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Involvement of epigenetic mechanisms in regulating expression of the calcium sensing receptor in colorectal cancer
Abstracts CaSR have been widely reported in malignant progression. One scenario is the loss of CaSR expression, reported in parathyroid adenoma and colon carcinoma, which could result in the loss of growth suppressive effects of elevated extracellular Ca2 + by CaSR on these two types of malignancies. Another situation appears to be the activation of CaSR and increased production of parathyroid hormone-related peptide (PTHrP) thereof. PTHrP is a causal factor in hypercalcemia of malignancy that importantly contributes to skeletal metastasis. The expression and signaling of CaSR have been studied in several cancers, including ovarian tumors, gastrinomas, and gliomas besides comparatively detailed studies in breast, prostate, and colon cancers. Studies on H-500 rat Leydig cells, a xenotransplantable model of humoral hypercalcemia of malignancy have revealed the mechanisms of CaSR-induced proliferation and survival of this type of tumors, particularly in relation of PTHrP production. Pharmacological agonists and antagonists of CaSR are promising therapeutic options depending on whether the activation of CaSR is favorable such as in case of colon cancer or inactivating the receptor, e.g. in the case of breast- and prostate tumors.
doi:10.1016/j.bone.2012.08.076
O58 Involvement of epigenetic mechanisms in regulating expression of the calcium sensing receptor in colorectal cancer I.Sh. Fetahuc, J. Höbausc, C. Gröschelc, S. Tennakoonc, T. Manhardtc, I. Mesteria, S. Baumgartner-Parzerb, E. Kállayc a Department of Pathology, Medical University of Vienna, Vienna, Austria b Department of Internal Medicine III, Division of Endocrinology and Metabolism, Medical University of Vienna, Vienna, Austria c Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria
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samples and respective adjacent mucosa (n = 57). To study the effect of Calcium in normal colon physiology, we fed C57BL/6 mice with a diet containing high (0.9%) or low (0.1%) levels of Calcium and assessed the influence of dietary Calcium on proliferation (Ki67 immunohistochemistry staining) and CaSR mRNA expression. In tumours we found significantly less CaSR expression and significantly higher expression of genes involved in replication licensing compared with respective adjacent mucosa (pb 0.001). Furthermore, in tumour samples we found negative correlation between CaSR and the licensing factors (CDC45 (p b 0.05, ρ = − 0.265) and MCM6 (p b 0.05, ρ = − 0.262) reaching significance). Surprisingly, CaSR showed a positive correlation with the licensing factors in ‘normal’ tissue (CDT1 (p b 0.05, ρ = 0.269), MCM2 (p b 0.003, ρ = 0.382) and MCM5 (p b 0.001, ρ = 0.490)). In vivo we saw that low dietary Calcium enlarged the proliferative zone of the crypts with a parallel increase in CaSR expression. The upregulation of CaSR is probably a defence mechanism of normal colonocytes to control epithelial growth and counteract hyper-proliferative signals. This control mechanism is lost in cancer where the CaSR expression is lost.
doi:10.1016/j.bone.2012.08.078
O60 The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis C. Casalàa, E. Gil-Guiñóna, J.L. Ordóñezb, S. Miguel-Queralta, E. Rodrígueza, P. Galvána, C. Lavarinoa, F. Munellc, E. de Alavab,d, J. Moraa, C. de Torresa a Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu, Barcelona, Spain b Centro de Investigación del Cáncer-IBMCC (USAL-CIC), Salamanca, Spain c Institut de Recerca Hospital Vall d'Hebron, Barcelona, Spain d Salamanca University Hospital, Spain
Abstract: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer (CRC). The antiproliferative action of calcium in colon might be mediated by the calcium sensing receptor (CaSR). The CaSR expression is lost in human CRC. We hypothesized that DNA hypermethylation and histone deacetylation may cause CaSR silencing in tumors. We analyzed CaSR mRNA and protein expression in colorectal tumors and cell lines by real time qRT-PCR and immunofluorescence. We determined the methylation pattern in the second promoter of CaSR that contains a large CpG island by bisulfite sequencing. To induce the expression of CaSR we treated four colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, and two different histone deacetylase inhibitors (HDIs) suberoylanilide hydroxamic acid and sodium butyrate. In our patient cohort we observed significantly less CaSR mRNA expression (Pb 0.001) in colorectal tumors compared with adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of CaSR protein in tumors. The CaSR promoter was strongly methylated in tumors compared with respective adjacent mucosa and we found a weak, but significant correlation between extent of methylation and mRNA expression (Spearman's rho = − 0.29, P b 0.05). Although in cell lines analyzed, CaSR promoter was densely methylated, treatment with 5-aza-dC caused only modest increase of CaSR expression. The HDIs restored the expression of CaSR in a compound and cell line dependent manner. In summary, DNA hypermethylation and histone deacetylation seem to be important players in silencing CaSR expression in colorectal cancer, although additional mechanism or factors might be also involved.
