Dopamine And cAMP-regulated Phosphoprotein, Mr 32000 Research Articles

Abstract 5956: New molecular mechanisms of small cell lung cancer growth

Abstract Lung cancer is the leading cause of cancer death among both men and women in the United States. Small cell lung cancer (SCLC) results in over 30,000 deaths of Americans annually and comprises 15-17% incidences of lung cancer. SCLC is the most aggressive form of lung cancer due to its rapid doubling time and early widespread metastasis. Most SCLC patients present with advanced disease, respond to initial systemic chemotherapy, and then treatment refractory progression usually occurs within one year due to acquired drug resistance. Consequently, the median survival time of SCLC patients is only 9 to 20 months and merely 7% of SCLC patients survive beyond five years. A better molecular understanding of SCLC is necessary to develop much needed new treatment strategies. For the first time, we demonstrate that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its splice variant t-DARPP stimulate tumors of neuroendocrine origin, and we show DARPP-32 proteins are aberrantly overexpressed in human SCLC. Upregulation of t-DARPP protein in a subset of SCLC patients positively correlates with overexpression of genes associated with Notch signaling, including achaete-scute homologue 1 (ASCL1). We reveal that DARPP-32 isoforms are transcriptionally activated by ASCL1 in human SCLC cells. To determine whether DARPP-32 drives SCLC growth in vivo, we utilized an orthotopic lung cancer xenograft mouse model. Mice challenged with DARPP-32-ablated DMS-53 cells show a substantial decrease in lung tumor growth relative to controls. Correspondingly, we demonstrate increased tumor growth in mice harboring lung tumors formed by administration of DMS-53 or H1048 cells overexpressing DARPP-32 and t-DARPP proteins. Mechanistically, we describe how DARPP-32 isoforms promote SCLC growth through increased proliferation, Akt/Erk-mediated survival, and anti-apoptotic signaling. Taken together, we define 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. Citation Format: Sk. Kayum Alam, Li Wang, Yanan Ren, Christina E. Hernandez, Farhad Kosari, Anja C. Roden, Rendong Yang, Luke H. Hoeppner. New molecular mechanisms of small cell lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5956.

Abstract 873: Molecular mechanisms of non-small cell lung cancer growth and drug resistance

Abstract Lung cancer is the deadliest and most frequently diagnosed type of tumor worldwide, with 1.6 million deaths reported annually. Non-small cell lung cancer (NSCLC) represents 85% of all lung cancer cases and carries a poor 5-year survival rate below 15%. Prognoses remain dismal due to the large number of patients diagnosed with advanced stage disease and the development of resistance to current therapies. A better understanding of acquired drug resistance will help to circumvent the progression of lung cancer and make significant strides in improving NSCLC patient treatment. Our recent work demonstrates that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its amino-terminally truncated splice variant (t-DARPP) promote lung tumor growth in orthotopic mouse models. IHC staining of 62 human lung adenocarcinoma tissues showed that t-DARPP expression is elevated with increasing tumor (T) staging score, which represents the size of the primary tumor and whether it has grown into nearby areas, as a metric of tumor progression and growth. Correspondingly, a computational biology analysis of 513 lung adenocarcinoma patients revealed upregulation of t-DARPP isoform expression correlates with advanced T stage and is associated with poor overall survival. We identified a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through activation of non-canonical NF-κB2 signaling. It has been shown that DARPP-32 overexpression inhibits gefitinib-induced apoptosis in gastric cancer and t-DARPP contributes to the trastuzumab resistance phenotype in breast cancer. Our recent findings suggest DARPP-32 and t-DARPP overexpression in NSCLC promotes resistance to specific molecular targeted inhibitors by enabling tumor cells to evade apoptosis via Akt- and Erk-dependent cell survival mechanisms. Current ongoing studies are focused on manipulating expression of DARPP-32 isoforms in lung tumor cells to prevent resistance to targeted therapies in NSCLC patients. Citation Format: SK Kayum Alam, Matteo Astone, Ping Liu, Li Wang, Abbygail M. Coyle, Erin N. Dankert, Dane K. Hoffman, Stephanie R. Hall, Wei Zhang, Rui Kuang, Anja C. Roden, Aaron S. Mansfield, Luke H. Hoeppner. Molecular mechanisms of non-small cell lung cancer growth and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 873.

Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells.

Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is frequently overexpressed in earlt stages of gastric cancers. We utilized in vitro assays, 3D gastric gland organoid cultures, mouse models, and human tissue samples to investigate the biological and molecular impact of DARPP-32 on activation of IGF1R and STAT3 signaling and gastric tumorigenesis. DARPP-32 enhanced phosphorylation of IGF1R (Y1135), a step that was critical for STAT3 phosphorylation at Y705, nuclear localization, and transcription activation. By using proximity ligation and co-immunoprecipitation assays, we found that IGF1R and DARPP-32 co-existed in the same protein complex. Binding of DARPP-32 to IGF1R promoted IGF1R phosphorylation with subsequent activation of downstream SRC and STAT3. Analysis of gastric tissues from the TFF1 knockout (KO) mouse model of gastric neoplasia, demonstrated phosphorylation of STAT3 in the early stages of gastric tumorigenesis. By crossing the TFF1 KO mice with DARPP-32 (DP) knockout (KO) mice, that have normal stomach, we obtained double knockout (TFF1 KO/DP KO). The gastric mucosa from the double KO mice did not show phosphorylation of IGF1R or STAT3. In addition, the TFF1 KO/DP KO mice had significant delay in developing neoplastic gastric lesions. Analysis of human gastric cancer tissue microarrays, showed high levels of DARPP-32 and positive immunostaining for nuclear STAT3 in cancer tissues, as compared to non-cancer histologically normal tissues. In summary, the presence of a signaling axis mediated by DARPP-32–IGF1R is a critical step in gastric tumorigenesis, playing an important role in activation of STAT3.

Abstract 5447: Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer

Abstract Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is a novel cancer gene overexpressed in 2/3 of gastric cancer patients. DARPP-32 promotes cancer cell survival, drug resistance, and invasion. Activation of STAT3, signal transducer and activator of transcription 3, is important for tumorigenesis and drug resistance. Among factors that regulate STAT3, SOCS3 acts as a negative regulator of STAT3 to prevent its uncontrolled oncogenic activation. In this study, we aimed to investigate if DARPP-32 regulates STAT3 and determine the underlying molecular mechanisms. Methods: The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays. Luciferase reporter and immunohistochemistry stain were used in this study. Results: Overexpression of DARPP-32 in AGS gastric cancer cells increased phosphorylation of STAT3 (Y705) and activation of STAT3 luciferase reporter (P<0.01), and decreased SOCS3 protein expression. DARPP-32 expression increased phosphorylation and ubiquitination of the SOCS3 protein, and subsequently shortened the SOCS3 protein half-life. We provide evidence showing that DARPP-32 promotes activation of IGF1R-SRC axis, critical for phosphorylation and degradation of SOCS3, a negative regulator of STAT3 activity. Proximity ligation and co-immunoprecipitation assays indicated that DARPP-32 and IGF1R co-exist in the same protein complex where DARPP-32 binding to IGF1R promotes its phosphorylation with activation of downstream SRC and STAT3. We obtained double knockouts mice (Darpp32KO/Tff1KO) to determine the histological changes at 3 age groups (3, 6, 9- 12 month). Using the different age groups, we found that the double knockout mouse model have significantly lower incidence of LGD at the age of 3 month, as compared to TFF1 KO (P<0.05) mice. The Darpp32KO/Tff1KO gastric mucosa demonstrated lack of phosphorylation and nuclear localization of STAT3 with significant delay in the onset of tumor lesions. Immunohistochemistry analysis confirmed overexpression of DARPP-32 and nuclear localization of STAT3 in human gastric cancers. Conclusion: The in vitro studies indicate that DARPP-32 plays a critical role in activation of STAT3 signaling through regulation of IGF1R and SRC, leading to SOCS3 degradation. In vivo results suggest that lack of DARPP-32 delays the development of tumors in the TFF1 knockout mouse model but is not sufficient alone to abrogate the full carcinogenesis cascade. These novel findings add to the oncogenic functions of DARPP-32 and highlight its potential role in the pro-inflammatory gastric carcinogenesis cascade. Citation Format: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5447.

