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309 Maintenance of S-nitrosothiol Homeostasis Plays an Important Role in Growth Suppression in Estrogen Receptor Positive Breast Tumors
S76
european journal of cancer 48, suppl. 5 (2012) S25–S288
309 Maintenance of S-nitrosothiol Homeostasis Plays an Important Role in Growth Suppression in Estrogen Receptor Positive Breast Tumors A. Canas ˜ 1 , L. Lopez-Sanchez1 , A. Valverde1 , V. Hernandez1 , C. LopezPedrera1 , J. De la Haba-Rodriguez1 , E. Aranda1 , A. Rodriguez-Ariza1 . 1 Hospital Reina Sof´ıa, Oncology, Cordoba, Spain Introduction: One of the mechanisms governing protein denitrosylation is the system thioredoxin/thioredoxin reductase (Trx/TrxR). Alteration of this enzymatic system may impair S-nitrosothiol (SNO) homeostasis in tumor cells, providing new insights into the role of nitric oxide in cancer and its role in tumor progression and antitumoral treatment. Material and Method: MCF-7, MDA-MB-231 and BT-474 cells were pretreated or not with the highly specific TrxR inhibitor auranofin and exposed to different doses of S-nitroso-L-Cysteine (CSNO). S-nitrosylated proteins were detected using the ’biotin-switch’ assay. Subcelullar localization of ER-alpha was analyzed by confocal microscopy. Oncomine database was explored for TrxR (TXNRD1) expression in ER− and ER+ breast tumors. Results: In all the three cell lines, a high CSNO dose (500 mM) reduced cell proliferation and this effect was potentiated by pretreatment with auranofin. Augmented levels of S-nitrosylated proteins were observed in MCF-7 cells treated with 500 mM CSNO, and this effect also was potentiated by pretreatment with auranofin. However, treatment with auranofin and 100 nM CSNO enhanced cell proliferation of estrogen receptor positive (ER+) MCF-7 cells, but not of MDA-MB-231 (ER−, mut p53), or BT-474 (ER+, mut p53) cells. This cell growth was associated with Akt and Erk 1/2 phosphorylation, augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-a. The specific silencing of ER-alpha expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation potentiated the pro-proliferative effect of SNO homeostasis impairment in MCF-7 cells. Moreover, the subcelullar distribution of ER-alpha was altered, with a predominantly nuclear localization in those cells with impaired SNO homeostasis. When Oncomine database was interrogated, positive ER status in breast tumors was found to be associated with significantly lower levels of TXNDR1 expression. Conclusion: ER status in breast cancer may dictate the tumor response to different nitrosative environments. Factors impairing the Trx/TrxR enzymatic system in tumor cells may confer survival advantages to ER+/p53 wt breast tumors. These molecular mechanisms may also play a significant role in the development of resistance against hormonal therapies that arise in this type of mammary tumors. 311 Lysyl Oxidase Antagonizes RAS Oncogene-mediated Transformation S. Liu1 , M.C. Ha-Thi1 , R. Schafer ¨ 1 . 1 Charite´ University Hospital, Institute of Pathology, Berlin, Germany Introduction: We have systemically identified the transcriptome alterations mediated by oncogenic RAS pathway signalling and analyzed the cellular functions of robustly down-regulated targets in RAS transformed cells. The targets include lysyl oxidase (LOX), thrombospondin 1 (THBS1), follistatinlike 1 (FSTL1) and cysteine-rich protein 61 (CYR61). LOX encodes a 53kDa enzyme which plays a key role in mediating the cross-linking of extracellular matrix proteins. Its smaller cleavage product, a propeptide of 18 KDa, inhibits NF-kB signalling. Previous publications have suggested a dual role for LOX as a tumor suppressor and a metastasis promoting gene. Material and Methods: Stable cell lines in which LOX was over-expressed or silenced were generated by lentiviral vector-mediated gene transfer in human telomerized, SV40-transformed preneoplastic fibroblasts (BJELB) and