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338: Smad ubiquitination regulatory factor 2 (Smurf2) regulates the gap junction protein connexin43 during mitosis
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
336 EDI3, a glycerophosphodiesterase linking metabolism to cellular migration and attachment R. Marchan1 , M.S. Lesjak1 , B. Buettner1 , J.D. Stewart2 , J. Lambert3 , H. Keun4 , J. Rahnenfuehrer5 , J.G. Hengstler1 . 1 Leibniz Research Centre for Working Environment and Human Factors (IfaDo), Systems Toxicology, Dortmund, Germany, 2 University of Southampton, Center for Biological Sciences, Southampton, United Kingdom, 3 Leibniz-Institut fur ¨ Analytische Wissenschaften − ISAS − e.V., Interface Processes, Dortmund, Germany, 4 Imperial College London, Surgery and Cancer, London, United Kingdom, 5 Technische Universitaet Dortmund, Statistics, Dortmund, Germany Introduction: Metastasis from the primary tumour remains a therapeutic challenge and is a major cause of death in cancer patients. We identified the glycerophosphodiesterase (GDE), EDI3 (GPCPD1; GDE5) in a study aimed to find markers of endometrial cancer metastasis. Patients with tumours expressing high EDI3 went on to develop metastasis and had worse prognosis. EDI3 was shown to hydrolyse glycerophosphocholine (GPC) to choline and glycerol-3-phosphate (G3P), with consequences in both lipid and choline metabolism. EDI3 expression was also associated with cellular migration via PKCa. Our work was the first to implicate a GDE in cancer, but the link between EDI3’s enzymatic activity and its role in migration or other processes important in cellular transformation remain unknown. Therefore, our aim was to identify other processes where EDI3 may be involved and investigate pathways downstream of EDI3 to determine which may be more critical for the observed phenotypic changes. Materials and Methods: Gene array analysis after EDI3 knockdown was performed and gene ontology analysis used to identify relevant biological motifs. RNA, protein and functional assays were performed after knockdown and overexpression to characterize EDI3’s potential role in integrin-mediated processes. Choline kinase alpha (CHKA), which phosphorylates choline to phosphocholine − a first step in phosphatidylcholine synthesis; G3P acyltransferase 1 (GPAT1), which adds an acyl group to G3P, producing the signalling lipid lysophosphatidic acid (LPA); and EDI3 were downregulated using siRNA and the effect on migration, key signalling proteins, and metabolism compared. Confirmation was obtained by overexpressing the respective proteins. Results and Discussion: An enrichment of genes involved in integrinmediated signalling led to the characterization of EDI3 in cellular attachment and spreading on a fibronectin matrix. Loss of EDI3 decreased expression of integrin a5/b1 expression, and was associated with delayed attachment and spreading, processes linked to migration. GPAT1 influenced cell migration, and the combined knockdown of both EDI3 and GPAT1 did not exacerbate the migration effect, suggesting that their influence on migration is linked. Furthermore, knocking down GPAT1 in EDI3-overexpressing cells ‘rescued’ EDI3’s mediated increase in migration. Interestingly, in contrast to previous reports, CHKA had no influence on migration or viability, even though loss of EDI3 leads to decreased phosphocholine levels. Conclusion: EDI3’s influence on cell migration appears to be mediated via the GPAT1 pathway. However no common signalling protein has been identified. Further investigation into the proteins and products of this pathway must be investigated to understand which of the several candidates are important for EDI3-mediated migration. No conflict of interest. 337 STAT3, IL-17 and COX-2 features in the transgenic adenocarcinoma of mouse prostate (TRAMP) model and in the aging mice (FVB) submitted to goniothalamin therapy
Goniothalamin treatment led to decrease of both cellular proliferation and inflammation in TRAMP and senile mice. The molecular results showed that Goniothalamin treatment displayed a reduction to STAT-3 and IL-17 in both models, mainly in senile mice. On the other hand COX-2 reactivity was not altered. Conclusions: Goniothalamin treatment demonstrated significant effect on the pro-inflammatory cytokine and on a transcription factor signal transducer, acting in many pro-oncogenic signals, possibly delaying prostate cancer progression. Moreover, this treatment suggested a preventive effect on senile prostatic microenvironment, considering the role of senescence in different clinical disorders and the potential to elderly mice to develop prostatic cancer. No conflict of interest. 