ABSTRACT
BACKGROUND: CC-115, a dual mTORC1/2 and DNA-PK inhibitor, has promising antitumour activity when combined with androgen receptor (AR) inhibition in pre-clinical models. METHODS: Phase 1b multicentre trial evaluating enzalutamide with escalating doses of CC-115 in AR inhibitor-naive mCRPC patients (n = 41). Primary endpoints were safety and RP2D. Secondary endpoints included PSA response, time-to-PSA progression, and radiographic progression. RESULTS: Common adverse effects included rash (31.7% Grades 1-2 (Gr); 31.7% Gr 3), pruritis (43.9% Gr 1-2), diarrhoea (37% Gr 1-2), and hypertension (17% Gr 1-2; 9.8% Gr 3). CC-115 RP2D was 5 mg twice a day. In 40 evaluable patients, 80% achieved ≥50% reduction in PSA (PSA50), and 58% achieved ≥90% reduction in PSA (PSA90) by 12 weeks. Median time-to-PSA progression was 14.7 months and median rPFS was 22.1 months. Stratification by PI3K alterations demonstrated a non-statistically significant trend towards improved PSA50 response (PSA50 of 94% vs. 67%, p = 0.08). Exploratory pre-clinical analysis suggested CC-115 inhibited mTOR pathway strongly, but may be insufficient to inhibit DNA-PK at RP2D. CONCLUSIONS: The combination of enzalutamide and CC-115 was well tolerated. A non-statistically significant trend towards improved PSA response was observed in patients harbouring PI3K pathway alterations, suggesting potential predictive biomarkers of response to a PI3K/AKT/mTOR pathway inhibitor. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02833883.
Subject(s)
Benzamides , Phenylthiohydantoin , Prostatic Neoplasms, Castration-Resistant , Pyrazines , Triazoles , Male , Humans , Prostatic Neoplasms, Castration-Resistant/pathology , Prostate-Specific Antigen/therapeutic use , Mechanistic Target of Rapamycin Complex 1 , Phosphatidylinositol 3-Kinases , Nitriles/therapeutic use , DNA/therapeutic useABSTRACT
The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (â¼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.
Subject(s)
Chromosome Deletion , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Prostate/cytology , Adaptor Proteins, Signal Transducing/metabolism , Animals , CRISPR-Associated Protein 9/genetics , Epithelial Cells , Genes, Tumor Suppressor , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Organoids , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Guide, Kinetoplastida , Ribonucleoproteins/genetics , Transcriptional Regulator ERG/genetics , Xenograft Model Antitumor AssaysABSTRACT
The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2â¶4â¶2 or 2â¶6â¶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA-protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST-DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA-binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA-binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.
Subject(s)
Candida glabrata/genetics , Cell Cycle Proteins , Fungal Proteins/chemistry , Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics , Telomere/genetics , Candida albicans/genetics , Candida albicans/metabolism , Candida glabrata/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , Protein Binding , Protein Folding , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Telomere/metabolism , Telomere-Binding Proteins/metabolismABSTRACT
Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.
Subject(s)
Prostatic Neoplasms , Animals , Humans , Male , Mice , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Lysosomes/metabolism , Mice, Knockout , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Prostatic Neoplasms/pathology , UbiquitinationABSTRACT
Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)18, (CUG)24 and (AAG)24 RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1(236X) protein. Heterologous ClC1(236X) expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C2C12 cells, the clc1 splice variants generated by (CCUG)18-RNA resembled those in DM2 muscle and differed from those generated by (CUG)24 and (AAG)24. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1(236X) splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C2C12 model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.
