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1.
Mol Biol Rep ; 51(1): 606, 2024 May 05.
Article in English | MEDLINE | ID: mdl-38704498

ABSTRACT

BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.


Subject(s)
Adenosine Triphosphate , Cell Differentiation , Induced Pluripotent Stem Cells , Mutation , Organoids , Retina , Retinoblastoma , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Organoids/metabolism , Retina/metabolism , Retina/cytology , Retinoblastoma/genetics , Retinoblastoma/metabolism , Adenosine Triphosphate/metabolism , Cell Differentiation/genetics , Mutation/genetics , Heterozygote , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Glycolysis/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism
2.
Cancer Sci ; 115(5): 1576-1586, 2024 May.
Article in English | MEDLINE | ID: mdl-38468443

ABSTRACT

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.


Subject(s)
Down-Regulation , Epithelial Cells , Retinoblastoma Binding Proteins , Signal Transduction , ras Proteins , Humans , Epithelial Cells/metabolism , Female , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Binding Proteins/genetics , ras Proteins/metabolism , ras Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Epithelial-Mesenchymal Transition/genetics , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Mammary Glands, Human/cytology , Cell Line, Tumor , Retinoblastoma Protein/metabolism , Retinoblastoma Protein/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics
3.
J Biol Chem ; 300(3): 105707, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38309505

ABSTRACT

Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the pro-tumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer.


Subject(s)
Bromodomain Containing Proteins , Cell Cycle Proteins , Liver Neoplasms , Nuclear Proteins , Transcription Factors , Ubiquitin-Specific Proteases , Humans , Bromodomain Containing Proteins/genetics , Bromodomain Containing Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Genes, Tumor Suppressor , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Retinoblastoma Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Specific Proteases/metabolism
4.
Gastroenterology ; 166(6): 1130-1144.e8, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38262581

ABSTRACT

BACKGROUND & AIMS: Despite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Here, we aim to develop new therapeutic approaches for liver cancer. METHODS: A compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with cyclin-dependent kinase (CDK) 4/6 inhibitor. The combination effects of CDK4/6 inhibitor and exportin 1 (XPO1) inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. RESULTS: The combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic drug screen, cereblon (CRBN)-based proteolysis targeting chimera (PROTAC) ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Up-regulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that ubiquitin specific peptidase 2 (USP2) directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. CONCLUSIONS: Our study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.


Subject(s)
Adaptor Proteins, Signal Transducing , Cellular Senescence , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Exportin 1 Protein , Karyopherins , Liver Neoplasms , Protein Kinase Inhibitors , Receptors, Cytoplasmic and Nuclear , Ubiquitin-Protein Ligases , Humans , Cellular Senescence/drug effects , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Karyopherins/antagonists & inhibitors , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Ubiquitin-Protein Ligases/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Binding Proteins/genetics , Drug Synergism , Senotherapeutics/pharmacology , Xenograft Model Antitumor Assays , Signal Transduction/drug effects , Proteolysis/drug effects , Hydrazines/pharmacology , Hydrazines/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Hep G2 Cells , Mice , Piperazines , Pyridines , Triazoles
5.
Mol Biotechnol ; 66(1): 102-111, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37041423

ABSTRACT

Retinoblastoma (RB) is a malignant ocular cancer that affects children. Several microRNAs (miRNAs) have been implicated in RB regulation. The present study aimed to investigate the role of miR-4529-3p in RB pathogenesis. Scratch, Transwell, and Cell Counting Kit (CCK)-8 assays were conducted to assess the migratory, invasive, and proliferative abilities of RB cells. The expression levels of miR-4529-3p, RB1, and ERK pathway-related proteins were analyzed using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Target relationships were verified using dual-luciferase reporter experiments. A murine RB model was developed to analyze the effects of miR-4529-3p on RB tumor growth in vivo. Our experiments revealed high levels of miR-4529-3p and low levels of RB1 in RB tissues. Functional analyses revealed that the migratory, invasive, and proliferative abilities of RB cells were repressed by miR-4529-3p inhibition. Similarly, p-ERK 1/2 protein levels were suppressed by miR-4529-3p inhibition. Furthermore, downregulation of miR-4529-3p limited tumor growth in vivo. Mechanistically, miR-4259-3p targets RB1. Interestingly, RB1 silencing abrogated the alleviative effects of miR-4529-3p downregulation in RB cells. MiR-4529-3p promotes RB progression by inhibiting RB1 and activating the ERK pathway. This evidence suggests that the miR-4529-3p/RB1 regulatory axis may be a prospective target for RB treatment in clinical settings.


