Molecular characterization and phylogenetic analysis of unusual x-type hmw glutenin subunits from 1s(l) genome of aegilops longissima

Wheat related diploid species Ae. longissima (2n=2x=14, (SSl)-S-l) has extensive storage protein variations that may provide useful gene resources for wheat quality improvement. In this work, five novel 1S(l)-encoded x-type high molecular glutenin subunits (HMW-GS) were identified and designated as 1S(l)x-123, 1S(l)x-129, 1S(l)x1-136, 1S(l)x2-136 and 1S(l)x2.2, respectively. Their complete open reading frames (ORFs) were cloned and sequenced by AS-PCR, which contained 2874 bp (956 aa) for 1S(l)x-123, 2946 bp (979 aa) for 1S(l)x-129, 2901 bp (965 aa) for 1S(l)x1-136, 2982bp (991 aa) for 1S(l)x2-136 and 2928 bp (974 aa) for 1S(l)x2.2. Molecular characterization demonstrated that five unusual subunits had greater repetitive domains resulted from a larger fragment insertion (74-113 aa). Particularly, 1S(l)x-129 had an extra cysteine residue at the position 109 due to a TAT -> TGT dot mutation, which may improve the formation of superior gluten macropolymer. Our results suggest that these unusual HMW-GS could be served as potential superior gene resources for improving wheat gluten quality. Phylogenetic analysis revealed that HMW-GS genes from Glu-1Sx genomes had close evolutionary relationships with those of Glu-1Dx genome while sequences from Ae. speltoides aligned with those of B genome

Abstract 1095: A combination of suberoylanilide hydroxamic acid and quinacrine is an effective therapeutic approach in preclinical settings of upper gastrointestinal cancers

Abstract Background: Quinacrine (QC), an antimalarial drug, has been shown to possess anticancer effects. Suberoylanilide hydroxamic acid (SAHA) inhibits class I and class II HDACs and is approved for cancer therapy. Developing novel approaches to overcome cancer drug resistance could significantly enhance current therapeutic approaches and improve patient care. Methods: ATP-GLO, clonogenic survival, Annexin-V apoptosis assay, comet assay and DNA double-strand breaks (DSB) kits were used. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analyses. Results: A combination of QC/SAHA significantly increased cell death in all cancer cell lines and had no effect on immortalized non-cancer cell lines (HFE145, NIH-3T3 and EPC2) (P20 fold, P<0.01). Comet assay data showed increased DNA damage compared with vehicle-treated cells (8-fold, P<0.01). Western blot analysis demonstrated a notable increase in activation of PARP, caspases 3, 9 and γ-H2AX following QC/SAHA co-treatment in all cancer cell lines. Interestingly, the combination of QC/SAHA substantially decreased the protein levels of both wtP53 and mutP53 in these cells. Tumor xenograft data confirmed that a combination of QC/SAHA is more effective than a single agent in abrogating tumor growth (P<0.05). Conclusion: Our novel findings show that QC and SAHA have a synergistic effect on cancer cell death. The results provide compelling evidence that increased DNA damage mediates the cytotoxic effect of combined QC/SAHA. Such effect is likely related to mutP53 and wtP53 protein degradation induced by QC/SAHA combination. Our findings provide a rationale for a clinical evaluation of combined QC/SAHA therapy in gastro-esophageal cancers. Ongoing studies are currently being undertaken to understand mechanisms that regulate the degradation of wtp53 and mutp53 proteins. Citation Format: Shoumin Zhu, Wael El-Rifai. A combination of suberoylanilide hydroxamic acid and quinacrine is an effective therapeutic approach in preclinical settings of upper gastrointestinal cancers [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 1095. doi:10.1158/1538-7445.AM2017-109

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

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<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<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<0.01, p<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

Abstract A20: DARPP-32-mediated EGFR protein stability leads to gefitinib resistance in gastric cancer

