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. 2016 Dec 20;7(51):84326-84337.
doi: 10.18632/oncotarget.12607.

Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C

Suthakar Ganapathy  1 , Johan B Fagman  2 , Ling Shen  1 , Tianqi Yu  1 , Xiaodong Zhou  1   3 , Wei Dai  4 , Alexandros Makriyannis  1 , Changyan Chen  1
Affiliations

Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C

Suthakar Ganapathy et al. Oncotarget. .

Abstract

Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

Keywords: Chk1; Nf1; Ral A; apoptosis; mitotic catastrophe.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Effects of HMG on the induction of apoptosis and cell cycle progression in Nf1-deficient cells treated with HMG
(A and B) Nf1-proficient and -deficient cells (A) or human fibroblasts (B) with different genetic manipulations were treated with HMG (10 uM) for various times as indicated. DNA fragmentation assay was then performed. The error bars represent the standard deviation (SD) over 5 independent experiments (n = 5, *p < 0.01). (C and D) Cells were treated with HMG for 24 h, and then subjected to cell cycle analysis, using a flow cytometer. The percentages of the cells accumulated in the G1 or G2/M phase were measured and plotted. The error bars represent SD from 5 independent experiments (n = 5, *p < 0.05).
Figure 2
Figure 2. Ral A activation and association with Ral/BP1 in HMG-treated Nf1-deficient or -knockdown cells
(A) Following HMG treatment, the Nf1-proficient or -deficient cell lysates were extracted and Ral A activity was measured. The even loadings were normalized by running another immunoblot gel with equal amount of total proteins per lane, which were then blotted with anti-β-actin antibody. (B) Ral A activity was analyzed in HF cells with or without the knockdown of Nf1 and ectopically expressing v-K-ras as described above. (C) Cell lysates were extracted from untreated or HMG-treated ST or ST/Nf1 cells as described above. Ral B activity was then examined. (D) After HMG treatment, cell lysates were prepared and immunoprecipitated with anti-Ral A antibody. Immunoprecipitates were then blotted with anti-Ral/BP1 antibody. The even loadings of total proteins were normalized by Ral A expression. (E) Reciprocal co-immunoprecipitation with anti-Ral/BP1 antibody and immunoblotting with anti-Ral A antibody. The even loadings of total proteins were normalized by Ral/BP1 expression. (F) Co-immunoprecipitation with anti-Ral B antibody and immunoblotting with anti-Ral/BP1 antibody were conducted. The even loadings of total proteins were normalized by Ral B expression.
Figure 3
Figure 3. Activation of Chk1 in Nf1-deficient or -knockdown cells treated with HMG
(A) Following HMG treatment, ST or ST/Nf1 cells were immunoblotted with the anti-phosphorylated-Chk1 antibody. The even loadings of each lanes were normalized by Chk1 expression. (B) After transient transfection of the sc or siRN-Rals, the expressions of p-Chk1, Ral A and Ral B were analyzed in the cells with or without HMG treatment by immunoblotting. The even loadings of total proteins were normalized by β-actin expression. (C) Chk1 phosphorylation was performed in HF cells with Nf1 knockdown or ectopically expressing v-K-ras. Even loadings of each lane were normalized by β-actin expression.
Figure 4
Figure 4. Nuclear translocation of Chk1 in Nf1 defective cells after HMG treatment
(A) Cells were treated with HMG and the cytosolic or nuclear fractions were prepared. Subsequently, immunoblotting were conducted, using anti-Chk1 antibody. The even loading of the cytosolic samples was normalized by tubulin and of the nuclear proteins by LamA. (B) After knockdown of Nf1 or ectopically expressing v-K-ras in HF cells, Chk1 expression in the cytosolic or nuclear fraction was examined. The even loading of the cytosolic samples was normalized by tubulin and of the nuclear proteins by LamA. (C) After the knockdown of Ral A or B, Chk1 expression was tested in each subcellular fraction as described above. (D) Immunofluorescent staining of Chk1 in untreated or HMG-treated ST/Nf1 or ST cells, with or without the transfection of siRNA-Ral A was performed. The slides mounted with the samples were incubated with an anti-Chk1 antibody, and then subjected to the second anti-mouse-IG antibody conjugated with fluorescein and stained with DAPI.
Figure 5
Figure 5. Ral A dependency of HMG-induced mitotic arrest and apoptosis in Nf1-deficient cells
(A) ST or ST/Nf1 cells were transfected with scRNA, siRNA-Ral A or B, prior to HMG treatment for 24 h, and then cell cycle progression was analyzed, using a flow cytometer. (B) After the same treatments as indicated above, DNA fragmentation assay was conducted. The error bars represent SD from 5 independent experiments (n = 5, *p < 0.05).
Figure 6
Figure 6. Induction of apoptosis in the xenografted ST tumors or soft tissue tumors isolated from Nf1-deficient mice upon HMG injection
(A) ST cells were inoculated subcutaneously into the nude mice (6 mice per group). HMG was injected peritoneally right after the inoculation and subsequently administrated every 3 days. Once became noticeable, the diameters of the tumors were measured every 7 days and plotted. The error bars represent SD (n = 6, *p < 0.05). Twenty-eight days later, the isolated tumors were weighed and data were plotted. The photos of the examples of the tumors were taken. (B) The slides mounted with the tumors were stained with TUNEL reagent as well as with anti-Ki67, PCNA, phosphorylated-Chk1, Ral A or Ral B antibody, respectively.
Figure 7
Figure 7. Stochastic signaling pathways for sensitizing Nf1-defective cells to apoptosis after PKC inhibition
In this apoptotic process, Ral A plays an important role in eliciting a persistent mitotic arrest, which partially contributes to the induction of apoptosis.
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