Abstract: We have analyzed genetic and epigenetic mechanisms potentially responsible for transcriptional silencing of the calcium-sensing receptor (CaSR) in unfavorable neuroblastomas (NB). Hypermethylation of a specific region within the CpG island encompassing the CaSR gene promoter 2 was detected in 25% primary NB, in association with reduced CaSR mRNA expression and several predictors of poor outcome in NB, including amplification of the oncogene MYCN. Hypermethylation of this region was also detected in MYCN-amplified NB cell lines in which the CaSR expression was reduced or absent. Treatment with 5′aza-2-deoxycitidine (Aza) and/or trichostatin A restored the expression of the CaSR in these cell lines and, following Aza exposure, concurrent decreased percentages of methylated CpG sites were observed at the abovementioned region. Also, by interphase fluorescence in situ hybridization, monosomy of chromosome 3, where the human CASR gene resides, was seen in N 90% of primary NTs of all clinical, histological and biological subgroups. Furthermore, ectopic overexpression of the CaSR in two MYCN-amplified NB cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, CaSR-overexpressing NB cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is not an in vitro artefact in NB cell lines, nor a passenger event in primary NB, but a mechanism relevant for NB survival.
doi:10.1016/j.bone.2012.08.077
doi:10.1016/j.bone.2012.08.079
O59 Involvement of the calcium sensing receptor in colorectal cancer proliferation A. Aggarwala, J. Höbausa, I.Sh. Fetahua, I. Mesterib, Enikö Kállaya a Department of Pathophysiology and Allergy Research, Medical University Vienna, Austria b Department of Pathology, Medical University Vienna, Austria
O61 Effect of increased dietary calcium on murine colonic CaSR expression and the effect of induced colitis on “rescued” CaSR−/PTH− (C−/P−) knockout mice R. Turkia, J.E. Kellya, I. Pachecob, R.J. MacLeoda a GIDRU/Physiology, DBMS, Queen's University, Canada b Pathology and Molecular Medicine, Queen's University, Canada
Abstract: Epidemiological studies suggest an inverse correlation between dietary Calcium intake and colorectal cancer (CRC) risk. During tumourigenesis, expression of the calcium-sensing receptor (CaSR) is diminished probably leading to loss of sensitivity to the tumour inhibiting effects of Calcium in transformed cells. We hypothesized that the CaSR mediates anti-proliferative effects of Calcium. We examined the expression of CaSR and of a cluster of genes regulating replication licensing (CDT1, CDC6, CDC45, MCM2-7, Geminin) at mRNA level by qRT-PCR in CRC
Abstract: Introduction: Increased consumption of milk reduces the risk of colon cancer. Colitis increases risk of developing colon cancer. We determined if the colonic CaSR was altered by increased dietary calcium and characterized the effect of induced colitis in control (C+/P+) and C−/P− knockout mice. Methods: PWK/PhJ and NIHSwiss mice were fed 0.5% (A), 2% calcium (B) or 2% with VitD3 (C) diets for 4 wks. Colitis induced by feeding 3% dextran sodium sulphate (DSS) to C+/P+ and C−/P− mice for 5 days. Myelin peroxidase (MPO) activity and TNFα levels were measured.
doi:10.1016/j.bone.2012.08.076
O58 Involvement of epigenetic mechanisms in regulating expression of the calcium sensing receptor in colorectal cancer I.Sh. Fetahuc, J. Höbausc, C. Gröschelc, S. Tennakoonc, T. Manhardtc, I. Mesteria, S. Baumgartner-Parzerb, E. Kállayc a Department of Pathology, Medical University of Vienna, Vienna, Austria b Department of Internal Medicine III, Division of Endocrinology and Metabolism, Medical University of Vienna, Vienna, Austria c Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria
S23
samples and respective adjacent mucosa (n = 57). To study the effect of Calcium in normal colon physiology, we fed C57BL/6 mice with a diet containing high (0.9%) or low (0.1%) levels of Calcium and assessed the influence of dietary Calcium on proliferation (Ki67 immunohistochemistry staining) and CaSR mRNA expression. In tumours we found significantly less CaSR expression and significantly higher expression of genes involved in replication licensing compared with respective adjacent mucosa (pb 0.001). Furthermore, in tumour samples we found negative correlation between CaSR and the licensing factors (CDC45 (p b 0.05, ρ = − 0.265) and MCM6 (p b 0.05, ρ = − 0.262) reaching significance). Surprisingly, CaSR showed a positive correlation with the licensing factors in ‘normal’ tissue (CDT1 (p b 0.05, ρ = 0.269), MCM2 (p b 0.003, ρ = 0.382) and MCM5 (p b 0.001, ρ = 0.490)). In vivo we saw that low dietary Calcium enlarged the proliferative zone of the crypts with a parallel increase in CaSR expression. The upregulation of CaSR is probably a defence mechanism of normal colonocytes to control epithelial growth and counteract hyper-proliferative signals. This control mechanism is lost in cancer where the CaSR expression is lost.