Subject(s)
Chloride Channels/genetics , Myotonic Disorders/genetics , Adult , Blotting, Western , Female , Humans , In Vitro Techniques , Male , Microscopy, Confocal , Myotonic Dystrophy , Patch-Clamp Techniques , Protein Transport , RNA Splicing , Real-Time Polymerase Chain ReactionABSTRACT
Previous studies have suggested that PTEN loss is associated with p110ß signaling dependency, leading to the clinical development of p110ß-selective inhibitors. Here we use a panel pre-clinical models to reveal that PI3K isoform dependency is not governed by loss of PTEN and is impacted by feedback inhibition and concurrent PIK3CA/PIK3CB alterations. Furthermore, while pan-PI3K inhibition in PTEN-deficient tumors is efficacious, upregulation of Insulin Like Growth Factor 1 Receptor (IGF1R) promotes resistance. Importantly, we show that this resistance can be overcome through targeting AKT and we find that AKT inhibitors are superior to pan-PI3K inhibition in the context of PTEN loss. However, in the presence of wild-type PTEN and PIK3CA-activating mutations, p110α-dependent signaling is dominant and selectively inhibiting p110α is therapeutically superior to AKT inhibition. These discoveries reveal a more nuanced understanding of PI3K isoform dependency and unveil novel strategies to selectively target PI3K signaling nodes in a context-specific manner.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Signal Transduction , Animals , Cell Line, Tumor , Feedback, Physiological , Humans , Isoenzymes/metabolism , Male , Mice , Models, Biological , Organoids/drug effects , Organoids/metabolism , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Receptor, IGF Type 1/metabolism , Up-Regulation/drug effectsABSTRACT
Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K-mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered ß-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors. IMPLICATIONS: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of ß-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial-mesenchymal transition.
Subject(s)
Breast Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , beta Catenin/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/physiology , Female , Humans , Mutation , Proto-Oncogene Proteins c-akt/genetics , Signal TransductionABSTRACT
Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yeast, or Rqh1 in fission yeast leads to inappropriate recombination, chromosome abnormalities, and disturbed replication fork progression. Studies with yeasts have demonstrated that auxiliary gene functions can contribute in overlapping ways with Sgs1 or Rqh1 to circumvent or overcome lesions in DNA caused by certain genotoxic agents. In the combined absence of these functions, recombination-mediated processes lead to severe loss of fitness. Here we performed a genetic study to determine the role of the Ustilago maydis Blm homolog in DNA repair and in alleviating replication stress. We characterized the single mutant as well as double mutants additionally deleted of genes encoding Srs2, Fbh1, Mus81, or Exo1. Unlike yeasts, neither the blm srs2, blm exo1, nor blm mus81 double mutant exhibited extreme loss of fitness. Inactivation of Brh2, the BRCA2 homolog, suppressed toxicity to hydroxyurea caused by loss of Blm function. However, differential suppression by Brh2 derivatives lacking the canonical DNA-binding region suggests that the particular domain structure comprising this DNA-binding region may be instrumental in promoting the observed hydroxyurea toxicity.
Subject(s)
DNA Replication , Fungal Proteins/genetics , RecQ Helicases/genetics , Recombination, Genetic , Ustilago/genetics , DNA Repair , Exodeoxyribonucleases/genetics , Hydroxyurea/pharmacology , Mutation/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Rad51 Recombinase/geneticsABSTRACT
Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution. SIGNIFICANCE: This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.
Subject(s)
Insulin Receptor Substrate Proteins/genetics , Oncogene Proteins/metabolism , Prostatic Neoplasms/genetics , Transcriptional Regulator ERG/metabolism , Animals , Carcinogenesis/genetics , Cell Line, Tumor , DNA Copy Number Variations , Disease Models, Animal , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Gene Knockout Techniques , Gene Rearrangement , Humans , Insulin Receptor Substrate Proteins/metabolism , Male , Mice , Oncogene Proteins/genetics , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Primary Cell Culture , Promoter Regions, Genetic/genetics , Prostate/pathology , Prostatic Neoplasms/pathology , RNA-Seq , Signal Transduction/genetics , Transcriptional Regulator ERG/genetics , Xenograft Model Antitumor AssaysABSTRACT
Although cancer is commonly perceived as a disease of dedifferentiation, the hallmark of early-stage prostate cancer is paradoxically the loss of more plastic basal cells and the abnormal proliferation of more differentiated secretory luminal cells. However, the mechanism of prostate cancer proluminal differentiation is largely unknown. Through integrating analysis of the transcription factors (TFs) from 806 human prostate cancers, we found that ERG was highly correlated with prostate cancer luminal subtyping. ERG overexpression in luminal epithelial cells inhibited those cells' normal plasticity to transdifferentiate into a basal lineage, and ERG superseded PTEN loss, which favored basal differentiation. ERG KO disrupted prostate cell luminal differentiation, whereas AR KO had no such effects. Trp63 is a known master regulator of the prostate basal lineage. Through analysis of 3D chromatin architecture, we found that ERG bound and inhibited the enhancer activity and chromatin looping of a Trp63 distal enhancer, thereby silencing its gene expression. Specific deletion of the distal ERG binding site resulted in the loss of ERG-mediated inhibition of basal differentiation. Thus, ERG, in its fundamental role in lineage differentiation in prostate cancer initiation, orchestrated chromatin interactions and regulated prostate cell lineage toward a proluminal program.
