ABSTRACT
The accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.
Subject(s)
B7-H1 Antigen , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16 , Protein Stability , Cellular Senescence/immunology , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Animals , Humans , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/genetics , Immunologic Surveillance , Mice, Inbred C57BL , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/genetics , Mice , Proteolysis , Receptors, IgG/metabolism , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Inflammation/geneticsABSTRACT
Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we find that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors. Mechanistically, O-GlcNAcylation of MITF at Serine 49 enhances its interaction with importin α/ß, thus promoting its translocation to nuclei, where it suppresses palbociclib-induced senescence. Inhibition of MITF or its O-GlcNAcylation re-sensitizes resistant cells to palbociclib. Moreover, clinical studies confirm the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on the mechanism regulating palbociclib resistance and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients.
Subject(s)
Breast Neoplasms , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Drug Resistance, Neoplasm , Microphthalmia-Associated Transcription Factor , N-Acetylglucosaminyltransferases , Piperazines , Pyridines , Humans , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Breast Neoplasms/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Microphthalmia-Associated Transcription Factor/metabolism , Microphthalmia-Associated Transcription Factor/genetics , Female , Drug Resistance, Neoplasm/drug effects , Piperazines/pharmacology , Pyridines/pharmacology , Cell Line, Tumor , N-Acetylglucosaminyltransferases/metabolism , N-Acetylglucosaminyltransferases/antagonists & inhibitors , N-Acetylglucosaminyltransferases/genetics , Animals , Mice , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Retinal vascular endothelial cell (RVECs) injury is a major cause of morbidity and mortality among the patients with diabetes. RVECs dysfunction is the predominant pathological manifestation of vascular complication in diabetic retinopathy. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in RVECs dysfunction is still unclear. METHODS: RT-qPCR analysis and western blot were conducted to detect the change of m6A RNA modification in diabetic retinopathy. CCK-8 assay, transwell experiment, wound healing assay, tube formation experiment, m6A-IP-qPCR were performed to determine the role of YTHDC1 in RVECs. Retinal trypsin digestion test and H&E staining were used to evaluate histopathological changes. RESULTS: The levels of m6A RNA methylation were significantly up-regulated in HG-induced RVECs, which were caused by increased expression of YTHDC1. YTHDC1 regulated the viability, proliferation, migration and tube formation ability in vitro. YTHDC1 overexpression impaired RVECs function by repressing CDK6 expression, which was mediated by YTHDC1-dependent mRNA decay. Moreover, it showed sh-YTHDC1 inhibited CDK6 nuclear export. Sh-YTHDC1 promotes the mRNA degradation of CDK6 in the nucleus but does not affect the cytoplasmic CDK6 mRNA. In vivo experiments showed that overexpression of CDK6 reversed the protective effect of sh-YTHDC1 on STZ-induced retinal tissue damage. CONCLUSION: YTHDC1-mediated m6A methylation regulates diabetes-induced RVECs dysfunction. YTHDC1-CDK6 signaling axis could be therapeutically targeted for treating DR.
Subject(s)
Adenosine , Cyclin-Dependent Kinase 6 , Diabetic Retinopathy , Endothelial Cells , Glucose , Endothelial Cells/metabolism , Animals , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/genetics , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/genetics , Adenosine/analogs & derivatives , Adenosine/metabolism , Glucose/metabolism , Glucose/pharmacology , Humans , Retina/metabolism , Male , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , Cell Proliferation , Nerve Tissue ProteinsABSTRACT
The synthesis, biochemical evaluation and radiosynthesis of a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor and radioligand was performed. NT431, a newly synthesized 4-fluorobenzyl-abemaciclib, exhibited high potency to CDK4/6 and against four cancer cell lines with IC50 similar to that of the parent abemaciclib. We performed a two-step one-pot radiosynthesis to produce [18F]NT431 with good radiochemical yield (9.6 ± 3%, n = 3, decay uncorrected), high radiochemical purity (>95%), and high molar activity (>370 GBq/µmol (>10.0 Ci/µmol). In vitro autoradiography confirmed the specific binding of [18F]NT431 to CDK4/6 in brain tissues. Dynamic PET imaging supports that both [18F]NT431 and the parent abemaciclib crossed the BBB albeit with modest brain uptake. Therefore, we conclude that it is unlikely that NT431 or abemaciclib (FDA approved drug) can accumulate in the brain in sufficient concentrations to be potentially effective against breast cancer brain metastases or brain cancers. However, despite the modest BBB penetration, [18F]NT431 represents an important step towards the development and evaluation of a new generation of CDK4/6 inhibitors with superior BBB penetration for the treatment and visualization of CDK4/6 positive tumors in the CNS. Also, [18F]NT431 may have potential application in peripheral tumors such as breast cancer and other CDK4/6 positive tumors.
