DARPP-32 and t-DARPP promote non-small cell lung cancer growth through regulation of IKKα-dependent cell migration.
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ABSTRACT:
SUBMITTER: Alam SK
PROVIDER: S-EPMC5959014 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundSmall cell lung cancer (SCLC) is the most aggressive form of lung cancer, and new molecular insights are necessary for prognostic and therapeutic advances.MethodsDopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its N-terminally truncated splice variant, t-DARPP, were stably overexpressed or ablated in human DMS-53 and H1048 SCLC cells. Functional assays and immunoblotting were used to assess how DARPP-32 isoforms regulate SCLC cell growth, proliferation, and apoptosis. DARPP-32-modulated SCLC cells were orthotopically injected into the lungs of SCID mice to evaluate how DARPP-32 and t-DARPP regulate neuroendocrine tumour growth. Immunostaining for DARPP-32 proteins was performed in SCLC patient-derived specimens. Bioinformatics analysis and subsequent transcription assays were used to determine the mechanistic basis of DARPP-32-regulated SCLC growth.ResultsWe demonstrate in mice that DARPP-32 and t-DARPP promote SCLC growth through increased Akt/Erk-mediated proliferation and anti-apoptotic signalling. DARPP-32 isoforms are overexpressed in SCLC patient-derived tumour tissue, but undetectable in physiologically normal lung. Achaete-scute homologue 1 (ASCL1) transcriptionally activates DARPP-32 isoforms in human SCLC cells.ConclusionsWe reveal new regulatory mechanisms of SCLC oncogenesis that suggest DARPP-32 isoforms may represent a negative prognostic indicator for SCLC and serve as a potential target for the development of new therapies.
| S-EPMC7463034 | biostudies-literature
Project description:DARPP-32 is a dual-function protein kinase/phosphatase inhibitor that is involved in striatal signaling. The phosphorylation of DARPP-32 at threonine 34 is essential for mediating the effects of both psychostimulant and antipsychotic drugs; however, these drugs are known to have opposing behavioral and clinical effects. We hypothesized that these drugs exert differential effects on striatonigral and striatopallidal neurons, which comprise distinct output pathways of the basal ganglia. To directly test this idea, we developed bacterial artificial chromosome transgenic mice that allowed the analysis of DARPP-32 phosphorylation selectively in striatonigral and striatopallidal neurons. Using this new methodology, we found that cocaine, a psychostimulant, and haloperidol, a sedation-producing antipsychotic, exert differential effects on DARPP-32 phosphorylation in the two neuronal populations that can explain their opposing behavioral effects. Furthermore, we found that a variety of drugs that target the striatum have cell type-specific effects that previous methods were not able to discern.
| S-EPMC2737705 | biostudies-literature
Project description:CD44E is a frequently overexpressed variant of CD44 in gastric cancer. Mechanisms that regulate CD44 splicing and expression in gastric cancer remain unknown. Herein, we investigated the role of DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000) in promoting tumor growth through regulation of CD44 splicing. Using western blot and quantitative real-time PCR analysis, our results indicated that knockdown of endogenous DARPP-32 markedly reduces the expression of CD44 V8-V10 (CD44E). Using a quantitative splicing luciferase reporter system, we detected a significant increase in the reporter activity following DARPP-32 overexpression (P<0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (P<0.001). Further experiments showed that DARPP-32 regulates the expression of SRp20 splicing factor and co-exists with it in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 has an important role in regulating SRp20 protein stability. The knockdown of endogenous DARPP-32 confirmed that DARPP-32 regulates the SRp20-dependent CD44E splicing. Using tumor xenograft mouse model, knocking down endogenous DARPP-32 markedly reduced SRp20 and CD44E protein levels with a decreased tumor growth. The reconstitution of SRp20 expression in these cells rescued tumor growth. In addition, we also demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E expression levels in human gastric primary tumors. Our novel findings establish for the first time the role of DARPP-32 in regulating splicing factors in gastric cancer cells. The DARPP-32-SRp20 axis has a key role in regulating the CD44E splice variant that promotes gastric tumorigenesis.
| S-EPMC4486340 | biostudies-literature
Project description:ObjectiveOverexpression of dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated isoform (t-DARPP) are associated with gastric tumorigenesis. Herein, we investigated the role of DARPP-32 proteins in regulating angiopoietin 2 (ANGPT2) and promoting tumour angiogenesis.DesignQuantitative real-time RT-PCR, immunoblotting, luciferase reporter, immunofluorescence, immunohistochemistry and angiogenesis assays were applied to investigate the regulation of angiogenesis by DARPP-32 proteins.ResultsOverexpression of DARPP-32 significantly increased the mRNA and protein levels of ANGPT2 in gastric cancer cells. The overexpression of DARPP-32 T34A mutant or the N-terminal truncated isoform, t-DARPP, led to similar effects ruling out the T34-dependent regulation of protein phosphatase 1 activity in regulating ANGPT2. DARPP-32 proteins induced a secreted form of ANGPT2, which was detectable in the media, functionally active, and able to induce angiogenesis, measured by the human umbilical vein endothelial cells tube formation assay. Antibody blocking of the secreted ANGPT2 abrogated its function. To identify the mechanism by which DARPP-32 regulates ANGPT2, we examined the activities of NF-κB and signal transducer and activator of transcription 3 (STAT3), known regulators of angiogenesis. The results ruled out NF-κB and showed induction of STAT3 phosphorylation, activation and nuclear localisation. Inhibition or knockdown of STAT3 significantly attenuated the induction of ANGPT2 by DARPP-32 proteins. In vivo xenograft models demonstrated that overexpression of DARPP-32 promotes angiogenesis and tumour growth. Analyses of human gastric cancer tissues showed a strong correlation between DARPP-32 and ANGPT2.ConclusionsOur novel findings establish the role of DARPP-32-STAT3 axis in regulating ANGPT2 in cancer cells to promote angiogenesis and tumorigenesis.
