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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 LIF regulation. A, WM278 cells were treated or not with TGFβfor 24 h and LIF expression was analyzed by qPCR. Error bars are standard deviations and z-test was performed (***p < 0.001). B, WM278 and WM793B cells were treated or not with TGFβ for 24 h and LIF expression was analyzed by Western blot (left panel) after concentration of conditioned media. β-tubulin was used as control. Right panel: Densitometric analysis of LIF protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p < 0.001). C, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Error bars are standard errors of mean and z-test was performed compared to non-treated control (**p < 0.01, ***p<0.001). D, WM278 cells transfected with scrambled or LIF 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 the 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 < 0.01). E, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h. LIF expression was analyzed by qPCR. Data is graphed as the mean of fold induction of gene expression in response to TGFβ for at least 3 independent experiments. Error bars are the standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (**p < 0.01).
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Fig2: TGFβ mediates its effects through LIF regulation. A, WM278 cells were treated or not with TGFβfor 24 h and LIF expression was analyzed by qPCR. Error bars are standard deviations and z-test was performed (***p < 0.001). B, WM278 and WM793B cells were treated or not with TGFβ for 24 h and LIF expression was analyzed by Western blot (left panel) after concentration of conditioned media. β-tubulin was used as control. Right panel: Densitometric analysis of LIF protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p < 0.001). C, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Error bars are standard errors of mean and z-test was performed compared to non-treated control (**p < 0.01, ***p<0.001). D, WM278 cells transfected with scrambled or LIF 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 the 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 < 0.01). E, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h. LIF expression was analyzed by qPCR. Data is graphed as the mean of fold induction of gene expression in response to TGFβ for at least 3 independent experiments. Error bars are the standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (**p < 0.01).

Mentions: Transcriptome analysis of WM793B and WM278 cells revealed that one particular gene, the leukemia inhibitory factor (LIF), appeared to be strongly upregulated by TGFβ (data not shown). This was further confirmed and quantified at both mRNA and protein levels. As shown in Figure 2A and B, TGFβ potently induced LIF expression both at the mRNA and protein levels in WM793B and WM278 cells, suggesting a role for LIF in mediating the TGFβ effects in melanoma cells. Both cell lines showed a 5 to 6 fold increase in protein levels, as quantified by densitometry analysis. Our results highlight LIF as a TGFβ target gene in melanoma, suggesting that it may play a role downstream of TGFβ-mediated growth inhibition in melanoma. As shown in Figure 2C, while a control scrambled siRNA showed no effect, blocking LIF expression was able to almost completely block the TGFβ effect on the induction of G1 arrest, indicating that LIF plays a major role in the TGFβ-induced cell cycle arrest. We then tested the effect of silencing LIF gene expression on the induction of caspase-mediated apoptosis by TGFβ in WM278 cells. As shown in Figure 2D, blocking LIF expression almost completely inhibited the TGFβ-mediated induction of apoptosis, showing that LIF is required for TGFβ-mediated apoptosis. Efficiency of the LIF siRNA knockdown was assessed by qPCR (Figure 2E). These results highlight the involvement of LIF in cell cycle arrest and caspase-mediated cell death upon TGFβ stimulation.Figure 2


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 LIF regulation. A, WM278 cells were treated or not with TGFβfor 24 h and LIF expression was analyzed by qPCR. Error bars are standard deviations and z-test was performed (***p < 0.001). B, WM278 and WM793B cells were treated or not with TGFβ for 24 h and LIF expression was analyzed by Western blot (left panel) after concentration of conditioned media. β-tubulin was used as control. Right panel: Densitometric analysis of LIF protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p < 0.001). C, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Error bars are standard errors of mean and z-test was performed compared to non-treated control (**p < 0.01, ***p<0.001). D, WM278 cells transfected with scrambled or LIF 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 the 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 < 0.01). E, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h. LIF expression was analyzed by qPCR. Data is graphed as the mean of fold induction of gene expression in response to TGFβ for at least 3 independent experiments. Error bars are the standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (**p < 0.01).
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Fig2: TGFβ mediates its effects through LIF regulation. A, WM278 cells were treated or not with TGFβfor 24 h and LIF expression was analyzed by qPCR. Error bars are standard deviations and z-test was performed (***p < 0.001). B, WM278 and WM793B cells were treated or not with TGFβ for 24 h and LIF expression was analyzed by Western blot (left panel) after concentration of conditioned media. β-tubulin was used as control. Right panel: Densitometric analysis of LIF protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p < 0.001). C, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Error bars are standard errors of mean and z-test was performed compared to non-treated control (**p < 0.01, ***p<0.001). D, WM278 cells transfected with scrambled or LIF 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 the 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 < 0.01). E, WM278 cells transfected with scrambled or LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h. LIF expression was analyzed by qPCR. Data is graphed as the mean of fold induction of gene expression in response to TGFβ for at least 3 independent experiments. Error bars are the standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (**p < 0.01).
Mentions: Transcriptome analysis of WM793B and WM278 cells revealed that one particular gene, the leukemia inhibitory factor (LIF), appeared to be strongly upregulated by TGFβ (data not shown). This was further confirmed and quantified at both mRNA and protein levels. As shown in Figure 2A and B, TGFβ potently induced LIF expression both at the mRNA and protein levels in WM793B and WM278 cells, suggesting a role for LIF in mediating the TGFβ effects in melanoma cells. Both cell lines showed a 5 to 6 fold increase in protein levels, as quantified by densitometry analysis. Our results highlight LIF as a TGFβ target gene in melanoma, suggesting that it may play a role downstream of TGFβ-mediated growth inhibition in melanoma. As shown in Figure 2C, while a control scrambled siRNA showed no effect, blocking LIF expression was able to almost completely block the TGFβ effect on the induction of G1 arrest, indicating that LIF plays a major role in the TGFβ-induced cell cycle arrest. We then tested the effect of silencing LIF gene expression on the induction of caspase-mediated apoptosis by TGFβ in WM278 cells. As shown in Figure 2D, blocking LIF expression almost completely inhibited the TGFβ-mediated induction of apoptosis, showing that LIF is required for TGFβ-mediated apoptosis. Efficiency of the LIF siRNA knockdown was assessed by qPCR (Figure 2E). These results highlight the involvement of LIF in cell cycle arrest and caspase-mediated cell death upon TGFβ stimulation.Figure 2

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