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NF2 blocks Snail-mediated p53 suppression in mesothelioma.

Cho JH, Lee SJ, Oh AY, Yoon MH, Woo TG, Park BJ - Oncotarget (2015)

Bottom Line: In addition, p53 and E-cadherin were decreased by silica-treatment.We found that NF2 (frequently deleted in MPM) inhibited Snail-mediated p53 suppression and was stabilized by RKIP.These results indicate that MPM can be induced by reduction of RKIP/NF2, which suppresses p53 through Snail.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduated School of System Biology, College of Natural Science, Pusan National University, Busan.

ABSTRACT
Although asbestos causes malignant pleural mesothelioma (MPM), rising from lung mesothelium, the molecular mechanism has not been suggested until now. Extremely low mutation rate in classical tumor suppressor genes (such as p53 and pRb) and oncogenes (including Ras or myc) indicates that there would be MPM-specific carcinogenesis pathway. To address this, we treated silica to mimic mesothelioma carcinogenesis in mesothelioma and non-small cell lung cancer cell lines (NSCLC). Treatment of silica induced p-Erk and Snail through RKIP reduction. In addition, p53 and E-cadherin were decreased by silica-treatment. Elimination of Snail restored p53 expression. We found that NF2 (frequently deleted in MPM) inhibited Snail-mediated p53 suppression and was stabilized by RKIP. Importantly, GN25, an inhibitor of p53-Snail interaction, induced p53 and apoptosis. These results indicate that MPM can be induced by reduction of RKIP/NF2, which suppresses p53 through Snail. Thus, the p53-Snail binding inhibitor such as GN25 is a drug candidate for MPM.

No MeSH data available.


Related in: MedlinePlus

Involvement of NF2 in RKIP-Snail-p53 network(A) NF2 expression is reduced by silica. When RKIP was reduced by silica (10 μg/ml, 6 hr), NF2 expression was also drop-downed. (B) RKIP transfection induces NF2 expression. FLAG-NF2 was co-transfected with RKIP and Snail into A549 cells. Each vectors were transfected for 24 hr. (C) Snail did not co-exist with NF2 expression. In co-transfected cell with NF2 and Snail, both proteins were disappeared. FLAG-NF2 was co-transfected with Snail into A549 cells. Each vectors were transfected for 24 hr, and then silica was treated for 24 hr. And WB analysis was performed with indicated antibodies. EV indicated the empty vector, and Actin was used as loading control. (D) NF2 induces p53 expression. A549 cells were transfected with FLAG-NF2 for 24 hr and incubated with cyclohexamide (CHX; 100 μg/ml) for 6 to 24 hr. WB analysis was performed. Actin was used as loading control. (E) Proteasome inhibitor blocks NF2-Snail reduction in co-transfected cells. 293 cells were transfected with Snail and/or NF2 for 3 hr and incubated with 6 hr with 100 μM of ALLN. (F) Snail knock down induces NF2 expression in H28. At the same sample of Figure 4f, we checked the expression of NF2. (G) NF2 transfection increases nuclear p53 expression in H28 cell line. FLAG-NF2 was transfected for 24 hr. The cells were stained with anti-p53 (DO-1; red), NF2 (green), DAPI (blue).
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Figure 5: Involvement of NF2 in RKIP-Snail-p53 network(A) NF2 expression is reduced by silica. When RKIP was reduced by silica (10 μg/ml, 6 hr), NF2 expression was also drop-downed. (B) RKIP transfection induces NF2 expression. FLAG-NF2 was co-transfected with RKIP and Snail into A549 cells. Each vectors were transfected for 24 hr. (C) Snail did not co-exist with NF2 expression. In co-transfected cell with NF2 and Snail, both proteins were disappeared. FLAG-NF2 was co-transfected with Snail into A549 cells. Each vectors were transfected for 24 hr, and then silica was treated for 24 hr. And WB analysis was performed with indicated antibodies. EV indicated the empty vector, and Actin was used as loading control. (D) NF2 induces p53 expression. A549 cells were transfected with FLAG-NF2 for 24 hr and incubated with cyclohexamide (CHX; 100 μg/ml) for 6 to 24 hr. WB analysis was performed. Actin was used as loading control. (E) Proteasome inhibitor blocks NF2-Snail reduction in co-transfected cells. 293 cells were transfected with Snail and/or NF2 for 3 hr and incubated with 6 hr with 100 μM of ALLN. (F) Snail knock down induces NF2 expression in H28. At the same sample of Figure 4f, we checked the expression of NF2. (G) NF2 transfection increases nuclear p53 expression in H28 cell line. FLAG-NF2 was transfected for 24 hr. The cells were stained with anti-p53 (DO-1; red), NF2 (green), DAPI (blue).