Abstract Background: Gastric cancer is an aggressive tumor that is often resistant to chemotherapeutics. The 17q amplicon genes, including dopamine and cAMP-regulated phosphoprotein MW 32 kDa (DARPP-32), are overexpressed in two-thirds of gastric adenocarcinomas and may induce chemotherapy resistance via abrogation of apoptosis. EGFR plays an important role in tumor growth and activation of the PI3K pathway. Gefitinib (Iressa) is a specific and effective EGFR inhibitor that has shown antitumor activity in clinical trials against several gastrointestinal cancers, including gastric cancers. However, molecular mechanisms that mediate resistance to gefitinib remain poorly understood. Methods and Results: The expression of DARPP-32 in the multistep carcinogenesis cascade was examined using IHC analysis on 533 samples. The composite expression score, calculated from immunostaining patterns, progressively increased significantly from normal or gastritis to metaplasia, dysplasia, and adenocarcinoma (p<0.001). Using clonogenic survival assays, and ATP-GLO cell viability assays, we found that overexpression of DARPP-32 leads to a 3-fold increase in gefitinib resistance in MKN-28 cells (25 μM gefitinib, treated overnight, P<0.01). We also found that DARPP-32 expressing MKN-28 cells had activated the PI3K-AKT pathway as compared to control cells. We next examined the binding of DARPP-32 to EGFR and ERBB3. The three-way co-immunoprecipitation experiments demonstrated the existence of DARPP-32, EGFR, and ERBB3 in the same protein complex. The enhanced EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, then activated the PI3K-AKT pathway, as indicated by p-p85 (Tyr458) subunit of PI3K and p-AKT (Ser473) levels. Following treatment with gefitinib (25 μM) overnight, MKN-28 cells expressing DARPP-32 displayed stable protein levels of EGFR. In contrast, control cells exhibited a significant reduction in EGFR protein levels, suggesting that DARPP-32 promotes stability and suppresses degradation of EGFR protein. After the gefitinib treatment of DARPP-32-expressing MKN-28 cells, p-ERBB3 and activated PI3K-AKT pathway were maintained at higher levels than in control cells. The knockdown of endogenous DARPP-32 by lentiviral DARPP-32 shRNA in SNU-16 cell lines reversed these signaling effects and increased sensitivity to gefitinib (p<0.01). Conclusion: Our results suggest that DARPP-32 overexpression may participate in the transition to intestinal metaplasia and progression to neoplasia. The in vitro studies indicate that DARPP-32 plays a role in increasing the stability of EGFR protein by delaying gefitinib-induced EGFR degradation; maintaining EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, which regulates the PI3K/Akt pathway. DARPP-32 may have potential as a predictive biomarker in gastric cancer prognosis and clinical response to treatment

Use of DARPP-32 to increase interactions between EGFR and ERBB3 and to promote gastric cancer resistance to gefitinib

18 Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP−32), is overexpressed during gastric carcinogenesis. Gefitinib is a specific and effective EGFR inhibitor that has shown antitumor activity in clinical trials against gastrointestinal cancers. Gastric tumors can become resistant to gefitinib. However, molecular mechanisms that mediate resistance to gefitinib remain poorly understood. Methods: The expression of DARPP−32 in the multi-step carcinogenesis cascade was examined using IHC analysis. Cell survival was determined by clonogenic survival and ATP−Glo assays. Apoptosis was assessed by Annexin-V and immunoblot analyses. The association between DARPP−32 and EGFR was evaluated by immunofluorescence and co-IP assays. Results: The composite expression score progressively increased significantly from normal or gastritis to adenocarcinoma (p<0.001). Overexpression of DARPP−32 in MKN-28 cells blocked gefitinib-induced apoptosis and increased the drug’s IC50 10−fold, compared to that of control cells (P<0.01). And DARPP-32 expressing MKN−28 cells had activated the PI3K-AKT pathway as compared to control cells. The three−way co-IP experiments demonstrated the existence of DARPP-32, EGFR, and ERBB3 in the same protein complex. DARPP-32 enhanced EGFR-ERBB3 heterodimerization promoting phosphorylation of ERBB3, and then activated the PI3K-AKT pathway. Following treatment with gefitinib (25 μM) overnight, MKN−28 cells expressing DARPP-32 displayed stable protein levels of EGFR. The knockdown of endogenous DARPP-32 by DARPP-32 shRNA in SNU-16 cell lines reversed these signaling effects and increased sensitivity to gefitinib (p<0.01). Conclusions: Our results suggest that DARPP-32 overexpression may participate in the progression to neoplasia. DARPP-32 also plays a role in increasing the stability of EGFR protein; DARPP-32 promotes resistance of gastric cancer cells to gefitinib by promoting interaction between EGFR and ERBB3 and activating PI3K–AKT signaling. DARPP-32 may have potential as a predictive biomarker in gastric cancer prognosis and clinical response to treatment

Flower-Like Au–CuO/Bi2WO6 Microsphere Catalysts: Synthesis, Characterization, and Their Catalytic Performances for CO Oxidation

The flower-like Bi2WO6 microsphere was synthesized through a simple hydrothermal route, and three catalysts, Au/Bi2WO6, CuO/Bi2WO6, and Au–CuO/Bi2WO6, were prepared by a deposition–precipitation method. The morphology and structure of the catalysts were characterized by X-ray powder diffraction, surface area analyzer, inductively coupled plasma optical emission spectrometer, scanning electron microscope, transmission electron microscopy, UV/Vis spectrometer, as well as X-ray photoelectron spectroscopy. Their catalytic performances in catalytic CO oxidation were evaluated. For Au/Bi2WO6 and CuO/Bi2WO6, Au and CuO nanoparticles highly dispersed on Bi2WO6 are 3 and 10 nm, respectively, in average size. For Au–CuO/Bi2WO6, a part of the Au nanoparticles (Au NPs) strongly adheres to the CuO, due to the strong interaction between Au NPs and CuO, which has a positive effect on catalytic activity of Au–CuO/Bi2WO6. Au–CuO/Bi2WO6 can convert CO into CO2 completely at 40 °C, as the contents of Au and Cu are 0.438 wt % and 4.85 wt %, respectively

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.AM2017-35