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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

Induction of p-Erk by silica(A) p-Erk is elevated in MPM cell lines as strong as K-Ras mutated cell lines. A549 and H460 are K-Ras mutated cell lines. SCLC cell lines (H69, H209, H146) were used for negative control, because of absence of Ras-related mutation. Indicated protein levels were analyzed by WB. Erk activation was determined by anti-p-Erk specific Ab. t-Erk indicated total Erk. Actin was used for loading control. (B) Silica induces p-Erk in A549. Activation of Erk was monitored by WB analysis with p-Erk after treatment for indicated time in A549 and MPM cell lines. (C) Silica promotes A549 growth at early phase and did not alter the MPM cell line's growth. Cells were seeded at 5×105 cell/well and incubated with 10 μg/ml silica for indicated time. Cell count was performed by tryphan blue dye exclusive using hemocytometer by two independent researchers. (D) Inhibition of RTK using Iressa/tarceva combination treatment did not block the p-Erk increase. RTK inhibitor (10 μM) was treated for 24hr and silica was treated after 1 hr. Actin was used as loading control. (E) MAPK inhibitors cannot suppress silica-induced p-Erk, and silica suppress DNA damage-induced p53. H28 showed the induction of p53 in response to Adr, indicating that this cell line possesses wild type p53. PD98059 (5 μg/ml), U0126 (2 μM), silica and Adriamycin (2 μg/ml) were treated for 24 hr. WB analysis was performed, and Actin was used as loading control.
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Figure 1: Induction of p-Erk by silica(A) p-Erk is elevated in MPM cell lines as strong as K-Ras mutated cell lines. A549 and H460 are K-Ras mutated cell lines. SCLC cell lines (H69, H209, H146) were used for negative control, because of absence of Ras-related mutation. Indicated protein levels were analyzed by WB. Erk activation was determined by anti-p-Erk specific Ab. t-Erk indicated total Erk. Actin was used for loading control. (B) Silica induces p-Erk in A549. Activation of Erk was monitored by WB analysis with p-Erk after treatment for indicated time in A549 and MPM cell lines. (C) Silica promotes A549 growth at early phase and did not alter the MPM cell line's growth. Cells were seeded at 5×105 cell/well and incubated with 10 μg/ml silica for indicated time. Cell count was performed by tryphan blue dye exclusive using hemocytometer by two independent researchers. (D) Inhibition of RTK using Iressa/tarceva combination treatment did not block the p-Erk increase. RTK inhibitor (10 μM) was treated for 24hr and silica was treated after 1 hr. Actin was used as loading control. (E) MAPK inhibitors cannot suppress silica-induced p-Erk, and silica suppress DNA damage-induced p53. H28 showed the induction of p53 in response to Adr, indicating that this cell line possesses wild type p53. PD98059 (5 μg/ml), U0126 (2 μM), silica and Adriamycin (2 μg/ml) were treated for 24 hr. WB analysis was performed, and Actin was used as loading control.

Mentions: To obtain the basic information about MPM, we measured the expression of p53 and Erk activation. In two kinds of MPM cell lines, p53 showed quite different expression pattern (Figure 1A). H28 showed very low p53 expression and H2452 expressed small sized p53, despite genetically wild type (Figure 1A) [15, 16]. However, p-Erk expression in MPM was elevated as strongly as K-ras mutated cell lines (A549 and H460), in spite of wild type Ras [17, 18]. Thus, we checked the effect of silica on expression of p-Erk in A549 and MPM cell lines. Treatment of silica on serum free condition could induce p-Erk at early phase and reduced at late phase in A549. However, p-Erk in MPM was not reduced even in late phase (Figure 1B). Consistently with p-Erk, cell proliferation in A549 was induced from 6 hr and declined from 24 hr (Figure 1C and Supplementary Figure 1A). However, silica did not alter the cell proliferation in MPM cell lines (Figure 1C and Supplementary Figure 1A). These results indicate that there is defect in regulation of Erk activity in MPM. So, we first tested the involvement of receptor tyrosine kinase (RTK) activation through the combination treatment of RTK inhibitors (Iressa and tarceva). However, although they could suppress basal p-Erk, silica-induced p-Erk was not blocked by them (Figure 1D). Next, we checked the activation of Erk under MEK1/2 inhibitors treated condition. Despite obvious reduction of basal p-Erk in both cell lines, p-Erk was induced in A549 by silica-treatment under MEK1/2 inhibitor-treated condition (Figure 1E). This result indicates that silica can regulate downstream factor of MEK1/2. Thus, PD98059 or U0126 did not show obvious growth suppression effect on MPM cell lines (Supplementary Figure 1B and 1C). Moreover, p53 was increased by treatment of Adriamycin in H28, similarly to A549 (Figure 1E), implying that H28 possesses wild type p53.