Subject(s)
Cellular Reprogramming , Epithelial Cells/metabolism , Oncogene Proteins/metabolism , Prostatic Neoplasms/metabolism , Transcriptional Regulator ERG/metabolism , Animals , Epithelial Cells/pathology , Gene Knockout Techniques , Male , Mice , Mice, Transgenic , Oncogene Proteins/genetics , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Transcriptional Regulator ERG/geneticsABSTRACT
Despite the development of second-generation antiandrogens, acquired resistance to hormone therapy remains a major challenge in treating advanced prostate cancer. We find that cancer-associated fibroblasts (CAFs) can promote antiandrogen resistance in mouse models and in prostate organoid cultures. We identify neuregulin 1 (NRG1) in CAF supernatant, which promotes resistance in tumor cells through activation of HER3. Pharmacological blockade of the NRG1/HER3 axis using clinical-grade blocking antibodies re-sensitizes tumors to hormone deprivation in vitro and in vivo. Furthermore, patients with castration-resistant prostate cancer with increased tumor NRG1 activity have an inferior response to second-generation antiandrogen therapy. This work reveals a paracrine mechanism of antiandrogen resistance in prostate cancer amenable to clinical testing using available targeted therapies.
Subject(s)
Androgen Antagonists/pharmacology , Drug Resistance, Neoplasm/genetics , Neuregulin-1/genetics , Prostatic Neoplasms/genetics , Tumor Microenvironment/genetics , Animals , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kaplan-Meier Estimate , Male , Mice, SCID , Neuregulin-1/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/prevention & control , Tumor Microenvironment/drug effects , Xenograft Model Antitumor Assays/methodsABSTRACT
Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.
Subject(s)
DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/chemistry , Ustilago/genetics , BRCA2 Protein/chemistry , DNA Damage/genetics , DNA Repair/genetics , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombination, GeneticABSTRACT
A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.
Subject(s)
DNA Repair , Fungal Proteins/metabolism , Rad52 DNA Repair and Recombination Protein/metabolism , Rec A Recombinases/metabolism , Recombination, Genetic , Ustilago/metabolism , Amino Acid Sequence , Animals , Fungal Proteins/genetics , Fungal Proteins/radiation effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Molecular Sequence Data , Mutagenesis , Phenotype , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Rad51 Recombinase/radiation effects , Rad52 DNA Repair and Recombination Protein/genetics , Rec A Recombinases/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Ultraviolet Rays , Ustilago/genetics , Ustilago/growth & development , Ustilago/radiation effectsABSTRACT
On the basis of our previous work defining the molecular rationale for combined targeting of the PI3K and AR pathways in PTEN loss prostate cancer, the first clinical trial was recently reported demonstrating a significant benefit for combination therapy in patients with metastatic prostate cancer. In this phase II trial, loss of PTEN was a biomarker predictive of response to combined AKT and AR inhibition. Given that PTEN loss prostate cancers are significantly enriched for ERG genomic rearrangements, we evaluated how the aberrant expression of ERG may impact response to PI3K/AR-targeted therapy. Here, we show that overexpression of ERG in the setting of Pten loss promotes resistance to combined PI3K and AR pathway inhibition with associated maintenance of AR target gene expression. Importantly, following AR knockout in the setting of ERG overexpression, there is maintenance of a subset of AR lineage-specific target genes, making AR dispensable in this context. This has important clinical implications as even in the setting of the androgen-regulated TMPRSS2:ERG genomic rearrangement, ERG expression is never abolished following AR inhibition and may allow for cell survival following AR (lineage)-targeted therapies.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Animals , Benzamides , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Imidazoles/pharmacology , Male , Mice, Knockout , Mice, SCID , Mice, Transgenic , Nitriles , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Quinolines/pharmacology , Receptors, Androgen/genetics , Signal Transduction/genetics , Transcriptional Regulator ERG/genetics , Transcriptional Regulator ERG/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulating the action of Rad51. In turn, BRCA2 appears to be regulated by other interacting proteins. Dss1, a small interacting protein that binds to the C-terminal domain, has a profound effect on activity as deduced from studies on the BRCA2-related protein Brh2 in Ustilago maydis. Evidence accumulating in mammalian systems suggests that BCCIP, another small interacting protein that binds to the C-terminal domain of BRCA2, also serves to regulate homologous recombination activity. Here we were interested in testing the role of the putative U. maydis BCCIP ortholog Bcp1 in DNA repair and recombination. In keeping with the mammalian paradigm, Bcp1 bound to the C-terminal region of Brh2. Mutants deleted of the gene were extremely slow growing, showed a delay passing through S phase and exhibited sensitivity to hydroxyurea, but were otherwise normal in DNA repair and homologous recombination. In the absence of Bcp1 cells were unable to maintain the wild type morphology when challenged by a DNA replication stress. These results suggest that Bcp1 could be involved in coordinating morphogenetic events with DNA processing during replication.
