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IGF-1R Modulates Colon Cancer Stem Cell Properties by Regulating Novel Tumor Suppressor MicroRNAs
show increased expression of activated form of IGF-1R [Patel et.al. Anticancer Res, 2010, 30:319; Yu et al. Transl Oncol, 2009, 2:321]. Hence, we hypothesized that IGF-1R plays an important regulatory role in CSC phenotype. Methods: Chemotherapy (oxaliplatin and 5-FU, referred to as FOLFOX) resistant colon cancer HCT-116 (p53 wild type) and HT-29 (p53 mutant) cells were generated by prolonged exposure to drugs. Immunohistochemical analysis was carried out for coexpression of IGF-1R and CD166. Colonosphere formation was used to examine growth and self-renewal while matrigel invasion assay was used to assess invasiveness of FOLFOX-resistant cells. Standard quantitative reverse transcription polymerase chain reaction was carried out for mRNA and microRNA (miR) expression. Results: We observed a colocalization of IGF-1R and CD166 (a colon CSC marker) in human CRCs which was higher in invasive cancer than adenoma. Moreover, degree of colocalization positively correlated with presence of lymphnode metastasis. Inhibition of IGF-1R with and IGF-1R tyrosine kinase inhibitor GSK1904529A resulted in a dose dependant decrease in primary (a measure of CSCs growth) and secondary (a measure of CSC self-renewal) colonosphere formation which was significantly more in p53 wild type colon cancer cells. The above phenomenon was greater for combination of GSK1904529 and FOLFOX than either modality alone. In addition, IGF-1R inhibition caused a 50% reduction in invasive ability of FOLFOX-resistant cells. In the pursuit to understand mechanism of the above phenomenon, we identified miR-363, a non p53 regulated miR, and miR-215, a p53 regulated miR and known to be downregulated in CRC, to be highly overexpressed (28-fold and 6-fold respectively) following IGF-1R inhibition while both miRs were significantly reduced in FOLFOX-resistant cells. We observed a significant reduction in CSCs markers expression following pre-miR-215 and pre-miR-363 transfection of FOLFOX-resistant colon cancer cells. More importantly, transfection of p53-mutant colon cancer cells with pre-miR-215, which caused downregulation of its target thymidylate synthase (TS), sensitized them to anchorage-independent growth inhibition by GSK1904529A. Finally, pre-miR-363 modulated cell invasion by downregulating its target integrin αV. Conclusion: IGF-1R modulates colon CSC phenotype via regulating novel miRs that regulate CSC growth and invasion. Status of p53 gene might be predictive of response to IGF-1R inhibitor on colorectal CSCs.
Developmental Origins of Irritable Bowel Syndrome (IBS)-Like Symptoms: Epigenetic Dysregulation Qingjie Li, Sushil K. Sarna Background and Aims: IBS is a complex disease with recurring clusters of symptoms. Genetic mutations and single nucleotide polymorphisms do not fit these traits. We reported previously that neonatal inflammatory insult enhances smooth muscle reactivity to ACh in adulthood, which enhances colonic motor function to induce diarrhea-like conditions. In this study, we tested the hypothesis that adverse early life events reprogram the expression of genes encoding vasoactive intestinal polypeptide (VIP) and pore-forming α1C-subunit of the Cav1.2b channels (Canca1c) by epigenetic mechanisms in colonic muscularis externa to induce colonic motility dysfunction in adulthood. Methods: Ten-day old rat neonates received intraluminal 130 mg/kg TNBS in the colon; control pups received saline. Colonic muscularis externae (ME) were obtained 6 to 8-week later. Results: qRT-PCR and western blotting showed significant increases in the mRNA and protein expressions of VIP and α1C-subunit in the ME of about 50% of adult rats that received neonatal inflammatory insult (IBS-rats). Chromatin immunoprecipitation assays showed marked increase in acetylation of histone H3 lysine 9 (H3K9Ac, a marker of transcriptional activation) (0.8±0.1 vs. 0.45±0.06 Ctr., p<0.05) and attenuation in trimethylation of histone H3 lysine 9 (H3K9me3, a marker of transcriptional repression) (0.8±0.07 vs.1.2±0.03 Ctr., p<0.05) in the core promoter region of Cacna1c in IBS-rats. As a result, the RNA