338 Smad ubiquitination regulatory factor 2 (Smurf2) regulates the gap junction protein connexin43 during mitosis T.A. Fykerud1 , S.D. Koirala1 , M.Z. Totland1 , L.M. Knudsen1 , Z. Yohannes1 , Y. Omori2 , A. Brech3 , E. Leithe1 . 1 The Norwegian Radium Hospital, Department of Cancer Prevention, Oslo, Norway, 2 Akita University School of Medicine, Department of Molecular and Tumor Pathology, Akita, Japan, 3 The Norwegian Radium Hospital, Department of Biochemistry, Oslo, Norway Background: The connexins are a family of integral membrane proteins that form channels between adjacent cells, which assemble into distinct plasma membrane domains called gap junctions. Gap junctions enable the adjacent cells to directly communicate with each other, by exchanging ions and small molecules between the cells. Gap junctions play important roles in cell growth control and in the maintenance of tissue homeostasis. There is significant evidence that aberrant regulation of gap junction intercellular communication is involved in carcinogenesis. The connexin protein family comprises 21 members in humans, of which connexin43 (Cx43) is the beststudied member. Cx43 has been shown to act as a tumor suppressor in various tissues. Previous studies have shown that Cdk1 phosphorylates Cx43 during mitosis, which is associated with a reduction in gap junction intercellular communication and relocalization of Cx43 from the plasma membrane to intracellular structures. Here, we have investigated the molecular mechanisms involved in the regulation of Cx43 during mitosis. Materials and Methods: IAR20 cells and HeLa cells stably transfected with Cx43 were used as model systems for studying Cx43 during mitosis, where both unsynchronized cells and cells synchronized with RO-3306 were analyzed. The subcellular localization of Cx43 in mitotic cells was determined by confocal microscopy. The binding partners to Cx43 and the changes in Cx43 phosphorylation and ubiquitination status during mitosis was determined by immunoprecipitation and western blotting. Results and Discussion: Cx43 was found to relocalize from the plasma membrane to early endosomes during mitotic progression. The increased endocytosis of Cx43 started in prophase and continued until the late mitotic phases. The E3 ubiquitin ligase Smurf2 (smad ubiquitination regulatory factor 2), which previously has been shown to regulate the mitotic spindle checkpoint, was found to colocalize with Cx43 gap junctions in the early phases of mitosis. Depletion of Smurf2 by siRNA counteracted gap junction endocytosis and trafficking of Cx43 to early endosomes in mitotic cells, resulting in increased levels of gap junctions at the plasma membrane during mitosis. Conclusions: This study identifies Smurf2 as a novel regulator of Cx43 gap junctions during mitosis. The data represent the first evidence that the intracellular trafficking of Cx43 during mitosis is regulated by an E3 ubiquitin ligase. No conflict of interest. 339 MALDI-MS peptide profiling of thyroid cancer cell lines to detect peptide signatures changes with the PI3-K inhibitor GDC-0941, in hypoxia and normoxia
L.A. Kido1 , F. Montico1 , D.B. Vendramini-Costa2 , J.E. Carvalho3 , R.A. Pilli2 , V.H.A. Cagnon1 . 1 Institute of Biology, Structural and Functional Biology, ˜ Paulo, Brazil, 2 Institute of Chemistry, Organic Chemistry, Campinas Sao ˜ Paulo, Brazil, 3 Institute of Biology − CPQBA, Structural and Campinas Sao ˜ Paulo, Brazil Functional Biology, Campinas Sao
F. Henderson1 , K. Williams1 . 1 University of Manchester, Manchester, United Kingdom
Background: Senescence is associated with significant changes in the hormonal environment causing prostatic disorders, including inflammatory and proliferative processes. TRAMP model has been widely used to study the molecular events in the progression of prostate cancer, since this animal develops prostate tumors that share many features with human cancer. The aim herewith was to characterize and compare IL-17, COX-2, and STAT-3 signaling in the ventral prostate from TRAMP and senile (FVB) mice treated with goniothalamin treatment, a promising synthetic substance which exhibits anti-inflammatory and anti-proliferative properties. Material and Methods: The animals were divided into: Control groups; Young (18 week-old FVB), Senile (52 week-old FVB) and TRAMP: 8 and 12-week-old mice. Treated groups; Senile-Goniothalamin (150 mg/kg orally) and TRAMP8Goniothalamin (150 mg/kg orally). The ventral lobe was collected after 4 weeks for light microscopy, immunohistochemistry and western blotting. Results: Structural analysis demonstrated that senescence led to changes in prostatic microenvironment, such atrophy, inflammation, and especially, proliferative lesions (PIN), which were similar to those observed in TRAMP mice.