Subject(s)
MicroRNAs , Retinal Neoplasms , Retinoblastoma , Child , Humans , Animals , Mice , Retinoblastoma/genetics , Retinoblastoma/metabolism , Retinoblastoma/pathology , MAP Kinase Signaling System/genetics , Cell Line, Tumor , MicroRNAs/genetics , MicroRNAs/metabolism , Signal Transduction , Retinal Neoplasms/genetics , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism
6.
Cell Oncol (Dordr) ; 47(1): 209-227, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37606819

ABSTRACT

PURPOSE: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken. METHODS: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells. RESULTS: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis. CONCLUSION: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.


Subject(s)
Retinal Neoplasms , Retinoblastoma , Chick Embryo , Animals , Humans , Child , Retinoblastoma/genetics , Retinoblastoma/pathology , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Chickens/metabolism , Carcinogenesis , Ubiquitin-Protein Ligases/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism
7.
Sci Transl Med ; 15(722): eadf6732, 2023 Nov 15.
Article in English | MEDLINE | ID: mdl-37967200

ABSTRACT

Aberrant DNA methylation has been implicated as a key driver of prostate cancer lineage plasticity and histologic transformation to neuroendocrine prostate cancer (NEPC). DNA methyltransferases (DNMTs) are highly expressed, and global DNA methylation is dysregulated in NEPC. We identified that deletion of DNMT genes decreases expression of neuroendocrine lineage markers and substantially reduced NEPC tumor development and metastasis in vivo. Decitabine, a pan-DNMT inhibitor, attenuated tumor growth in NEPC patient-derived xenograft models, as well as retinoblastoma gene (RB1)-deficient castration-resistant prostate adenocarcinoma (CRPC) models compared with RB1-proficient CRPC. We further found that DNMT inhibition increased expression of B7 homolog 3 (B7-H3), an emerging druggable target, via demethylation of B7-H3. We tested DS-7300a (i-DXd), an antibody-drug conjugate targeting B7-H3, alone and in combination with decitabine in models of advanced prostate cancer. There was potent single-agent antitumor activity of DS-7300a in both CRPC and NEPC bearing high expression of B7-H3. In B7-H3-low models, combination therapy of decitabine plus DS-7300a resulted in enhanced response. DNMT inhibition may therefore be a promising therapeutic target for NEPC and RB1-deficient CRPC and may sensitize B7-H3-low prostate cancer to DS-7300a through increasing target expression. NEPC and RB1-deficient CRPC represent prostate cancer subgroups with poor prognosis, and the development of biomarker-driven therapeutic strategies for these populations may ultimately help improve patient outcomes.


Subject(s)
Antineoplastic Agents , Neuroendocrine Tumors , Prostatic Neoplasms, Castration-Resistant , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms, Castration-Resistant/drug therapy , DNA Methylation/genetics , Decitabine/pharmacology , Decitabine/therapeutic use , Cell Line, Tumor , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Neuroendocrine Tumors/drug therapy , Transcription Factors/metabolism , Antineoplastic Agents/therapeutic use , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism
8.
JCI Insight ; 8(21)2023 Nov 08.
Article in English | MEDLINE | ID: mdl-37937640

ABSTRACT

Poly (ADP-ribose) polymerase inhibitors (PARPis) are approved for cancer therapy according to their synthetic lethal interactions, and clinical trials have been applied in non-small cell lung cancer. However, the therapeutic efficacy of PARPis in lung adenocarcinoma (LUAD) is still unknown. We explored the effect of a mutated retinoblastoma gene (RB1) on PARPi sensitivity in LUAD. Bioinformatic screening was performed to identify PARPi-sensitive biomarkers. Here, we showed that viability of LUAD cell lines with mutated RB1 was significantly decreased by PARPis (niraparib, rucaparib, and olaparib). RB1 deficiency induced genomic instability, prompted cytosolic double-stranded DNA (dsDNA) formation, activated the cGAS/STING pathway, and upregulated downstream chemokines CCL5 and CXCL10, triggering immune cell infiltration. Xenograft experiments indicated that PARPi treatment reduced tumorigenesis in RB1-KO mice. Additionally, single-cell RNA sequencing analysis showed that malignant cells with downregulated expression of RB1 had more communications with other cell types, exhibiting activation of specific signaling such as GAS, IFN response, and antigen-presenting and cytokine activities. Our findings suggest that RB1 mutation mediates the sensitivity to PARPis through a synthetic lethal effect by triggering the cGAS/STING pathway and upregulation of immune infiltration in LUAD, which may be a potential therapeutic strategy.