Abstract 356: DARPP-32, a bridge between pro-inflammatory NF-kB and STAT3 signaling in gastric cancer

Abstract Background: Gastric cancer is one of the most frequently diagnosed malignancies with poor prognosis in the world. We have previously shown that Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is a novel cancer gene, which is overexpressed in 2/3 of gastric cancers patients; and associated with increased cancer cell survival, drug resistance, and invasion. However, the mechanisms of how DARPP-32 promotes gastric carcinogenesis remain unclear. Methods: DARPP-32 promoter activity and stat3 activity were measured by luciferase. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays. Results: Promoter analysis of DARPP-32 indicated the presence of NF-κB transcription factor putative binding sites in the DARPP-32 promoter region. We cloned the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). TNF-α treatment induced DARPP-32 reporter activity (P < 0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence (-996 to -1008 bp), containing putative NF-κB binding sites, is the most active region. We confirmed that activation of NF-kB by TNF-α and H.pylori infection leads to induction of DARPP-32 mRNA and protein level. Interestingly, we found that DARPP-32 induction leads to increased phosphorylation of STAT3 and activation of STAT3 luciferase reporter (P<0.01). We detected a decrease in the protein level of SOC3, a negative regulator of STAT3. DARPP-32 shortened the SOCS3 protein half-life by mediating an increase in phosphorylation and ubiquitination of the SOCS3 protein following treatment with MG132 and IL6. Furthermore, we detected an increase in SRC and IGF1R phosphorylation in DARPP-32-overexpressing cells. SRC inhibitor (Dasatinib) and SRC-siRNA blocked DARPP-32-induced activation of STAT3. We further examined our hypothesis that DARPP-32 could interact with IGF1R and enhance IGF1R autophosphorylation. Using co-immunoprecipitation, we found that DARPP-32 and IGF1R co-exist in the same protein complex. Conclusion: We demonstrated, for the first time, the role of NF-κB in DARPP-32 transcriptional regulation. The in vitro studies indicate that DARPP-32 plays a role in activation of STAT3 signaling through inhibition of SOCS3 and activation of IGF1R and SRC. DARPP-32 interacted with IGF1R, which increasing the phosphorylation of IGF1R and SRC, and induced SOCS3 phosphorylation and degradation. This cascade of events leads to STAT3 activation in gastric cancer cells. We propose DARPP-32 as an important signaling bridge between NF-kB and STAT3 in response to pro-inflammatory signaling in gastric carcinogenesis. Citation Format: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. DARPP-32, a bridge between pro-inflammatory NF-kB and STAT3 signaling in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 356. doi:10.1158/1538-7445.AM2017-356

PPARα modulation of mesolimbic dopamine transmission rescues depression-related behaviors

Depressive disorders cause a substantial burden for the individual and the society. Key depressive symptoms can be modeled in animals and enable the development of novel therapeutic interventions. Chronic unavoidable stress disrupts rats' competence to escape noxious stimuli and self-administer sucrose, configuring a depression model characterized by escape deficit and motivational anhedonia associated to impaired dopaminergic responses to sucrose in the nucleus accumbens shell (NAcS). Repeated treatments that restore these responses also relieve behavioral symptoms. Ventral tegmental area (VTA) dopamine neurons encode reward and motivation and are implicated in the neuropathology of depressive-like behaviors. Peroxisome proliferator-activated receptors type-α (PPARα) acutely regulate VTA dopamine neuron firing via β2 subunit-containing nicotinic acetylcholine receptors (β2*nAChRs) through phosphorylation and this effect is predictive of antidepressant-like effects.Here, by combining behavioral, electrophysiological and biochemical techniques, we studied the effects of repeated PPARα stimulation by fenofibrate on mesolimbic dopamine system. We found decreased β2*nAChRs phosphorylation levels and a switch from tonic to phasic activity of dopamine cells in the VTA, and increased phosphorylation of dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32) in the NAcS. We then investigated whether long-term fenofibrate administration to stressed rats reinstated the decreased DARPP-32 response to sucrose and whether this effect translated into antidepressant-like properties. Fenofibrate restored dopaminergic responses to appetitive stimuli, reactivity to aversive stimuli and motivation to self-administer sucrose. Overall, this study suggests PPARα as new targets for antidepressant therapies endowed with motivational anti-anhedonic properties, further supporting the role of an unbalanced mesolimbic dopamine system in pathophysiology of depressive disorders.