Introduction: Tumour hypoxia is a state of deprived oxygen due to cancer cells over-proliferating with insufficient blood vessel formation, and is associated with aggressive tumours and a poor prognosis. The transcription factor Hif-1a is a central regulator for pathways involved in the tumour hypoxia. The PI3K/Akt pathway is commonly upregulated in a variety of cancers, and has been associated with the activation of Hif-1a. Therefore, inhibiting the PI3-K/Akt pathway offers a potential anti-cancer therapeutic target. MALDI-MS is a soft ionization technique which uses an untargeted approach to look at large number of peptides/proteins in a single experiment. MALDIMS will be used to detect protein changes in thyroid cancer cell lines with and without the presence of the PI3-K inhibitor GDC-0941, in hypoxia and normoxia. This will be carried out by generating peptide profiles of cell lysates, and using MS/MS to identify these peptides and thus the protein from which it’s derived. Western blots will be carried out for Akt and Hif-1a to ensure the drug is acting as expected, and to investigate the interactions of GDC-0941, hypoxia and Hif-1a.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
336 EDI3, a glycerophosphodiesterase linking metabolism to cellular migration and attachment R. Marchan1 , M.S. Lesjak1 , B. Buettner1 , J.D. Stewart2 , J. Lambert3 , H. Keun4 , J. Rahnenfuehrer5 , J.G. Hengstler1 . 1 Leibniz Research Centre for Working Environment and Human Factors (IfaDo), Systems Toxicology, Dortmund, Germany, 2 University of Southampton, Center for Biological Sciences, Southampton, United Kingdom, 3 Leibniz-Institut fur ¨ Analytische Wissenschaften − ISAS − e.V., Interface Processes, Dortmund, Germany, 4 Imperial College London, Surgery and Cancer, London, United Kingdom, 5 Technische Universitaet Dortmund, Statistics, Dortmund, Germany Introduction: Metastasis from the primary tumour remains a therapeutic challenge and is a major cause of death in cancer patients. We identified the glycerophosphodiesterase (GDE), EDI3 (GPCPD1; GDE5) in a study aimed to find markers of endometrial cancer metastasis. Patients with tumours expressing high EDI3 went on to develop metastasis and had worse prognosis. EDI3 was shown to hydrolyse glycerophosphocholine (GPC) to choline and glycerol-3-phosphate (G3P), with consequences in both lipid and choline metabolism. EDI3 expression was also associated with cellular migration via PKCa. Our work was the first to implicate a GDE in cancer, but the link between EDI3’s enzymatic activity and its role in migration or other processes important in cellular transformation remain unknown. Therefore, our aim was to identify other processes where EDI3 may be involved and investigate pathways downstream of EDI3 to determine which may be more critical for the observed phenotypic changes. Materials and Methods: Gene array analysis after EDI3 knockdown was performed and gene ontology analysis used to identify relevant biological motifs. RNA, protein and functional assays were performed after knockdown and overexpression to characterize EDI3’s potential role in integrin-mediated processes. Choline kinase alpha (CHKA), which phosphorylates choline to phosphocholine − a first step in phosphatidylcholine synthesis; G3P acyltransferase 1 (GPAT1), which adds an acyl group to G3P, producing the signalling lipid lysophosphatidic acid (LPA); and EDI3 were downregulated using siRNA and the effect on migration, key signalling proteins, and metabolism compared. Confirmation was obtained by overexpressing the respective proteins. Results and Discussion: An enrichment of genes involved in integrinmediated signalling led to the characterization of EDI3 in cellular attachment and spreading on a fibronectin matrix. Loss of EDI3 decreased expression of integrin a5/b1 expression, and was associated with delayed