Subject(s)
Adenocarcinoma of Lung , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Animals , Humans , Mice , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
9.
Gene ; 887: 147724, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37604323

ABSTRACT

Retinoblastoma gene1 (RB1) is the first tumor suppressor gene that stands as the guardian of the gate of the G1 period and plays a central role in proliferation and differentiation. However, no reports focused on the possible internal ribosome entry site (IRES) function of the RB1 gene flanking sequence. In this study, we constructed a bicistronic reporter with the RB1 5'untranslated region (5́UTR) inserted between two reporter coding regions. We found RB1 5'UTR harbors an IRES and has higher activity in cancer cell lines than normal cells. Besides, RB1 IRES acquired the highest activity in the G0/G1 phase of the cell cycle, and the RB1 5'UTR mutation collected from retinoblastoma decreased IRES activity compared with RB1 5'UTR wild-type. These data indicated that RB1 IRES is a mechanism of stress regulation and is related to cell cycle control and cancer progression.


Subject(s)
Retinal Neoplasms , Retinoblastoma , Humans , Internal Ribosome Entry Sites/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , Retinoblastoma/genetics , 5' Untranslated Regions/genetics , Retinal Neoplasms/genetics , Cell Cycle Checkpoints , Ubiquitin-Protein Ligases/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism
10.
Int J Mol Sci ; 24(11)2023 Jun 02.
Article in English | MEDLINE | ID: mdl-37298606

ABSTRACT

The general notion of complete hydatidiform moles is that most of them consist entirely of paternal DNA; hence, they do not express p57, a paternally imprinted gene. This forms the basis for the diagnosis of hydatidiform moles. There are about 38 paternally imprinted genes. The aim of this study is to determine whether other paternally imprinted genes could also assist in the diagnostic approach of hydatidiform moles. This study comprised of 29 complete moles, 15 partial moles and 17 non-molar abortuses. Immunohistochemical study using the antibodies of paternal-imprinted (RB1, TSSC3 and DOG1) and maternal-imprinted (DNMT1 and GATA3) genes were performed. The antibodies' immunoreactivity was evaluated on various placental cell types, namely cytotrophoblasts, syncytiotrophoblasts, villous stromal cells, extravillous intermediate trophoblasts and decidual cells. TSSC3 and RB1 expression were observed in all cases of partial moles and non-molar abortuses. In contrast, their expression in complete moles was identified in 31% (TSSC3) and 10.3% (RB1), respectively (p < 0.0001). DOG1 was consistently negative in all cell types in all cases. The expressions of maternally imprinted genes were seen in all cases, except for one case of complete mole where GATA3 was negative. Both TSSC3 and RB1 could serve as a useful adjunct to p57 for the discrimination of complete moles from partial moles and non-molar abortuses, especially in laboratories that lack comprehensive molecular service and in cases where p57 staining is equivocal.


Subject(s)
Hydatidiform Mole , Moles , Animals , Female , Humans , Pregnancy , Antibodies/metabolism , Cyclin-Dependent Kinase Inhibitor p57/genetics , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Hydatidiform Mole/diagnosis , Hydatidiform Mole/genetics , Immunohistochemistry , Moles/metabolism , Placenta/metabolism , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
11.
Appl Immunohistochem Mol Morphol ; 31(6): 371-378, 2023 07 01.
Article in English | MEDLINE | ID: mdl-37126387

ABSTRACT

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive, primary neuroendocrine carcinoma of the skin whose main risk factors are immunosuppression, UV radiation exposure, and Merkel cell polyomavirus. Programmed death-1/programmed death ligand-1 (PD-L1)-based immunotherapy is currently the first choice for treating patients with metastatic MCC. METHODS: MCC biopsies (17) were evaluated for their nucleus and cytoplasm characteristics and growth patterns, as well as for intratumor lymphocytes, mitotic number, and lymphovascular invasion. Paraffin-embedded tissue samples of the biopsies were stained with MCPyV large T-antigen (LTag), RB1, p53, and PD-L1. RESULTS: We observed MCPyV LTag expression in 9 out of the 17 tumors, and all 9 cases were positive for RB1 ( P <0.000). p53 staining was not significantly correlated with MCPyV LTag. We observed no relationship between p53 expression and any other parameters, and PD-L1 expression was low in the MCC samples. We evaluated PD-L1 using both the combined positive score and tumor proportion score (TPS), and found that TPS was correlated with MCPyV LTag expression ( P =0.016). Tumors with tumor-infiltrating lymphocytes showed a better prognosis than those without these lymphocytes ( P =0.006). DISCUSSION: Our data demonstrated that RB1 was effective for immunohistochemically investigating the MCPyV status of tumors. TPS was superior to the combined positive score in evaluating PD-L1 in MCC. Tumor-infiltrating lymphocytes were the only parameters that were associated with survival. Further studies with larger series are required to confirm these results.


Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Humans , Carcinoma, Merkel Cell/pathology , B7-H1 Antigen/metabolism , Merkel cell polyomavirus/metabolism , Tumor Suppressor Protein p53 , Skin Neoplasms/pathology , Polyomavirus Infections/complications , Polyomavirus Infections/metabolism , Ubiquitin-Protein Ligases , Retinoblastoma Binding Proteins/metabolism
12.
Molecules ; 28(7)2023 Mar 30.
Article in English | MEDLINE | ID: mdl-37049846

ABSTRACT

Type 2 diabetes (T2D) is characterized by insulin resistance (IR), often accompanied by inflammation. Macrophage activation acts as an inflammatory response, which is characterized by macrophage recruitment in the initial stage. Ginsenoside Rb1 (Rb1) is a main active ingredient, which is known for its fat-reducing, anti-inflammatory effects. To clarify that Rb1 regulates macrophage activation in adipose tissue and improves tissue inflammation, network pharmacology and molecular docking were used for target prediction and preliminary validation. By constructing the co-culture model of adipose-derived stem cells (ADSC) and primary macrophage (PM), the body adipose tissue microenvironment was simulated to observe the adipogenesis degree of adipocytes under the effect of Rb1. The levels of cytokines, macrophage polarization, and protein or RNA expression in the inflammatory signaling pathway were finally detected. The results showed that 89 common targets of T2D-Rb1 were obtained after their intersection. Furthermore, according to the results of the KEGG pathway and PPI analysis, PTGS2 (COX-2) is the downstream protein of PPARγ-NF-κB. The molecular binding energy of PPARγ-Rb1 is -6.8 kcal/mol. Rb1 significantly inhibited the increase in MCP-1, TNF-α, and IL-1ß induced by hypertrophic adipocytes supernatant and promoted the expression of IL-10. Rb1 inhibited the activation of inflammatory macrophages and PM migration and upregulated PPARγ expression with the blocking of NF-κB activation. Additionally, Rb1 promoted the expression of IRS1 and PI3K in the insulin signal pathway, which had a similar effect with ROS. Therefore, Rb1 might affect macrophage activation through PPARγ, which might alleviate obese insulin resistance in T2D early stage.


Subject(s)
Diabetes Mellitus, Type 2 , Insulin Resistance , Humans , PPAR gamma/metabolism , Macrophage Activation , NF-kappa B/metabolism , Diabetes Mellitus, Type 2/complications , Molecular Docking Simulation , Obesity/metabolism , Inflammation/metabolism , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Binding Proteins/metabolism
13.
Int J Mol Sci ; 24(6)2023 Mar 12.
Article in English | MEDLINE | ID: mdl-36982482

ABSTRACT

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.


Subject(s)
Neuroblastoma , Child , Humans , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Gene Amplification , Gene Expression Regulation, Neoplastic , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
14.
J Clin Invest ; 133(10)2023 05 15.
Article in English | MEDLINE | ID: mdl-36928314

ABSTRACT

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RB1-deficient cancers remain elusive. Here, we showed that RB1 loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid-containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss-induced sensitization to ferroptosis. Importantly, using cell line-derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a promising approach for the treatment of RB1-deficient malignancies.