in BJELR cells, a HRAS-transformed derivative of BJELB. Protein and mRNA expression of LOX, HRAS and components of the MEK/ERK signaling pathway were examined by Western blot and qPCR. Anchorage-independent growth of parental cells and derivatives was determined by cultivation in semi-solid agar medium. Cell cycle alterations were assayed by flow cytometry. Results and Discussion: The expression of LOX was found consistently repressed in RAS transformed cells of human, mouse and rat origin. Transient silencing of LOX expression in BJELB cells resulted in morphological transformation and anchorage independent proliferation. Stable LOX knockdown had similar effects in BJELB cells and enhanced endogenous RAS expression as well as phosphorylated MEK1/2 levels. Upon serum stimulation, ERK activation was faster in LOX-depleted than in LOX-expressing cells. Treatment of cells with the LOX inhibitor beta-aminopropionitrile fumarate (BAPN) resulted in elevated levels of activated MEK1/2. Stable cell populations transduced with the recombinant LOX vector expressed the transgene 18-fold compared to vector-only controls. LOX overexpression in BJELR cells reversed the transformed morphology, inhibited proliferation by 30%, and reduced anchorage independent growth by more than 90%. Ectopic stable LOX expression controlled by an ubiquitin promoter induced cell cycle arrest in G0/G1 in BJELR cells. In RAS-transformed rat fibroblasts, LOX expression also significantly inhibited focus formation.
Sunday 8 − Tuesday 10 July 2012
Conclusion: The function of LOX as a transformation-suppressing gene in human and rat fibroblasts was confirmed by overexpression in HRAStransformed cells as well as by silencing in preneoplastic cells which did not express the HRAS oncogene. Analysis of the MEK/ERK pathway suggested that robust signaling mediated the transformed phenotype in the absence of LOX activity. It is likely that crosstalk with other signaling pathways, e.g. the NF-kB pathway, contributes to cellular transformation in BJELB cells. 313 Role of Intracellular LOXL2 in the Regulation of Epithelial Plasticity E.P. Cuevas1 , G. Canesin1 , V. Santos1 , A. Montes1 , G. Moreno-Bueno1 , F. Portillo1 , A. Cano1 . 1 Department of Biochemistry UAM Instituto de ´ Investigaciones Biomedicas ‘Alberto Sols’, (CSIC-UAM) Madrid Spain. IdiPAZ, Madrid, Spain Background: Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family that catalyze the cross-linking of collagens or elastins in the extracellular matrix regulating the tensile strength of tissues [1]. LOX members share a highly conserved C-terminal catalytic domain but differ in the N-terminal region. LOXL2 is characterized by an N-terminal part that contains four scavenger receptor cysteine-rich (SRCR) domains, proposed to mediate ligand binding of several secreted and receptor proteins [1]. We previously reported that LOXL2 is an interacting partner of Snail1, mediates epithelialto-mesenchymal transition (EMT) [2] and controls epidermal differentiation by Snail1-dependent and -independent pathways [3]. We also described intracellular LOXL2 as a poor prognosis marker in squamous cell carcinomas [3] and as a marker of metastatic basal-like breast carcinomas [4]. LOXL2 is required for the metastatic dissemination of basal-like breast carcinoma cells and downregulates cell polarity components at transcriptional level [4]. However, the mechanism of LOXL2 action on gene regulation and the specific role of its catalytic activity remains largely unknown. Material and Methods: Several LOXL2 mutants were generated in the catalytic domain: DCatLOXL2 (aa 547–667) and point mutants in the His residues required for Cu binding, as well as in different SRCR domains. LOXL2 mutants were analyzed for their repression activity on E-cadherin, tight junction and cell polarity genes compared to w.t. LOXL2. Stable transfection of DCatLOXL2 was also performed on MDCK cells and the phenotypic