DARPP-32: from neurotransmission to cancer.

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.

409 Factors Relating to GI Bleeding Induced by Dual Antiplatelet Therapy (DAPT) in Japanese Patients With Percutaneous Coronary Intervention - Retrospective Cohort and Case Control Studies

Background: Splicing and expression of alternative exons is a tissue specific feature that affects the cancer genome. Notably, pre-mRNA-splicing factor SRp20 plays an important role in regulating alternative splicing. Expression of CD44 and its splicing variants have been shown to regulate carcinogenesis and invasion. Herein, we investigated factors that regulate splicing of CD44 and expression of its variant CD44E in gastric cancer. We identified a novel role for dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), in regulating CD44 splicing and promoting gastric tumorigenesis. Methods:Pre-mRNA splicing was measured by utilizing a luciferase-based quantitative reporter system. Quantitative realtime RT-PCR and Western blot analysis were used to measure the mRNA and protein levels. Co-immunoprecipitation, invasion, and ubiquitination assays were performed to investigate the mechanism by which DARPP-32 regulates CD44 splicing . Results: Knockdown of endogenous DARPP-32 in MKN45 gastric cancer cells markedly reduced expression of CD44 V8-V10 (CD44E) splice variant. We found that overexpression of DARPP-32 in AGS gastric cancer cells, with low endogenous levels, significantly increased the splicing activity (P<0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (P<0.001). Mechanistic investigations revealed that modulation of DARPP-32 was associated with changes in splicing factor SRp20 expression through a posttranslational mechanism that involves the binding of DARPP-32 and SRp20 in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 enhances SRp20 protein stability through regulation of ubiquitination. In contrast, the knockdown of endogenous DARPP-32 reversed these effects and indicated that DARPP-32 regulated the SRp20-dependent CD44E splicing. In a xenograft tumor mouse model, knocking down endogenous DARPP-32 markedly decreased tumor growth, and overexpression of SRp20 rescued tumor growth. Importantly, our clinical data demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E in human gastric primary tumors, strongly supporting a role for DARPP-32 in regulating CD44E splicing in gastric cancer. Conclusion: These novel findings establish for the first time that DARPP-32-SRp20 axis plays a key role in regulating the expression of CD44E splice variant in gastric tumorigenesis. This novel DARPP-32-mediated splicingdependent mechanism provides a new paradigm in cancer invasion.

Gastric tumour-derived ANGPT2 regulation by DARPP-32 promotes angiogenesis

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...

Tu1654 Pro-Inflammatory Signaling Transcriptional Regulates DARPP-32 in Gastric Cancer

Background: Helicobacter pylori (H.pylori) infection plays an important role in gastric carcinogenesis and exemplifies the strong association between inflammation and cancer. DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, Mr 32000) is a novel cancer gene. The aim of this study was to investigate the role of inflammation and H.pylori in regulating DARPP-32 expression. Methods: Immunohistochemistry (IHC) were used to determine protein expression. DARPP-32 promoter activity was measured by a luciferase reporter system. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. Results: The IHC analysis demonstrated overexpression of p-NFκB and DARPP-32 in the early stages of human gastric cancer. Therefore, we examined the relationship between NF-kB and DARPP-32, and found that DARPP-32 does not regulate the transcriptional activity of NF-κB as measured by pNF-κB luciferase reporter and western blot analysis. On the other hand, we found that TNF-α treatment leads to activation of NFκB and mediates upregulation of mRNA and protein levels of DARPP-32 in several gastric cancer cell lines (P 10-fold, P<0.001) and protein levels. In addition, treatment with an NF-κB inhibitor (BAY 11-7082) decreased DARPP-32 mRNA and protein levels. In an attempt to study the transcriptional regulation, we cloned a 2500 bp of the DARPP-32 promoter into the luciferase reporter (DARPP-32-Luc). The TNF-α treatment of AGS and MKN-45 cells led to a significant induction of luciferase activity of DARPP-32 reporter (P<0.01), and treatment of these cells with BAY 11-7082 (5 μM) reversed these effects (P<0.01). Furthermore, transient expression of NF-kB-p65 in AGS cells led to a 3fold induction in the luciferase activity of DARPP-32 reporter (P<0.01). Using deletion constructs, we have found that the -1000bp of promoter area is the most active region. We confirmed that NF-kB-p65 binds to DARPP-32 promoter by ChIP assay. In vitro data using gastric cancer cell lines and H.pylori infection (J166 and 7.13 strains) demonstrated simultaneous increase in the p-NF-kB and increase in the mRNA and protein levels of DARPP-32. Using the Tff1-KO gastric cancer mouse model that is characterized by NF-kB activation, we found that DARPP-32 was overexpressed at the protein and mRNA levels. Treatment of wild-type and Tff1-KO mice with H.pylori (PMSS1 strain) led to significant induction of both p-NF-kB and DARPP-32. Conclusion: Our results demonstrated, for the first time, that H. pylori infection leads to induction of NF-κB-dependent DARPP-32 expression. Given the oncogenic functions of DARPP-32 in regulating the PI3K/AKT and CXCR4 signaling, this novel DARPP-32 regulationmechanism provides a new paradigm in gastric carcinogenesis.

