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The leukemia inhibitory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in human cutaneous melanoma.

Humbert L, Ghozlan M, Canaff L, Tian J, Lebrun JJ - BMC Cancer (2015)

Bottom Line: Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays.Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration.Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada. laure.humbert@mail.mcgill.ca.

ABSTRACT

Background: Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFβ) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion.

Methods: In this study, we aimed at elucidating the molecular mechanisms underlying TGFβ-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays.

Results: We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFβ in melanoma cells, defining LIF as a novel TGFβ downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration. Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes.

Conclusions: Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFβ to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.

No MeSH data available.


Related in: MedlinePlus

TGFβ mediates its effects through p21 regulation. A, WM278 and WM793B were treated or not with TGFβ for 24 h and expression of LIF, p21, p15, and c-MYC was analyzed by Western blot(left panel). β-tubulin was used as control. Right panel: Densitometry of LIF and p21 protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p<0.001). B, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as mean of percentages of cells in G1 phase for 3 independent experiments. Error bars are standard errors of mean and t-test was performed compared to non-treated control (**p < 0.01, *p < 0.05). C, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin was used as control. D, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Data is graphed as geometric mean of relative luciferase units normalized to non-treated control for at least 3 independent experiments. Error bars are the standard errors of mean and z-test was performed compared to non-treated control (*p < 0.05). E, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and gene expression was analyzed by qPCR. Data is graphed as mean of fold induction of gene expression in response to TGFβ for at least 3 replicates. Error bars are standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (*p < 0.05).
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Fig3: TGFβ mediates its effects through p21 regulation. A, WM278 and WM793B were treated or not with TGFβ for 24 h and expression of LIF, p21, p15, and c-MYC was analyzed by Western blot(left panel). β-tubulin was used as control. Right panel: Densitometry of LIF and p21 protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p<0.001). B, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as mean of percentages of cells in G1 phase for 3 independent experiments. Error bars are standard errors of mean and t-test was performed compared to non-treated control (**p < 0.01, *p < 0.05). C, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin was used as control. D, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Data is graphed as geometric mean of relative luciferase units normalized to non-treated control for at least 3 independent experiments. Error bars are the standard errors of mean and z-test was performed compared to non-treated control (*p < 0.05). E, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and gene expression was analyzed by qPCR. Data is graphed as mean of fold induction of gene expression in response to TGFβ for at least 3 replicates. Error bars are standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (*p < 0.05).

Mentions: To further understand how TGFβ/LIF regulate growth inhibition in melanomas, we examined the expression levels of several cell cycle regulators that have been shown to be involved downstream of TGFβ-mediated growth arrest in other tissues. These include p15 and p21 that were shown to be induced by TGFβ [36-38] and the oncogene c-MYC that was found to be downregulated by TGFβ in keratinocytes [39]. We examined the TGFβ effects on the expression levels of these downstream mediators in WM793B and WM278 cells. As observed by immunoblot analysis in Figure 3A, while there was no change in the protein expression levels of p15 or c-MYC, TGFβ significantly induced p21 protein, suggesting that p21 may act as the main cell cycle regulator downstream of TGFβ in human melanoma. To further address the role of p21 in these effects, we transfected WM278 cells with a specific p21 siRNA or a scrambled sequence as a negative control and examined the TGFβ effects on cell cycle arrest. As shown in Figure 3B, TGFβ significantly induced G1 arrest in the mock and scrambled siRNA conditions. However, when p21 expression was silenced, the TGFβ effect was completely abolished, suggesting that p21 not only is required downstream of TGFβ to mediate cell cycle arrest in melanoma cells but also plays a central role in the regulation of these events. The efficiency of the p21 siRNA was demonstrated by Western blot, using a specific p21 monoclonal antibody (Figure 3C). p21 has also been linked to the apoptotic process, however its exact function remains unclear and controversial, as it was shown to inhibit apoptosis in lymphoma cells [29], primary fibroblasts [30], and hepatoma cells [31], while it promotes apoptosis in ovarian cancer cells [32], hepatocytes [33] and hepatocarcinoma cells [34], and thymocytes [35]. Thus, we investigated whether TGFβ-mediated p21 expression in melanoma cells was required for the mediation of the TGFβ pro-apoptotic effects. As shown in Figure 3D, silencing p21 expression with a specific siRNA almost completely blocked TGFβ-mediated caspase-mediated cell death, defining a new role for p21 in melanoma as a pro-apoptotic factor. While the mechanisms through which p21 regulates the cell cycle have been relatively well documented, its effects on apoptosis are poorly understood. In order to elucidate how p21 promotes apoptosis downstream of TGFβ, we analyzed the expression of several pro-apoptotic genes known to be regulated by TGFβ (Bax, Bim, Bak and Apaf-1) [40]. As shown in Figure 3E, TGFβ induced the expression of all tested genes in melanoma cells. However, using a RNA interference approach, while we found TGFβ-mediated Bax and Bim gene expression to be p21-dependent, regulation of Bak and Apaf-1 by TGFβ does not involve p21. This indicates that p21 mediates some of the pro-apoptotic effects of TGFβ by inducing the expression of specific pro-apoptotic genes in melanoma. Together, our results highlight p21 as an important regulator of both TGFβ-mediated cell cycle arrest and apoptosis in human melanoma.Figure 3


The leukemia inhibitory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in human cutaneous melanoma.