doi:10.1016/j.bone.2012.08.078
O60 The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis C. Casalàa, E. Gil-Guiñóna, J.L. Ordóñezb, S. Miguel-Queralta, E. Rodrígueza, P. Galvána, C. Lavarinoa, F. Munellc, E. de Alavab,d, J. Moraa, C. de Torresa a Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu, Barcelona, Spain b Centro de Investigación del Cáncer-IBMCC (USAL-CIC), Salamanca, Spain c Institut de Recerca Hospital Vall d'Hebron, Barcelona, Spain d Salamanca University Hospital, Spain
Abstract: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer (CRC). The antiproliferative action of calcium in colon might be mediated by the calcium sensing receptor (CaSR). The CaSR expression is lost in human CRC. We hypothesized that DNA hypermethylation and histone deacetylation may cause CaSR silencing in tumors. We analyzed CaSR mRNA and protein expression in colorectal tumors and cell lines by real time qRT-PCR and immunofluorescence. We determined the methylation pattern in the second promoter of CaSR that contains a large CpG island by bisulfite sequencing. To induce the expression of CaSR we treated four colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, and two different histone deacetylase inhibitors (HDIs) suberoylanilide hydroxamic acid and sodium butyrate. In our patient cohort we observed significantly less CaSR mRNA expression (Pb 0.001) in colorectal tumors compared with adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of CaSR protein in tumors. The CaSR promoter was strongly methylated in tumors compared with respective adjacent mucosa and we found a weak, but significant correlation between extent of methylation and mRNA expression (Spearman's rho = − 0.29, P b 0.05). Although in cell lines analyzed, CaSR promoter was densely methylated, treatment with 5-aza-dC caused only modest increase of CaSR expression. The HDIs restored the expression of CaSR in a compound and cell line dependent manner. In summary, DNA hypermethylation and histone deacetylation seem to be important players in silencing CaSR expression in colorectal cancer, although additional mechanism or factors might be also involved.
Abstract: We have analyzed genetic and epigenetic mechanisms potentially responsible for transcriptional silencing of the calcium-sensing receptor (CaSR) in unfavorable neuroblastomas (NB). Hypermethylation of a specific region within the CpG island encompassing the CaSR gene promoter 2 was detected in 25% primary NB, in association with reduced CaSR mRNA expression and several predictors of poor outcome in NB, including amplification of the oncogene MYCN. Hypermethylation of this region was also detected in MYCN-amplified NB cell lines in which the CaSR expression was reduced or absent. Treatment with 5′aza-2-deoxycitidine (Aza) and/or trichostatin A restored the expression of the CaSR in these cell lines and, following Aza exposure, concurrent decreased percentages of methylated CpG sites were observed at the abovementioned region. Also, by interphase fluorescence in situ hybridization, monosomy of chromosome 3, where the human CASR gene resides, was seen in N 90% of primary NTs of all clinical, histological and biological subgroups. Furthermore, ectopic overexpression of the CaSR in two MYCN-amplified NB cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, CaSR-overexpressing NB cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is not an in vitro artefact in NB cell lines, nor a passenger event in primary NB, but a mechanism relevant for NB survival.
doi:10.1016/j.bone.2012.08.077
doi:10.1016/j.bone.2012.08.079
O59 Involvement of the calcium sensing receptor in colorectal cancer proliferation A. Aggarwala, J. Höbausa, I.Sh. Fetahua, I. Mesterib, Enikö Kállaya a Department of Pathophysiology and Allergy Research, Medical University Vienna, Austria b Department of Pathology, Medical University Vienna, Austria
O61 Effect of increased dietary calcium on murine colonic CaSR expression and the effect of induced colitis on “rescued” CaSR−/PTH− (C−/P−) knockout mice R. Turkia, J.E. Kellya, I. Pachecob, R.J. MacLeoda a GIDRU/Physiology, DBMS, Queen's University, Canada b Pathology and Molecular Medicine, Queen's University, Canada
Abstract: Epidemiological studies suggest an inverse correlation between dietary Calcium intake and colorectal cancer (CRC) risk. During tumourigenesis, expression of the calcium-sensing receptor (CaSR) is diminished probably leading to loss of sensitivity to the tumour inhibiting effects of Calcium in transformed cells. We hypothesized that the CaSR mediates anti-proliferative effects of Calcium. We examined the expression of CaSR and of a cluster of genes regulating replication licensing (CDT1, CDC6, CDC45, MCM2-7, Geminin) at mRNA level by qRT-PCR in CRC
Abstract: Introduction: Increased consumption of milk reduces the risk of colon cancer. Colitis increases risk of developing colon cancer. We determined if the colonic CaSR was altered by increased dietary calcium and characterized the effect of induced colitis in control (C+/P+) and C−/P− knockout mice. Methods: PWK/PhJ and NIHSwiss mice were fed 0.5% (A), 2% calcium (B) or 2% with VitD3 (C) diets for 4 wks. Colitis induced by feeding 3% dextran sodium sulphate (DSS) to C+/P+ and C−/P− mice for 5 days. Myelin peroxidase (MPO) activity and TNFα levels were measured.