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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-mediated LIF upregulation regulates p21 expression at the transcriptional level. A, WM278 cells were treated or not with TGFβ and LIF for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. B, WM278 cells transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by qPCR. Data is graphed as the mean of the fold induction of p21 gene expression in response to TGFβ for at least 3 biological replicates. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the mock and scrambled siRNA treated conditions (*p < 0.05, **p<0.01). C, WM278 cells were transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. D, WM278 and WM793B cells transfected with a scrambled or a LIF siRNA 24 h earlier were transfected with the p21 luciferase reporter and the Renilla luciferase constructs, treated or not with TGFβ for 24 h, lysed and assessed for luciferase activity. Data is graphed as the arithmetic mean of relative luciferase units normalized to Renilla luciferase activity for 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated control (***p<0.001). E, WM278 cells were treated with LIF (100 ng/mL) or F, with TGFβ for different period of times or G, with scrambled or LIF siRNA in the presence or absence of TGFβ, and phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 content was used as a control.
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Fig4: TGFβ-mediated LIF upregulation regulates p21 expression at the transcriptional level. A, WM278 cells were treated or not with TGFβ and LIF for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. B, WM278 cells transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by qPCR. Data is graphed as the mean of the fold induction of p21 gene expression in response to TGFβ for at least 3 biological replicates. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the mock and scrambled siRNA treated conditions (*p < 0.05, **p<0.01). C, WM278 cells were transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. D, WM278 and WM793B cells transfected with a scrambled or a LIF siRNA 24 h earlier were transfected with the p21 luciferase reporter and the Renilla luciferase constructs, treated or not with TGFβ for 24 h, lysed and assessed for luciferase activity. Data is graphed as the arithmetic mean of relative luciferase units normalized to Renilla luciferase activity for 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated control (***p<0.001). E, WM278 cells were treated with LIF (100 ng/mL) or F, with TGFβ for different period of times or G, with scrambled or LIF siRNA in the presence or absence of TGFβ, and phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 content was used as a control.

Mentions: Our results indicate that both p21 and LIF play an important regulatory role downstream of TGFβ in regulating melanoma growth inhibition. Interestingly, Oncostatin M, a member of the LIF family had previously been shown to induce p21 expression in osteoblastic cells [27,28]. This led us to investigate whether LIF could also regulate p21 gene expression, thereby linking LIF and p21 to TGFβ-mediated cell growth inhibition. We first assessed the link between LIF and p21 by treating WM278 cells with LIF and showed that LIF stimulation led to p21 upregulation comparable to TGFβ treatment (Figure 4A). To then investigate whether TGFβ-mediated p21 gene expression was LIF-dependent, we silenced LIF gene expression in WM278 cells and analyzed the effect of p21 regulation by TGFβ on both mRNA and protein levels. While p21 mRNA and protein expression were upregulated by TGFβ in the mock and scrambled siRNA conditions (Figure 4B and C), blocking LIF expression using a siRNA completely blocked this effect, indicating that LIF is required for TGFβ-mediated p21 upregulation. Furthermore, we found this effect to take place at the transcriptional level, as LIF gene expression knockdown using a specific LIF siRNA completely blocked TGFβ-induced p21 gene promoter activity. Indeed, as shown in Figure 4D, in WM278 and WM793 cells transfected with the p21-luciferase reporter construct (p21-luc) in the presence or the absence of a scrambled or LIF specific siRNA, the TGFβ-induced luciferase activity was completely blocked when LIF expression was knockdown. As LIF signaling is mediated through activation of the transcription factor STAT3, and as the p21-luc construct contains a STAT3 binding element [27], we next assessed whether TGFβ-induced p21 expression in melanoma was STAT3-dependent. For this, we used a second reporter construct (p21inr-luc) in which the STAT3 binding element has been removed [41]. Interestingly, TGFβ was unable to activate the p21 gene promoter in the absence of the STAT3 binding element (Figure 4D). These results indicate that LIF and its downstream effector STAT3 are required for TGFβ to induce p21 gene expression at the transcriptional level. STAT3 is the major effector of LIF and as shown in Figure 4E, stimulation of WM278 cells with LIF rapidly induces phosphorylation of STAT3. As we found TGFβ to increase LIF expression levels, we then investigated whether TGFβ could lead to STAT3 activation. As shown in Figure 4F, TGFβ stimulation of WM278 cells resulted in a significant increase in STAT3 phosphorylation, thus indicating that STAT3 is a downstream effector of the TFGβ pathway in these cells. Finally, to show that this increase in STAT3 activation by TGFβ was mediated through LIF, we treated WM278 and WM793B cells with LIF siRNA and treated or not with TGFβ. As shown in Figure 4G, knocking down LIF blocked the phosphorylation of STAT3 by TFGβ, further demonstrating that LIF is required for this regulation.Figure 4