Tu1658 Inhibition of Peritoneal Metastasis by Overcoming Death Receptor Resistance by Small Molecule Targeting of C-FLIP

Background: Helicobacter pylori (H.pylori) infection plays an important role in gastric carcinogenesis and exemplifies the strong association between inflammation and cancer. DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, Mr 32000) is a novel cancer gene. The aim of this study was to investigate the role of inflammation and H.pylori in regulating DARPP-32 expression. Methods: Immunohistochemistry (IHC) were used to determine protein expression. DARPP-32 promoter activity was measured by a luciferase reporter system. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. Results: The IHC analysis demonstrated overexpression of p-NFκB and DARPP-32 in the early stages of human gastric cancer. Therefore, we examined the relationship between NF-kB and DARPP-32, and found that DARPP-32 does not regulate the transcriptional activity of NF-κB as measured by pNF-κB luciferase reporter and western blot analysis. On the other hand, we found that TNF-α treatment leads to activation of NFκB and mediates upregulation of mRNA and protein levels of DARPP-32 in several gastric cancer cell lines (P 10-fold, P<0.001) and protein levels. In addition, treatment with an NF-κB inhibitor (BAY 11-7082) decreased DARPP-32 mRNA and protein levels. In an attempt to study the transcriptional regulation, we cloned a 2500 bp of the DARPP-32 promoter into the luciferase reporter (DARPP-32-Luc). The TNF-α treatment of AGS and MKN-45 cells led to a significant induction of luciferase activity of DARPP-32 reporter (P<0.01), and treatment of these cells with BAY 11-7082 (5 μM) reversed these effects (P<0.01). Furthermore, transient expression of NF-kB-p65 in AGS cells led to a 3fold induction in the luciferase activity of DARPP-32 reporter (P<0.01). Using deletion constructs, we have found that the -1000bp of promoter area is the most active region. We confirmed that NF-kB-p65 binds to DARPP-32 promoter by ChIP assay. In vitro data using gastric cancer cell lines and H.pylori infection (J166 and 7.13 strains) demonstrated simultaneous increase in the p-NF-kB and increase in the mRNA and protein levels of DARPP-32. Using the Tff1-KO gastric cancer mouse model that is characterized by NF-kB activation, we found that DARPP-32 was overexpressed at the protein and mRNA levels. Treatment of wild-type and Tff1-KO mice with H.pylori (PMSS1 strain) led to significant induction of both p-NF-kB and DARPP-32. Conclusion: Our results demonstrated, for the first time, that H. pylori infection leads to induction of NF-κB-dependent DARPP-32 expression. Given the oncogenic functions of DARPP-32 in regulating the PI3K/AKT and CXCR4 signaling, this novel DARPP-32 regulationmechanism provides a new paradigm in gastric carcinogenesis.

Induction of MT1-MMP expression and enhancement of gastric cancer cells invasion by DARPP-32-CXCR4 interaction.

