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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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Integrin αv-dependent MEK1 activity is required for melanoma cell survival in 3D-collagen. (A) M21 (αv+) cells and M21L (αv−) cells were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. The levels of active MEK1 and ERK1/2 as well as of total MEK1 and ERK1/2 were detected by Western blotting, with actin as control and the displayed blots are representative among five experiments. (B and C) M21 (αv+) cells within 3D-collagen were treated with the MEK1 inhibitors PD98059 (B) and U0126 (C) or DMSO as a vehicle control. Annexin-V staining detected apoptosis and the bar graphs show the mean ± SD of apoptotic cells among three independent experiments (** −P < 0.01, as compared with vehicle control using unpaired two-tailed t test). Phosphorylated ERK1/2 was detected by Western blotting to control for the suppressive effect of the MEK1 inhibitors. (D) p53 DNA-binding activities were detected by EMSA and p53 protein levels determined by Western blotting in M21 (αv+) cells treated with PD98059 within 3D-collagen for 3–7 d. Phosphorylated ERK1/2 was detected to monitor the inhibitory effect of PD98059, and total ERK1/2 and actin levels were analyzed as controls. Note that the exposure time in this EMSA was longer than for EMSAs displayed in other figures (Fig. 2, B and D).
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fig5: Integrin αv-dependent MEK1 activity is required for melanoma cell survival in 3D-collagen. (A) M21 (αv+) cells and M21L (αv−) cells were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. The levels of active MEK1 and ERK1/2 as well as of total MEK1 and ERK1/2 were detected by Western blotting, with actin as control and the displayed blots are representative among five experiments. (B and C) M21 (αv+) cells within 3D-collagen were treated with the MEK1 inhibitors PD98059 (B) and U0126 (C) or DMSO as a vehicle control. Annexin-V staining detected apoptosis and the bar graphs show the mean ± SD of apoptotic cells among three independent experiments (** −P < 0.01, as compared with vehicle control using unpaired two-tailed t test). Phosphorylated ERK1/2 was detected by Western blotting to control for the suppressive effect of the MEK1 inhibitors. (D) p53 DNA-binding activities were detected by EMSA and p53 protein levels determined by Western blotting in M21 (αv+) cells treated with PD98059 within 3D-collagen for 3–7 d. Phosphorylated ERK1/2 was detected to monitor the inhibitory effect of PD98059, and total ERK1/2 and actin levels were analyzed as controls. Note that the exposure time in this EMSA was longer than for EMSAs displayed in other figures (Fig. 2, B and D).

Mentions: Integrin αvβ3 ligation promotes sustained activation of the MEK1–ERK1/2 signaling pathway in vascular cells during angiogenesis (Eliceiri et al., 1998). Because of an active mutation of BRAF, MEK1 and ERK1/2 are constitutively activated in most melanoma cell lines under conventional 2D culture conditions (Satyamoorthy et al., 2003). However, it is unclear if integrin αvβ3 may play a role in regulation of MEK1 and ERK1/2 activities in melanoma cells within a 3D environment. To address this question, we examined MEK1 and ERK1/2 activities in integrin αv-positive M21 and integrin αv-negative M21L cells cultured in 3D-collagen. We detected no differences in MEK1 or ERK1/2 activities between M21 (αv+) and M21L (αv–) cells under 2D culture conditions (Fig. 5 A, d 0). However, both MEK1 and ERK1/2 activities were markedly reduced in αv-negative M21L cells in contrast to αv-positive M21 cells after 3–7 d of exposure in 3D-collagen (Fig. 5 A). This indicates that whereas MEK1 and ERK1/2 is active in melanoma cells under 2D culture conditions, integrin αv appears to be needed for activation of MEK1 and ERK1/2 within a 3D environment. To examine the potential role of MEK1 signaling in integrin αv-mediated melanoma cell survival, we blocked MEK1 activity by treatment of integrin αv-positive M21 cells with two specific MEK1 inhibitors, PD98059 and U0126. Both inhibitors strongly induced apoptosis in M21 (αv+) cells within 3D-collagen (Fig. 5, B and C), but with no changes in integrin αvβ3 cell surface expression levels (not depicted). This shows that integrin αv-dependent MEK1 signaling is required for melanoma cell survival within a 3D environment.


