT, DLD, PDHA1, PDHB, GLS, MTF1, and CDKN2A) were analyzed to identify ESCC-relevant targets. Weighted correlation network analysis (WGCNA) was performed to obtain CUG-related genes (CRGs). A total of seven differentially expressed genes were identified (FDX1, DLAT, LIAS, PDHB, MTF1, GLS, and CDKN2A). DLAT was upregulated in stage III, and LIPT1 was upregulated in N0 + N1 cancers. The high expression of CDKN2A, and PDHA1, was related to better overall survival, whereas the low expression of LIAS was related to better clinical outcomes. WGCNA was performed to get CUG-related genes (CRGs) and showed three key modules that related to FDX1, DLAT, and LIPT1. Moreover, CRGs (BTLA, CT47A1, and PRRX1) were selected to construct a risk score model in order to predict the survival and prognosis of patients with ESCC. Additionally, the cuproptosis score based on CUGs and a nomogram constructed based on it helped accurately predict the prognosis of patients with ESCC; thus, maybe it can be used for the clinical diagnosis of ESCC. The results also showed that milciclib might inhibit the proliferation and migration of KYSE150 and KYSE510 cells by targeting CDKN2A. In conclusion, the abovementioned CUGs and CRGs play a crucial role in tumorigenesis and cancer progression in ESCC, indicating their potential as therapeutic targets.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Zerumbone liquid crystalline nanoparticles protect against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro

The purpose of this study was to evaluate the potential of zerumbone-loaded liquid crystalline nanoparticles (ZER-LCNs) in the protection of broncho-epithelial cells and alveolar macrophages against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro. The effect of the treatment of ZER-LCNs on in vitro cell models of cigarette smoke extract (CSE)-treated mouse RAW264.7 and human BCi-NS1.1 basal epithelial cell lines was evaluated for their anti-inflammatory, antioxidant and anti-senescence activities using colorimetric and fluorescence-based assays, fluorescence imaging, RT-qPCR and proteome profiler kit. The ZER-LCNs successfully reduced the expression of pro-inflammatory markers including Il-6, Il-1β and Tnf-α, as well as the production of nitric oxide in RAW 264.7 cells. Additionally, ZER-LCNs successfully inhibited oxidative stress through reduction of reactive oxygen species (ROS) levels and regulation of genes, namely GPX2 and GCLC in BCi-NS1.1 cells. Anti-senescence activity of ZER-LCNs was also observed in BCi-NS1.1 cells, with significant reductions in the expression of SIRT1, CDKN1A and CDKN2A. This study demonstrates strong in vitro anti-inflammatory, antioxidative and anti-senescence activities of ZER-LCNs paving the path for this formulation to be translated into a promising therapeutic agent for chronic respiratory inflammatory conditions including COPD and asthma.