polymerase II (RNAP II) association with the Cacna1c promoter significantly increased, indicating enhanced Cacna1c gene transcription. We investigated the role of VIP in the epigenetic modification of Cacna1c by incubating ME from naïve rats with 100 ng/ml VIP for 24 hours. VIP-treatment markedly increased H3K9Ac and decreased H3K9me3 at the upstream CREB-binding site (-2062/-2055) and around the transcription start site (+21 ~ +168), and hence enhanced RNAP II interaction with this promoter region suggesting that VIP modulates epigenetic components at the Cacna1c promoter to stimulate Cacna1c gene transcription. VIP antagonist, (p-Chloro-D-Phe6,Leu17)VIP, abrogated VIP-mediated RNAP II binding, H3K9 acetylation and trimethylation at the Cacna1c promoter. Intraperitoneal administration of 100 mM sodium butyrate, a histone deacetylase inhibitor, significantly elevated Cacna1c mRNA in ME of naïve adult rats (1.5±0.06 vs. 1±.01 Ctr., p<0.05), indicating that histone hyperacetylation is necessary for α1C gene transcription. Conclusions: Our findings reveal the epigenetic mechanisms by which adverse early life events alter the gene expression of specific cell signaling proteins to cause colonic motor dysfunction in adulthood. The intensities of epigenetic modifications are modifiable by environmental influences, such as stress, which explains relapses and remissions of symptoms in IBS patients.
666 MLN-8237 Treatment Alone and in Combination With Cisplatin Suppresses In Vitro and In Vivo Progression of Gastrointestinal Cancer Cells Vikas Sehdev, Mohammed Soutto, Abbes Belkhiri, Jeffrey Ecsedy, Wael El-Rifai Background: Gastric cancers respond poorly to conventional chemotherapy indicating active intrinsic mechanisms against drug induced cell death. AURKA overexpression facilitates oncogenic transformation by causing chromosomal instability, aberrant mitosis and aneuploidy. We have previously reported that AURKA is overexpressed in >50% of upper gastrointestinal adenocarcinomas. Additionally, we have confirmed that AURKA induces pro-survival mechanisms through activation of AKT and β-catenin, and inhibition of p53- and p73mediated apoptotic pathways in gastrointestinal adenocarcinomas. Methods and Results: In this study, we evaluated the effect of AURKA specific inhibitor (MLN-8237) treatment alone and/or in combination with Cisplatin (CDDP) on gastrointestinal cancer cell survival, viability, apoptotic markers and tumor volume. The ATP-glow cell viability assay and clonogenic cell survival assay data indicated that in comparison to treatment with MLN (0.5μM) or CDDP (2.5μM) alone; the combination treatment with MLN-8237 (0.5μM) and CDDP (2.5μM) significantly increased (p<0.05) inhibition of cellular viability and survival in AGS, KATOIII, MKN28, FLO-1 and OE33 gastrointestinal cancer cell lines. Similarly, the western blot data after treatment with MLN (0.5μM) and/or CDDP (2.5μM) for 48h exhibits higher levels of cleaved forms of caspase 3 and PARP with MLN (0.5μM) and CDDP (2.5μM) combination treatment in AGS, FLO-1 and OE33 cell lines. The tumor growth results from OE33 mouse xenograft study indicate that treatment with MLN-8237 (40mg/kg) and CDDP (2mg/kg) combination is significantly (p<0.01) more potent than MLN-8237 (40mg/kg) or CDDP (2mg/kg) treatment alone with the following order of effective ne ss : C on tr ol (321.0±18.3mm3), CDDP (101.4±6.2 mm3), MLN-8237 (178.8±16.9 mm3)\, and MLN8237/CDDP(41.1±7.7 mm3). Moreover, the qRT-PCR data from tumor xenograft samples following treatment with MLN-8237/CDDP combination showed significantly higher mRNA levels of p73 and its downstream transcriptional target genes (p21, puma, NOXA and BAX) than MLN-8237 or CDDP alone (P<0.01). Conclusions: Our In Vitro and In Vivo data indicate that inhibition of AURKA with MLN-8237 is an effective therapeutic strategy for inducing apoptosis and suppressing growth of gastrointestinal cancer cells. Additionally, MLN-8237 in combination with CDDP enhances the anti-tumor activity of MLN-8237 against gastrointestinal cancer cells. Therefore, our study demonstrates that MLN-8237 is an effective antitumor agent which can be potentially combined with CDDP for better therapeutic outcome in gastrointestinal cancer patients.