NF2 blocks Snail-mediated p53 suppression in mesothelioma.

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

Induction of p-Erk by silica(A) p-Erk is elevated in MPM cell lines as strong as K-Ras mutated cell lines. A549 and H460 are K-Ras mutated cell lines. SCLC cell lines (H69, H209, H146) were used for negative control, because of absence of Ras-related mutation. Indicated protein levels were analyzed by WB. Erk activation was determined by anti-p-Erk specific Ab. t-Erk indicated total Erk. Actin was used for loading control. (B) Silica induces p-Erk in A549. Activation of Erk was monitored by WB analysis with p-Erk after treatment for indicated time in A549 and MPM cell lines. (C) Silica promotes A549 growth at early phase and did not alter the MPM cell line's growth. Cells were seeded at 5×105 cell/well and incubated with 10 μg/ml silica for indicated time. Cell count was performed by tryphan blue dye exclusive using hemocytometer by two independent researchers. (D) Inhibition of RTK using Iressa/tarceva combination treatment did not block the p-Erk increase. RTK inhibitor (10 μM) was treated for 24hr and silica was treated after 1 hr. Actin was used as loading control. (E) MAPK inhibitors cannot suppress silica-induced p-Erk, and silica suppress DNA damage-induced p53. H28 showed the induction of p53 in response to Adr, indicating that this cell line possesses wild type p53. PD98059 (5 μg/ml), U0126 (2 μM), silica and Adriamycin (2 μg/ml) were treated for 24 hr. WB analysis was performed, and Actin was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Induction of p-Erk by silica(A) p-Erk is elevated in MPM cell lines as strong as K-Ras mutated cell lines. A549 and H460 are K-Ras mutated cell lines. SCLC cell lines (H69, H209, H146) were used for negative control, because of absence of Ras-related mutation. Indicated protein levels were analyzed by WB. Erk activation was determined by anti-p-Erk specific Ab. t-Erk indicated total Erk. Actin was used for loading control. (B) Silica induces p-Erk in A549. Activation of Erk was monitored by WB analysis with p-Erk after treatment for indicated time in A549 and MPM cell lines. (C) Silica promotes A549 growth at early phase and did not alter the MPM cell line's growth. Cells were seeded at 5×105 cell/well and incubated with 10 μg/ml silica for indicated time. Cell count was performed by tryphan blue dye exclusive using hemocytometer by two independent researchers. (D) Inhibition of RTK using Iressa/tarceva combination treatment did not block the p-Erk increase. RTK inhibitor (10 μM) was treated for 24hr and silica was treated after 1 hr. Actin was used as loading control. (E) MAPK inhibitors cannot suppress silica-induced p-Erk, and silica suppress DNA damage-induced p53. H28 showed the induction of p53 in response to Adr, indicating that this cell line possesses wild type p53. PD98059 (5 μg/ml), U0126 (2 μM), silica and Adriamycin (2 μg/ml) were treated for 24 hr. WB analysis was performed, and Actin was used as loading control.
Mentions: To obtain the basic information about MPM, we measured the expression of p53 and Erk activation. In two kinds of MPM cell lines, p53 showed quite different expression pattern (Figure 1A). H28 showed very low p53 expression and H2452 expressed small sized p53, despite genetically wild type (Figure 1A) [15, 16]. However, p-Erk expression in MPM was elevated as strongly as K-ras mutated cell lines (A549 and H460), in spite of wild type Ras [17, 18]. Thus, we checked the effect of silica on expression of p-Erk in A549 and MPM cell lines. Treatment of silica on serum free condition could induce p-Erk at early phase and reduced at late phase in A549. However, p-Erk in MPM was not reduced even in late phase (Figure 1B). Consistently with p-Erk, cell proliferation in A549 was induced from 6 hr and declined from 24 hr (Figure 1C and Supplementary Figure 1A). However, silica did not alter the cell proliferation in MPM cell lines (Figure 1C and Supplementary Figure 1A). These results indicate that there is defect in regulation of Erk activity in MPM. So, we first tested the involvement of receptor tyrosine kinase (RTK) activation through the combination treatment of RTK inhibitors (Iressa and tarceva). However, although they could suppress basal p-Erk, silica-induced p-Erk was not blocked by them (Figure 1D). Next, we checked the activation of Erk under MEK1/2 inhibitors treated condition. Despite obvious reduction of basal p-Erk in both cell lines, p-Erk was induced in A549 by silica-treatment under MEK1/2 inhibitor-treated condition (Figure 1E). This result indicates that silica can regulate downstream factor of MEK1/2. Thus, PD98059 or U0126 did not show obvious growth suppression effect on MPM cell lines (Supplementary Figure 1B and 1C). Moreover, p53 was increased by treatment of Adriamycin in H28, similarly to A549 (Figure 1E), implying that H28 possesses wild type p53.

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