Mentions: Although we revealed that reduction of RKIP promoted Snail-mediated p53 suppression, relevance between RKIP and Snail has not been clearly demonstrated. So, we checked the interaction between RKIP and Snail. However, we did not observe the interaction (Supplementary Figure S4A). So we searched additional factor that could link RKIP and Snail and tested the involvement of NF2 that is also known to be frequently mutated in MPM and related with MAPK signaling and migration [26-28]. In A549, silica could suppressed the expression of transfected NF2 as strongly as RKIP (Figure 5A). In addition, NF2 in H28 was rapidly degraded by proteasome (Supplementary Figure S4B). In fact, RKIP could induce NF2 expression, whereas Snail suppress it (Figure 5B). We also observed the elimination of Snail when NF2 was co-transfected (Figure 5B). To confirm this, we co-transfected NF2 and Snail in A549 and 293 cells and found that NF2 as well as Snail were obviously reduced when they were co-transfected (Figure 5C and Supplementary Figure S4C). To get more detail evidence about reduction of Snail and NF2, we transfected Snail as dose dependently into NF2 transfected cells and found that NF2 expression was gradually decreased following expression of Snail (Supplementary Figure S4D). Reversely, dose-dependent decrease of Snail could be detected by increase of NF2 transfection (Supplementary Figure S4E). In addition, p53 reduction (Figure 5C) and p-Erk induction by silica and Snail (Supplementary Figure S4C) were abolished by NF2 transfection. These results indicate that reduction of NF2 is important for silica/snail-mediated Erk activation and p53 suppression. In fact, NF2 could induce p53 expression (Figure 5D). Considering that proteasome inhibitor (ALLN) could block the reduction of Snail and NF2 (Figure 5E), low expression of NF2 in MPM (in particular, H28) would be resulted from Snail expression. To test this, we checked the expression of NF2 in si-Snail transfected H28 and found that consistently with p53 induction, NF2 was increased by Si-Snail (Figure 5F). Moreover, transfection of NF2 into H28 could induce p53 expression in nucleus (Figure 5G). These results indicate that, in MPM, low expression of NF2, which would be resulted from RKIP reduction, would be reason for p53 inactivation and snail elevation.


NF2 blocks Snail-mediated p53 suppression in mesothelioma.

Cho JH, Lee SJ, Oh AY, Yoon MH, Woo TG, Park BJ - Oncotarget (2015)