664 2-Methoxy-5-Amino-N-Hydroxybenzamide Sensitizes Colon Cancer (CC) Cells to TRAIL-Mediated Apoptosis Through Modulation of Death Receptor (DR)5 and Survivin Carmine Stolfi, Roberta Caruso, Eleonora Franzè, Ivan Monteleone, Daniela De Nitto, Angelamaria Rizzo, Giuseppe S. Sica, Francesco Pallone, Giovanni Monteleone Background and aim: TRAIL-induced apoptosis is a crucial event in the negative regulation of tumor growth. However, colon cancer (CC) cells are largely resistant to TRAIL-driven cell death. 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14) induces endoplasmic reticulum stress (ERS) and inhibits CC cell growth. Since ERS-induced intracellular signals regulate TRAIL pathway-associated proteins, we examined whether 2-14 makes CC cells sensitive to TRAIL-driven apoptosis In Vitro and In Vivo. Methods: Human (HCT-116, HT115, DLD-1 and HT-29) and murine (CT26) CC cell lines were cultured with or without 2-14 and/or TRAIL. Death receptor (DR)4 and DR5 were analyzed by real-time PCR, flowcytometry and Western blotting. TRAIL pathway-associated proteins were assessed by Western blotting. To assess the role of DRs and survivin on 2-14-mediated TRAIL sensitization, DLD-1 and HT-29 cells were transfected with specific DR4, DR5, survivin or control siRNA and then treated with 2-14 and/or TRAIL. The effect of 2-14 treatment on survivin expression was also assessed in primary CC mucosal explants. The In Vivo capability of 2-14 to sensitize CC cells to TRAIL-induced apoptosis was evaluated in a syngenic CC model, in which CT26-derived xenografts were induced in mice. Additionally, the effects of 2-14 on DR5 and survivin expression were evaluated in a murine model of colon carcinogenesis induced by dextran sodium sulfate (DSS)/azoxymethane. Results: 2-14 sensitized DLD-1 and HT-29 cells to TRAIL-induced apoptosis and this effect was associated with increased DR5 expression. Silencing of DR5 partially reverted the 2-14-mediated TRAIL-driven cell apoptosis raising the possibility that 2-14 might target further TRAIL signaling-associated molecules. Analysis of pro- and anti-apoptotic factors and silencing studies showed that survivin confers protection in DLD-1 and HT-29 cells against TRAIL-induced apoptosis. In these cells, 2-14 promoted ubiquitination and proteasome-mediated degradation of survivin. Moreover, 214 significantly reduced survivin protein expression in CC mucosal explants. Using a murine model of TRAIL-resistant CC, we also showed that 2-14 up-regulated DR5 and reduced survivin expression, thereby synergizing with TRAIL in inhibiting tumor growth and promoting apoptosis of established colon tumors. Finally, intra-peritoneal administration of 2-14 up-regulated DR5 and down-regulated survivin in the DSS/azoxymethane-induced colon tumors. Conclusions: 2-14 acts as a sensitizer for TRAIL-induced apoptosis and this may be a novel therapeutic strategy for CC that are resistant against TRAIL-based therapies.
