RESUMO
Repetitive elements (REs) compose â¼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.
Assuntos
Replicação do DNA/genética , Proteínas F-Box/metabolismo , Neoplasias/genética , Sequências Repetitivas de Ácido Nucleico/genética , Adulto , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade , Interferons/metabolismo , Lisina/metabolismo , Masculino , Metilação , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Neoplasias/imunologia , Nucleossomos/metabolismo , Transdução de Sinais , Transcrição Gênica , Resultado do TratamentoRESUMO
Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.
Assuntos
Anemia de Fanconi , Neoplasias , Humanos , Sobrevivência Celular/genética , Cromatina/genética , Ciclina E/genética , Ciclina E/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Neoplasias/genéticaRESUMO
A mesenchymal tumor phenotype associates with immunotherapy resistance, although the mechanism is unclear. Here, we identified FBXO7 as a maintenance regulator of mesenchymal and immune evasion phenotypes of cancer cells. FBXO7 bound and stabilized SIX1 co-transcriptional regulator EYA2, stimulating mesenchymal gene expression and suppressing IFNα/ß, chemokines CXCL9/10, and antigen presentation machinery, driven by AXL extracellular ligand GAS6. Ubiquitin ligase SCFFBXW7 antagonized this pathway by promoting EYA2 degradation. Targeting EYA2 Tyr phosphatase activity decreased mesenchymal phenotypes and enhanced cancer cell immunogenicity, resulting in attenuated tumor growth and metastasis, increased infiltration of cytotoxic T and NK cells, and enhanced anti-PD-1 therapy response in mouse tumor models. FBXO7 expression correlated with mesenchymal and immune-suppressive signatures in patients with cancer. An FBXO7-immune gene signature predicted immunotherapy responses. Collectively, the FBXO7/EYA2-SCFFBXW7 axis maintains mesenchymal and immune evasion phenotypes of cancer cells, providing rationale to evaluate FBXO7/EYA2 inhibitors in combination with immune-based therapies to enhance onco-immunotherapy responses.
Assuntos
Proteínas F-Box , Proteína 7 com Repetições F-Box-WD , Neoplasias , Animais , Linhagem Celular Tumoral , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD/genética , Proteínas de Homeodomínio/genética , Humanos , Evasão da Resposta Imune , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Tirosina Fosfatases/genética , Ubiquitina/metabolismoRESUMO
SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Meduloblastoma/metabolismo , Fatores de Transcrição SOX9/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Compostos de Anilina/farmacologia , Animais , Benzamidas , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Cromonas/farmacologia , Cisplatino/farmacologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Camundongos Nus , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Fatores de Transcrição SOX9/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that ß-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of ß-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In ß-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type ß-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of ß-actin in Pol I transcription. The rRNA synthesis defects in the ß-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (monomethylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated ß-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.-Almuzzaini, B., Sarshad, A. A. , Rahmanto, A. S., Hansson, M. L., Von Euler, A., Sangfelt, O., Visa, N., Farrants, A.-K. Ö., Percipalle, P. In ß-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.
Assuntos
Actinas/metabolismo , Reprogramação Celular/fisiologia , DNA Ribossômico/metabolismo , Epigênese Genética/fisiologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Actinas/genética , Animais , Células Cultivadas , Cromatina , DNA Ribossômico/genética , Camundongos , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/fisiologia , Transcrição Gênica/fisiologiaRESUMO
BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Proteína de Leucina Linfoide-Mieloide/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genéticaRESUMO
Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , NF-kappa B/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Apoptose/genética , Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Deleção Cromossômica , Cromossomos Humanos Par 13 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mieloma Múltiplo/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse. SIGNIFICANCE: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Humanos , Camundongos , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cutaneous apocrine carcinoma is an extreme rare malignancy derived from a sweat gland. Histologically sweat gland cancers resemble metastatic mammary apocrine carcinomas, but the genetic landscape remains poorly understood. Here, we report a rare metastatic case with a PALB2 aberration identified previously as a familial susceptibility gene for breast cancer in the Finnish population. As PALB2 exhibits functions in the BRCA1/2-RAD51-dependent homologous DNA recombination repair pathway, we sought to use ex vivo functional screening to explore sensitivity of the tumor cells to therapeutic targeting of DNA repair. Drug screening suggested sensitivity of the PALB2 deficient cells to BET-bromodomain inhibition, and modest sensitivity to DNA-PKi, ATRi, WEE1i and PARPi. A phenotypic RNAi screen of 300 DNA repair genes was undertaken to assess DNA repair targeting in more detail. Core members of the HR and MMEJ pathways were identified to be essential for viability of the cells. RNAi inhibition of RAD52-dependent HR on the other hand potentiated the efficacy of a novel BETi ODM-207. Together these results describe the first ever CAC case with a BRCAness genetic background, evaluate combinatorial DNA repair targeting, and provide a data resource for further analyses of DNA repair targeting in PALB2 deficient cancers.
RESUMO
BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Neoplasias Cutâneas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vemurafenib/farmacologia , Melanoma Maligno CutâneoRESUMO
Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.