Involvement of NF2 in RKIP-Snail-p53 network(A) NF2 expression is reduced by silica. When RKIP was reduced by silica (10 μg/ml, 6 hr), NF2 expression was also drop-downed. (B) RKIP transfection induces NF2 expression. FLAG-NF2 was co-transfected with RKIP and Snail into A549 cells. Each vectors were transfected for 24 hr. (C) Snail did not co-exist with NF2 expression. In co-transfected cell with NF2 and Snail, both proteins were disappeared. FLAG-NF2 was co-transfected with Snail into A549 cells. Each vectors were transfected for 24 hr, and then silica was treated for 24 hr. And WB analysis was performed with indicated antibodies. EV indicated the empty vector, and Actin was used as loading control. (D) NF2 induces p53 expression. A549 cells were transfected with FLAG-NF2 for 24 hr and incubated with cyclohexamide (CHX; 100 μg/ml) for 6 to 24 hr. WB analysis was performed. Actin was used as loading control. (E) Proteasome inhibitor blocks NF2-Snail reduction in co-transfected cells. 293 cells were transfected with Snail and/or NF2 for 3 hr and incubated with 6 hr with 100 μM of ALLN. (F) Snail knock down induces NF2 expression in H28. At the same sample of Figure 4f, we checked the expression of NF2. (G) NF2 transfection increases nuclear p53 expression in H28 cell line. FLAG-NF2 was transfected for 24 hr. The cells were stained with anti-p53 (DO-1; red), NF2 (green), DAPI (blue).
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Figure 5: Involvement of NF2 in RKIP-Snail-p53 network(A) NF2 expression is reduced by silica. When RKIP was reduced by silica (10 μg/ml, 6 hr), NF2 expression was also drop-downed. (B) RKIP transfection induces NF2 expression. FLAG-NF2 was co-transfected with RKIP and Snail into A549 cells. Each vectors were transfected for 24 hr. (C) Snail did not co-exist with NF2 expression. In co-transfected cell with NF2 and Snail, both proteins were disappeared. FLAG-NF2 was co-transfected with Snail into A549 cells. Each vectors were transfected for 24 hr, and then silica was treated for 24 hr. And WB analysis was performed with indicated antibodies. EV indicated the empty vector, and Actin was used as loading control. (D) NF2 induces p53 expression. A549 cells were transfected with FLAG-NF2 for 24 hr and incubated with cyclohexamide (CHX; 100 μg/ml) for 6 to 24 hr. WB analysis was performed. Actin was used as loading control. (E) Proteasome inhibitor blocks NF2-Snail reduction in co-transfected cells. 293 cells were transfected with Snail and/or NF2 for 3 hr and incubated with 6 hr with 100 μM of ALLN. (F) Snail knock down induces NF2 expression in H28. At the same sample of Figure 4f, we checked the expression of NF2. (G) NF2 transfection increases nuclear p53 expression in H28 cell line. FLAG-NF2 was transfected for 24 hr. The cells were stained with anti-p53 (DO-1; red), NF2 (green), DAPI (blue).
Mentions: Although we revealed that reduction of RKIP promoted Snail-mediated p53 suppression, relevance between RKIP and Snail has not been clearly demonstrated. So, we checked the interaction between RKIP and Snail. However, we did not observe the interaction (Supplementary Figure S4A). So we searched additional factor that could link RKIP and Snail and tested the involvement of NF2 that is also known to be frequently mutated in MPM and related with MAPK signaling and migration [26-28]. In A549, silica could suppressed the expression of transfected NF2 as strongly as RKIP (Figure 5A). In addition, NF2 in H28 was rapidly degraded by proteasome (Supplementary Figure S4B). In fact, RKIP could induce NF2 expression, whereas Snail suppress it (Figure 5B). We also observed the elimination of Snail when NF2 was co-transfected (Figure 5B). To confirm this, we co-transfected NF2 and Snail in A549 and 293 cells and found that NF2 as well as Snail were obviously reduced when they were co-transfected (Figure 5C and Supplementary Figure S4C). To get more detail evidence about reduction of Snail and NF2, we transfected Snail as dose dependently into NF2 transfected cells and found that NF2 expression was gradually decreased following expression of Snail (Supplementary Figure S4D). Reversely, dose-dependent decrease of Snail could be detected by increase of NF2 transfection (Supplementary Figure S4E). In addition, p53 reduction (Figure 5C) and p-Erk induction by silica and Snail (Supplementary Figure S4C) were abolished by NF2 transfection. These results indicate that reduction of NF2 is important for silica/snail-mediated Erk activation and p53 suppression. In fact, NF2 could induce p53 expression (Figure 5D). Considering that proteasome inhibitor (ALLN) could block the reduction of Snail and NF2 (Figure 5E), low expression of NF2 in MPM (in particular, H28) would be resulted from Snail expression. To test this, we checked the expression of NF2 in si-Snail transfected H28 and found that consistently with p53 induction, NF2 was increased by Si-Snail (Figure 5F). Moreover, transfection of NF2 into H28 could induce p53 expression in nucleus (Figure 5G). These results indicate that, in MPM, low expression of NF2, which would be resulted from RKIP reduction, would be reason for p53 inactivation and snail elevation.

Bottom Line: In addition, p53 and E-cadherin were decreased by silica-treatment.We found that NF2 (frequently deleted in MPM) inhibited Snail-mediated p53 suppression and was stabilized by RKIP.These results indicate that MPM can be induced by reduction of RKIP/NF2, which suppresses p53 through Snail.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduated School of System Biology, College of Natural Science, Pusan National University, Busan.

ABSTRACT
Although asbestos causes malignant pleural mesothelioma (MPM), rising from lung mesothelium, the molecular mechanism has not been suggested until now. Extremely low mutation rate in classical tumor suppressor genes (such as p53 and pRb) and oncogenes (including Ras or myc) indicates that there would be MPM-specific carcinogenesis pathway. To address this, we treated silica to mimic mesothelioma carcinogenesis in mesothelioma and non-small cell lung cancer cell lines (NSCLC). Treatment of silica induced p-Erk and Snail through RKIP reduction. In addition, p53 and E-cadherin were decreased by silica-treatment. Elimination of Snail restored p53 expression. We found that NF2 (frequently deleted in MPM) inhibited Snail-mediated p53 suppression and was stabilized by RKIP. Importantly, GN25, an inhibitor of p53-Snail interaction, induced p53 and apoptosis. These results indicate that MPM can be induced by reduction of RKIP/NF2, which suppresses p53 through Snail. Thus, the p53-Snail binding inhibitor such as GN25 is a drug candidate for MPM.

No MeSH data available.


Related in: MedlinePlus