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, 2 (11), 1034-43

Increased Wild-Type N-ras Activation by Neurofibromin Down-Regulation Increases Human Neuroblastoma Stem Cell Malignancy

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Increased Wild-Type N-ras Activation by Neurofibromin Down-Regulation Increases Human Neuroblastoma Stem Cell Malignancy

Dan Han et al. Genes Cancer.

Abstract

Cellular heterogeneity is a well-known feature of human neuroblastoma tumors and cell lines. Of the 3 phenotypes (N-, I-, and S-type) isolated and characterized, the I-type cancer stem cell of neuroblastoma is the most malignant. Here, we report that, although wild-type N-Ras protein is expressed at the same level in all 3 neuroblastoma cell phenotypes, activated N-Ras-GTP level is significantly higher in I-type cancer stem cells. When activated N-Ras levels were decreased by transfection of a dominant-negative N-Ras construct, the malignant potential of I-type cancer stem cells decreased significantly. Conversely, when weakly malignant N-type cells were transfected with a constitutively active N-Ras construct, activated N-Ras levels, and malignant potential, were significantly increased. Thus, high levels of N-Ras-GTP are required for the increased malignancy of I-type neuroblastoma cancer stem cells. Moreover, increased activation of N-Ras results from significant down-regulation of neurofibromin (NF1), an important RasGAP. This specific down-regulation is mediated by an ubiquitin-proteasome-dependent pathway. Thus, decreased expression of NF1 in I-type neuroblastoma cancer stem cells causes a high level of activated N-Ras that is, at least in part, responsible for their higher tumorigenic potential.