Subject(s)
Ferroptosis , Prostatic Neoplasms , Male , Humans , Mice , Animals , Ferroptosis/genetics , Prostatic Neoplasms/pathology , Cell Line , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism
15.
Histopathology ; 82(5): 767-778, 2023 Apr.
Article in English | MEDLINE | ID: mdl-36647795

ABSTRACT

AIMS: Onychomatricoma (OM), an uncommon benign fibroepithelial neoplasm of the nail unit, is sometimes diagnostically challenging for clinicians and pathologists. OM consistently expresses CD34, but no specific immunohistohemical markers or recurrent genetic alterations have been identified to date. Recent studies have suggested that Wnt signalling is a key molecular characteristic of OM. METHODS AND RESULTS: Ten cases were analysed: four classical OM including two with pleomorphic cells; two superficial acral fibromyxoma-like variants of OM; three micropapilliferum variants of OM including one with pleomorphic cells; and one proliferating variant of OM. Immunohistochemically, the spindle cells were positive with CD34 (n = 10) and CD99 (n = 1), with focal reactivity for CD10 (n = 5). The epithelial component of the tumours expressed immunopositivity for LEF-1. Using array comparative genomic hybridization (aCGH), we demonstrated that all OM, including its variants that were tested (n = 8), harboured a few copy number alterations with losses of whole or part of chromosome 13 including the RB1 gene (n = 8) and chromosome 16 (n = 6). CONCLUSION: We report a recurrent loss of RB1 (13q) as a possible driver molecular event in OM indicating a relationship between OM and other lesions of the spectrum of the so-called '13q/RB1' family of tumours. We did not identify a role for the Wnt/beta-catenin signalling pathway, as has been proposed in a recent study. LEF-1 could be a potential sensitive and specific marker of OM and should be used in the differential diagnosis between OM, superficial acral fibromyxoma, and the CD34-positive fibrosing family of tumours.


Subject(s)
Fibroma , Nail Diseases , Skin Neoplasms , Humans , Antigens, CD34/metabolism , Biomarkers, Tumor/metabolism , Cell Adhesion Molecules/metabolism , Comparative Genomic Hybridization , Fibroma/pathology , Nail Diseases/pathology , Retinoblastoma Binding Proteins/metabolism , Skin Neoplasms/pathology , Ubiquitin-Protein Ligases/metabolism
16.
Histopathology ; 82(6): 885-898, 2023 May.
Article in English | MEDLINE | ID: mdl-36720791

ABSTRACT

AIMS: Recently, YAP1 fusion genes have been demonstrated in eccrine poroma and porocarcinoma, and the diagnostic use of YAP1 immunohistochemistry has been highlighted in this setting. In other organs, loss of YAP1 expression can reflect YAP1 rearrangement or transcriptional repression, notably through RB1 inactivation. In this context, our objective was to re-evaluate the performance of YAP1 immunohistochemistry for the diagnosis of poroma and porocarcinoma. METHODS AND RESULTS: The expression of the C-terminal part of the YAP1 protein was evaluated by immunohistochemistry in 543 cutaneous epithelial tumours, including 27 poromas, 14 porocarcinomas and 502 other cutaneous tumours. Tumours that showed a lack of expression of YAP1 were further investigated for Rb by immunohistochemistry and for fusion transcripts by real-time PCR (YAP1::MAML2 and YAP1::NUTM1). The absence of YAP1 expression was observed in 24 cases of poroma (89%), 10 porocarcinoma (72%), 162 Merkel cell carcinoma (98%), 14 squamous cell carcinoma (SCC) (15%), one trichoblastoma and one sebaceoma. Fusions of YAP1 were detected in only 16 cases of poroma (n = 66%), 10 porocarcinoma (71%) all lacking YAP1 expression, and in one sebaceoma. The loss of Rb expression was detected in all cases except one of YAP1-deficient SCC (n = 14), such tumours showing significant morphological overlap with porocarcinoma. In-vitro experiments in HaCat cells showed that RB1 knockdown resulted in repression of YAP1 protein expression. CONCLUSION: In addition to gene fusion, we report that transcriptional repression of YAP1 can be observed in skin tumours with RB1 inactivation, including MCC and a subset of SCC.


Subject(s)
Carcinoma , Eccrine Porocarcinoma , Poroma , Skin Neoplasms , Sweat Gland Neoplasms , Humans , Poroma/genetics , Poroma/metabolism , Poroma/pathology , Sweat Gland Neoplasms/diagnosis , Eccrine Porocarcinoma/genetics , Eccrine Porocarcinoma/pathology , Skin Neoplasms/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Binding Proteins/metabolism
17.
Oncogene ; 42(6): 449-460, 2023 02.
Article in English | MEDLINE | ID: mdl-36513743

