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N-Myc-induced up-regulation of TRPM6/TRPM7 channels promotes neuroblastoma cell proliferation.

Zhang Z, Faouzi M, Huang J, Geerts D, Yu H, Fleig A, Penner R - Oncotarget (2014)

Bottom Line: The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism.Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition.These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Research, The Queen's Medical Center, University of Hawaii Cancer Center and John A. Burns School of Medicine, University of Hawaii, Honolulu, U.S.A. Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China. Contributed equally to this work.

ABSTRACT
Intracellular levels of the divalent cations Ca2+ and Mg2+ are important regulators of cell cycle and proliferation. However, the precise mechanisms by which they are regulated in cancer remain incompletely understood. The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism. Here, we investigated the human neuroblastoma cell line SHEP-21N in which the MYCN oncogene (encoding N-Myc) can be reversibly expressed under control of an inducible repressor. We report that N-Myc expression increases cell growth and up-regulates both TRPM6 and TRPM7 expression. Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition. These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake. Genetic suppression of TRPM6/TRPM7 through siRNA inhibits cell proliferation, suggesting that N-Myc can promote neuroblastoma cell proliferation through up-regulation of divalent cation-transporting channels.

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Related in: MedlinePlus

TRPM6 and TRPM7 correlation with MYCN in neuroblastomaA-C, N-Myc, TRPM7 and TRPM6 mRNA expression correlation with MYCN amplification in the Kocak-649 cohort. Microarray analysis of N-Myc, TRPM7 and TRPM6 mRNA expression in Kocak-649 (GSE45547), the largest neuroblastoma cohort in the public domain. The graphs present N-Myc (A), TRPM7 (B) and TRPM6 (C) expression in tumors without (n=550) and with (n=93) MYCN amplification. Y-axes represent sample ranks in a non-parametric Kruskal-Wallis t test; actual mean ± s.e.m. expression values were: 17,839 ± 1,137 (MYCN), 104.9 ± 3.7 (TRPM6), 807.0 ± 321.1 (TRPM7). Both N-Myc and TRPM7 expression are significantly higher in tumors with MYCN amplification (p=1.5 • 10−51 and p=2.3 • 10−7 respectively; Kruskal-Wallis t test). D-F, qRT-PCR analysis of N-Myc (D), TRPM7 (E) and TRPM6 (F) expression levels in the SHEP-21N cell line where MYCN transgene is controlled by tetracycline, i.e., N-Myc expression is repressed in the presence of tetracycline (control), but removal of tetracycline induces N-Myc expression (N-Myc). The graphs show normalized N-Myc, TRPM7 and TRPM6 expression in SHEP-21N cells with (N-Myc, n = 9) or without (control, n = 9) N-Myc expression. *, p<0.01.
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Figure 1: TRPM6 and TRPM7 correlation with MYCN in neuroblastomaA-C, N-Myc, TRPM7 and TRPM6 mRNA expression correlation with MYCN amplification in the Kocak-649 cohort. Microarray analysis of N-Myc, TRPM7 and TRPM6 mRNA expression in Kocak-649 (GSE45547), the largest neuroblastoma cohort in the public domain. The graphs present N-Myc (A), TRPM7 (B) and TRPM6 (C) expression in tumors without (n=550) and with (n=93) MYCN amplification. Y-axes represent sample ranks in a non-parametric Kruskal-Wallis t test; actual mean ± s.e.m. expression values were: 17,839 ± 1,137 (MYCN), 104.9 ± 3.7 (TRPM6), 807.0 ± 321.1 (TRPM7). Both N-Myc and TRPM7 expression are significantly higher in tumors with MYCN amplification (p=1.5 • 10−51 and p=2.3 • 10−7 respectively; Kruskal-Wallis t test). D-F, qRT-PCR analysis of N-Myc (D), TRPM7 (E) and TRPM6 (F) expression levels in the SHEP-21N cell line where MYCN transgene is controlled by tetracycline, i.e., N-Myc expression is repressed in the presence of tetracycline (control), but removal of tetracycline induces N-Myc expression (N-Myc). The graphs show normalized N-Myc, TRPM7 and TRPM6 expression in SHEP-21N cells with (N-Myc, n = 9) or without (control, n = 9) N-Myc expression. *, p<0.01.