667 Activation of the Tumor Suppressor MicroRNA-29c by the Selective Cyclooxygenase-2 Inhibitor, Celecoxib in Human Gastric Cancer Hidekazu Suzuki, Yoshimasa Saito, Hiroyuki Imaeda, Juntaro Matsuzaki, Kenro Hirata, Hitoshi Tsugawa, Yae Kanai, Toshifumi Hibi Background : We have previously reported that microRNAs (miRNAs), small non-coding RNAs that function as endogenous silencers of target genes, play critical roles during human carcinogenesis (Cancer Cell 9: 435, 2006; Oncogene 28:2738, 2009). The selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib has been highlighted as a potential drug for chemoprevention of gastrointestinal tumors. To investigate the molecular mechanism of gastric carcinogenesis and a new therapeutic approach for gastric cancer, miRNA expression profiles were examined in gastric cancer cells treated with celecoxib. Methods : Human gastric cancer cell lines were exposed to celecoxib. Fifty three patients with gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers were examined with an informed consent (UMIN-CTR 000001057). miRNA expression profiles in gastric cancer cell lines and gastric tumor tissues were analyzed using microarray and quantitative RT-PCR. Expression of miRNA target genes was analyzed by Western blotting and immunohistochemistry. Results : miRNA microarray analysis revealed that miR-29c was significantly activated by celecoxib in gastric cancer cells. miR-29c was significantly
665 IGF-1R Modulates Colon Cancer Stem Cell Properties by Regulating Novel Tumor Suppressor MicroRNAs Bhaumik B. Patel, Yingjie Yu, Jianhua Du, Edi Levi, Adhip P. Majumdar Background: Poor outcomes in advanced colorectal cancer (CRC) is mainly attributable to survival of a small population of cancer cells called cancer stem cells (CSCs) with the ability to self-renew, resist chemotherapy killing and metastasize (broadly speaking CSC phenotype). We have shown that chemosurviving/chemoresistant CRC cells are enriched in CSCs and
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663
Developmental Origins of Irritable Bowel Syndrome (IBS)-Like Symptoms: Epigenetic Dysregulation Qingjie Li, Sushil K. Sarna Background and Aims: IBS is a complex disease with recurring clusters of symptoms. Genetic mutations and single nucleotide polymorphisms do not fit these traits. We reported previously that neonatal inflammatory insult enhances smooth muscle reactivity to ACh in adulthood, which enhances colonic motor function to induce diarrhea-like conditions. In this study, we tested the hypothesis that adverse early life events reprogram the expression of genes encoding vasoactive intestinal polypeptide (VIP) and pore-forming α1C-subunit of the Cav1.2b channels (Canca1c) by epigenetic mechanisms in colonic muscularis externa to induce colonic motility dysfunction in adulthood. Methods: Ten-day old rat neonates received intraluminal 130 mg/kg TNBS in the colon; control pups received saline. Colonic muscularis externae (ME) were obtained 6 to 8-week later. Results: qRT-PCR and western blotting showed significant increases in the mRNA and protein expressions of VIP and α1C-subunit in the ME of about 50% of adult rats that received neonatal inflammatory insult (IBS-rats). Chromatin immunoprecipitation assays showed marked increase in acetylation of histone H3 lysine 9 (H3K9Ac, a marker of transcriptional activation) (0.8±0.1 vs. 0.45±0.06 Ctr., p<0.05) and attenuation in trimethylation of histone H3 lysine 9 (H3K9me3, a marker of transcriptional repression) (0.8±0.07 vs.1.2±0.03 Ctr., p<0.05) in the core promoter region of Cacna1c in IBS-rats. As a result, the RNA polymerase II (RNAP II) association with the Cacna1c promoter significantly increased, indicating enhanced Cacna1c gene transcription. We investigated the role of VIP in the epigenetic modification of Cacna1c by incubating ME from naïve rats with 100 ng/ml VIP for 24 hours. VIP-treatment markedly increased H3K9Ac and decreased H3K9me3 at the upstream CREB-binding site (-2062/-2055) and around the transcription start site (+21 ~ +168), and hence enhanced RNAP II interaction with this promoter region suggesting that VIP modulates epigenetic components at the Cacna1c promoter to stimulate Cacna1c gene transcription. VIP antagonist, (p-Chloro-D-Phe6,Leu17)VIP, abrogated VIP-mediated RNAP II binding, H3K9 acetylation and trimethylation at the Cacna1c promoter. Intraperitoneal administration of 100 mM sodium butyrate, a histone deacetylase inhibitor, significantly elevated Cacna1c mRNA in ME of naïve adult rats (1.5±0.06 vs. 1±.01 Ctr., p<0.05), indicating that histone hyperacetylation is necessary for α1C gene transcription. Conclusions: Our findings reveal the epigenetic mechanisms by which adverse early life events alter the gene expression of specific cell signaling proteins to cause colonic motor dysfunction in adulthood. The intensities of epigenetic modifications are modifiable by environmental influences, such as stress, which explains relapses and remissions of symptoms in IBS patients.
