ABSTRACT
The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.
Subject(s)
CDC2 Protein Kinase/metabolism , Embryonic Stem Cells/physiology , Epigenesis, Genetic , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Animals , CDC2 Protein Kinase/genetics , Cell Differentiation , Cells, Cultured , Chromatin Immunoprecipitation/methods , Female , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , MCF-7 Cells , Male , Mice , Mice, Knockout , Phosphorylation , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
OBJECTIVE: To investigate alterations of homologous recombination repair (HRR) and especially BReast CAncer 1/2 (BRCA1/2) gene on overall survival (OS). Moreover, to explore the effect of inhibition of poly(ADP-ribose)-polymerase (PARPi) as systemic therapy for metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS: Of all HRR-screened patients with metastatic prostate cancer, baseline characteristics were sampled. Kaplan-Meier estimates and multivariable Cox regression models predicted the effect of HRR/BRCA1/2 alterations on OS. RESULTS: Of 196 eligible patients, 61 (31%) harboured any HRR and 40 (20%) BRCA1/2 alterations. Of HRR alterations, 40 (66%) vs six (10%) vs five (8.2%) vs four (6.6%) vs two (3.3%) vs four (6.6%) were BRCA1/2 vs Ataxia-telangiectasia mutated kinase (ATM) vs checkpoint kinase 2 (CHEK2) vs cyclin-dependent kinase 12 (CDK12) vs Fanconi anaemia complementation Group A (FANCA) vs positive for other mutations. Of these, 30% received a PARPi. OS differed significantly between HRR-positive vs -negative patients. Specifically in hormone-sensitive prostate cancer, the median OS was 63 (HRR positive) vs 57 (BRCA1/2 positive) vs 113 months (HRR negative) (P ≤ 0.01). In mCRPC, OS was 42 (HRR positive) vs 41 (BRCA1/2 positive) vs 70 months (HRR negative) (P ≤ 0.01). HRR and BRCA1/2 alterations were associated with worse OS after multivariable adjustment. Finally, patients with mCRPC with BRCA1/2 mutation treated without PARPi harboured worse OS than patients with BRCA1/2 mutation and PARPi therapy (median OS: 33 vs 48 months, P < 0.03). CONCLUSION: Incidence of HRR alteration in a clinical real-world setting is high when using blood- and tissue-based tests. Patients with HRR/BRCA alterations have worse outcomes resulting in significant OS differences between HRR/BRCA-positive patients with mCRPC with and without PARPi usage vs HRR/BRCA-negative patients.
ABSTRACT
The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.
Subject(s)
Cyclin-Dependent Kinase 5/metabolism , Melanoma, Experimental/pathology , Melanoma/pathology , Skin Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Cyclin-Dependent Kinase 5/genetics , Female , Gene Dosage , Humans , Male , Melanoma/drug therapy , Melanoma/genetics , Melanoma/mortality , Melanoma, Experimental/drug therapy , Melanoma, Experimental/genetics , Mice , Mice, Knockout , Phosphorylation/drug effects , Phosphorylation/genetics , Prognosis , Skin/pathology , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Vimentin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Kinases govern many cellular responses through the reversible transfer of a phosphate moiety to their substrates. However, pairing a substrate with a kinase is challenging. In proximity labeling experiments, proteins proximal to a target protein are marked by biotinylation, and mass spectrometry can be used for their identification. Here, we combine ascorbate peroxidase (APEX) proximity labeling and a phosphorylation enrichment-based workflow, Phospho-APEX (pAPEX), to rapidly identify phosphorylated and biotinylated neighbor proteins which can be considered for candidate substrates. The pAPEX strategy enriches and quantifies differences in proximity for proteins and phosphorylation sites proximal to an APEX2-tagged kinase under the kinase "ON" and kinase "OFF" conditions. As a proof of concept, we identified candidate substrates of MAPK1 in HEK293T and HCT116 cells and candidate substrates of PKA in HEK293T cells. In addition to many known substrates, C15orf39 was identified and confirmed as a novel MAPK1 substrate. In all, we adapted the proximity labeling-based platform to accommodate phosphorylation analysis for kinase substrate identification.