Mentions: We set out to examine the expression level of the Mg2+-influx channels TRPM6 and TRPM7 in the largest publicly available neuroblastoma expression profiling dataset, the Kocak-649 cohort [29]. On the basis of their MYCN gene copy number status, these tumor samples can be divided into MYCN-amplified (n=93) or non-MYCN-amplified (n=550) tumors. We first examined the expression level of N-Myc in these two groups and found that the expression level is, as expected, significantly higher in the MYCN-amplified samples (p = 1.5 × 10−51, Fig. 1A). Interestingly, TRPM7 mRNA expression was found in all tumor samples, and was significantly correlated with both MYCN amplification (p = 2.3 × 10−7, Fig. 1B) and mRNA expression (p = 3.5 × 10−3 in a 2logPearson test). No significant correlations were found for TRPM6 (Fig. 1C), most likely due to the small numbers of tumors with significant TRPM6 expression (average TRPM6 expression was 8-times lower than that of TRPM7). In order to investigate the role of N-Myc in TRPM7/TRPM6 regulation, we chose the SHEP-21N cell line, a clone derived from the SHEP-2 neuroblastoma, in which N-Myc is constitutively expressed but can be experimentally repressed [30]. These cells contain a MYCN trans-gene under the control of a tetracycline-responsive repressor element, so that tetracycline exposure turns off N-Myc expression. Quantitative RT-PCR (qRT-PCR) analysis showed that SHEP-21N cells without N-Myc expression had basal expression of TRPM7 and TRPM6, which was considerably enhanced by N-Myc up-regulation (Figs. 1D-F). Induction of TRPM6 and TRPM7 expression by N-Myc was significant for both channel kinases, but higher for TRPM6 (2.1-fold for TRPM6 and 1.5-fold for TRPM7). This suggests that N-Myc up-regulates both TRPM genes and concomitantly increases the ratio of TRPM6 over TRPM7.


N-Myc-induced up-regulation of TRPM6/TRPM7 channels promotes neuroblastoma cell proliferation.

Zhang Z, Faouzi M, Huang J, Geerts D, Yu H, Fleig A, Penner R - Oncotarget (2014)

