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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

LIF mediates the TGFβ-dependent inhibition of melanoma cell migration. A, WM278 cells treated with LIF (100 or 500 ng/mL) or B, transfected with a scrambled or a LIF or a p21 siRNA 48 h earlier were plated in starvation medium on top of 24-well cell culture Transwell inserts, stimulated or not with TGFβ for 48 h, and the migratory cells were labeled with crystal violet, photographed using phase contrast light microscopy. A representative image is shown for each cell line (left panel). Migratory cells were counted using the ImageJ software and graphed (right panel). Data is graphed as the mean of at least 3 biological replicates. Error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated mock condition (*p < 0.05).
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Fig5: LIF mediates the TGFβ-dependent inhibition of melanoma cell migration. A, WM278 cells treated with LIF (100 or 500 ng/mL) or B, transfected with a scrambled or a LIF or a p21 siRNA 48 h earlier were plated in starvation medium on top of 24-well cell culture Transwell inserts, stimulated or not with TGFβ for 48 h, and the migratory cells were labeled with crystal violet, photographed using phase contrast light microscopy. A representative image is shown for each cell line (left panel). Migratory cells were counted using the ImageJ software and graphed (right panel). Data is graphed as the mean of at least 3 biological replicates. Error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated mock condition (*p < 0.05).

Mentions: We previously showed that TGFβ is not only a tumor suppressor in cutaneous melanoma but also acts as an anti-metastatic agent, inhibiting both the migratory and invasive properties of melanoma cells [12]. Recent literature showed that LIF-R is a metastasis suppressor in breast cancer [42]. We thus investigated whether LIF signaling might also play such a role in melanoma, by regulating the TGFβ-mediated inhibition of migration and invasion. We thus aimed at determining whether TGFβ-mediated LIF gene expression could also mediate the TGFβ anti-migratory/invasive activities. For this, we assessed cell migration using the Transwell assay, as previously described [12]. As shown in Figure 5A, TGFβ strongly inhibited cell migration in melanoma cells. Moreover, treatment with LIF mimicked this TGFβ effect, indicating that LIF, in addition to mediate the TGFβ-mediated growth inhibition, may also be involved in the TGFβ anti-metastatic effects. This was confirmed using a specific LIF siRNA: while a scrambled siRNA showed no effect, blocking LIF expression significantly inhibit the TGFβ effect on melanoma cell migration by about 60% (Figure 5B). We then assessed if p21 had a role in this process using a specific p21 siRNA. Interestingly, blocking p21 expression had no effect on migration, indicating that LIF mediates TGFβ anti-migratory activities independently of p21 (Figure 5B). Overall these results demonstrate that LIF is not only involved in the tumor suppressive effects of TGFβ but also in its anti-metastatic activities, and that different pathways downstream of LIF are involved in these processes.Figure 5


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)

LIF mediates the TGFβ-dependent inhibition of melanoma cell migration. A, WM278 cells treated with LIF (100 or 500 ng/mL) or B, transfected with a scrambled or a LIF or a p21 siRNA 48 h earlier were plated in starvation medium on top of 24-well cell culture Transwell inserts, stimulated or not with TGFβ for 48 h, and the migratory cells were labeled with crystal violet, photographed using phase contrast light microscopy. A representative image is shown for each cell line (left panel). Migratory cells were counted using the ImageJ software and graphed (right panel). Data is graphed as the mean of at least 3 biological replicates. Error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated mock condition (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: LIF mediates the TGFβ-dependent inhibition of melanoma cell migration. A, WM278 cells treated with LIF (100 or 500 ng/mL) or B, transfected with a scrambled or a LIF or a p21 siRNA 48 h earlier were plated in starvation medium on top of 24-well cell culture Transwell inserts, stimulated or not with TGFβ for 48 h, and the migratory cells were labeled with crystal violet, photographed using phase contrast light microscopy. A representative image is shown for each cell line (left panel). Migratory cells were counted using the ImageJ software and graphed (right panel). Data is graphed as the mean of at least 3 biological replicates. Error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated mock condition (*p < 0.05).
Mentions: We previously showed that TGFβ is not only a tumor suppressor in cutaneous melanoma but also acts as an anti-metastatic agent, inhibiting both the migratory and invasive properties of melanoma cells [12]. Recent literature showed that LIF-R is a metastasis suppressor in breast cancer [42]. We thus investigated whether LIF signaling might also play such a role in melanoma, by regulating the TGFβ-mediated inhibition of migration and invasion. We thus aimed at determining whether TGFβ-mediated LIF gene expression could also mediate the TGFβ anti-migratory/invasive activities. For this, we assessed cell migration using the Transwell assay, as previously described [12]. As shown in Figure 5A, TGFβ strongly inhibited cell migration in melanoma cells. Moreover, treatment with LIF mimicked this TGFβ effect, indicating that LIF, in addition to mediate the TGFβ-mediated growth inhibition, may also be involved in the TGFβ anti-metastatic effects. This was confirmed using a specific LIF siRNA: while a scrambled siRNA showed no effect, blocking LIF expression significantly inhibit the TGFβ effect on melanoma cell migration by about 60% (Figure 5B). We then assessed if p21 had a role in this process using a specific p21 siRNA. Interestingly, blocking p21 expression had no effect on migration, indicating that LIF mediates TGFβ anti-migratory activities independently of p21 (Figure 5B). Overall these results demonstrate that LIF is not only involved in the tumor suppressive effects of TGFβ but also in its anti-metastatic activities, and that different pathways downstream of LIF are involved in these processes.Figure 5

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