| S-EPMC4573388 | biostudies-literature
Project description:Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay, DARPP-32-overexpressing AGS cells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.
| S-EPMC3552044 | biostudies-literature
Project description:Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC.
| S-EPMC6940020 | biostudies-literature
Project description:Dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32), also known as phosphoprotein phosphatase-1 regulatory subunit 1B (PPP1R1B), was initially discovered as a substrate of dopamine-activated protein kinase A (PKA) in the neostriatum in the brain. While phosphorylation at Thr-34 by PKA converts DARPP-32 into a potent inhibitor of protein phosphatase 1 (PP1), phosphorylation at Thr-75 transforms DARPP-32 into an inhibitor of PKA. Through regulation of DARPP-32 phosphorylation and modulation of protein phosphatase and kinase activities, DARPP-32 plays a critical role in mediating the biochemical, electrophysiological, and behavioral effects controlled by dopamine and other neurotransmitters in response to drugs of abuse and psychostimulants. Altered expression of DARPP-32 and its truncated isoform (t-DARPP), specifically in the prefrontal cortex, has been associated with schizophrenia and bipolar disorder. Moreover, cleavage of DARPP-32 by calpain has been implicated in Alzheimer's disease. Amplification of the genomic locus of DARPP-32 at 17q12 has been described in several cancers. DARPP-32 and t-DARPP are frequently overexpressed at the mRNA and protein levels in adenocarcinomas of the breast, prostate, colon, and stomach. Several studies demonstrated the pro-survival, pro-invasion, and pro-angiogenic functions of DARPP-32 in cancer. Overexpression of DARPP-32 and t-DARPP also promotes chemotherapeutic drug resistance and cell proliferation in gastric and breast cancers through regulation of pro-oncogenic signal transduction pathways. The expansion of DARPP-32 research from neurotransmission to cancer underscores the broad scope and implication of this protein in disparate human diseases.
| S-EPMC4951238 | biostudies-literature
Project description:CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.
| S-EPMC4096130 | biostudies-literature
Project description:Bcl-2 interacts with the inositol 1,4,5-trisphosphate receptor (InsP3R) and thus prevents InsP3-induced Ca(2+) elevation that induces apoptosis. Here we report that Bcl-2 binds dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a protein kinase A (PKA)-activated and calcineurin (CaN)-deactivated inhibitor of protein phosphatase 1 (PP1). Bcl-2 docks DARPP-32 and CaN in a complex on the InsP3R, creating a negative feedback loop that prevents exaggerated Ca(2+) release by decreasing PKA-mediated InsP3R phosphorylation. T-cell activation increases PKA activity, phosphorylating both the InsP3R and DARPP-32. Phosphorylated DARPP-32 inhibits PP1, enhancing InsP3R phosphorylation and Ca(2+) release. Elevated Ca(2+) activates CaN, which dephosphorylates DARPP-32 to dampen Ca(2+) release by eliminating PP1 inhibition to enable it to dephosphorylate the InsP3R. Knocking down either Bcl-2 or DARPP-32 abrogates this feedback mechanism, resulting in increased Ca(2+) elevation and apoptosis. This feedback mechanism appears to be exploited by high levels of Bcl-2 in chronic lymphocytic leukemia cells, repressing B-cell receptor-induced Ca(2+) elevation and apoptosis.
| S-EPMC3903247 | biostudies-literature
Project description:BackgroundHerceptin (trastuzumab) is a humanized monoclonal antibody that is approved for the treatment of metastatic breast cancer patients whose tumors overexpress Her2 (erbB2/neu). Up to 70% of Her2-positive breast cancers demonstrate a response to Herceptin-based therapies, but resistance almost inevitably arises within a year of the initial response. To help understand the mechanism of Herceptin resistance, we isolated clonal variants of Her2-positive BT474 human breast cancer cells (BT/Her(R)) that are highly resistant to Herceptin. These cell lines exhibit sustained PI3K/Akt signaling as an essential component of Herceptin-resistant proliferation. Several genes in the protein kinase A (PKA) signaling network have altered expression in BT/Her(R) cells, including PPP1R1B, which encodes a 32 kDa protein known as Darpp-32 and its amino-terminal truncated variant, t-Darpp. The purpose of the current work was to determine the role of Darpp-32 and t-Darpp in Herceptin resistance.Methodology and resultsWe determined expression of Darpp-32 and t-Darpp in BT/Her(R) cells selected for resistance to Herceptin. Subsequently, cDNAs encoding the two isoforms of Darpp-32 were transfected, separately and together, into Her2-positive SK-Br-3 breast cancer cells. Transfected cells were tested for resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. DNA binding activity by the cAMP response element binding protein (CREB) was also measured. We found that BT/Her(R) cells overexpressed t-Darpp but not Darpp-32. Moreover, t-Darpp overexpression in SK-Br-3 cells was sufficient for conferring resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. Darpp-32 co-expression reversed t-Darpp's effects on Herceptin resistance and Akt phosphorylation. t-Darpp overexpression led to increased CREB binding activity, which was also reversible by Darpp-32.Conclusionst-Darpp and Darpp-32 appear to have antagonistic effects on Herceptin resistance. We present a unified model by which these effects might be mediated via the PKA regulatory network.
| S-EPMC2704867 | biostudies-literature