Subject(s)
Phosphorylation , Ascorbate Peroxidases/chemistry , Ascorbate Peroxidases/metabolism , Biotinylation , HEK293 Cells , Humans , Mass Spectrometry , Substrate SpecificityABSTRACT
MicroRNAs (miRNAs) have been known to affect various biological processes by repressing expression of specific genes. Here we describe an essential function of the miR-34/449 family during differentiation of epithelial cells. We found that miR-34/449 suppresses the cell-cycle machinery in vivo and promotes cell-cycle exit, thereby allowing epithelial cell differentiation. Constitutive ablation of all six members of this miRNA family causes derepression of multiple cell cycle-promoting proteins, thereby preventing epithelial cells from exiting the cell cycle and entering a quiescent state. As a result, formation of motile multicilia is strongly inhibited in several tissues such as the respiratory epithelium and the fallopian tube. Consequently, mice lacking miR-34/449 display infertility as well as severe chronic airway disease leading to postnatal death. These results demonstrate that miRNA-mediated repression of the cell cycle is required to allow epithelial cell differentiation.
Subject(s)
Cell Cycle Proteins/biosynthesis , Cell Cycle/physiology , Cell Differentiation/physiology , MicroRNAs/metabolism , Mouse Embryonic Stem Cells/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line , Cilia/genetics , Cilia/metabolism , Fallopian Tubes/cytology , Fallopian Tubes/metabolism , Female , Mice , MicroRNAs/genetics , Mouse Embryonic Stem Cells/cytology , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolismABSTRACT
Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Interleukin-10/biosynthesis , Myeloid Cells/drug effects , Small Molecule Libraries/pharmacology , Animals , Cells, Cultured , Cyclin-Dependent Kinase 8/immunology , Dose-Response Relationship, Drug , Humans , Interleukin-10/immunology , Mice , Mice, Inbred C57BL , Molecular Structure , Myeloid Cells/immunology , Myeloid Cells/metabolism , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: Deregulation of miRNA-210 is a common event in several types of cancer. However, increased expression levels in the cancer tissue have been associated with both poor and good prognosis of patients. Similarly, the function of miR-210 with regard to cell growth and apoptosis is still controversial. METHODS: Overexpression of miR-210 was performed in HCT116, SW480 and SW707 colorectal cancer (CRC) cell lines. Functional effects of a modulated miR-210 expression were analyzed with regard to proliferation, clonogenicity, cell cycle distribution, reactive oxygen species (ROS) generation, and apoptosis. Furthermore, quantitative real time (RT)-PCR and immunoblot analyses were performed to investigate signaling pathways affected by miR-210. RESULTS: We show that in CRC cells miR-210 inhibits clonogenicity and proliferation which was accompanied by an accumulation of cells in the G2/M phase of the cell cycle. Additionally, overexpression of miR-210 results in an increase of ROS generation. Moreover, miR-210 mediated the induction of apoptosis which was associated with an upregulation of pro-apoptotic Bim expression and enhanced processing of Caspase 2. Importantly, inhibition of ROS generation rescued cells from miR-210-induced apoptosis. CONCLUSIONS: Taken together, miR-210 induces apoptosis in CRC cells via a ROS-dependent mechanism.
ABSTRACT
The expression of cyclin proteins is tightly regulated during the cell cycle, to allow precise activation of cyclin-dependent kinases. In this issue, Pan et al. (https://doi.org/10.1083/jcb.202308066) identify an RNA-binding protein, PC4, as a regulator of cyclin D1 mRNA stability in hepatocellular carcinoma cells. This study provides a new mechanism regulating the levels of a key cell cycle protein, cyclin D1, in human cells.
Subject(s)
Cyclin D1 , DNA-Binding Proteins , RNA Stability , Transcription Factors , Humans , Cell Cycle , Cell Cycle Proteins/metabolism , Cyclin D1/metabolism , Cyclin-Dependent Kinases/metabolism , Phosphorylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Line, TumorABSTRACT
Current treatments for advanced prostate cancer (PCa) primarily target androgen receptor (AR)-pathways. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR signaling inhibitors (ARSI) remains a significant clinical challenge. This study introduces BSJ-5-63, a novel triple degrader targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, with potential to transform CRPC therapy. BSJ-5-63 effectively downregulates homologous recombination repair (HRR) genes, including BRCA1 and BRCA2, through CDK12 degradation, and attenuates AR signaling through CDK7 and CDK9 degradation, further enhancing its therapeutic impact. Importantly, BSJ-5-63 induces a "BRCAness" state that persists for a significant duration, enabling sequential combination therapy with PARP inhibitors (PARPis) while potentially minimizing drug-related toxicity and resistance. In both in vitro and in vivo studies, BSJ-5-63 exhibited potent antiproliferative effects in both AR-positive and AR-negative CRPC models. This study presents a promising multi-pronged approach for CRPC treatment, addressing both DNA repair mechanisms and AR signaling, with the potential to benefit a wide range of patients regardless of their BRCA1/2 mutational status. SIGNIFICANCE: This study introduces BSJ-5-63, a triple degrader designed to target CDK12, CDK7, and CDK9, making a significant advancement in CRPC therapy. The distinctive mechanism of BSJ-5-63 involves downregulating HRR genes and inhibiting AR signaling, thereby inducing a BRCAness state. This enhances sensitivity to PARP inhibition, effectively addressing ARSI resistance and improving the overall efficacy of treatment. The development of BSJ-5-63 represents a promising therapeutic approach, with the potential to benefit a broad spectrum of CRPC patients.
