The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis

Abstract

Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma 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, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma 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 neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.

Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens

“…Among available neuroblastoma cell lines, only LAN1 cells exhibited detectable endogenous CaSR mRNA expression [15]. Low levels of CaSR protein were present in these cells as well (Supplementary Figure S1A).…”

“…Low levels of CaSR protein were present in these cells as well (Supplementary Figure S1A). Two MYCN-amplified cell lines were previously transfected with pCMVCaSRGFP or pCMVGFP [15]. CaSR protein expression was also present in the neuroblastoma metastasis from which a PDX model was generated (Supplementary Figure S1A).…”

“…We have previously reported that acute exposure to high extracellular calcium (Ca 2+ o ) concentrations following serum deprivation [18] induces apoptotic cell death in CaSRoverexpressing neuroblastoma cells [15]. To assess whether cinacalcet is able to increase this effect, SKNLP cells were exposed to DMSO or cinacalcet in serum deprivation media.…”

“…Retrotranscription, PCR and qPCR were carried out as described [14,15]. Realtime PCR runs were performed in a 7500 SDS system using gene specific Assays on Demand and Taqman Universal PCR Master Mix, or specific primers (Supplementary Table S1) and SYBRGreen (Applied Biosystems, Forster City, CA).…”

“…Our initial work showed that CaSR is expressed in benign, differentiated neuroblastic tumors and upregulated upon differentiation induction [14]. Next, we reported that the CaSR gene is silenced by genetic and epigenetic mechanisms in MYCN-amplified, unfavorable neuroblastomas [15]. Accordingly, ectopic overexpression of the CaSR significantly reduced the proliferative and tumorigenic capacities of neuroblastoma cells.…”