14 Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is amplified and overexpressed in approximately 70% of gastric cancers. The prognosis for gastric cancer patients remains poor, especially in more advanced stages. Recently, it was suggested that CXCL-12 and its receptor, CXCR4, are involved in gastric cancer metastasis. However, the detailed mechanism of gastric cancer metastasis is still not completely understood. Methods: Cells invasive activity was determined by invasion assay and HUVEC invasion assay. The association between DARPP-32 and CXCR4 was evaluated by immunofluorescence and co-immunoprecipitation assays. CXCR4 degradation was analyzed by Ubiquitination Assay. Results: Overexpression of DARPP-32 in AGS cells increased cell invasion with about as three-fold invasive cells as the vector control (p&lt;0.01). As measured by HUVEC invasion assay, overexpression of DARPP-32 in AGS cells also had a significant increase in the invasive activity (p&lt;0.001). We found that DARPP-32 led to increased CXCR4 and MT1-MMP protein levels in DARPP-32 expressing AGS cells. The co-immunoprecipitation and immunofluorescence experiments demonstrated the existence of DARPP-32 and CXCR4 in the same protein complex. AGS cells expressing DARPP-32 displayed stable protein levels of CXCR4. IP-Western blot showed reduced ubiquitination of CXCR4 protein following the overexpression of DARPP-32 and treatment with CXCL-12, as compared to controls. Using AMD3100 (0.2 ng/ml) overnight blocked DARPP-32-induced cell invasion. The knockdown of endogenous DARPP-32 by lentiviral DARPP-32 shRNA in MKN-45 cell line reversed these signaling effects and decreased cell invasive activity, as measured by invasion and HUVEC invasion Assay (p&lt;0.01, p&lt;0.05). Conclusions: The in vitro studies indicate that DARPP-32 plays a role in invasion and metastasis; DARPP-32 promotes invasion and metastasis of gastric cancer cells by interacted with CXCR4, delaying CXCL-12-induced CXCR4 Ubiquitination and blocking CXCR4 degradation, activating MMP2 by increasing MT1-MMP expression. The in vivo experiments are ongoing to determine the efficacy of DARPP-32 in mediating CXCR4 overexpression and metastasis.

Influence of palatability on motivation to operate for caloric and non-caloric food in non food-deprived and food-deprived rats

Palatability is the hedonic food component that is considered to override the homeostatic mechanisms that control food intake, and we compared how much effort non food-deprived and food-deprived rats were willing to spend in order to earn a palatable caloric (sucrose) or non-caloric (saccharin) snack. We first studied the dopaminergic response, in terms of dopamine levels and dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32) phosphorylation pattern, to two consecutive palatable caloric or non-caloric snacks in the nucleus accumbens shell (NAcS) of non food-deprived and fasted rats. We report that non food-deprived rats developed rapid habituation in the NAcS dopaminergic response to the second consumption of both caloric and non-caloric palatable food, while food-deprived rats developed rapid habituation only to saccharin. Next, we show that in self-administration experiments, non food-deprived rats spent a similar effort when operating for sucrose or saccharin. However, the same rats showed an increased response specifically for sucrose after 18-h fasting. After pre-feeding devaluation, rats reduced their response to sucrose but not for saccharin.These results strengthen the hypothesis that food intake is mainly controlled by palatability in non food-deprived rats and by caloric content in food-deprived rats. Moreover, they show that rapid habituation development was associated with a similar, basal working activity aimed at ingesting both caloric and non-caloric food, as observed in non food-deprived rats consuming sucrose or saccharin and in fasted rats consuming saccharin. Conversely, lack of habituation, as present in fasted rats consuming a caloric food, was associated with extra energy expenditure.

Regulation of CXCR4-Mediated Invasion by DARPP-32 in Gastric Cancer Cells

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.