ABSTRACT
BACKGROUND: Fluorescence-based confocal microscopy (FCM) can be used to create virtual H&E sections in real time. So far, FCM has been used in dermato-, uro-, and gynecopathology. FCM allows the creation of a completely digitized frozen section, which could potentially replace conventional frozen sections in the future. OBJECTIVE: The aim of the current work is to implement FCM technology as a component of fully digitized processes in the pathological workflow. For this purpose, the current use of FCM in liver transplant pathology will be extended to other disciplines such as urology and otorhinolaryngology. MATERIALS AND METHODS: The FCM technique continues to be used prospectively on native tissue samples from potential donor livers. Conventional frozen sections are used comparatively to virtual FCM scans. RESULTS: The data show a nearly perfect agreement for the detection of cholangitis, fibrosis, and malignancy, and a high level of agreement for, e.g., macrovesicular steatosis, inflammation, steatohepatitis, and necrosis between virtual FCM scans and conventional routine diagnostic frozen sections. CONCLUSION: Since the availability of time- and cost-intensive frozen section diagnostics in the context of transplant pathology in continuous operation (24/7) is now only established at very few university centers in Germany due to an increasing shortage of specialists, the use of FCM could be an important building block in the current process leading towards a fully digitized pathology workflow and should thus be extended to various disciplines.
Subject(s)
Microscopy, Confocal , Microscopy, Confocal/methods , Humans , Liver Transplantation , Frozen Sections/methods , Microscopy, Fluorescence/methods , Liver/pathology , Liver/diagnostic imagingABSTRACT
BACKGROUND AND OBJECTIVE: With European Medicines Agency approval of PARP inhibitors in metastatic castration-resistant prostate cancer and ongoing trials in metastatic hormone-sensitive prostate cancer, detection of genetic alterations in BRCA1/2 and other homologous recombination repair genes has gained an important role. Our aim was to investigate the feasibility and comparability of comprehensive next-generation sequencing (NGS) of liquid biopsy (LB; circulating tumor DNA) and tumor tissue (TT) samples in a real-world clinical setting. METHODS: The study cohort consisted of 50 patients with metastatic prostate cancer (mPC) who had TT NGS performed for BRCA1/2 alterations and consent for additional LB NGS. The Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for TT NGS. The Guardant360 83-gene assay (Guardant Health, Palo Alto, CA, USA) was used for LB NGS, including all types of somatic alterations, microsatellite instability, and blood tumor mutational burden. We calculated BRCA1/2 alteration rates and the negative percentage agreement (NPA) and positive percentage agreement (PPA) between TT and LB results. KEY FINDINGS AND LIMITATIONS: TT NGS was successful in 44/50 patients (88%), with pathogenic BRCA1/2 alterations detected in four (9%). LB NGS was successful in all 50 patients (100%), with BRCA1/2 alterations detected in ten (20%). In a subgroup analysis for the 44 patients with successful TT NGS, NPA was 85% and PPA was 50%. The median time between TT sample collection and blood sampling for NGS was 132 wk (IQR 94-186). The limited sample size and differences in the time of NGS assessment are limitations. CONCLUSIONS AND CLINICAL IMPLICATIONS: LB NGS resulted in a higher detection rate for BRCA1/2 alterations in comparison to conventional TT NGS (20% vs 9%). Ideally, BRCA1/2 testing should be based on both approaches to identify all patients with mPC eligible for PARP inhibitor therapy. PATIENT SUMMARY: Our study shows that genetic tests for both tumor tissue and blood samples results in higher rates of detection of BRCA1/2 gene alterations in patients with metastatic prostate cancer.
