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Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen.

Bao W, Strömblad S - J. Cell Biol. (2004)

Bottom Line: We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo.Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis.Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Stockholm, 141 86, Sweden.

ABSTRACT
Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.

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p53 controls a MEK1-dependent melanoma cell survival pathway. (A and B) The levels of activated and total MEK1 as well as activated and total ERK1/2 were detected by Western blotting in M21 (αv+), M21L (αv−), and in M21L-p53His175 clones (Lp53His175) (A) and M21L-p53-siRNA clones (Lp53siRNA) (B) after 5 d of culture within 3D-collagen. The p53 protein levels were measured as control for the effect of p53-siRNA and actin levels as control. (C) M21L (αv−) clones stably expressing p53-siRNA (Lp53siRNA) were treated with the MEK1 inhibitor PD98059 in 3D-collagen for 5 d and compared with untreated M21L (αv−) and M21 (αv+) cells. Apoptosis was detected by Annexin-V staining. The displayed results are representative among three independent experiments.
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fig6: p53 controls a MEK1-dependent melanoma cell survival pathway. (A and B) The levels of activated and total MEK1 as well as activated and total ERK1/2 were detected by Western blotting in M21 (αv+), M21L (αv−), and in M21L-p53His175 clones (Lp53His175) (A) and M21L-p53-siRNA clones (Lp53siRNA) (B) after 5 d of culture within 3D-collagen. The p53 protein levels were measured as control for the effect of p53-siRNA and actin levels as control. (C) M21L (αv−) clones stably expressing p53-siRNA (Lp53siRNA) were treated with the MEK1 inhibitor PD98059 in 3D-collagen for 5 d and compared with untreated M21L (αv−) and M21 (αv+) cells. Apoptosis was detected by Annexin-V staining. The displayed results are representative among three independent experiments.

Mentions: Given that MEK1 signaling did not act as a mediator of integrin αv-dependent regulation of p53, we instead tested if suppression of p53 might affect MEK1–ERK1/2 activity within 3D-collagen. We used the M21L (αv−) clones stably expressing dn p53-His175 or p53-siRNA. Under 2D culture conditions (d 0), we observed no influence on MEK1 by expression of p53-His175 or p53-siRNA (unpublished data). However, surprisingly, suppression of p53 by both p53-His175 and p53-siRNA expression rescued MEK1 and ERK1/2 activities in integrin αv-negative M21L cells to similar levels as in integrin αv-positive M21 cells within 3D-collagen (Fig. 6, A and B). However, no changes were observed at MEK1 or ERK1/2 protein levels (Fig. 6, A and B), indicating that the suppression of p53 in cells lacking integrin αv instead only influenced MEK1 and ERK1/2 activities. Furthermore, we examined whether MEK1 activity was still required for melanoma cell survival after knockdown of p53 by siRNA and thus whether MEK1 may functionally act downstream of p53 in regulation of melanoma cell survival. As shown in Fig. 6 C, M21L-p53siRNA (αv−) clones that normally survived to the same extent as M21 (αv+) cells underwent apoptosis to a high degree when treated with the MEK1 inhibitor PD98059 within 3D-collagen, indicating that these cells were still dependent on MEK1 for survival. This indicates that MEK1 is critical and functionally acts downstream of p53 in integrin αv-mediated melanoma cell survival. However, overexpression of ca MEK1 (S218D/S222D) did not rescue M21L (αv−) cell survival in 3D-collagen (unpublished data). Given that block of p53 rescued the same M21L (αv−) cells, this suggests that p53 may act not only through MEK1 regulation, but also through additional downstream pathways to induce melanoma cell apoptosis.


Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen.

Bao W, Strömblad S - J. Cell Biol. (2004)

p53 controls a MEK1-dependent melanoma cell survival pathway. (A and B) The levels of activated and total MEK1 as well as activated and total ERK1/2 were detected by Western blotting in M21 (αv+), M21L (αv−), and in M21L-p53His175 clones (Lp53His175) (A) and M21L-p53-siRNA clones (Lp53siRNA) (B) after 5 d of culture within 3D-collagen. The p53 protein levels were measured as control for the effect of p53-siRNA and actin levels as control. (C) M21L (αv−) clones stably expressing p53-siRNA (Lp53siRNA) were treated with the MEK1 inhibitor PD98059 in 3D-collagen for 5 d and compared with untreated M21L (αv−) and M21 (αv+) cells. Apoptosis was detected by Annexin-V staining. The displayed results are representative among three independent experiments.
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fig6: p53 controls a MEK1-dependent melanoma cell survival pathway. (A and B) The levels of activated and total MEK1 as well as activated and total ERK1/2 were detected by Western blotting in M21 (αv+), M21L (αv−), and in M21L-p53His175 clones (Lp53His175) (A) and M21L-p53-siRNA clones (Lp53siRNA) (B) after 5 d of culture within 3D-collagen. The p53 protein levels were measured as control for the effect of p53-siRNA and actin levels as control. (C) M21L (αv−) clones stably expressing p53-siRNA (Lp53siRNA) were treated with the MEK1 inhibitor PD98059 in 3D-collagen for 5 d and compared with untreated M21L (αv−) and M21 (αv+) cells. Apoptosis was detected by Annexin-V staining. The displayed results are representative among three independent experiments.
Mentions: Given that MEK1 signaling did not act as a mediator of integrin αv-dependent regulation of p53, we instead tested if suppression of p53 might affect MEK1–ERK1/2 activity within 3D-collagen. We used the M21L (αv−) clones stably expressing dn p53-His175 or p53-siRNA. Under 2D culture conditions (d 0), we observed no influence on MEK1 by expression of p53-His175 or p53-siRNA (unpublished data). However, surprisingly, suppression of p53 by both p53-His175 and p53-siRNA expression rescued MEK1 and ERK1/2 activities in integrin αv-negative M21L cells to similar levels as in integrin αv-positive M21 cells within 3D-collagen (Fig. 6, A and B). However, no changes were observed at MEK1 or ERK1/2 protein levels (Fig. 6, A and B), indicating that the suppression of p53 in cells lacking integrin αv instead only influenced MEK1 and ERK1/2 activities. Furthermore, we examined whether MEK1 activity was still required for melanoma cell survival after knockdown of p53 by siRNA and thus whether MEK1 may functionally act downstream of p53 in regulation of melanoma cell survival. As shown in Fig. 6 C, M21L-p53siRNA (αv−) clones that normally survived to the same extent as M21 (αv+) cells underwent apoptosis to a high degree when treated with the MEK1 inhibitor PD98059 within 3D-collagen, indicating that these cells were still dependent on MEK1 for survival. This indicates that MEK1 is critical and functionally acts downstream of p53 in integrin αv-mediated melanoma cell survival. However, overexpression of ca MEK1 (S218D/S222D) did not rescue M21L (αv−) cell survival in 3D-collagen (unpublished data). Given that block of p53 rescued the same M21L (αv−) cells, this suggests that p53 may act not only through MEK1 regulation, but also through additional downstream pathways to induce melanoma cell apoptosis.

Bottom Line: We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo.Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis.Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Stockholm, 141 86, Sweden.

ABSTRACT
Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.

Show MeSH
Related in: MedlinePlus