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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 is required for melanoma cell survival in 3D-collagen. (A) Integrin αvβ3 (left) and β3 subunit (right) expression characterized by flow cytometry in the melanoma cells used in this work. The M1 gate marks positive integrin staining as compared with the negative control (not depicted). (B) Apoptosis was detected with Annexin-V staining in integrin αv-positive M21 and αv-negative M21L cells cultured in 3D-collagen for the indicated times of 3–7 d. The displayed staining profiles are representative among three independent experiments. M1 marks Annexin-V positive cells as compared with the control (left row) and the given numbers represent the quantification of the fraction of Annexin-V positive cells in the displayed representative experiment. (C and D) Bar graphs display apoptosis in M21 (αv+) and M21L (αv−) cells (C) and in M0 (αv−) and M0-αv (αv+) (D) cells cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Each graph in C and D represents the mean ± SD of Annexin-V positive cells among three independent experiments (* −P < 0.05; ** −P < 0.01; as compared with M21 (C) or M0-αv (D) cells using unpaired two tailed t test).
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fig1: Integrin αv is required for melanoma cell survival in 3D-collagen. (A) Integrin αvβ3 (left) and β3 subunit (right) expression characterized by flow cytometry in the melanoma cells used in this work. The M1 gate marks positive integrin staining as compared with the negative control (not depicted). (B) Apoptosis was detected with Annexin-V staining in integrin αv-positive M21 and αv-negative M21L cells cultured in 3D-collagen for the indicated times of 3–7 d. The displayed staining profiles are representative among three independent experiments. M1 marks Annexin-V positive cells as compared with the control (left row) and the given numbers represent the quantification of the fraction of Annexin-V positive cells in the displayed representative experiment. (C and D) Bar graphs display apoptosis in M21 (αv+) and M21L (αv−) cells (C) and in M0 (αv−) and M0-αv (αv+) (D) cells cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Each graph in C and D represents the mean ± SD of Annexin-V positive cells among three independent experiments (* −P < 0.05; ** −P < 0.01; as compared with M21 (C) or M0-αv (D) cells using unpaired two tailed t test).

Mentions: Previous studies indicated a role for integrin αvβ3 in melanoma cell survival both in 3D collagen in vitro and in human skin in vivo (Montgomery et al., 1994; Petitclerc et al., 1999). To elucidate potential molecular mechanisms for integrin αvβ3-mediated melanoma cell survival, we used M21 and M0 melanoma cell lines with or without integrin αv. We characterized and compared the cell surface expression of integrin αvβ3 and of the integrin β3 subunit in these cells by flow cytometry (Fig. 1 A). These melanoma cells were then applied in a 3D dermal collagen gel model enriched in collagen type I. Integrin α2β1 is a major receptor for collagen type I, mediating initial M21 cell adhesion and spreading onto collagen type I, but when collagen is degraded, the exposure of cryptic RGD sites renders collagen type I to also allow binding of integrin αvβ3 (Montgomery et al., 1994). To control the potential effect of integrin αv expression on integrin α2β1, the cell surface expression of integrin α2β1 was analyzed by flow cytometry. However, we detected no differences in integrin α2β1 levels between the different integrin αv-positive and αv-negative M21 subpopulations (unpublished data), indicating that alterations of integrin αv did not affect integrin α2β1 and thereby did not affect the capability to attach to collagen type I. M21 (integrin αv+) and M21L (integrin αv−) cells were then incubated in 3D-collagen and analyzed for apoptosis using Annexin-V staining. We found that M21L (αv−) cells underwent apoptosis to a much higher degree as compared with M21 (αv+) cells after 3–7 d of exposure in 3D-collagen (Fig. 1, B and C), confirming previous findings that integrin αv is critical for M21 melanoma cell survival in 3D-collagen (Montgomery et al., 1994). It should be noted that only low levels of apoptosis were observed in M21 (αv+) and M21L (αv−) cells under 2D culture conditions (d 0). Among different experiments, the number of apoptotic M21L (αv−) cells ranged between 30 and 70% and the onset of apoptosis in these cells varied somewhat in time and was typically clearly visible after 5 d in 3D-collagen (unpublished data). In addition, similar results were obtained by TUNEL staining (unpublished data). The role of integrin αv in melanoma cell survival was also addressed in M0 cell variants, an independent set of melanoma cells either lacking integrin αv (M0) or expressing high levels of wt integrin αv (M0-αv) (Chen et al., 1995). M0 (αv−) and M0-αv (αv+) cells displayed low levels of apoptosis under 2D culture conditions (d 0) (Fig. 1 D). However, M0 (αv−) cells became apoptotic to a clearly higher degree as compared with M0-αv (αv+) after culture in 3D-collagen for 3–5 d, indicating a role for integrin αv also in the survival of these melanoma cells in 3D-collagen. Altogether, these results suggest an essential role for integrin αv in control of melanoma cell survival.