Subject(s)
Circulating Tumor DNA , Feasibility Studies , High-Throughput Nucleotide Sequencing , Prostatic Neoplasms , Humans , Male , Liquid Biopsy , High-Throughput Nucleotide Sequencing/methods , Circulating Tumor DNA/blood , Circulating Tumor DNA/genetics , Aged , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/blood , Prostatic Neoplasms/drug therapy , Germany , Middle Aged , Neoplasm Metastasis , BRCA2 Protein/genetics , Aged, 80 and overABSTRACT
Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.
Subject(s)
Breast Neoplasms , Inflammatory Breast Neoplasms , Humans , Female , Inflammatory Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Signal Transduction , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Stem Cells/metabolism , STAT3 Transcription Factor/metabolismABSTRACT
Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/pathology , Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/metabolismABSTRACT
Treatment with the Bcl-2/Bcl-XL inhibitor ABT-737 is a promising novel strategy to therapeutically induce apoptotic cell death in malignant tumors such as glioblastomas. Although many studies have demonstrated that ABT-737 acts synergistically with chemotherapeutic drugs, the possibility of a combined treatment with ionizing radiation (IR) and ABT-737 has not yet been thoroughly investigated. Similarly, the relationship between p53 function and the pro-apoptotic effects of ABT-737 are still obscure. Here, we demonstrate that IR and ABT-737 synergistically induce apoptosis in glioblastoma cells. The sensitivity to ABT-737-mediated cell death is significantly increased by the IR-dependent accumulation of cells in the G2/M cell cycle phase. Wild type p53 function inhibits the efficacy of a combined IR and ABT-737 treatment via a p21-dependent G1 cell cycle arrest. Moreover, mutant as well as wild type p53 counteract the pro-apoptotic activity of ABT-737 by maintaining the expression levels of the Mcl-1 protein. Thus, p53 regulates the sensitivity to ABT-737 of glioblastoma cells. Our results warrant a further evaluation of a novel combination therapy using IR and ABT-737. The efficacy of such a therapy could be substantially enhanced by Mcl-1-lowering strategies.
Subject(s)
Apoptosis/drug effects , Apoptosis/radiation effects , Biphenyl Compounds/administration & dosage , Glioblastoma/metabolism , Nitrophenols/administration & dosage , Sulfonamides/administration & dosage , Tumor Suppressor Protein p53/metabolism , Apoptosis/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Piperazines/administration & dosage , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Small Interfering/genetics , Radiation, Ionizing , Tumor Suppressor Protein p53/genetics , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/geneticsABSTRACT
Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin DCDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptorpositive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin DCDK4/6 inhibitors in cancer treatment.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Neoplasms/drug therapy , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinogenesis , Cell Proliferation/drug effects , Cellular Senescence , Clinical Trials as Topic , Drug Resistance, Neoplasm , Drug Synergism , Humans , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.
Subject(s)
Autophagy , Brain Neoplasms/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic , Glioma/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Apoptosis , Apoptosis Regulatory Proteins , Cell Line, Tumor , Flow Cytometry , Humans , Models, Biological , Phosphorylation , RNA, Small Interfering/metabolism , TransfectionABSTRACT
Breast cancer (BC) is the most common cause of cancer-related death in women worldwide. Therapies targeting molecular pathways altered in BC had significantly enhanced treatment options for BC over the last decades, which ultimately improved the lives of millions of women worldwide. Among various molecular pathways accruing substantial interest for the development of targeted therapies are cyclin-dependent kinases (CDKs)-in particular, the two closely related members CDK4 and CDK6. CDK4/6 inhibitors indirectly trigger the dephosphorylation of retinoblastoma tumor suppressor protein by blocking CDK4/6, thereby blocking the cell cycle transition from the G1 to S phase. Although the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib gained FDA approval for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative BC as they significantly improved progression-free survival (PFS) in randomized clinical trials, regrettably, some patients showed resistance to these therapies. Though multiple molecular pathways could be mechanistically responsible for CDK4/6 inhibitor therapy resistance, one of the most predominant ones seems to be the fibroblast growth factor receptor (FGFR) pathway. FGFRs are involved in many aspects of cancer formation, such as cell proliferation, differentiation, and growth. Importantly, FGFRs are frequently mutated in BC, and their overexpression and/or hyperactivation correlates with CDK4/6 inhibitor resistance and shortened PFS in BC. Intriguingly, the inhibition of aberrant FGFR activity is capable of reversing the resistance to CDK4/6 inhibitors. This review summarizes the molecular background of FGFR signaling and discusses the role of aberrant FGFR signaling during cancer development in general and during the development of CDK4/6 inhibitor resistance in BC in particular, together with other possible mechanisms for resistance to CDK4/6 inhibitors. Subsequently, future directions on novel therapeutic strategies targeting FGFR signaling to overcome such resistance during BC treatment will be further debated.