ABSTRACT

Current clinical therapies targeting receptor tyrosine kinases including focal adhesion kinase (FAK) have had limited or no effect on esophageal squamous cell carcinoma (ESCC). Unlike esophageal adenocarcinomas, ESCC acquire glucose in excess of their anabolic need. We recently reported that glucose-induced growth factor-independent proliferation requires the phosphorylation of FAKHis58. Here, we confirm His58 phosphorylation in FAK immunoprecipitates of glucose-stimulated, serum-starved ESCC cells using antibodies specific for 3-phosphohistidine and mass spectrometry. We also confirm a role for the histidine kinase, NME1, in glucose-induced FAKpoHis58 and ESCC cell proliferation, correlating with increased levels of NME1 in ESCC tumors versus normal esophageal tissues. Unbiased screening identified glucose-induced retinoblastoma transcriptional corepressor 1 (RB1) binding to FAK, mediated through a "LxCxE" RB1-binding motif in FAK's FERM domain. Importantly, in the absence of growth factors, glucose increased FAK scaffolding of RB1 in the cytoplasm, correlating with increased ESCC G1→S phase transition. Our data strongly suggest that this glucose-mediated mitogenic pathway is novel and represents a unique targetable opportunity in ESCC.


Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Focal Adhesion Protein-Tyrosine Kinases , Humans , Cell Line, Tumor , Cell Proliferation , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/pathology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Glucose , Intercellular Signaling Peptides and Proteins/metabolism , Phosphorylation , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
18.
J Med Virol ; 95(1): e28338, 2023 01.
Article in English | MEDLINE | ID: mdl-36418188

ABSTRACT

Epstein-Barr virus (EBV) was the first tumor virus discovered in humans and can cause various types of tumors. Molecular classification suggests that EBV-associated gastric cancer (EBVaGC) is a unique subtype of gastric cancer.EBV was also the first virus found to encode its own microRNAs. However, the functions of many miRNAs remain unknown. This study investigated the roles and targets of miR-BART2-5p (BART2-5p) and miR-BART11-5p (BART11-5p) in EBVaGC. The expression of RB and p21 in EBVaGC and EBV negative GC (EBVnGC) cells was evaluated by western blotting. Expression of BART2-5p and BART11-5p in EBVaGC cells was evaluated by droplet digital PCR. The effects of BART2-5p or BART11-5p and their potential mechanisms were further investigated using cell counting kit-8, colony formation assay, flow cytometry analysis, and transwell assay. BART2-5p and BART11-5p were abundantly expressed and RB and p21 were downregulated in EBVaGC cells. BART2-5p regulates RB and p21 expression by directly targeting them. BART11-5p regulates RB expression by directly targeting RB. Both BART2-5p and BART11-5p promoted proliferation and migration of gastric cancer cells, while inhibiting apoptosis and promoting S-phase arrest of the cell cycle. Thus, BART2-5p and BART11-5p play important roles in promoting proliferation and migration, and inhibiting apoptosis in EBVaGC by targeting RB and p21, thus providing new potential therapeutic targets for EBVaGC.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21 , Epstein-Barr Virus Infections , MicroRNAs , RNA, Viral , Retinoblastoma Binding Proteins , Stomach Neoplasms , Humans , Apoptosis , Carcinoma/metabolism , Carcinoma/pathology , Cell Proliferation , Epstein-Barr Virus Infections/genetics , Herpesvirus 4, Human/genetics , MicroRNAs/metabolism , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , RNA, Viral/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Retinoblastoma Binding Proteins/metabolism
19.
Nat Commun ; 13(1): 5258, 2022 09 07.
Article in English | MEDLINE | ID: mdl-36071033

ABSTRACT

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.


Subject(s)
Antineoplastic Agents , Breast Neoplasms , Protein Kinase Inhibitors , Antineoplastic Agents/therapeutic use , Biomarkers , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Drug Resistance, Neoplasm/genetics , Female , Humans , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/therapeutic use , Receptors, Estrogen/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
20.
Mol Genet Genomics ; 297(6): 1671-1687, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36076047

ABSTRACT

We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 (RBBP8) and MutS homologue 4 (MSH4), between primary tumors and normal tissues of TCGA-BLCA were validated by methylation-specific PCR (MSP) in UBC (n = 70) compared to normal tissues (n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8- and MSH4-methylation along with patients' clinical features. Then, we assessed the prognostic significance of RBBP8 and MSH4. RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients' response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4-methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy.


Subject(s)
Carcinoma , Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Cisplatin/pharmacology , Cisplatin/therapeutic use , Prognosis , Urinary Bladder/metabolism , Urinary Bladder/pathology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Epigenesis, Genetic , DNA Repair/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Carcinoma/drug therapy , Carcinoma/genetics , Carcinoma/metabolism , DNA Methylation/genetics
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