Subject(s)
Aminopyridines , Benzimidazoles , Brain Neoplasms , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Positron-Emission Tomography , Protein Kinase Inhibitors , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Humans , Positron-Emission Tomography/methods , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/enzymology , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cell Line, Tumor , Benzimidazoles/pharmacology , Benzimidazoles/chemistry , Aminopyridines/chemistry , Aminopyridines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Animals , Radiopharmaceuticals/chemistry , Fluorine Radioisotopes/chemistry , Brain/diagnostic imaging , Brain/metabolism , Mice , FemaleABSTRACT
This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated ß-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated ß-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.
Subject(s)
Aminopyridines , Breast Neoplasms , Cell Proliferation , Piperazines , Purines , Pyridines , Humans , Aminopyridines/pharmacology , Piperazines/pharmacology , Purines/pharmacology , Pyridines/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Cell Proliferation/drug effects , Cell Line, Tumor , Receptors, Estrogen/metabolism , Fulvestrant/pharmacology , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Cyclin-Dependent Kinase 4/metabolism , Receptors, Progesterone/metabolism , Protein Kinase Inhibitors/pharmacology , Cyclin-Dependent Kinase 6/metabolism , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1.
Subject(s)
CRISPR-Cas Systems , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Drug Resistance, Neoplasm , Head and Neck Neoplasms , Piperazines , Pyridines , Squamous Cell Carcinoma of Head and Neck , Humans , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Piperazines/pharmacology , Piperazines/therapeutic use , Pyridines/pharmacology , Mice , Animals , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/metabolism , Cell Line, Tumor , MTOR Inhibitors/pharmacology , MTOR Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , TOR Serine-Threonine Kinases/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Xenograft Model Antitumor Assays , Synthetic Lethal Mutations , Oncogene ProteinsABSTRACT
Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.
Subject(s)
Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Humans , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Anaphase-Promoting Complex-Cyclosome/metabolism , Anaphase-Promoting Complex-Cyclosome/genetics , Cell Line, Tumor , S Phase/drug effects , Pyridines/pharmacology , Piperazines/pharmacology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , E2F Transcription Factors/metabolism , E2F Transcription Factors/genetics , Cell Cycle Checkpoints/drug effects , Cyclins/metabolism , Cyclins/genetics , F-Box ProteinsABSTRACT
Breast cancer (BC) cells have a high risk of metastasis due to epithelial-mesenchymal transition (EMT). Palbociclib (CDK4/6 inhibitor) is an approved drug for BC treatment. However, the drug resistance and metastasis can impair the treatment outcome of Palbociclib. Understanding the mechanisms of EMT and Palbociclib drug resistance in BC is conducive to the formulation of novel therapeutic strategy. Here, we investigated the role of circHIAT1/miR-19a-3p/CADM2 axis in modulating EMT and Palbociclib resistance in BC. circHIAT1 and CADM2 were down-regulated in BC tissues and cell lines, and miR-19a-3p showed an up-regulation. circHIAT1 could interact with miR-19a-3p and suppress its activity, while miR-19a-3p functioned to negatively regulate CADM2. Forced over-expression of circHIAT1 could impaired the EMT status and migratory ability of BC cells, and this effect was inhibited by miR-19a-3p mimic. In addition, we also generated Palbociclib resistant BC cells, and showed that circHIAT1 and CADM2 were down-regulated in the resistant BC cells while miR-19a-3p showed an up-regulation. Forced circHIAT1 over-expression re-sensitized BC cells to Palbociclib treatment. Quercetin, a bioactive flavonoid, could suppressed the migration and invasion of BC cells, and re-sensitized BC cells to Palbociclib. The anti-cancer effect of quercetin could be attributed to its regulatory effect on circHIAT1/miR-19a-3p/CADM2 axis. In vivo tumorigenesis experiment further revealed that quercetin administration enhanced the anti-cancer effect of Palbociclib, an effect was dependent on the up-regulation of circHIAT1 by quercetin. In summary, this study identified quercetin as a potential anti-cancer compound to reverse Palbociclib resistance and impair EMT in BC cells by targeting circHIAT1/miR-19a-3p/CADM2 axis.