666 MLN-8237 Treatment Alone and in Combination With Cisplatin Suppresses In Vitro and In Vivo Progression of Gastrointestinal Cancer Cells Vikas Sehdev, Mohammed Soutto, Abbes Belkhiri, Jeffrey Ecsedy, Wael El-Rifai Background: Gastric cancers respond poorly to conventional chemotherapy indicating active intrinsic mechanisms against drug induced cell death. AURKA overexpression facilitates oncogenic transformation by causing chromosomal instability, aberrant mitosis and aneuploidy. We have previously reported that AURKA is overexpressed in >50% of upper gastrointestinal adenocarcinomas. Additionally, we have confirmed that AURKA induces pro-survival mechanisms through activation of AKT and β-catenin, and inhibition of p53- and p73mediated apoptotic pathways in gastrointestinal adenocarcinomas. Methods and Results: In this study, we evaluated the effect of AURKA specific inhibitor (MLN-8237) treatment alone and/or in combination with Cisplatin (CDDP) on gastrointestinal cancer cell survival, viability, apoptotic markers and tumor volume. The ATP-glow cell viability assay and clonogenic cell survival assay data indicated that in comparison to treatment with MLN (0.5μM) or CDDP (2.5μM) alone; the combination treatment with MLN-8237 (0.5μM) and CDDP (2.5μM) significantly increased (p<0.05) inhibition of cellular viability and survival in AGS, KATOIII, MKN28, FLO-1 and OE33 gastrointestinal cancer cell lines. Similarly, the western blot data after treatment with MLN (0.5μM) and/or CDDP (2.5μM) for 48h exhibits higher levels of cleaved forms of caspase 3 and PARP with MLN (0.5μM) and CDDP (2.5μM) combination treatment in AGS, FLO-1 and OE33 cell lines. The tumor growth results from OE33 mouse xenograft study indicate that treatment with MLN-8237 (40mg/kg) and CDDP (2mg/kg) combination is significantly (p<0.01) more potent than MLN-8237 (40mg/kg) or CDDP (2mg/kg) treatment alone with the following order of effective ne ss : C on tr ol (321.0±18.3mm3), CDDP (101.4±6.2 mm3), MLN-8237 (178.8±16.9 mm3)\, and MLN8237/CDDP(41.1±7.7 mm3). Moreover, the qRT-PCR data from tumor xenograft samples following treatment with MLN-8237/CDDP combination showed significantly higher mRNA levels of p73 and its downstream transcriptional target genes (p21, puma, NOXA and BAX) than MLN-8237 or CDDP alone (P<0.01). Conclusions: Our In Vitro and In Vivo data indicate that inhibition of AURKA with MLN-8237 is an effective therapeutic strategy for inducing apoptosis and suppressing growth of gastrointestinal cancer cells. Additionally, MLN-8237 in combination with CDDP enhances the anti-tumor activity of MLN-8237 against gastrointestinal cancer cells. Therefore, our study demonstrates that MLN-8237 is an effective antitumor agent which can be potentially combined with CDDP for better therapeutic outcome in gastrointestinal cancer patients.