effects compared to w.t. LOXL2 following standard procedures [2]. Results: Promoter studies indicated that LOXL2 represses target genes independently of its enzymatic activity by a mechanism that can involve the cooperation with transcription factors as Snail1 or E47 in the case of E-cadherin, but also by mechanisms independent of these factors on other genes. Promoter assays with different LOXL2 catalytic and SRCR domain mutants identify the protein region essential for its repressor activity. In addition, we have identified several signaling pathways required for the transcriptional repression of LOXL2, including MAPK, PI3K and mTOR. Several studies with the catalytic mutants indicate the implication of the LOXL2 catalytic domain in the regulation of its extracellular secretion and the requirement of the intact LOXL2 for induction of a full EMT process. Conclusions: Intracellular LOXL2 seems to act as a multifunctional protein regulating several target genes through diverse mechanisms as well as regulating signaling pathways dependently or independently of its catalytic activity. Reference(s) [1] Payne SL et al. J Cell Biochem 101:1338−54 (2007). [2] Peinado H et al. EMBO J 24:3446−58 (2005). [3] Peinado H et al. Cancer Res 68:4541−50 (2008). [4] Moreno-Bueno G et al. EMBO Mol Med 3: 528−44 (2011). 314 Ascorbic Acid Effect in Two Different Cell Lines of Colorectal Adenocarcinoma − in Vitro and in Vivo Studies A.S. Pires1 , A.C. Mamede2 , V.S. Silva1 , A.M. Abrantes1 , A.C. Gon¸calves3 , A.B. Sarmento-Ribeiro3 , M.F. Botelho1 . 1 Biophysics Unit, IBILI − Faculty of Medicine − University of Coimbra, Coimbra, Portugal, 2 CICS-UBI − Health Sciences Research Centre, University of Beira Interior, Coimbra, Portugal, 3 Applied Molecular Biology and Hematology Group, Faculty of Medicine − University of Coimbra, Coimbra, Portugal Background: Colorectal cancer (CC) is one of the most common malignancies in the western world that over the last years has been demonstrated as a progressive and multistep genetic disease. Vitamin C is available in its reduced form (ascorbic acid − AA) and in its oxidized form (dehydroascorbic acid − DHA). Although AA is most commonly known for its antioxidant properties, reducing the oxidative stress, it is also known to function as a pro-oxidant, promoting the formation of reactive oxygen species (ROS) in physiological conditions. The increased production of hydrogen peroxide, coupled with the breakdown of the activity of antioxidant enzymes and the presence of transition metals in cancer cells, may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential. The aim of this study
european journal of cancer 48, suppl. 5 (2012) S25–S288
309 Maintenance of S-nitrosothiol Homeostasis Plays an Important Role in Growth Suppression in Estrogen Receptor Positive Breast Tumors A. Canas ˜ 1 , L. Lopez-Sanchez1 , A. Valverde1 , V. Hernandez1 , C. LopezPedrera1 , J. De la Haba-Rodriguez1 , E. Aranda1 , A. Rodriguez-Ariza1 . 1 Hospital Reina Sof´ıa, Oncology, Cordoba, Spain Introduction: One of the mechanisms governing protein denitrosylation is the system thioredoxin/thioredoxin reductase (Trx/TrxR). Alteration of this enzymatic system may impair S-nitrosothiol (SNO) homeostasis in tumor cells, providing new insights into the role of nitric oxide in cancer and its role in tumor progression and antitumoral treatment. Material and Method: MCF-7, MDA-MB-231 and BT-474 cells were pretreated or not with the highly specific TrxR inhibitor auranofin and exposed to different doses of S-nitroso-L-Cysteine (CSNO). S-nitrosylated proteins were detected using the ’biotin-switch’ assay. Subcelullar localization of ER-alpha was analyzed by confocal microscopy. Oncomine database was explored for TrxR (TXNRD1) expression in ER− and ER+ breast tumors. Results: In all the three cell lines, a high CSNO dose (500 mM) reduced cell proliferation and this effect was potentiated by pretreatment with