Subject(s)
Breast Neoplasms , Cyclin-Dependent Kinase 6 , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , MicroRNAs , Piperazines , Pyridines , Quercetin , Epithelial-Mesenchymal Transition/drug effects , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Female , Drug Resistance, Neoplasm/drug effects , Pyridines/pharmacology , Piperazines/pharmacology , Cell Line, Tumor , Quercetin/pharmacology , Animals , Mice , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/genetics , Gene Expression Regulation, Neoplastic/drug effects , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cell Movement/drug effects , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Mice, Nude , Xenograft Model Antitumor AssaysABSTRACT
Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, including abemaciclib, have been approved for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced, and metastatic breast cancer. Despite the high therapeutic efficacy of CDK4/6 inhibitors, they are associated with various adverse effects, including potentially fatal interstitial lung disease. Therefore, a combination of CDK4/6 inhibitors with letrozole or fulvestrant has been attempted but has demonstrated limitations in reducing adverse effects, highlighting the need to develop new combination therapies. This study proposes a combination strategy using CDK4/6 inhibitors and tricyclic antidepressants to enhance the therapeutic outcomes of these inhibitors while reducing their side effects. The therapeutic efficacies of abemaciclib and desipramine were tested in different cancer cell lines (H460, MCF7, and HCT-116). The antitumor effects of the combined abemaciclib and desipramine treatment were evaluated in a xenograft colon tumor model. In vitro cell studies have shown the synergistic anticancer effects of combination therapy in the HCT-116 cell line. The combination treatment significantly reduced tumor size compared with control or single treatment without causing apparent toxicity to normal tissues. Although additional in vivo studies are necessary, this study suggests that the combination therapy of abemaciclib and desipramine may represent a novel therapeutic approach for treating solid tumors.
Subject(s)
Aminopyridines , Benzimidazoles , Desipramine , Drug Synergism , Xenograft Model Antitumor Assays , Humans , Benzimidazoles/pharmacology , Benzimidazoles/administration & dosage , Aminopyridines/pharmacology , Aminopyridines/administration & dosage , Animals , Mice , Desipramine/pharmacology , Cell Line, Tumor , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Female , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cell Proliferation/drug effects , Mice, Nude , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , MCF-7 Cells , HCT116 Cells , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/administration & dosage , Mice, Inbred BALB CABSTRACT
In search of potential anticancer agents, we synthesized SNO-donor salicylaldimine main ligand-based Pt(II) complexes bearing NH3 as co-ligand at trans-position (C1-C6). These complexes showed similarity in structure with transplatin as the two N donor atoms of the main ligand and NH3 co-ligand were coordinated to Pt in trans position to each other. Each complex with different substituents on the main ligand was characterized thoroughly by detailed spectroscopic and spectrophotometric methods. Four of these complexes were studied in solid state by single crystal X-ray analysis. The stability of reference complex C1 was measured in solution state in DMSOd6 or its mixture with D2O using 1H NMR methods. These complexes were further investigated for their anticancer activity in triple-negative-breast (TNBC) cells including MDA-MB-231, MDA-MB-468 and MDA-MB-436 cells. All these complexes showed satisfactory cytotoxic effect as revealed by the MTT results. Importantly, the highly active complex C4 anticancer effect was compared to the standard chemotherapeutic agents including cisplatin, oxaliplatin and 5-fluorouracil (5-FU). Functionally, C4 suppressed invasion, spheroids formation ability and clonogenic potential of cancer cells. C4 showed synergistic anticancer effect when used in combination with palbociclib, JQ1 and paclitaxel in TNBC cells. Mechanistically, C4 inhibited cyclin-dependent kinase (CDK)4/6 pathway and targeted the expressions of MYC/STAT3/CCND1/CNNE1 axis. Furthermore, C4 suppressed the EMT signaling pathway that suggested a role of C4 in the inhibition of TNBC metastasis. Our findings may pave further in detailed mechanistic study on these complexes as potential chemotherapeutic agents in different types of human cancers.