664 2-Methoxy-5-Amino-N-Hydroxybenzamide Sensitizes Colon Cancer (CC) Cells to TRAIL-Mediated Apoptosis Through Modulation of Death Receptor (DR)5 and Survivin Carmine Stolfi, Roberta Caruso, Eleonora Franzè, Ivan Monteleone, Daniela De Nitto, Angelamaria Rizzo, Giuseppe S. Sica, Francesco Pallone, Giovanni Monteleone Background and aim: TRAIL-induced apoptosis is a crucial event in the negative regulation of tumor growth. However, colon cancer (CC) cells are largely resistant to TRAIL-driven cell death. 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14) induces endoplasmic reticulum stress (ERS) and inhibits CC cell growth. Since ERS-induced intracellular signals regulate TRAIL pathway-associated proteins, we examined whether 2-14 makes CC cells sensitive to TRAIL-driven apoptosis In Vitro and In Vivo. Methods: Human (HCT-116, HT115, DLD-1 and HT-29) and murine (CT26) CC cell lines were cultured with or without 2-14 and/or TRAIL. Death receptor (DR)4 and DR5 were analyzed by real-time PCR, flowcytometry and Western blotting. TRAIL pathway-associated proteins were assessed by Western blotting. To assess the role of DRs and survivin on 2-14-mediated TRAIL sensitization, DLD-1 and HT-29 cells were transfected with specific DR4, DR5, survivin or control siRNA and then treated with 2-14 and/or TRAIL. The effect of 2-14 treatment on survivin expression was also assessed in primary CC mucosal explants. The In Vivo capability of 2-14 to sensitize CC cells to TRAIL-induced apoptosis was evaluated in a syngenic CC model, in which CT26-derived xenografts were induced in mice. Additionally, the effects of 2-14 on DR5 and survivin expression were evaluated in a murine model of colon carcinogenesis induced by dextran sodium sulfate (DSS)/azoxymethane. Results: 2-14 sensitized DLD-1 and HT-29 cells to TRAIL-induced apoptosis and this effect was associated with increased DR5 expression. Silencing of DR5 partially reverted the 2-14-mediated TRAIL-driven cell apoptosis raising the possibility that 2-14 might target further TRAIL signaling-associated molecules. Analysis of pro- and anti-apoptotic factors and silencing studies showed that survivin confers protection in DLD-1 and HT-29 cells against TRAIL-induced apoptosis. In these cells, 2-14 promoted ubiquitination and proteasome-mediated degradation of survivin. Moreover, 214 significantly reduced survivin protein expression in CC mucosal explants. Using a murine model of TRAIL-resistant CC, we also showed that 2-14 up-regulated DR5 and reduced survivin expression, thereby synergizing with TRAIL in inhibiting tumor growth and promoting apoptosis of established colon tumors. Finally, intra-peritoneal administration of 2-14 up-regulated DR5 and down-regulated survivin in the DSS/azoxymethane-induced colon tumors. Conclusions: 2-14 acts as a sensitizer for TRAIL-induced apoptosis and this may be a novel therapeutic strategy for CC that are resistant against TRAIL-based therapies.
667 Activation of the Tumor Suppressor MicroRNA-29c by the Selective Cyclooxygenase-2 Inhibitor, Celecoxib in Human Gastric Cancer Hidekazu Suzuki, Yoshimasa Saito, Hiroyuki Imaeda, Juntaro Matsuzaki, Kenro Hirata, Hitoshi Tsugawa, Yae Kanai, Toshifumi Hibi Background : We have previously reported that microRNAs (miRNAs), small non-coding RNAs that function as endogenous silencers of target genes, play critical roles during human carcinogenesis (Cancer Cell 9: 435, 2006; Oncogene 28:2738, 2009). The selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib has been highlighted as a potential drug for chemoprevention of gastrointestinal tumors. To investigate the molecular mechanism of gastric carcinogenesis and a new therapeutic approach for gastric cancer, miRNA expression profiles were examined in gastric cancer cells treated with celecoxib. Methods : Human gastric cancer cell lines were exposed to celecoxib. Fifty three patients with gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers were examined with an informed consent (UMIN-CTR 000001057). miRNA expression profiles in gastric cancer cell lines and gastric tumor tissues were analyzed using microarray and quantitative RT-PCR. Expression of miRNA target genes was analyzed by Western blotting and immunohistochemistry. Results : miRNA microarray analysis revealed that miR-29c was significantly activated by celecoxib in gastric cancer cells. miR-29c was significantly
665 IGF-1R Modulates Colon Cancer Stem Cell Properties by Regulating Novel Tumor Suppressor MicroRNAs Bhaumik B. Patel, Yingjie Yu, Jianhua Du, Edi Levi, Adhip P. Majumdar Background: Poor outcomes in advanced colorectal cancer (CRC) is mainly attributable to survival of a small population of cancer cells called cancer stem cells (CSCs) with the ability to self-renew, resist chemotherapy killing and metastasize (broadly speaking CSC phenotype). We have shown that chemosurviving/chemoresistant CRC cells are enriched in CSCs and
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