attachment and spreading, processes linked to migration. GPAT1 influenced cell migration, and the combined knockdown of both EDI3 and GPAT1 did not exacerbate the migration effect, suggesting that their influence on migration is linked. Furthermore, knocking down GPAT1 in EDI3-overexpressing cells ‘rescued’ EDI3’s mediated increase in migration. Interestingly, in contrast to previous reports, CHKA had no influence on migration or viability, even though loss of EDI3 leads to decreased phosphocholine levels. Conclusion: EDI3’s influence on cell migration appears to be mediated via the GPAT1 pathway. However no common signalling protein has been identified. Further investigation into the proteins and products of this pathway must be investigated to understand which of the several candidates are important for EDI3-mediated migration. No conflict of interest. 337 STAT3, IL-17 and COX-2 features in the transgenic adenocarcinoma of mouse prostate (TRAMP) model and in the aging mice (FVB) submitted to goniothalamin therapy
Goniothalamin treatment led to decrease of both cellular proliferation and inflammation in TRAMP and senile mice. The molecular results showed that Goniothalamin treatment displayed a reduction to STAT-3 and IL-17 in both models, mainly in senile mice. On the other hand COX-2 reactivity was not altered. Conclusions: Goniothalamin treatment demonstrated significant effect on the pro-inflammatory cytokine and on a transcription factor signal transducer, acting in many pro-oncogenic signals, possibly delaying prostate cancer progression. Moreover, this treatment suggested a preventive effect on senile prostatic microenvironment, considering the role of senescence in different clinical disorders and the potential to elderly mice to develop prostatic cancer. No conflict of interest. 338 Smad ubiquitination regulatory factor 2 (Smurf2) regulates the gap junction protein connexin43 during mitosis T.A. Fykerud1 , S.D. Koirala1 , M.Z. Totland1 , L.M. Knudsen1 , Z. Yohannes1 , Y. Omori2 , A. Brech3 , E. Leithe1 . 1 The Norwegian Radium Hospital, Department of Cancer Prevention, Oslo, Norway, 2 Akita University School of Medicine, Department of Molecular and Tumor Pathology, Akita, Japan, 3 The Norwegian Radium Hospital, Department of Biochemistry, Oslo, Norway Background: The connexins are a family of integral membrane proteins that form channels between adjacent cells, which assemble into distinct plasma membrane domains called gap junctions. Gap junctions enable the adjacent cells to directly communicate with each other, by exchanging ions and small molecules between the cells. Gap junctions play important roles in cell growth control and in the maintenance of tissue homeostasis. There is significant evidence that aberrant regulation of gap junction intercellular communication is involved in carcinogenesis. The connexin protein family comprises 21 members in humans, of which connexin43 (Cx43) is the beststudied member. Cx43 has been shown to act as a tumor suppressor in various tissues. Previous studies have shown that Cdk1 phosphorylates Cx43 during mitosis, which is associated with a reduction in gap junction intercellular communication and relocalization of Cx43 from the plasma membrane to intracellular structures. Here, we have investigated the molecular mechanisms involved in the regulation of Cx43 during mitosis. Materials and Methods: IAR20 cells and HeLa cells stably transfected with Cx43 were used as model systems for studying Cx43 during mitosis, where both unsynchronized cells and cells synchronized with RO-3306 were analyzed. The subcellular localization of Cx43 in mitotic cells was determined by confocal microscopy. The binding partners to Cx43 and the changes in Cx43 phosphorylation and ubiquitination status during mitosis was determined by immunoprecipitation and western blotting. Results and Discussion: Cx43 