Subject(s)
Antineoplastic Agents , Cyclin D1 , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Epithelial-Mesenchymal Transition , STAT3 Transcription Factor , Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Epithelial-Mesenchymal Transition/drug effects , Cell Line, Tumor , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Proto-Oncogene Proteins c-myc/metabolism , Female , Coordination Complexes/pharmacology , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesis , Ligands , Carcinogenesis/drug effects , Cell Proliferation/drug effects , Platinum/chemistry , Platinum/pharmacology , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/chemical synthesisABSTRACT
Myocardial infarction (MI) seriously threatens the health of elderly people, and reducing myocardial injury is of great significance for the treatment of MI. LncRNA-TTN-AS1 shows protective effects on cardiomyocyte injury, while the role of TTN-AS1 in MI remains unknown. CCK8, flow cytometry, and JC-1 staining assessed cell viability, apoptosis and mitochondrial membrane potential (MMP), respectively. Cellular reactive oxygen species (ROS) and secreted lactate dehydrogenase (LDH) levels were measured. The interactions between ELF5, TTN-AS1, PCBP2 and CDK6 were explored using ChIP, luciferase reporter assay, RIP, and pull-down. The severity of MI in mice was evaluated using TTC, H&E, and TUNEL staining. The data revealed that OGD/R significantly induced ROS, mitochondrial injury and apoptosis in AC16 cells, while overexpression of ELF5 or TTN-AS1 reversed these phenomena. ELF5 transcriptionally activated TTN-AS1 through binding with its promoter. TTN-AS1 increased CDK6 stability via recruiting PCBP2. CDK6 knockdown abolished the inhibitory effects of TTN-AS1 overexpression on OGD/R-induced myocardial injury. Furthermore, overexpression of TTN-AS1 or ELF5 alleviated MI progression in mice by upregulating CDK6. Collectively, TTN-AS1 transcriptionally regulated by ELF5 alleviated myocardial apoptosis and injury during MI via recruiting PCBP2 to increase CDK6 stability, which shed new lights on exploring new strategies against MI.