Assuntos
Carcinogênese/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Proteases Específicas de Ubiquitina/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Neuroblastoma/genética , Neuroblastoma/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Vorinostat/farmacologia , Peixe-Zebra/genéticaRESUMO
INTRODUCTION: Mutational inactivation of the FBXW7/hCDC4 tumor suppressor gene (TSG) is common in many cancer types, but infrequent in breast cancers. This study investigates the presence and impact of FBXW7/hCDC4 promoter methylation in breast cancer. METHODS: FBXW7/hCDC4-ß expression and promoter methylation was assessed in 161 tumors from two independent breast cancer cohorts. Associations between methylation status and clinicopathologic characteristics were assessed by Fisher's exact test. Survival was analyzed using the Kaplan-Meier method in addition to modeling the risk by use of a multivariate proportional hazard (Cox) model adjusting for possible confounders of survival. RESULTS: Methylation of the promoter and loss of mRNA expression was found both in cell lines and primary tumors (43% and 51%, respectively). Using Cox modeling, a trend was found towards decreased hazard ratio (HR) for death in women with methylation of FBXW7/hCDC4-ß in both cohorts (HR 0.53 (95% CI 0.23 to 1.23) and HR 0.50 (95% CI 0.23 to 1.08), respectively), despite an association between methylation and high-grade tumors (P = 0.017). Interestingly, in subgroups of patients whose tumors are p53 mutated or lymph-node positive, promoter methylation identified patients with significantly improved survival (P = 0.048 and P = 0.017, respectively). CONCLUSIONS: We demonstrate an alternative mechanism for inactivation of the TSG FBXW7/hCDC4, namely promoter specific methylation. Importantly, in breast cancer, methylation of FBXW7/hCDC4-ß is related to favorable prognosis despite its association with poorly differentiated tumors. Future work may define whether FBXW7/hCDC4 methylation is a biomarker of the response to chemotherapy and a target for epigenetic modulation therapy.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Metilação de DNA , Proteínas F-Box/genética , Regiões Promotoras Genéticas , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Ilhas de CpG , Proteína 7 com Repetições F-Box-WD , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes p53 , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Análise Multivariada , Reação em Cadeia da Polimerase , Prognóstico , RNA Mensageiro/genética , SobrevidaRESUMO
E-type cyclins (E1 and E2) regulate the S phase program in the mammalian cell division cycle. Expression of cyclin E1 and E2 is frequently deregulated in a variety of cancer types and a wealth of experimental evidence supports an oncogenic role of these proteins in human tumorigenesis. Although the molecular mechanisms responsible for cyclin E1 deregulation in cancer are well defined, little is known regarding cyclin E2. Here we report that cyclin E2 is targeted for ubiquitin-dependent proteolysis by the ubiquitin ligase SCF(Fbxw7/hCdc4). Ubiquitylation is triggered by phosphorylation of cyclin E2 on residues Thr392 and Ser396, and to a lesser extent Thr74, contained in two consensus Cdc4-phosphodegrons. Furthermore, we found that ectopic expression of cyclin E1 enhances the ubiquitin-dependent proteolysis of cyclin E2 in vivo, suggesting a potential cross-talk in the regulation of E-type cyclin activity. Since SCF(Fbxw7/hCdc4) is functionally inactivated in several human cancer types, alteration of this molecular pathway could contribute to the deregulation of cyclin E2 in tumorigenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Ciclo Celular , Linhagem Celular Tumoral , Ciclina E/antagonistas & inibidores , Ciclina E/genética , Ciclina E/metabolismo , Ciclinas/química , Ciclinas/genética , Proteína 7 com Repetições F-Box-WD , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Especificidade por Substrato , Treonina/químicaRESUMO
The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.
Assuntos
Ciclo Celular/genética , MicroRNAs/genética , Neoplasias/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Medula Óssea/fisiologia , Linhagem Celular , Cromatina/genética , Ensaio de Unidades Formadoras de Colônias , Ciclina D1/genética , Ciclina E/genética , Ciclinas/genética , DNA/genética , Primers do DNA , Citometria de Fluxo , Deleção de Genes , Humanos , Rim/embriologia , MicroRNAs/fisiologia , Reação em Cadeia da Polimerase , RNA/genética , RNA Longo não Codificante , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransferasesRESUMO
RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/química , Proteína com ValosinaRESUMO
Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
Assuntos
Mitocôndrias/genética , Mitofagia/genética , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Poliubiquitina/genética , Proteína Sequestossoma-1/genética , Ubiquitinação/genéticaRESUMO
Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteína Quinase Ativada por DNA/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Tirosina Quinases/genética , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Tirosina Quinases/metabolismo , ProteomaRESUMO
Epithelial-myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial-myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial-myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC.
Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Everolimo/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular , Mutação , Mioepitelioma/tratamento farmacológico , Neoplasias das Glândulas Salivares/tratamento farmacológico , Adulto , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/patologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Mioepitelioma/genética , Mioepitelioma/patologia , Prognóstico , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.