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)

Integrin αv-dependent MEK1 activity is required for melanoma cell survival in 3D-collagen. (A) M21 (αv+) cells and M21L (αv−) cells were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. The levels of active MEK1 and ERK1/2 as well as of total MEK1 and ERK1/2 were detected by Western blotting, with actin as control and the displayed blots are representative among five experiments. (B and C) M21 (αv+) cells within 3D-collagen were treated with the MEK1 inhibitors PD98059 (B) and U0126 (C) or DMSO as a vehicle control. Annexin-V staining detected apoptosis and the bar graphs show the mean ± SD of apoptotic cells among three independent experiments (** −P < 0.01, as compared with vehicle control using unpaired two-tailed t test). Phosphorylated ERK1/2 was detected by Western blotting to control for the suppressive effect of the MEK1 inhibitors. (D) p53 DNA-binding activities were detected by EMSA and p53 protein levels determined by Western blotting in M21 (αv+) cells treated with PD98059 within 3D-collagen for 3–7 d. Phosphorylated ERK1/2 was detected to monitor the inhibitory effect of PD98059, and total ERK1/2 and actin levels were analyzed as controls. Note that the exposure time in this EMSA was longer than for EMSAs displayed in other figures (Fig. 2, B and D).
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fig5: Integrin αv-dependent MEK1 activity is required for melanoma cell survival in 3D-collagen. (A) M21 (αv+) cells and M21L (αv−) cells were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. The levels of active MEK1 and ERK1/2 as well as of total MEK1 and ERK1/2 were detected by Western blotting, with actin as control and the displayed blots are representative among five experiments. (B and C) M21 (αv+) cells within 3D-collagen were treated with the MEK1 inhibitors PD98059 (B) and U0126 (C) or DMSO as a vehicle control. Annexin-V staining detected apoptosis and the bar graphs show the mean ± SD of apoptotic cells among three independent experiments (** −P < 0.01, as compared with vehicle control using unpaired two-tailed t test). Phosphorylated ERK1/2 was detected by Western blotting to control for the suppressive effect of the MEK1 inhibitors. (D) p53 DNA-binding activities were detected by EMSA and p53 protein levels determined by Western blotting in M21 (αv+) cells treated with PD98059 within 3D-collagen for 3–7 d. Phosphorylated ERK1/2 was detected to monitor the inhibitory effect of PD98059, and total ERK1/2 and actin levels were analyzed as controls. Note that the exposure time in this EMSA was longer than for EMSAs displayed in other figures (Fig. 2, B and D).
Mentions: Integrin αvβ3 ligation promotes sustained activation of the MEK1–ERK1/2 signaling pathway in vascular cells during angiogenesis (Eliceiri et al., 1998). Because of an active mutation of BRAF, MEK1 and ERK1/2 are constitutively activated in most melanoma cell lines under conventional 2D culture conditions (Satyamoorthy et al., 2003). However, it is unclear if integrin αvβ3 may play a role in regulation of MEK1 and ERK1/2 activities in melanoma cells within a 3D environment. To address this question, we examined MEK1 and ERK1/2 activities in integrin αv-positive M21 and integrin αv-negative M21L cells cultured in 3D-collagen. We detected no differences in MEK1 or ERK1/2 activities between M21 (αv+) and M21L (αv–) cells under 2D culture conditions (Fig. 5 A, d 0). However, both MEK1 and ERK1/2 activities were markedly reduced in αv-negative M21L cells in contrast to αv-positive M21 cells after 3–7 d of exposure in 3D-collagen (Fig. 5 A). This indicates that whereas MEK1 and ERK1/2 is active in melanoma cells under 2D culture conditions, integrin αv appears to be needed for activation of MEK1 and ERK1/2 within a 3D environment. To examine the potential role of MEK1 signaling in integrin αv-mediated melanoma cell survival, we blocked MEK1 activity by treatment of integrin αv-positive M21 cells with two specific MEK1 inhibitors, PD98059 and U0126. Both inhibitors strongly induced apoptosis in M21 (αv+) cells within 3D-collagen (Fig. 5, B and C), but with no changes in integrin αvβ3 cell surface expression levels (not depicted). This shows that integrin αv-dependent MEK1 signaling is required for melanoma cell survival within a 3D environment.

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