Subject(s)
Apoptosis , Cyclin-Dependent Kinase 6 , Myocardial Infarction , Myocytes, Cardiac , RNA, Long Noncoding , RNA-Binding Proteins , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/genetics , Mice , Apoptosis/genetics , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/genetics , Myocytes, Cardiac/metabolism , Reactive Oxygen Species/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Male , Mice, Inbred C57BL , Cell Line , Membrane Potential, MitochondrialABSTRACT
Tissue repair, immune defence and cancer progression rely on a vital cellular decision between quiescence and proliferation1,2. Mammalian cells proliferate by triggering a positive feedback mechanism3,4. The transcription factor E2F activates cyclin-dependent kinase 2 (CDK2), which in turn phosphorylates and inactivates the E2F inhibitor protein retinoblastoma (Rb). This action further increases E2F activity to express genes needed for proliferation. Given that positive feedback can inadvertently amplify small signals, understanding how cells keep this positive feedback in check remains a puzzle. Here we measured E2F and CDK2 signal changes in single cells and found that the positive feedback mechanism engages only late in G1 phase. Cells spend variable and often extended times in a reversible state of intermediate E2F activity before committing to proliferate. This intermediate E2F activity is proportional to the amount of phosphorylation of a conserved T373 residue in Rb that is mediated by CDK2 or CDK4/CDK6. Such T373-phosphorylated Rb remains bound on chromatin but dissociates from it once Rb is hyperphosphorylated at many sites, which fully activates E2F. The preferential initial phosphorylation of T373 can be explained by its relatively slower rate of dephosphorylation. Together, our study identifies a primed state of intermediate E2F activation whereby cells sense external and internal signals and decide whether to reverse and exit to quiescence or trigger the positive feedback mechanism that initiates cell proliferation.
Subject(s)
Cell Proliferation , Cyclin-Dependent Kinase 2 , E2F Transcription Factors , Retinoblastoma Protein , Phosphorylation , Cyclin-Dependent Kinase 2/metabolism , Retinoblastoma Protein/metabolism , E2F Transcription Factors/metabolism , Humans , Animals , Mice , Single-Cell Analysis , Chromatin/metabolism , G1 Phase , Feedback, Physiological , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cell LineABSTRACT
CDK6 plays a key role in the regulation of the cell cycle and is considered a crucial target for cancer therapy. In this work, conformational transitions of CDK6 were identified by using Gaussian accelerated molecular dynamics (GaMD), deep learning (DL), and free energy landscapes (FELs). DL finds that the binding pocket as well as the T-loop binding to the Vcyclin protein are involved in obvious differences of conformation contacts. This result suggests that the binding pocket of inhibitors (LQQ and AP9) and the binding interface of CDK6 to the Vcyclin protein play a key role in the function of CDK6. The analyses of FELs reveal that the binding pocket and the T-loop of CDK6 have disordered states. The results from principal component analysis (PCA) indicate that the binding of the Vcyclin protein affects the fluctuation behavior of the T-loop in CDK6. Our QM/MM-GBSA calculations suggest that the binding ability of LQQ to CDK6 is stronger than AP9 with or without the binding of the Vcyclin protein. Interaction networks of inhibitors with CDK6 were analyzed and the results reveal that LQQ contributes more hydrogen binding interactions (HBIs) and hot interaction spots with CDK6. In addition, the binding pocket endures flexibility changes from opening to closing states and the Vcyclin protein plays an important role in the stabilizing conformation of the T-loop. We anticipate that this work could provide useful information for further understanding the function of CDK6 and developing new promising inhibitors targeting CDK6.
Subject(s)
Cyclin-Dependent Kinase 6 , Deep Learning , Molecular Dynamics Simulation , Protein Binding , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/chemistry , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Humans , Protein Conformation , Binding Sites , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Principal Component Analysis , Thermodynamics , Normal DistributionABSTRACT
BACKGROUND: The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients. METHODS: BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored. RESULTS: Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs. CONCLUSIONS: Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.
Subject(s)
Breast Neoplasms , Cyclin-Dependent Kinase 4 , Drug Resistance, Neoplasm , Piperazines , Pyridines , Receptors, Estrogen , Humans , Pyridines/pharmacology , Pyridines/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Piperazines/pharmacology , Piperazines/therapeutic use , Female , Receptors, Estrogen/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cell Line, Tumor , Receptors, G-Protein-Coupled/metabolism , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Tumor MicroenvironmentABSTRACT
Antimicrobial peptides (AMPs) have sparked significant interest as potential anti-cancer agents, thereby becoming a focal point in pursuing novel cancer-fighting strategies. These peptides possess distinctive properties, underscoring the importance of developing more potent and selectively targeted versions with diverse mechanisms of action against human cancer cells. Such advancements would offer notable advantages compared to existing cancer therapies. This research aimed to examine the toxicity and selectivity of the nrCap18 peptide in both cancer and normal cell lines. Furthermore, the rate of cellular death was assessed using apoptosis and acridine orange/ethidium bromide (AO/EB) double staining at three distinct incubation times. Additionally, the impact of this peptide on the cancer cell cycle and migration was evaluated, and ultimately, the expression of cyclin-dependent kinase 4/6 (CDK4/6) genes was investigated. The results obtained from the study demonstrated significant toxicity and selectivity in cancer cells compared to normal cells. Moreover, a strong progressive increase in cell death was observed over time. Furthermore, the peptide exhibited the ability to halt the progression of cancer cells in the G1 phase of the cell cycle and impede their migration by suppressing the expression of CDK4/6 genes.