Highlights of This Issue

Gastric cancer remains a leading cause of cancer mortality worldwide. Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers. In this report, Zhu and colleagues uncovered a new function for DARPP-32 by demonstrating its role in cancer cell invasion. The results indicate that DARPP-32–CXCR4 interaction stabilizes the CXCR4 protein by attenuating the CXCL-12–induced CXCR4 protein ubiquitination and degradation. The ability of DARPP-32 to bind CXCR4 and activate MT1-MMP/MMP-2 signaling provides a novel mechanism that regulates CXCR4 protein expression and cancer cell invasion.Yi and colleagues found that activated STAT3 increases RANTES expression and secreted RANTES activates STAT3 via an autocrine pathway, which promotes resistance to 4-hydroxytamoxifen. These findings suggest that combination chemotherapy with tamoxifen and a STAT3 or RANTES inhibitor may be a promising treatment for overcoming breast cancer chemoresistance in the clinic. This study therefore provides new insights that may contribute to the proposed autocrine loop of RANTES, in which tamoxifen-resistant breast cancer cells become more aggressive and maintain drug resistance through STAT3 phosphorylation and increased antiapoptotic signals.Recently, histone deacetylase 2 (HDAC2) was reported to be increased in gastric, colon, and liver cancers. However, little is known about the role of HDAC2 in carcinogenesis. The present study discovered that overexpression of HDAC2 was accompanied with the loss of cyclin-dependent kinase inhibitor CDKN2A (p16INK4a). Further studies showed that the expression of p16INK4a was transcriptionally regulated by HDAC2, as demonstrated by the direct binding of HDAC2 on the promoter of p16INK4a. Moreover, inhibition of HDAC2 restored the expression of p16INK4a; induced the apoptosis, autophagic cell death, and cell-cycle arrest in human MKN-1 gastric cancer cells in vitro; and reduced the gastric tumor growth in vivo. These findings provide molecular basis for developing novel therapeutic strategies in gastric cancer through HDAC2 inhibition.SUSD2 (Sushi Domain Containing 2) encodes a membrane surface protein with weak or no expression in normal breast epithelial cells, yet high expression in breast carcinomas. SUSD2 interacts with galectin-1, a secreted protein that promotes tumor immune evasion, angiogenesis, and metastasis. Localization of galectin-1 on the surface of cells is dependent on the presence of SUSD2. Using a syngeneic mouse model, significantly fewer CD4 tumor-infiltrating lymphocytes were present in tumors expressing Susd2 compared with the vector control. SUSD2 contributes to an immune evasion mechanism and may represent a promising therapeutic target for breast cancer.

Resistance to TRAIL Is Mediated by DARPP-32 in Gastric Cancer

Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL. In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays. Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8. Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.

Striatal Medium-Sized Spiny Neurons: Identification by Nuclear Staining and Study of Neuronal Subpopulations in BAC Transgenic Mice

Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs), which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D1-receptors (D1Rs) project preferentially to the substantia nigra pars reticulata (SNr), whereas those expressing dopamine D2- receptors (D2Rs) project preferentially to the lateral part of the globus pallidus (LGP). The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr∼32,000), and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%). In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also unambiguously confirms the degree of segregation of MSNs in the mouse striatum and allows the full exploitation of results obtained with BAC-transgenic mice.

Negative regulation of cyclin-dependent kinase 5 targets by protein kinase C

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed protein serine/threonine kinase essential for brain development and implicated in synaptic plasticity, dopaminergic neurotransmission, drug addiction, and neurodegenerative disorders. Relatively little is known about the molecular mechanisms that regulate the activity of Cdk5 in vivo. In order to determine whether protein kinase C (PKC) regulates Cdk5 activity in the central nervous system, the phosphorylation levels of two Cdk5 substrates were evaluated under conditions of altered PKC activity in vivo. Treatment of acute striatal slices with a PKC-activating phorbol ester caused a time- and dose-dependent decrease in the levels of phospho-Ser6 inhibitor-1, phospho-Ser67 inhibitor-1, and phospho-Thr75 dopamine- and cAMP-regulated phosphoprotein, M r 32,000 (DARPP-32). This effect was reversed by the PKC inhibitor, Ro-32-0432. Moreover, phospho-Ser6 inhibitor-1, phospho-Ser67 inhibitor-1, and phospho-Thr75 DARPP-32 levels were elevated in brain tissue from mice lacking the gene for PKC-α. PKC did not phosphorylate Cdk5 or its cofactor, p25, in vitro. Striatal levels of the Cdk5 cofactor, p35, did not change in response to phorbol ester treatment. Furthermore, Cdk5 immunoprecipitated from striatal slices treated with phorbol ester had unaltered activity toward a control substrate in vitro. These results suggest that PKC exerts its effects on the phosphorylation state of Cdk5 substrates through an indirect mechanism that may involve the regulatory binding partners of Cdk5 other than its neuronal cofactors.

Inhibitor-1 Is Not Required for the Activation of Glycogen Synthase by Insulin in Skeletal Muscle

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.

Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.