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-mediated LIF upregulation regulates p21 expression at the transcriptional level. A, WM278 cells were treated or not with TGFβ and LIF for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. B, WM278 cells transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by qPCR. Data is graphed as the mean of the fold induction of p21 gene expression in response to TGFβ for at least 3 biological replicates. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the mock and scrambled siRNA treated conditions (*p < 0.05, **p<0.01). C, WM278 cells were transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. D, WM278 and WM793B cells transfected with a scrambled or a LIF siRNA 24 h earlier were transfected with the p21 luciferase reporter and the Renilla luciferase constructs, treated or not with TGFβ for 24 h, lysed and assessed for luciferase activity. Data is graphed as the arithmetic mean of relative luciferase units normalized to Renilla luciferase activity for 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated control (***p<0.001). E, WM278 cells were treated with LIF (100 ng/mL) or F, with TGFβ for different period of times or G, with scrambled or LIF siRNA in the presence or absence of TGFβ, and phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 content was used as a control.
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Fig4: TGFβ-mediated LIF upregulation regulates p21 expression at the transcriptional level. A, WM278 cells were treated or not with TGFβ and LIF for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. B, WM278 cells transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by qPCR. Data is graphed as the mean of the fold induction of p21 gene expression in response to TGFβ for at least 3 biological replicates. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the mock and scrambled siRNA treated conditions (*p < 0.05, **p<0.01). C, WM278 cells were transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. D, WM278 and WM793B cells transfected with a scrambled or a LIF siRNA 24 h earlier were transfected with the p21 luciferase reporter and the Renilla luciferase constructs, treated or not with TGFβ for 24 h, lysed and assessed for luciferase activity. Data is graphed as the arithmetic mean of relative luciferase units normalized to Renilla luciferase activity for 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated control (***p<0.001). E, WM278 cells were treated with LIF (100 ng/mL) or F, with TGFβ for different period of times or G, with scrambled or LIF siRNA in the presence or absence of TGFβ, and phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 content was used as a control.
Mentions: Our results indicate that both p21 and LIF play an important regulatory role downstream of TGFβ in regulating melanoma growth inhibition. Interestingly, Oncostatin M, a member of the LIF family had previously been shown to induce p21 expression in osteoblastic cells [27,28]. This led us to investigate whether LIF could also regulate p21 gene expression, thereby linking LIF and p21 to TGFβ-mediated cell growth inhibition. We first assessed the link between LIF and p21 by treating WM278 cells with LIF and showed that LIF stimulation led to p21 upregulation comparable to TGFβ treatment (Figure 4A). To then investigate whether TGFβ-mediated p21 gene expression was LIF-dependent, we silenced LIF gene expression in WM278 cells and analyzed the effect of p21 regulation by TGFβ on both mRNA and protein levels. While p21 mRNA and protein expression were upregulated by TGFβ in the mock and scrambled siRNA conditions (Figure 4B and C), blocking LIF expression using a siRNA completely blocked this effect, indicating that LIF is required for TGFβ-mediated p21 upregulation. Furthermore, we found this effect to take place at the transcriptional level, as LIF gene expression knockdown using a specific LIF siRNA completely blocked TGFβ-induced p21 gene promoter activity. Indeed, as shown in Figure 4D, in WM278 and WM793 cells transfected with the p21-luciferase reporter construct (p21-luc) in the presence or the absence of a scrambled or LIF specific siRNA, the TGFβ-induced luciferase activity was completely blocked when LIF expression was knockdown. As LIF signaling is mediated through activation of the transcription factor STAT3, and as the p21-luc construct contains a STAT3 binding element [27], we next assessed whether TGFβ-induced p21 expression in melanoma was STAT3-dependent. For this, we used a second reporter construct (p21inr-luc) in which the STAT3 binding element has been removed [41]. Interestingly, TGFβ was unable to activate the p21 gene promoter in the absence of the STAT3 binding element (Figure 4D). These results indicate that LIF and its downstream effector STAT3 are required for TGFβ to induce p21 gene expression at the transcriptional level. STAT3 is the major effector of LIF and as shown in Figure 4E, stimulation of WM278 cells with LIF rapidly induces phosphorylation of STAT3. As we found TGFβ to increase LIF expression levels, we then investigated whether TGFβ could lead to STAT3 activation. As shown in Figure 4F, TGFβ stimulation of WM278 cells resulted in a significant increase in STAT3 phosphorylation, thus indicating that STAT3 is a downstream effector of the TFGβ pathway in these cells. Finally, to show that this increase in STAT3 activation by TGFβ was mediated through LIF, we treated WM278 and WM793B cells with LIF siRNA and treated or not with TGFβ. As shown in Figure 4G, knocking down LIF blocked the phosphorylation of STAT3 by TFGβ, further demonstrating that LIF is required for this regulation.Figure 4

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