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 is required for melanoma cell survival in 3D-collagen. (A) Integrin αvβ3 (left) and β3 subunit (right) expression characterized by flow cytometry in the melanoma cells used in this work. The M1 gate marks positive integrin staining as compared with the negative control (not depicted). (B) Apoptosis was detected with Annexin-V staining in integrin αv-positive M21 and αv-negative M21L cells cultured in 3D-collagen for the indicated times of 3–7 d. The displayed staining profiles are representative among three independent experiments. M1 marks Annexin-V positive cells as compared with the control (left row) and the given numbers represent the quantification of the fraction of Annexin-V positive cells in the displayed representative experiment. (C and D) Bar graphs display apoptosis in M21 (αv+) and M21L (αv−) cells (C) and in M0 (αv−) and M0-αv (αv+) (D) cells cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Each graph in C and D represents the mean ± SD of Annexin-V positive cells among three independent experiments (* −P < 0.05; ** −P < 0.01; as compared with M21 (C) or M0-αv (D) cells using unpaired two tailed t test).
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fig1: Integrin αv is required for melanoma cell survival in 3D-collagen. (A) Integrin αvβ3 (left) and β3 subunit (right) expression characterized by flow cytometry in the melanoma cells used in this work. The M1 gate marks positive integrin staining as compared with the negative control (not depicted). (B) Apoptosis was detected with Annexin-V staining in integrin αv-positive M21 and αv-negative M21L cells cultured in 3D-collagen for the indicated times of 3–7 d. The displayed staining profiles are representative among three independent experiments. M1 marks Annexin-V positive cells as compared with the control (left row) and the given numbers represent the quantification of the fraction of Annexin-V positive cells in the displayed representative experiment. (C and D) Bar graphs display apoptosis in M21 (αv+) and M21L (αv−) cells (C) and in M0 (αv−) and M0-αv (αv+) (D) cells cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Each graph in C and D represents the mean ± SD of Annexin-V positive cells among three independent experiments (* −P < 0.05; ** −P < 0.01; as compared with M21 (C) or M0-αv (D) cells using unpaired two tailed t test).
Mentions: Previous studies indicated a role for integrin αvβ3 in melanoma cell survival both in 3D collagen in vitro and in human skin in vivo (Montgomery et al., 1994; Petitclerc et al., 1999). To elucidate potential molecular mechanisms for integrin αvβ3-mediated melanoma cell survival, we used M21 and M0 melanoma cell lines with or without integrin αv. We characterized and compared the cell surface expression of integrin αvβ3 and of the integrin β3 subunit in these cells by flow cytometry (Fig. 1 A). These melanoma cells were then applied in a 3D dermal collagen gel model enriched in collagen type I. Integrin α2β1 is a major receptor for collagen type I, mediating initial M21 cell adhesion and spreading onto collagen type I, but when collagen is degraded, the exposure of cryptic RGD sites renders collagen type I to also allow binding of integrin αvβ3 (Montgomery et al., 1994). To control the potential effect of integrin αv expression on integrin α2β1, the cell surface expression of integrin α2β1 was analyzed by flow cytometry. However, we detected no differences in integrin α2β1 levels between the different integrin αv-positive and αv-negative M21 subpopulations (unpublished data), indicating that alterations of integrin αv did not affect integrin α2β1 and thereby did not affect the capability to attach to collagen type I. M21 (integrin αv+) and M21L (integrin αv−) cells were then incubated in 3D-collagen and analyzed for apoptosis using Annexin-V staining. We found that M21L (αv−) cells underwent apoptosis to a much higher degree as compared with M21 (αv+) cells after 3–7 d of exposure in 3D-collagen (Fig. 1, B and C), confirming previous findings that integrin αv is critical for M21 melanoma cell survival in 3D-collagen (Montgomery et al., 1994). It should be noted that only low levels of apoptosis were observed in M21 (αv+) and M21L (αv−) cells under 2D culture conditions (d 0). Among different experiments, the number of apoptotic M21L (αv−) cells ranged between 30 and 70% and the onset of apoptosis in these cells varied somewhat in time and was typically clearly visible after 5 d in 3D-collagen (unpublished data). In addition, similar results were obtained by TUNEL staining (unpublished data). The role of integrin αv in melanoma cell survival was also addressed in M0 cell variants, an independent set of melanoma cells either lacking integrin αv (M0) or expressing high levels of wt integrin αv (M0-αv) (Chen et al., 1995). M0 (αv−) and M0-αv (αv+) cells displayed low levels of apoptosis under 2D culture conditions (d 0) (Fig. 1 D). However, M0 (αv−) cells became apoptotic to a clearly higher degree as compared with M0-αv (αv+) after culture in 3D-collagen for 3–5 d, indicating a role for integrin αv also in the survival of these melanoma cells in 3D-collagen. Altogether, these results suggest an essential role for integrin αv in control of melanoma cell survival.

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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Related in: MedlinePlus