TRPM6 and TRPM7 correlation with MYCN in neuroblastomaA-C, N-Myc, TRPM7 and TRPM6 mRNA expression correlation with MYCN amplification in the Kocak-649 cohort. Microarray analysis of N-Myc, TRPM7 and TRPM6 mRNA expression in Kocak-649 (GSE45547), the largest neuroblastoma cohort in the public domain. The graphs present N-Myc (A), TRPM7 (B) and TRPM6 (C) expression in tumors without (n=550) and with (n=93) MYCN amplification. Y-axes represent sample ranks in a non-parametric Kruskal-Wallis t test; actual mean ± s.e.m. expression values were: 17,839 ± 1,137 (MYCN), 104.9 ± 3.7 (TRPM6), 807.0 ± 321.1 (TRPM7). Both N-Myc and TRPM7 expression are significantly higher in tumors with MYCN amplification (p=1.5 • 10−51 and p=2.3 • 10−7 respectively; Kruskal-Wallis t test). D-F, qRT-PCR analysis of N-Myc (D), TRPM7 (E) and TRPM6 (F) expression levels in the SHEP-21N cell line where MYCN transgene is controlled by tetracycline, i.e., N-Myc expression is repressed in the presence of tetracycline (control), but removal of tetracycline induces N-Myc expression (N-Myc). The graphs show normalized N-Myc, TRPM7 and TRPM6 expression in SHEP-21N cells with (N-Myc, n = 9) or without (control, n = 9) N-Myc expression. *, p<0.01.
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Figure 1: TRPM6 and TRPM7 correlation with MYCN in neuroblastomaA-C, N-Myc, TRPM7 and TRPM6 mRNA expression correlation with MYCN amplification in the Kocak-649 cohort. Microarray analysis of N-Myc, TRPM7 and TRPM6 mRNA expression in Kocak-649 (GSE45547), the largest neuroblastoma cohort in the public domain. The graphs present N-Myc (A), TRPM7 (B) and TRPM6 (C) expression in tumors without (n=550) and with (n=93) MYCN amplification. Y-axes represent sample ranks in a non-parametric Kruskal-Wallis t test; actual mean ± s.e.m. expression values were: 17,839 ± 1,137 (MYCN), 104.9 ± 3.7 (TRPM6), 807.0 ± 321.1 (TRPM7). Both N-Myc and TRPM7 expression are significantly higher in tumors with MYCN amplification (p=1.5 • 10−51 and p=2.3 • 10−7 respectively; Kruskal-Wallis t test). D-F, qRT-PCR analysis of N-Myc (D), TRPM7 (E) and TRPM6 (F) expression levels in the SHEP-21N cell line where MYCN transgene is controlled by tetracycline, i.e., N-Myc expression is repressed in the presence of tetracycline (control), but removal of tetracycline induces N-Myc expression (N-Myc). The graphs show normalized N-Myc, TRPM7 and TRPM6 expression in SHEP-21N cells with (N-Myc, n = 9) or without (control, n = 9) N-Myc expression. *, p<0.01.
Mentions: We set out to examine the expression level of the Mg2+-influx channels TRPM6 and TRPM7 in the largest publicly available neuroblastoma expression profiling dataset, the Kocak-649 cohort [29]. On the basis of their MYCN gene copy number status, these tumor samples can be divided into MYCN-amplified (n=93) or non-MYCN-amplified (n=550) tumors. We first examined the expression level of N-Myc in these two groups and found that the expression level is, as expected, significantly higher in the MYCN-amplified samples (p = 1.5 × 10−51, Fig. 1A). Interestingly, TRPM7 mRNA expression was found in all tumor samples, and was significantly correlated with both MYCN amplification (p = 2.3 × 10−7, Fig. 1B) and mRNA expression (p = 3.5 × 10−3 in a 2logPearson test). No significant correlations were found for TRPM6 (Fig. 1C), most likely due to the small numbers of tumors with significant TRPM6 expression (average TRPM6 expression was 8-times lower than that of TRPM7). In order to investigate the role of N-Myc in TRPM7/TRPM6 regulation, we chose the SHEP-21N cell line, a clone derived from the SHEP-2 neuroblastoma, in which N-Myc is constitutively expressed but can be experimentally repressed [30]. These cells contain a MYCN trans-gene under the control of a tetracycline-responsive repressor element, so that tetracycline exposure turns off N-Myc expression. Quantitative RT-PCR (qRT-PCR) analysis showed that SHEP-21N cells without N-Myc expression had basal expression of TRPM7 and TRPM6, which was considerably enhanced by N-Myc up-regulation (Figs. 1D-F). Induction of TRPM6 and TRPM7 expression by N-Myc was significant for both channel kinases, but higher for TRPM6 (2.1-fold for TRPM6 and 1.5-fold for TRPM7). This suggests that N-Myc up-regulates both TRPM genes and concomitantly increases the ratio of TRPM6 over TRPM7.

Bottom Line: The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism.Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition.These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Research, The Queen's Medical Center, University of Hawaii Cancer Center and John A. Burns School of Medicine, University of Hawaii, Honolulu, U.S.A. Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China. Contributed equally to this work.

ABSTRACT
Intracellular levels of the divalent cations Ca2+ and Mg2+ are important regulators of cell cycle and proliferation. However, the precise mechanisms by which they are regulated in cancer remain incompletely understood. The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism. Here, we investigated the human neuroblastoma cell line SHEP-21N in which the MYCN oncogene (encoding N-Myc) can be reversibly expressed under control of an inducible repressor. We report that N-Myc expression increases cell growth and up-regulates both TRPM6 and TRPM7 expression. Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition. These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake. Genetic suppression of TRPM6/TRPM7 through siRNA inhibits cell proliferation, suggesting that N-Myc can promote neuroblastoma cell proliferation through up-regulation of divalent cation-transporting channels.

Show MeSH
Related in: MedlinePlus