was found to relocalize from the plasma membrane to early endosomes during mitotic progression. The increased endocytosis of Cx43 started in prophase and continued until the late mitotic phases. The E3 ubiquitin ligase Smurf2 (smad ubiquitination regulatory factor 2), which previously has been shown to regulate the mitotic spindle checkpoint, was found to colocalize with Cx43 gap junctions in the early phases of mitosis. Depletion of Smurf2 by siRNA counteracted gap junction endocytosis and trafficking of Cx43 to early endosomes in mitotic cells, resulting in increased levels of gap junctions at the plasma membrane during mitosis. Conclusions: This study identifies Smurf2 as a novel regulator of Cx43 gap junctions during mitosis. The data represent the first evidence that the intracellular trafficking of Cx43 during mitosis is regulated by an E3 ubiquitin ligase. No conflict of interest. 339 MALDI-MS peptide profiling of thyroid cancer cell lines to detect peptide signatures changes with the PI3-K inhibitor GDC-0941, in hypoxia and normoxia
L.A. Kido1 , F. Montico1 , D.B. Vendramini-Costa2 , J.E. Carvalho3 , R.A. Pilli2 , V.H.A. Cagnon1 . 1 Institute of Biology, Structural and Functional Biology, ˜ Paulo, Brazil, 2 Institute of Chemistry, Organic Chemistry, Campinas Sao ˜ Paulo, Brazil, 3 Institute of Biology − CPQBA, Structural and Campinas Sao ˜ Paulo, Brazil Functional Biology, Campinas Sao
F. Henderson1 , K. Williams1 . 1 University of Manchester, Manchester, United Kingdom
Background: Senescence is associated with significant changes in the hormonal environment causing prostatic disorders, including inflammatory and proliferative processes. TRAMP model has been widely used to study the molecular events in the progression of prostate cancer, since this animal develops prostate tumors that share many features with human cancer. The aim herewith was to characterize and compare IL-17, COX-2, and STAT-3 signaling in the ventral prostate from TRAMP and senile (FVB) mice treated with goniothalamin treatment, a promising synthetic substance which exhibits anti-inflammatory and anti-proliferative properties. Material and Methods: The animals were divided into: Control groups; Young (18 week-old FVB), Senile (52 week-old FVB) and TRAMP: 8 and 12-week-old mice. Treated groups; Senile-Goniothalamin (150 mg/kg orally) and TRAMP8Goniothalamin (150 mg/kg orally). The ventral lobe was collected after 4 weeks for light microscopy, immunohistochemistry and western blotting. Results: Structural analysis demonstrated that senescence led to changes in prostatic microenvironment, such atrophy, inflammation, and especially, proliferative lesions (PIN), which were similar to those observed in TRAMP mice.
Introduction: Tumour hypoxia is a state of deprived oxygen due to cancer cells over-proliferating with insufficient blood vessel formation, and is associated with aggressive tumours and a poor prognosis. The transcription factor Hif-1a is a central regulator for pathways involved in the tumour hypoxia. The PI3K/Akt pathway is commonly upregulated in a variety of cancers, and has been associated with the activation of Hif-1a. Therefore, inhibiting the PI3-K/Akt pathway offers a potential anti-cancer therapeutic target. MALDI-MS is a soft ionization technique which uses an untargeted approach to look at large number of peptides/proteins in a single experiment. MALDIMS will be used to detect protein changes in thyroid cancer cell lines with and without the presence of the PI3-K inhibitor GDC-0941, in hypoxia and normoxia. This will be carried out by generating peptide profiles of cell lysates, and using MS/MS to identify these peptides and thus the protein from which it’s derived. Western blots will be carried out for Akt and Hif-1a to ensure the drug is acting as expected, and to investigate the interactions of GDC-0941, hypoxia and Hif-1a.