Subject(s)
Breast Neoplasms/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Animals , Breast Neoplasms/genetics , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/genetics , Female , Humans , Receptor, Fibroblast Growth Factor, Type 1/genetics , Signal TransductionABSTRACT
Although inhibitors targeting CDK4/6 kinases (CDK4/6i) have shown promising clinical prospect in treating ER+/HER2- breast cancers, acquired drug resistance is frequently observed and mechanistic knowledge is needed to harness their full clinical potential. Here, we report that inhibition of CDK4/6 promotes ßTrCP1-mediated ubiquitination and proteasomal degradation of RB1, and facilitates SP1-mediated CDK6 transcriptional activation. Intriguingly, suppression of CK1ε not only efficiently prevents RB1 from degradation, but also prevents CDK4/6i-induced CDK6 upregulation by modulating SP1 protein stability, thereby enhancing CDK4/6i efficacy and overcoming resistance to CDK4/6i in vitro. Using xenograft and PDX models, we further demonstrate that combined inhibition of CK1ε and CDK4/6 results in marked suppression of tumor growth in vivo. Altogether, these results uncover the molecular mechanisms by which CDK4/6i treatment alters RB1 and CDK6 protein abundance, thereby driving the acquisition of CDK4/6i resistance. Importantly, we identify CK1ε as an effective target for potentiating the therapeutic efficacy of CDK4/6 inhibitors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/drug therapy , Casein Kinase 1 epsilon/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Protein Kinase Inhibitors/therapeutic use , Protein Stability/drug effects , Proteolysis/drug effects , Retinoblastoma Binding Proteins/metabolism , Sp1 Transcription Factor/metabolism , Transcriptional Activation/drug effects , Ubiquitin-Protein Ligases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Chemotherapy remains the main treatment option for patients with several tumor types. In this issue of Cancer Cell, Salvador-Barbero et al. demonstrate that treatment with CDK4/6 inhibitors after application of taxanes (or other chemotherapeutic compounds) strongly potentiates the anti-tumor effect due to repression of DNA repair machinery.
Subject(s)
Adenocarcinoma , Pancreatic Neoplasms , Cell Line, Tumor , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , HumansABSTRACT
Basal-like triple-negative breast cancers frequently express high levels of c-Myc. This oncoprotein signals to the core cell cycle machinery by impinging on cyclin E. High levels of E-type cyclins (E1 and E2) are often seen in human triple-negative breast tumors. In the current study, we examined the requirement for E-type cyclins in the c-Myc-driven mouse model of breast cancer (MMTV-c-Myc mice). To do so, we crossed cyclin E1- (E1-/-) and E2- (E2-/-) deficient mice with MMTV-c-Myc animals, and observed the resulting cyclin E1-/-/MMTV-c-Myc and cyclin E2-/-/MMTV-c-Myc females for breast cancer incidence. We found that mice lacking cyclins E1 or E2 developed breast cancers like their cyclin Ewild-type counterparts. In contrast, further reduction of the dosage of E-cyclins in cyclin E1-/-E2+/-/MMTV-c-Myc and cyclin E1+/-E2-/-/MMTV-c-Myc animals significantly decreased the incidence of mammary carcinomas, revealing arole for E-cyclins in tumor initiation. We also observed that depletion of E-cyclins in human triple-negative breast cancer cell lines halted cell cycle progression, indicating that E-cyclins are essential for tumor cell proliferation. In contrast, we found that the catalytic partner of E-cyclins, the cyclin-dependent kinase 2 (CDK2), is dispensable for the proliferation of these cells. These results indicate that E-cyclins, but not CDK2, play essential and rate-limiting roles in driving the proliferation of c-Myc overexpressing breast cancer cells.