Subject(s)
Apoptosis , Breast Neoplasms , Cathelicidins , Cyclin-Dependent Kinase 4 , Humans , Animals , Cell Line, Tumor , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Apoptosis/drug effects , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/genetics , Female , Rabbits , Cell Movement/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antimicrobial Cationic Peptides/pharmacology , Cyclin-Dependent Kinase 6/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Peptides/pharmacology , Peptides/chemistry , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Degradation of target proteins has been considered to be a promising therapeutic approach, but the rational design of compounds for degradation remains a challenge. In this study, we reasonably designed and synthesized only 10 compounds to discover effective CDK4/6 protein degraders. Among the newly synthesized compounds, 7f achieved dual degradation of CDK4/6 protein, with DC50 values of 10.5 and 2.5 nM, respectively. Compound 7f also exhibited inhibitory proliferative activity against Jurkat cells with an IC50 value of 0.18 µM. Furthermore, 7f induced cell apoptosis and G1 phase cell cycle arrest in a dose-dependent manner in Jurkat cells. In conclusion, these findings demonstrate the potential of 7f as a CDK4/6 degrader and a potential therapeutic strategy against cancer, thereby expanding the potential of CDK4/6 dual PROTACs.
Subject(s)
Antineoplastic Agents , Apoptosis , Cell Proliferation , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Drug Design , Humans , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cell Proliferation/drug effects , Apoptosis/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Jurkat Cells , Structure-Activity Relationship , Proteolysis/drug effects , Molecular StructureABSTRACT
AIM: Neuroblastoma (NB) is, in spite of current intensive therapy with severe side effects, still not cured so new therapies are needed. Recently, we showed combining phosphoinositide 3-kinase (PI3K) (BYL719), fibroblast growth factor receptor (FGFR) (JNJ-42756493) and cyclin-dependent kinase 4/6 (CDK4/6) (PD-0332991) inhibitors, in vitro in NB cell lines grown as monolayers had synergistic effects. However, there were variations depending on the combinations used and the targeted NB cell lines. To obtain further information and to mimic more natural circumstances, we investigated the effects of single and combined administrations of the above inhibitors in spheroid NB-cultures. MATERIAL AND METHODS: Spheroid cultures of NB cell lines SK-N-AS, SK-N-BE(2)-C, SK-N-FI and SK-N-SH were established and treated with single and combined administrations of BYL719, JNJ-42756493, and PD-0332991 and followed for growth, viability, proliferation, cytotoxicity and migration. KEY FINDINGS: Single inhibitor administrations gave dose dependent responses with regard to growth and viability and their combinations were efficient and resulted in a range of additive and synergistic effects. The responses to individual drugs and their various combinations were predominantly alike regardless of whether the cells were cultivated in monolayer or D spheroid NB models. However, in general, slightly higher drug concentrations were necessary in spheroidcultures. SIGNIFICANCE: This study provides pre-clinical evidence that single PI3K, FGFR, and CDK4/6, inhibitors exhibit promising anti-NB activity and when combined lower doses of the drugs could be also used in spheroid NB-cultures, supporting the pursuit of further in vitro and in vivo studies in preparation for future potential clinical use.