auranofin. Augmented levels of S-nitrosylated proteins were observed in MCF-7 cells treated with 500 mM CSNO, and this effect also was potentiated by pretreatment with auranofin. However, treatment with auranofin and 100 nM CSNO enhanced cell proliferation of estrogen receptor positive (ER+) MCF-7 cells, but not of MDA-MB-231 (ER−, mut p53), or BT-474 (ER+, mut p53) cells. This cell growth was associated with Akt and Erk 1/2 phosphorylation, augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-a. The specific silencing of ER-alpha expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation potentiated the pro-proliferative effect of SNO homeostasis impairment in MCF-7 cells. Moreover, the subcelullar distribution of ER-alpha was altered, with a predominantly nuclear localization in those cells with impaired SNO homeostasis. When Oncomine database was interrogated, positive ER status in breast tumors was found to be associated with significantly lower levels of TXNDR1 expression. Conclusion: ER status in breast cancer may dictate the tumor response to different nitrosative environments. Factors impairing the Trx/TrxR enzymatic system in tumor cells may confer survival advantages to ER+/p53 wt breast tumors. These molecular mechanisms may also play a significant role in the development of resistance against hormonal therapies that arise in this type of mammary tumors. 311 Lysyl Oxidase Antagonizes RAS Oncogene-mediated Transformation S. Liu1 , M.C. Ha-Thi1 , R. Schafer ¨ 1 . 1 Charite´ University Hospital, Institute of Pathology, Berlin, Germany Introduction: We have systemically identified the transcriptome alterations mediated by oncogenic RAS pathway signalling and analyzed the cellular functions of robustly down-regulated targets in RAS transformed cells. The targets include lysyl oxidase (LOX), thrombospondin 1 (THBS1), follistatinlike 1 (FSTL1) and cysteine-rich protein 61 (CYR61). LOX encodes a 53kDa enzyme which plays a key role in mediating the cross-linking of extracellular matrix proteins. Its smaller cleavage product, a propeptide of 18 KDa, inhibits NF-kB signalling. Previous publications have suggested a dual role for LOX as a tumor suppressor and a metastasis promoting gene. Material and Methods: Stable cell lines in which LOX was over-expressed or silenced were generated by lentiviral vector-mediated gene transfer in human telomerized, SV40-transformed preneoplastic fibroblasts (BJELB) and in BJELR cells, a HRAS-transformed derivative of BJELB. Protein and mRNA expression of LOX, HRAS and components of the MEK/ERK signaling pathway were examined by Western blot and qPCR. Anchorage-independent growth of parental cells and derivatives was determined by cultivation in semi-solid agar medium. Cell cycle alterations were assayed by flow cytometry. Results and Discussion: The expression of LOX was found consistently repressed in RAS transformed cells of human, mouse and rat origin. Transient silencing of LOX expression in BJELB cells resulted in morphological transformation and anchorage independent proliferation. Stable LOX knockdown had similar effects in BJELB cells and enhanced endogenous RAS expression as well as phosphorylated MEK1/2 levels. Upon serum stimulation, ERK activation was faster in LOX-depleted than in LOX-expressing cells. Treatment of cells with the LOX inhibitor beta-aminopropionitrile fumarate (BAPN) resulted in elevated levels of activated MEK1/2. Stable cell populations transduced with the recombinant LOX vector expressed the transgene 18-fold compared to vector-only controls. LOX overexpression in BJELR cells reversed the transformed morphology, inhibited proliferation by 30%, and reduced anchorage independent growth by more than 90%. Ectopic stable LOX expression controlled by an ubiquitin promoter induced cell cycle arrest in G0/G1 in BJELR cells. In RAS-transformed rat fibroblasts, LOX expression also significantly inhibited focus formation.
