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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 exerts strong growth inhibitory effects in various cutaneous melanoma cell lines. A, WM793B and WM278 cells were treated or not with TGFβ and their cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as the mean of the percentages of cells in each phase for at least 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, *p < 0.05). B, WM793B and WM278 cells were treated or not with TGFβ and apoptosis was determined by measuring the caspase3/7 activity. Data is graphed as the geometric mean of relative luciferase units normalized to the non-treated control for at least 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the z-test was performed compared to the non-treated control (***p < 0.001).
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Fig1: TGFβ exerts strong growth inhibitory effects in various cutaneous melanoma cell lines. A, WM793B and WM278 cells were treated or not with TGFβ and their cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as the mean of the percentages of cells in each phase for at least 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, *p < 0.05). B, WM793B and WM278 cells were treated or not with TGFβ and apoptosis was determined by measuring the caspase3/7 activity. Data is graphed as the geometric mean of relative luciferase units normalized to the non-treated control for at least 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the z-test was performed compared to the non-treated control (***p < 0.001).

Mentions: We previously found TGFβ to decrease cell viability in multiple melanoma cell lines, isolated from different patients [12]. To further investigate the mechanisms by which TGFβ regulates cell growth, we analyzed its effects on both cell cycle regulation and apoptosis. First, we analyzed the cell cycle profile of two cutaneous melanoma cell lines, WM793B and WM278, treated or not with TGFβ for 24 h. Following ethanol fixation of the cells and propidium iodide staining, the cell cycle profile was analyzed by flow cytometry. As shown in Figure 1A, WM793B and WM278 responded well to TGFβ showing a significant induction of G1 arrest. These results indicate that, while TGFβ growth inhibitory responses are lost in some melanomas, this growth factor still efficiently induces cell cycle arrest. We then analyzed the TGFβ effects on the regulation of apoptosis in these melanoma cell lines. Briefly, melanoma cells were treated or not with TGFβ for 72 h and caspase 3/7 activity was measured using a luminescent assay. Interestingly, as shown in Figure 1B, TGFβ significantly induced cell death, indicating that it acts as a pro-apoptotic factor in human melanoma and that its tumor suppressive effects are mediated through both cell cycle arrest in the G1 phase and caspase-mediated cell death.Figure 1


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 exerts strong growth inhibitory effects in various cutaneous melanoma cell lines. A, WM793B and WM278 cells were treated or not with TGFβ and their cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as the mean of the percentages of cells in each phase for at least 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, *p < 0.05). B, WM793B and WM278 cells were treated or not with TGFβ and apoptosis was determined by measuring the caspase3/7 activity. Data is graphed as the geometric mean of relative luciferase units normalized to the non-treated control for at least 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the z-test was performed compared to the non-treated control (***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389797&req=5

Fig1: TGFβ exerts strong growth inhibitory effects in various cutaneous melanoma cell lines. A, WM793B and WM278 cells were treated or not with TGFβ and their cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as the mean of the percentages of cells in each phase for at least 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, *p < 0.05). B, WM793B and WM278 cells were treated or not with TGFβ and apoptosis was determined by measuring the caspase3/7 activity. Data is graphed as the geometric mean of relative luciferase units normalized to the non-treated control for at least 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the z-test was performed compared to the non-treated control (***p < 0.001).
Mentions: We previously found TGFβ to decrease cell viability in multiple melanoma cell lines, isolated from different patients [12]. To further investigate the mechanisms by which TGFβ regulates cell growth, we analyzed its effects on both cell cycle regulation and apoptosis. First, we analyzed the cell cycle profile of two cutaneous melanoma cell lines, WM793B and WM278, treated or not with TGFβ for 24 h. Following ethanol fixation of the cells and propidium iodide staining, the cell cycle profile was analyzed by flow cytometry. As shown in Figure 1A, WM793B and WM278 responded well to TGFβ showing a significant induction of G1 arrest. These results indicate that, while TGFβ growth inhibitory responses are lost in some melanomas, this growth factor still efficiently induces cell cycle arrest. We then analyzed the TGFβ effects on the regulation of apoptosis in these melanoma cell lines. Briefly, melanoma cells were treated or not with TGFβ for 72 h and caspase 3/7 activity was measured using a luminescent assay. Interestingly, as shown in Figure 1B, TGFβ significantly induced cell death, indicating that it acts as a pro-apoptotic factor in human melanoma and that its tumor suppressive effects are mediated through both cell cycle arrest in the G1 phase and caspase-mediated cell death.Figure 1

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