Subject(s)
Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Drug Synergism , Neuroblastoma , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors , Receptors, Fibroblast Growth Factor , Spheroids, Cellular , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Humans , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Spheroids, Cellular/drug effects , Cell Line, Tumor , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Receptors, Fibroblast Growth Factor/metabolism , Protein Kinase Inhibitors/pharmacology , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cell Proliferation/drug effects , Pyridines/pharmacology , Molecular Targeted Therapy , Phosphatidylinositol 3-Kinases/metabolism , Cell Survival/drug effects , Piperazines/pharmacology , Piperazines/administration & dosage , Dose-Response Relationship, DrugABSTRACT
Objective: To evaluate the efficacy of chemotherapy and endocrine therapy combined with targeted drugs after progression on cyclin-dependent kinase 4/6 (CDK4/6) inhibitor treatment in hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2)-low metastatic breast cancer. Methods: Patients with metastatic breast cancer diagnosed with HR positive/HER2 low expression at the Fifth Medical Center of PLA General Hospital from October 1, 2018 to September 30, 2023 were retrospectively included. All patients received sequential chemotherapy or sequential endocrine therapy combined with targeted drugs after progression on CDK4/6 inhibitor treatment.The median follow-up was 9 months, and the follow-up ended on October 31, 2023. The patients were divided into chemotherapy group (receiving sequential chemotherapy) and endocrine therapy group (receiving sequential endocrine therapy combined with targeted drugs), according to the treatment plan. Information on demographic data, clinical and pathological diagnosis, treatment regimen, and efficacy evaluation was collected. The basic conditions of patients who may affect the curative effect of different treatment schemes were preset as stratified subgroups, including age, progesterone receptor (PR) status, HER2 status, disease-free survival, number of previous endocrine therapy and chemotherapy, and visceral metastasis. The primary endpoint was progression-free survival (PFS), the secondary endpoints were objective response rate (ORR), clinical benefit rate(CBR) and PFS based on stratification factors. The survival curve was plotted by Kaplan-Meier method, the comparison of PFS between groups was performed by log-rank test, and the comparison of ORR and CBR between groups were performed by χ2 test. Results: A total of 188 patients were included, including 126 patients in the chemotherapy group [all females, aged 29-74 (51±10) years] and 62 patients in the endocrine therapy group [1 male and 61 female, aged 29-77 (51±12) years]. ORR of chemotherapy group was 23.0% (29/126), higher than that of endocrine treatment group [3.2% (2/62)] (P<0.001); The CBR of chemotherapy group and endocrine therapy group were 46.8% (59/126) and 33.9% (21/62), respectively, with no statistical significance (P=0.091). The median PFS of chemotherapy group and endocrine therapy group were 5.0 (95%CI: 4.3-5.7) and 4.0 (95%CI: 1.6-6.4) months, respectively, with no statistical significance (P=0.484). In the preset stratified subgroups, the median PFS of chemotherapy [6.0 (95%CI: 5.4-6.6) months] was longer than that of endocrine combined with targeted therapy [2.0 (95%CI: 1.8-2.2) months] (P<0.001) in PR negative patients; In patients who had progressed on over 2 previous endocrine treatments, the median PFS of chemotherapy [5.0 (95%CI: 3.8-6.2) months] was longer than that of endocrine combined with targeted therapy [2.0 (95%CI: 0.6-3.4) months] (P=0.045). Conclusions: After progression on treatment with CDK4/6 inhibitors for HR-positive/HER2-low expression metastatic breast cancer, both chemotherapy and endocrine therpy combined with targeted drugs are viable treatment options. However, for patients with PR negative or ≥2 lines of endocrine therapy previously, priority should be accorded to chemotherapy.
Subject(s)
Breast Neoplasms , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Receptor, ErbB-2 , Adult , Aged , Female , Humans , Middle Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Neoplasm Metastasis , Protein Kinase Inhibitors/therapeutic use , Receptor, ErbB-2/metabolism , Receptors, Progesterone/metabolismABSTRACT
Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).