Humbert L, Ghozlan M, Canaff L, Tian J, Lebrun JJ - BMC Cancer (2015)

TGFβ mediates its effects through p21 regulation. A, WM278 and WM793B were treated or not with TGFβ for 24 h and expression of LIF, p21, p15, and c-MYC was analyzed by Western blot(left panel). β-tubulin was used as control. Right panel: Densitometry of LIF and p21 protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p<0.001). B, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as mean of percentages of cells in G1 phase for 3 independent experiments. Error bars are standard errors of mean and t-test was performed compared to non-treated control (**p < 0.01, *p < 0.05). C, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin was used as control. D, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Data is graphed as geometric mean of relative luciferase units normalized to non-treated control for at least 3 independent experiments. Error bars are the standard errors of mean and z-test was performed compared to non-treated control (*p < 0.05). E, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and gene expression was analyzed by qPCR. Data is graphed as mean of fold induction of gene expression in response to TGFβ for at least 3 replicates. Error bars are standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (*p < 0.05).
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Fig3: TGFβ mediates its effects through p21 regulation. A, WM278 and WM793B were treated or not with TGFβ for 24 h and expression of LIF, p21, p15, and c-MYC was analyzed by Western blot(left panel). β-tubulin was used as control. Right panel: Densitometry of LIF and p21 protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p<0.001). B, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as mean of percentages of cells in G1 phase for 3 independent experiments. Error bars are standard errors of mean and t-test was performed compared to non-treated control (**p < 0.01, *p < 0.05). C, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin was used as control. D, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Data is graphed as geometric mean of relative luciferase units normalized to non-treated control for at least 3 independent experiments. Error bars are the standard errors of mean and z-test was performed compared to non-treated control (*p < 0.05). E, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and gene expression was analyzed by qPCR. Data is graphed as mean of fold induction of gene expression in response to TGFβ for at least 3 replicates. Error bars are standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (*p < 0.05).
Mentions: To further understand how TGFβ/LIF regulate growth inhibition in melanomas, we examined the expression levels of several cell cycle regulators that have been shown to be involved downstream of TGFβ-mediated growth arrest in other tissues. These include p15 and p21 that were shown to be induced by TGFβ [36-38] and the oncogene c-MYC that was found to be downregulated by TGFβ in keratinocytes [39]. We examined the TGFβ effects on the expression levels of these downstream mediators in WM793B and WM278 cells. As observed by immunoblot analysis in Figure 3A, while there was no change in the protein expression levels of p15 or c-MYC, TGFβ significantly induced p21 protein, suggesting that p21 may act as the main cell cycle regulator downstream of TGFβ in human melanoma. To further address the role of p21 in these effects, we transfected WM278 cells with a specific p21 siRNA or a scrambled sequence as a negative control and examined the TGFβ effects on cell cycle arrest. As shown in Figure 3B, TGFβ significantly induced G1 arrest in the mock and scrambled siRNA conditions. However, when p21 expression was silenced, the TGFβ effect was completely abolished, suggesting that p21 not only is required downstream of TGFβ to mediate cell cycle arrest in melanoma cells but also plays a central role in the regulation of these events. The efficiency of the p21 siRNA was demonstrated by Western blot, using a specific p21 monoclonal antibody (Figure 3C). p21 has also been linked to the apoptotic process, however its exact function remains unclear and controversial, as it was shown to inhibit apoptosis in lymphoma cells [29], primary fibroblasts [30], and hepatoma cells [31], while it promotes apoptosis in ovarian cancer cells [32], hepatocytes [33] and hepatocarcinoma cells [34], and thymocytes [35]. Thus, we investigated whether TGFβ-mediated p21 expression in melanoma cells was required for the mediation of the TGFβ pro-apoptotic effects. As shown in Figure 3D, silencing p21 expression with a specific siRNA almost completely blocked TGFβ-mediated caspase-mediated cell death, defining a new role for p21 in melanoma as a pro-apoptotic factor. While the mechanisms through which p21 regulates the cell cycle have been relatively well documented, its effects on apoptosis are poorly understood. In order to elucidate how p21 promotes apoptosis downstream of TGFβ, we analyzed the expression of several pro-apoptotic genes known to be regulated by TGFβ (Bax, Bim, Bak and Apaf-1) [40]. As shown in Figure 3E, TGFβ induced the expression of all tested genes in melanoma cells. However, using a RNA interference approach, while we found TGFβ-mediated Bax and Bim gene expression to be p21-dependent, regulation of Bak and Apaf-1 by TGFβ does not involve p21. This indicates that p21 mediates some of the pro-apoptotic effects of TGFβ by inducing the expression of specific pro-apoptotic genes in melanoma. Together, our results highlight p21 as an important regulator of both TGFβ-mediated cell cycle arrest and apoptosis in human melanoma.Figure 3

Bottom Line: Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays.Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration.Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada. laure.humbert@mail.mcgill.ca.

ABSTRACT

Background: Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFβ) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion.

Methods: In this study, we aimed at elucidating the molecular mechanisms underlying TGFβ-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays.

Results: We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFβ in melanoma cells, defining LIF as a novel TGFβ downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration. Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes.

Conclusions: Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFβ to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.

No MeSH data available.


Related in: MedlinePlus