ABSTRACT
Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.
Subject(s)
Cell Cycle Proteins , Lung Neoplasms , Prostatic Neoplasms , Proto-Oncogene Proteins c-myc , Humans , Male , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/drug therapy , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Line, Tumor , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Mice , Animals , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathology , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/drug therapy , Proteolysis/drug effects , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein LigasesABSTRACT
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
Subject(s)
Antigens, Polyomavirus Transforming , Antigens, Viral, Tumor , Merkel cell polyomavirus , Neoplastic Stem Cells , Retinoblastoma Binding Proteins , Humans , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Carcinoma, Merkel Cell/virology , Carcinoma, Merkel Cell/genetics , Cell Differentiation , Cell Line, Tumor , Gene Knockdown Techniques , Gene Regulatory Networks , Merkel cell polyomavirus/genetics , Neoplastic Stem Cells/virology , Neoplastic Stem Cells/metabolism , Neurons/virology , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Skin Neoplasms/virology , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Antigens, Polyomavirus Transforming/genetics , Antigens, Polyomavirus Transforming/metabolismABSTRACT
BACKGROUND: SGI-1027 is a recognized inhibitor of DNA methyltransferase 1 (DNMT1), and earlier investigations have indicated an inverse correlation between dysregulated DNMT1 expression in gastric cancer (GC) and retinoblastoma 1 (RB1) gene expression. Despite this knowledge, the precise mechanisms underlying the action of SGI-1027 in GC cells remain inadequately comprehended. The primary objective of this study is to elucidate the impact of SGI-1027 on the behavior of GC cells, encompassing aspects such as growth and metastatic potential, by intervening in DNMT1, thereby influencing the regulation of RB1 gene expression. METHOD: The acquisition of the normal gastric mucosal cell line GES-1 and the human gastric cancer cell line MKN45 was followed by employing Western blot (WB) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques to evaluate the expression levels of RB1 and DNMT1 in these two cell lines. Subsequently, the MKN45 cell line was cultured in medium containing varying concentrations of SGI-1027, and the impact of SGI-1027 on the regulation of RB1 and DNMT1 in GC cells was reassessed using WB and qRT-PCR techniques. To scrutinize the effect of SGI-1027 on GC cells, we utilized the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay to determine cell proliferation and performed Transwell experiments to assess cell migration and invasion capabilities. Throughout this process, we also employed WB to assess the levels of cell cycle-associated proteins (Cyclin D1, Cyclin E1, and Cyclin B1) and proteins related to apoptosis (BCL-2 associated protein X apoptosis regulator (BAX) and B-cell lymphoma 2 apoptosis regulator (BCL-2)). Furthermore, we injected the MKN45 cell line and MKN45 cell line cultured with the optimal concentration of SGI-1027 for 5 days and 10 days into mice subcutaneously and through the tail vein, dividing them into the Model group, Model+SGI-1027 5d group, and Model+SGI-1027 10d group. We monitored changes in tumor size and volume in mice, and tumor tissues as well as lung tissues were collected for hematoxylin and eosin (HE) staining. Finally, DNMT1 expression levels in GC tissues were detected using both WB and immunohistochemistry (IHC) techniques. Additionally, RB1 expression levels in GC tissues were assessed using WB. RESULT: In contrast to GES-1 cells, MKN45 cells displayed a distinctive profile characterized by increased DNMT1 expression and decreased RB1 expression (p < 0.05). However, upon the introduction of SGI-1027, a notable decrease in DNMT1 levels within GC cells was observed, concomitant with an elevation in RB1 gene expression, with 25 µmol/L SGI-1027 identified as the optimal concentration (p < 0.05). Functional assays demonstrated that SGI-1027-treated GC cells exhibited pronounced features of inhibited proliferation, migration, and invasion when compared to untreated MKN45 cells (p < 0.05). Moreover, in SGI-1027-treated GC cells, the levels of Cyclin D1, Cyclin E1, Cyclin B1, and BCL-2 were significantly reduced, while the expression level of BAX increased (p < 0.05). Notably, the most pronounced impact was observed at 25 µmol/L SGI-1027, further underscoring its regulatory effects on tumor cell behavior (p < 0.05). In animal experiments, the Model group exhibited a substantial increase in tumor volume, with HE staining results indicating extensive necrosis in most gastric tissues and noticeable signs of lung metastasis, accompanied by increased DNMT1 expression and decreased RB1 gene expression. In contrast, the SGI-1027 group displayed a reduction in gastric tumor volume, decreased necrosis, and reduced lung tumor metastasis (p < 0.05). Additionally, the expression of DNMT1 was significantly reduced in SGI-1027-treated GC cells, while RB1 expression increased (p < 0.05), further confirming the inhibitory effects of SGI-1027 on tumor growth and metastasis. CONCLUSIONS: SGI-1027 effectively hinders the proliferation and dissemination of GC cells by downregulating DNMT1 and promoting the expression of RB1.
