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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 Snail in p53 suppression(A) Snail is induced by silica-treatment. Silica was treated for 24 hr in SF condition. The expression of endogenous snail was determined by anti-snail specific Ab. (B) Mutual exclusive expression pattern between RKIP and Snail. The expression of RKIP and Snail were monitored by WB analysis after treatment for indicated time in A549. (C) Snail overexpression cannot alter the RKIP expression. Flag-Snail was transfected for 24 hr in A549 cells. Silica treated for 24 hr in SF condition, and WB was performed. EV indicates the empty vector. Actin was used as loading control. (D) RKIP suppressed Snail expression. HA-RKIP was co-transfected in Snail-transfected A549 cells for 24 hr, and then silica was treated for 24 hr in SF condition, and WB was performed. (E) Snail is elevated in MPM cell lines. Snail expression level was monitored by WB analysis. (F) Snail knock down induces p53 expression in H28. Si-Snail was transfected for 24 hr in H28 cells, and then silica was treated for 12 to 24 hr in SF condition. WB analysis was performed with indicated antibodies, and Actin was used as loading control. (G) GN25 can increase p53 in A549 and H28. After treatment of GN25 (2-7.5 μM) for 12 hr, cells were subjected into WB analysis with indicated antibodies. Actin was used as loading control. (H) p53 expression is increased in nucleus by GN25 treatment, in regardless of silica-treatment. H28 was treated with GN25 (2.5 μM) before an hr to be treated silica. Silica was treated for 24 hr. The cells were stained with anti-p53 (DO-1; green), DAPI (blue). (I) GN25 suppresses H28 viability. After incubation with GN25 (2.5 μM) for 24 hr, the cell viability was monitored by MTT assay.
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Figure 4: Involvement of Snail in p53 suppression(A) Snail is induced by silica-treatment. Silica was treated for 24 hr in SF condition. The expression of endogenous snail was determined by anti-snail specific Ab. (B) Mutual exclusive expression pattern between RKIP and Snail. The expression of RKIP and Snail were monitored by WB analysis after treatment for indicated time in A549. (C) Snail overexpression cannot alter the RKIP expression. Flag-Snail was transfected for 24 hr in A549 cells. Silica treated for 24 hr in SF condition, and WB was performed. EV indicates the empty vector. Actin was used as loading control. (D) RKIP suppressed Snail expression. HA-RKIP was co-transfected in Snail-transfected A549 cells for 24 hr, and then silica was treated for 24 hr in SF condition, and WB was performed. (E) Snail is elevated in MPM cell lines. Snail expression level was monitored by WB analysis. (F) Snail knock down induces p53 expression in H28. Si-Snail was transfected for 24 hr in H28 cells, and then silica was treated for 12 to 24 hr in SF condition. WB analysis was performed with indicated antibodies, and Actin was used as loading control. (G) GN25 can increase p53 in A549 and H28. After treatment of GN25 (2-7.5 μM) for 12 hr, cells were subjected into WB analysis with indicated antibodies. Actin was used as loading control. (H) p53 expression is increased in nucleus by GN25 treatment, in regardless of silica-treatment. H28 was treated with GN25 (2.5 μM) before an hr to be treated silica. Silica was treated for 24 hr. The cells were stained with anti-p53 (DO-1; green), DAPI (blue). (I) GN25 suppresses H28 viability. After incubation with GN25 (2.5 μM) for 24 hr, the cell viability was monitored by MTT assay.

Mentions: Since silica promoted the formation of cytoplasmic small vesicular p53 (Figure 3C) and H28 also showed the similar feature (Figure 3E), we assumed that the Snail would be involved in p53 suppression mechanism. In fact, we have revealed that Snail can eliminate p53 by direct interaction and exocytosis [24, 25]. We also observed that the reduction of p53 was not blocked by MG132 (Supplementary Figure 2B), also supporting our hypothesis [25]. As we expected, treatment of silica could induce Snail (Figure 4A) and Snail expression showed the mutual exclusive pattern with RKIP following incubation time (Figure 4B). In addition, we observed the increase of exogenously transfected Snail by silica (Figure 4C), indicating that Snail would be regulated at post-transcription level. Moreover, Snail transfection did not alter the RKIP reduction, whereas RKIP transfection could block the Snail induction (Figure 4D). These results strongly suggest that RKIP is first and most-upstream regulator in silica-induced signaling cascade. Indeed, MPM cell lines showed the elevated expression of Snail (Figure 4E). Moreover, elimination of Snail using siRNA [25] could induce p53 expression in H28 (Figure 4F).


NF2 blocks Snail-mediated p53 suppression in mesothelioma.

