ABSTRACT
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.
Subject(s)
Fanconi Anemia , Neoplasms , Humans , Cell Survival/genetics , Chromatin/genetics , Cyclin E/genetics , Cyclin E/metabolism , DNA Damage , DNA Repair , DNA Replication/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Neoplasms/geneticsABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , F-Box Proteins/metabolism , Medulloblastoma/metabolism , SOX9 Transcription Factor/metabolism , Ubiquitin-Protein Ligases/metabolism , Aniline Compounds/pharmacology , Animals , Benzamides , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Movement , Chromones/pharmacology , Cisplatin/pharmacology , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Glycogen Synthase Kinase 3/metabolism , HEK293 Cells , Humans , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Mice, Nude , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , SOX9 Transcription Factor/genetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle Proteins/genetics , DNA-Activated Protein Kinase/genetics , PTEN Phosphohydrolase/genetics , Protein-Tyrosine Kinases/genetics , Pyrazoles/pharmacology , Pyrimidinones/pharmacology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA-Activated Protein Kinase/metabolism , Female , Gene Expression Profiling , Humans , PTEN Phosphohydrolase/metabolism , Protein-Tyrosine Kinases/metabolism , ProteomeABSTRACT
The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC-induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X-linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B-cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event-free survival in patients treated with spindle poison-containing chemotherapy. Accordingly, aggressive B-cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eµ-Myc lymphoma model. Together, we specify the USP9X-XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B-cell lymphoma.
Subject(s)
Antineoplastic Agents/pharmacology , B-Lymphocytes/drug effects , Cell Death , Drug Resistance , Lymphoma, B-Cell/pathology , Ubiquitin Thiolesterase/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Animals , B-Lymphocytes/physiology , Cells, Cultured , Disease Models, Animal , Humans , Mice , Mitosis , Protein Processing, Post-Translational , Ubiquitin/metabolismABSTRACT
Plant seedlings have either one or two cotyledons. The mechanisms that regulate this organ number are poorly understood. Mutations in the RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) gene of the dicot Arabidopsis have only one cotyledon, with low penetrance due to complex genetic redundancy. An analysis of patterning genes required for cotyledon initiation showed that these have normal expression patterns, defining the cotyledon anlagen, in rpk1. This was also true for key genes, which organize the shoot apical meristem (SAM). By contrast, epidermal cell shape and polarity were compromised in rpk1 embryos, as evidenced by disturbed polarity of the auxin efflux carrier PIN1. PIN1 is required for the establishment of auxin maxima, which induce and maintain organ primordia. The effects in rpk1 mutants manifest in a spatially and timely stochastic fashion probably due to redundancy of RPK1-like functions. Consistently, auxin maxima showed a stochastic distribution in rpk1 embryos, being at times entirely absent and at other times supernumerary. This variability may explain how monocotyledonous seedlings and cotyledon shape variants can developmentally arise in Arabidopsis and possibly in other plants.