Subject(s)
DNA Replication/physiology , Fungal Proteins/physiology , Ustilago/growth & development , Amino Acid Sequence , Fungal Proteins/genetics , Fungal Proteins/metabolism , Molecular Sequence Data , Morphogenesis , Recombination, Genetic , Sequence Alignment , Ustilago/metabolismABSTRACT
Prostate-specific membrane antigen (PSMA) or folate hydrolase 1 (FOLH1) is highly expressed on prostate cancer. Its expression correlates inversely with survival and increases with tumor grade. However, the biological role of PSMA has not been explored, and its role in prostate cancer remained elusive. Filling this gap, we demonstrate that in prostate cancer, PSMA initiates signaling upstream of PI3K through G protein-coupled receptors, specifically via the metabotropic glutamate receptor (mGluR). PSMA's carboxypeptidase activity releases glutamate from vitamin B9 and other glutamated substrates, which activate mGluR I. Activated mGluR I subsequently induces activation of phosphoinositide 3-kinase (PI3K) through phosphorylation of p110ß independent of PTEN loss. The p110ß isoform of PI3K plays a particularly important role in the pathogenesis of prostate cancer, but the origin of its activation was so far unknown. PSMA expression correlated with PI3K-Akt signaling in cells, animal models, and patients. We interrogated the activity of the PSMA-PI3K axis through positron emission tomography and magnetic resonance imaging. Inhibition of PSMA in preclinical models inhibited PI3K signaling and promoted tumor regression. Our data present a novel oncogenic signaling role of PSMA that can be exploited for therapy and interrogated with imaging.
ABSTRACT
A multigenic locus at 3p13-14, spanning FOXP1 to SHQ1, is commonly deleted in prostate cancer and lost broadly in a range of cancers but has unknown significance to oncogenesis or prognosis. Here, we report that FOXP1-SHQ1 deletion cooperates with PTEN loss to accelerate prostate oncogenesis and that loss of component genes correlates with prostate, breast, and head and neck cancer recurrence. We demonstrate that Foxp1-Shq1 deletion accelerates prostate tumorigenesis in mice in combination with Pten loss, consistent with the association of FOXP1-SHQ1 and PTEN loss observed in human cancers. Tumors with combined Foxp1-Shq1 and Pten deletion show increased proliferation and anaplastic dedifferentiation, as well as mTORC1 hyperactivation with reduced Akt phosphorylation. Foxp1-Shq1 deletion restores expression of AR target genes repressed in tumors with Pten loss, circumventing PI3K-mediated repression of the androgen axis. Moreover, FOXP1-SHQ1 deletion has prognostic relevance, with cancer recurrence associated with combined loss of PTEN and FOXP1-SHQ1 genes.
Subject(s)
Carrier Proteins/metabolism , Forkhead Transcription Factors/metabolism , PTEN Phosphohydrolase/metabolism , Repressor Proteins/metabolism , Animals , Carrier Proteins/genetics , Forkhead Transcription Factors/genetics , Humans , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Prostate/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Repressor Proteins/geneticsABSTRACT
Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Lung Neoplasms/diet therapy , Mutation , Adenocarcinoma/enzymology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolismABSTRACT
Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.