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

Involvement of Snail in p53 suppression(A) Snail is induced by silica-treatment. Silica was treated for 24 hr in SF condition. The expression of endogenous snail was determined by anti-snail specific Ab. (B) Mutual exclusive expression pattern between RKIP and Snail. The expression of RKIP and Snail were monitored by WB analysis after treatment for indicated time in A549. (C) Snail overexpression cannot alter the RKIP expression. Flag-Snail was transfected for 24 hr in A549 cells. Silica treated for 24 hr in SF condition, and WB was performed. EV indicates the empty vector. Actin was used as loading control. (D) RKIP suppressed Snail expression. HA-RKIP was co-transfected in Snail-transfected A549 cells for 24 hr, and then silica was treated for 24 hr in SF condition, and WB was performed. (E) Snail is elevated in MPM cell lines. Snail expression level was monitored by WB analysis. (F) Snail knock down induces p53 expression in H28. Si-Snail was transfected for 24 hr in H28 cells, and then silica was treated for 12 to 24 hr in SF condition. WB analysis was performed with indicated antibodies, and Actin was used as loading control. (G) GN25 can increase p53 in A549 and H28. After treatment of GN25 (2-7.5 μM) for 12 hr, cells were subjected into WB analysis with indicated antibodies. Actin was used as loading control. (H) p53 expression is increased in nucleus by GN25 treatment, in regardless of silica-treatment. H28 was treated with GN25 (2.5 μM) before an hr to be treated silica. Silica was treated for 24 hr. The cells were stained with anti-p53 (DO-1; green), DAPI (blue). (I) GN25 suppresses H28 viability. After incubation with GN25 (2.5 μM) for 24 hr, the cell viability was monitored by MTT assay.
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Figure 4: Involvement of Snail in p53 suppression(A) Snail is induced by silica-treatment. Silica was treated for 24 hr in SF condition. The expression of endogenous snail was determined by anti-snail specific Ab. (B) Mutual exclusive expression pattern between RKIP and Snail. The expression of RKIP and Snail were monitored by WB analysis after treatment for indicated time in A549. (C) Snail overexpression cannot alter the RKIP expression. Flag-Snail was transfected for 24 hr in A549 cells. Silica treated for 24 hr in SF condition, and WB was performed. EV indicates the empty vector. Actin was used as loading control. (D) RKIP suppressed Snail expression. HA-RKIP was co-transfected in Snail-transfected A549 cells for 24 hr, and then silica was treated for 24 hr in SF condition, and WB was performed. (E) Snail is elevated in MPM cell lines. Snail expression level was monitored by WB analysis. (F) Snail knock down induces p53 expression in H28. Si-Snail was transfected for 24 hr in H28 cells, and then silica was treated for 12 to 24 hr in SF condition. WB analysis was performed with indicated antibodies, and Actin was used as loading control. (G) GN25 can increase p53 in A549 and H28. After treatment of GN25 (2-7.5 μM) for 12 hr, cells were subjected into WB analysis with indicated antibodies. Actin was used as loading control. (H) p53 expression is increased in nucleus by GN25 treatment, in regardless of silica-treatment. H28 was treated with GN25 (2.5 μM) before an hr to be treated silica. Silica was treated for 24 hr. The cells were stained with anti-p53 (DO-1; green), DAPI (blue). (I) GN25 suppresses H28 viability. After incubation with GN25 (2.5 μM) for 24 hr, the cell viability was monitored by MTT assay.
Mentions: Since silica promoted the formation of cytoplasmic small vesicular p53 (Figure 3C) and H28 also showed the similar feature (Figure 3E), we assumed that the Snail would be involved in p53 suppression mechanism. In fact, we have revealed that Snail can eliminate p53 by direct interaction and exocytosis [24, 25]. We also observed that the reduction of p53 was not blocked by MG132 (Supplementary Figure 2B), also supporting our hypothesis [25]. As we expected, treatment of silica could induce Snail (Figure 4A) and Snail expression showed the mutual exclusive pattern with RKIP following incubation time (Figure 4B). In addition, we observed the increase of exogenously transfected Snail by silica (Figure 4C), indicating that Snail would be regulated at post-transcription level. Moreover, Snail transfection did not alter the RKIP reduction, whereas RKIP transfection could block the Snail induction (Figure 4D). These results strongly suggest that RKIP is first and most-upstream regulator in silica-induced signaling cascade. Indeed, MPM cell lines showed the elevated expression of Snail (Figure 4E). Moreover, elimination of Snail using siRNA [25] could induce p53 expression in H28 (Figure 4F).

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