Keywords: N-Ras; malignancy; neuroblastoma cancer stem cell; neurofibromin.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Ras isoforms expressed in neuroblastoma cell variants. (A) Representative Western blot of total Ras protein in neuroblastoma cells using a pan-Ras antibody. Cell lines are N-type: 1) SH-SY5Y, 2) LA1-55n, 3) SK-N-BE(1)n; I-type: 4) BE(2)-C, 5) SK-N-HM, 6) SK-N-MM, 7) CB-JMN; and S-type: 8) SH-EP1, 9) LA1-5s, 10) SMS-KCNs. Actin is used as the loading control. (B) Representative Western blot of H/N-Ras, K-Ras, and actin in neuroblastoma cell lines of different phenotypes. Cell lines are N-type: 1) SH-SY5Y, 2) LA1-55n; I-type: 3) BE(2)-C, 4) SK-N-LD, 5) CB-JMN, 6) SK-N-MM; S-type: 7) SH-EP1, 8) LA1-5s; and 9) control K-Ras–transfected LA1-5s. (C) Semiquantitative RT-PCR shows that N-Ras mRNA levels are much higher than H-Ras. DNA from plasmids containing N- or H-Ras coding sequence is included as controls (lane 12) to show a similar efficiency of primers. The cell lines are N-type: 1) SH-SY5Y, 2) LA1-55n, 3) SK-N-BE(1)n, 4) BE(2)-M17V; I-type: 5) SK-N-LD, 6) BE(2)-C, 7) SK-N-HM, 8) CB-JMN; and S-type: 9) SH-EP1, 10) LA1-5s, 11) SMS-KCNs. GAPD is used as the loading control. All lanes are from the same gel. (D) Four cell lines, SH-SY5Y (N-type), BE(2)-C (I-type), SK-N-LD (I-type), and LA1-5s (S-type), were chosen for quantitative real-time RT-PCR analysis. Serially diluted H-Ras and N-Ras plasmid constructs were used to generate standard curves. In all 4 cell lines, N-Ras mRNA levels are significantly 10- to 40-fold higher than H-Ras (**P < 0.001). (E) Representative Western blot of N-Ras, H-Ras, and actin in neuroblastoma cell lines of different phenotypes. Cell lines are the same as in B. Note that H-Ras mRNA and protein levels are much lower than those of N-Ras in all of the cell lines.
Figure 2.
Figure 2.
Activated Ras-GTP levels are high in I-type neuroblastoma cells. (A) Representative Western blot of N-Ras–GTP levels in neuroblastoma cells. Cell lines are N-type: 1) SH-SY5Y, 2) LA1-55n, 3) SK-N-BE(1)n, 4) IMR-32; I-type: 5) BE(2)-C, 6) SK-N-LD, 7) SK-N-MM, 8) CB-JMN, 9) SK-N-HM; and S-type: 10) SH-EP1, 11) LA1-5s, 12) SMS-KCNs. (B) Ras-GTP levels are significantly higher in I-type cells. n = number of different cell lines (*P < 0.01). Each cell line was examined in at least 3 independent experiments with different protein lysates and Ras-GTP levels normalized to total ras protein. (C) Representative Western blot shows that Ras-GTP levels decrease significantly in both RA- and BrdU-induced differentiated BE(2)-C cells (2.3- and 3.8-fold, respectively; P < 0.01).
Figure 3.
Figure 3.
Changing Ras-GTP level by dominant-negative (DN) and constitutively active (CA) N-Ras constructs alters malignancy in neuroblastoma cells. DN-N-Ras–transfected I-type BE(2)-C clones have a (A) >2-fold decrease in Ras-GTP level and (B) 3.8- to 5.0-fold decrease in CFE in soft agar compared to the vector-transfected control (*P < 0.01). (C) Representative photomicrographs of the DN-N-Ras– or control vector–transfected BE(2)-C cells from the soft agar assay. Conversely, CA-N-Ras expression in N-type SH-SY5Y cells causes a (D) dramatic increase in Ras-GTP level (>10-fold) and (E) significant increase in CFE in soft agar (7.5-fold; **P < 0.001). (F) Representative photomicrographs of the CA-N-Ras– or control vector–transfected SH-SY5Y cells from the soft agar assay.
Figure 4.
Figure 4.
High levels of Ras-GTP protect neuroblastoma cells from apoptosis. (A) Cell death was investigated using an apoptosis and necrosis assay, in which apoptotic cells exhibit green fluorescence, necrotic cells show red fluorescence, late-phase apoptotic cells show both green and red fluorescence, and living cells are not fluorescent. (Left) Representative phase-contrast photomicrograph used to determine the total cell number. (Right) A fluorescent photomicrograph of the same field showing the much higher rate of apoptosis in the DN-N-Ras–transfected BE(2)-C cells compared to the vector control. (B) In each experiment, apoptotic and necrotic cells were counted in 10 randomly chosen fields and expressed as a percentage of the total cell number. DN-N-Ras–transfected BE(2)-C cells show a significant, 4.1-fold, increase in apoptosis rate (*P < 0.01) but no change in necrosis. (C) Representative phase-contrast photomicrograph (left) and its fluorescent photomicrograph (right) showing the much lower rate of cell death in the CA-N-Ras–transfected SH-SY5Y cells compared to the vector control. (D) Percentage of apoptotic cells in CA-N-Ras–transfected SH-SY5Y cells is significantly, 4.0-fold, lower than the control (*P < 0.02), with no change in necrosis.
Figure 5.
Figure 5.
Amounts of neurofibromin protein, but not p120GAP, are lower specifically in I-type neuroblastoma cells. (A) Representative Western blot of p120GAP in human neuroblastoma cell lines: N-type: 1) SH-SY5Y, 2) LA1-55n, 3) SK-N-BE(1)n; I-type: 4) BE(2)-C, 5) SK-N-LD, 6) CB-JMN; and S-type: 7) SH-EP1, 8) LA1-5s. Actin was used as the loading control. (B) Representative Western blot of neurofibromin in 11 human neuroblastoma cell variants: N-type: 1) IMR32, 2) SH-SY5Y, 3) LA1-55n, 4) SK-N-BE(1)n; I-type: 5) NBL-S, 6) SK-N-LD, 7) BE(2)-C, 8) CB-JMN, 9) SK-N-MM, 10) SK-N-HM; and S-type: 11) SK-N-BE(2)s, 12) SH-EP1, 13) LA1-5s. Actin was used as the loading control. Each cell line was examined in at least 3 independent experiments with different protein lysates. All lanes are from the same gel. Note that whereas there are no phenotypic differences in p120GAP amounts, I-type cell lines contain significantly lower amounts of neurofibromin compared to either N- or S-type cells. (C) Linear regression analysis of amounts of neurofibromin and Ras-GTP in the neuroblastoma cell lines. r2 = 0.9232, P < 0.001. (D) RA- and BrdU-induced differentiation of I-type BE(2)-C cells lead to increased neurofibromin protein amounts of 3.2- and 2.9-fold, respectively (P > 0.01).
Figure 6.
Figure 6.
Down-regulation of neurofibromin protein increases both Ras-GTP level and malignant potential in neuroblastoma cells. (A) Transfection of specific NF1 shRNA into SH-SY5Y cells decreases neurofibromin 2.0-fold and increases Ras-GTP level 3.8-fold compared to the control shRNA. (B) shNF1-transfected SH-SY5Y cells also show significant >2-fold increased CFE in soft agar (*P < 0.01).
Figure 7.
Figure 7.
Down-regulation of neurofibromin in I-type cells occurs at the posttranslational level. (A) Semiquantitative RT-PCR analysis using primers that recognize exons 58 and 59 of NF1 mRNA, coding for the epitope specifically recognized by the neurofibromin antibody, shows similar levels of NF1 mRNA in all cell variants. The cell lines are N-type: 1) SH-SY5Y, 2) LA1-55n, 3) IMR-32, 4) KCN-83n; I-type: 5) SK-N-LD, 6) BE(2)-C, 7) CB-JMN, 8) SK-N-MM; and S-type: 9) SH-EP1, 10) LA1-5s, 11) SMS-KCNs. All lanes are from the same gel. (B) Real-time RT-PCR analyses using primers that amplify the N-terminal region (exon 2) of NF1 mRNA show that NF1 mRNA levels do not differ significantly among phenotypes. Relative NF1 mRNA level was normalized to GAPD mRNA and compared to SH-SY5Y set = 1.0. Each cell line was examined in 3 to 6 independent experiments with different RNA isolations (NS = not significantly different from N cell level; n = number of different cell lines). (C) I-type SK-N-MM and N-type SH-SY5Y cells were treated with MG132 at 10 μM for 3 and 6 hours. DMSO-treated cells were used as controls. Neurofibromin protein was detected by Western blotting. Actin is the negative control; hsp72 is the positive control. MG132 treatment significantly increases the neurofibromin amount in SK-N-MM cells 2.0-fold after 3 hours (P < 0.01) and 3.2-fold after 6 hours (P < 0.01). The neurofibromin amounts of SH-SY5Y cells are not significantly changed after MG132 treatment.

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