Subject(s)
Cell Proliferation , DNA (Cytosine-5-)-Methyltransferase 1 , Gene Expression Regulation, Neoplastic , Retinoblastoma Binding Proteins , Stomach Neoplasms , Humans , Stomach Neoplasms/pathology , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Cell Line, Tumor , Animals , Cell Proliferation/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Mice , Neoplasm Metastasis , Cell Movement/genetics , Mice, Nude , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Retinoblastoma Protein/metabolism , Retinoblastoma Protein/genetics , Mice, Inbred BALB C , Repressor ProteinsABSTRACT
Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.
Subject(s)
Casein Kinase II , Retinoblastoma Binding Proteins , Humans , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/metabolism , Casein Kinase II/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Binding Proteins/genetics , Female , Cell Line, Tumor , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Carboplatin/pharmacology , Synthetic Lethal Mutations , DNA Replication/drug effects , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Antineoplastic Agents/pharmacologyABSTRACT
RB1 stands as the pioneering discovery in tumour-suppressor genes, marking a pivotal breakthrough in comprehending cancer development. This overview delves into the role of RB1 in both health and disease, exploring its association with the tumourigenesis of various cancers and a distinct subset of soft-tissue neoplasms. Additionally, we discuss the application of immunohistochemistry and fluorescence in situ hybridisation to detect RB1 alterations.
Subject(s)
Neoplasms , Retinoblastoma Binding Proteins , Humans , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Cell Cycle , Pathologists , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Immunohistochemistry , In Situ Hybridization, Fluorescence , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/geneticsABSTRACT
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 , Organoids , Retina , Retinoblastoma Binding Proteins , Retinoblastoma , Ubiquitin-Protein Ligases , Humans , Adenosine Triphosphate/metabolism , Cell Differentiation/genetics , Glycolysis/genetics , Heterozygote , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Mutation/genetics , Organoids/metabolism , Retina/metabolism , Retina/cytology , Retinoblastoma/genetics , Retinoblastoma/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
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/geneticsABSTRACT
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/metabolismABSTRACT
Prostate cancer is the second most common malignancy among men in the world, posing a serious threat to men's health and lives. RB1 is the first human tumor suppressor gene to be described, and it is closely associated with the development, progression, and suppression of a variety of tumors. It was found that the loss of RB1 is an early event in prostate cancer development and is closely related to prostate cancer development, progression and treatment resistance. This paper reviews the current status of research on the relationship between RB1 and prostate cancer from three aspects: RB1 and prostate cell lineage plasticity; biological behavior; and therapeutic resistance. Providing a novel perspective for developing new therapeutic strategies for RB1-loss prostate cancer.
Subject(s)
Drug Resistance, Neoplasm , Prostatic Neoplasms , Retinoblastoma Binding Proteins , Ubiquitin-Protein Ligases , Humans , Male , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Drug Resistance, Neoplasm/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Cell Plasticity/drug effects , Cell LineageABSTRACT
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.