Sunday 8 − Tuesday 10 July 2012
Conclusion: The function of LOX as a transformation-suppressing gene in human and rat fibroblasts was confirmed by overexpression in HRAStransformed cells as well as by silencing in preneoplastic cells which did not express the HRAS oncogene. Analysis of the MEK/ERK pathway suggested that robust signaling mediated the transformed phenotype in the absence of LOX activity. It is likely that crosstalk with other signaling pathways, e.g. the NF-kB pathway, contributes to cellular transformation in BJELB cells. 313 Role of Intracellular LOXL2 in the Regulation of Epithelial Plasticity E.P. Cuevas1 , G. Canesin1 , V. Santos1 , A. Montes1 , G. Moreno-Bueno1 , F. Portillo1 , A. Cano1 . 1 Department of Biochemistry UAM Instituto de ´ Investigaciones Biomedicas ‘Alberto Sols’, (CSIC-UAM) Madrid Spain. IdiPAZ, Madrid, Spain Background: Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family that catalyze the cross-linking of collagens or elastins in the extracellular matrix regulating the tensile strength of tissues [1]. LOX members share a highly conserved C-terminal catalytic domain but differ in the N-terminal region. LOXL2 is characterized by an N-terminal part that contains four scavenger receptor cysteine-rich (SRCR) domains, proposed to mediate ligand binding of several secreted and receptor proteins [1]. We previously reported that LOXL2 is an interacting partner of Snail1, mediates epithelialto-mesenchymal transition (EMT) [2] and controls epidermal differentiation by Snail1-dependent and -independent pathways [3]. We also described intracellular LOXL2 as a poor prognosis marker in squamous cell carcinomas [3] and as a marker of metastatic basal-like breast carcinomas [4]. LOXL2 is required for the metastatic dissemination of basal-like breast carcinoma cells and downregulates cell polarity components at transcriptional level [4]. However, the mechanism of LOXL2 action on gene regulation and the specific role of its catalytic activity remains largely unknown. Material and Methods: Several LOXL2 mutants were generated in the catalytic domain: DCatLOXL2 (aa 547–667) and point mutants in the His residues required for Cu binding, as well as in different SRCR domains. LOXL2 mutants were analyzed for their repression activity on E-cadherin, tight junction and cell polarity genes compared to w.t. LOXL2. Stable transfection of DCatLOXL2 was also performed on MDCK cells and the phenotypic effects compared to w.t. LOXL2 following standard procedures [2]. Results: Promoter studies indicated that LOXL2 represses target genes independently of its enzymatic activity by a mechanism that can involve the cooperation with transcription factors as Snail1 or E47 in the case of E-cadherin, but also by mechanisms independent of these factors on other genes. Promoter assays with different LOXL2 catalytic and SRCR domain mutants identify the protein region essential for its repressor activity. In addition, we have identified several signaling pathways required for the transcriptional repression of LOXL2, including MAPK, PI3K and mTOR. Several studies with the catalytic mutants indicate the implication of the LOXL2 catalytic domain in the regulation of its extracellular secretion and the requirement of the intact LOXL2 for induction of a full EMT process. Conclusions: Intracellular LOXL2 seems to act as a multifunctional protein regulating several target genes through diverse mechanisms as well as regulating signaling pathways dependently or independently of its catalytic activity. Reference(s) [1] Payne SL et al. J Cell Biochem 101:1338−54 (2007). [2] Peinado H et al. EMBO J 24:3446−58 (2005). [3] Peinado H et al. Cancer Res 68:4541−50 (2008). [4] Moreno-Bueno G et al. EMBO Mol Med 3: 528−44 (2011). 314 Ascorbic Acid Effect in Two Different Cell Lines of Colorectal Adenocarcinoma − in Vitro and in Vivo Studies A.S. Pires1 , A.C. Mamede2 , V.S. Silva1 , A.M. Abrantes1 , A.C. Gon¸calves3 , A.B. Sarmento-Ribeiro3 , M.F. Botelho1 . 1 Biophysics Unit, IBILI − Faculty of Medicine − University of Coimbra, Coimbra, Portugal, 2 CICS-UBI − Health Sciences Research Centre, University of Beira Interior, Coimbra, Portugal, 3 Applied Molecular Biology and Hematology Group, Faculty of Medicine − University of Coimbra, Coimbra, Portugal Background: Colorectal cancer (CC) is one of the most common malignancies in the western world that over the last years has been demonstrated as a progressive and multistep genetic disease. Vitamin C is available in its reduced form (ascorbic acid − AA) and in its oxidized form (dehydroascorbic acid − DHA). Although AA is most commonly known for its antioxidant properties, reducing the oxidative stress, it is also known to function as a pro-oxidant, promoting the formation of reactive oxygen species (ROS) in physiological conditions. The increased production of hydrogen peroxide, coupled with the breakdown of the activity of antioxidant enzymes and the presence of transition metals in cancer cells, may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential. The aim of this study