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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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Suppression of p53 compensates for the lack of integrin αv for melanoma cell survival and tumor growth. (A) Apoptosis was analyzed by Annexin-V staining in M21L-p53His175 clones (Lp53His175), M21L (αv−) and M21 (αv+) cells, cultured under 2D conditions (day 0) and within 3D-collagen for the indicated times. The bar graph (top) shows the quantifications of Annexin-V staining of one representative among three independent experiments. p53 activities were detected by EMSA in the indicated cells cultured in 3D-collagen for 5 d (bottom) to control for suppression of p53 activity by p53-His175 within the 3D collagen model used. (B–D) Melanoma cells were injected s.c. into the back of C57/BL mice as described in Materials and methods. (B) Photographs show representative mice from each group 25 d after tumor cell injections. (C) The graph shows the mean tumor volumes for each group of mice over time (n = 7). (D) The wet weights were measured in dissected tumors after 25 d. The graph shows mean weight ± S.D. for each group (n = 7; P < 0.05, unpaired two-tailed t test). (E) M21 (αv+), M21L (αv−), and five individual M21L-p53-siRNA clones (Lp53siRNA) were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Apoptosis was detected by Annexin-V staining. The Western blots for p53 and actin protein levels are shown in the vertical display for the cells indicated immediately to the left of each lane. The displayed results are representative among three independent experiments. (F) The protein levels of PUMA, Bax, and Apaf1 were detected by Western blotting in M21L-p53-siRNA clones, M21L (αv−) and M21 (αv+) cells, cultured in 3D-collagen for the indicated times, controlled by p53 and actin levels.
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fig4: Suppression of p53 compensates for the lack of integrin αv for melanoma cell survival and tumor growth. (A) Apoptosis was analyzed by Annexin-V staining in M21L-p53His175 clones (Lp53His175), M21L (αv−) and M21 (αv+) cells, cultured under 2D conditions (day 0) and within 3D-collagen for the indicated times. The bar graph (top) shows the quantifications of Annexin-V staining of one representative among three independent experiments. p53 activities were detected by EMSA in the indicated cells cultured in 3D-collagen for 5 d (bottom) to control for suppression of p53 activity by p53-His175 within the 3D collagen model used. (B–D) Melanoma cells were injected s.c. into the back of C57/BL mice as described in Materials and methods. (B) Photographs show representative mice from each group 25 d after tumor cell injections. (C) The graph shows the mean tumor volumes for each group of mice over time (n = 7). (D) The wet weights were measured in dissected tumors after 25 d. The graph shows mean weight ± S.D. for each group (n = 7; P < 0.05, unpaired two-tailed t test). (E) M21 (αv+), M21L (αv−), and five individual M21L-p53-siRNA clones (Lp53siRNA) were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Apoptosis was detected by Annexin-V staining. The Western blots for p53 and actin protein levels are shown in the vertical display for the cells indicated immediately to the left of each lane. The displayed results are representative among three independent experiments. (F) The protein levels of PUMA, Bax, and Apaf1 were detected by Western blotting in M21L-p53-siRNA clones, M21L (αv−) and M21 (αv+) cells, cultured in 3D-collagen for the indicated times, controlled by p53 and actin levels.

Mentions: We then examined whether the regulation of p53 activity by integrin αv could functionally mediate the role of integrin αv in melanoma cell survival in 3D-collagen and tumor growth in vivo. To this end, we blocked integrin αv-negative M21L cell p53 activity by stably transfecting a dominant negative (dn) p53-His175 cDNA. A number of p53-His175 expressing clones were identified using Western blotting (unpublished data). The M21L-p53-His175 clones, M21L (αv−) and M21 (αv+) cells were cultured in 3D-collagen and analyzed for apoptosis. In contrast to the parental M21L (αv−) cells, all four M21L-p53His175 clones survived to a similar degree as the integrin αv-positive M21 cells (Fig. 4 A, top). To control for the inhibitory effect of p53-His175 in these clones within 3D-collagen, p53 DNA-binding activities were analyzed after 5 d in 3D-collagen using EMSA (Fig. 4 A, bottom). These results indicate that block of p53 function by p53-His175 compensates for the lack of integrin αv in melanoma cell survival. Furthermore, M21 (αv+) and M21L (αv−) cells as well as two of the M21L-p53-His175 clones were injected subcutaneously (s.c.) into the back of C57/BL nude mice, to test if block of p53 function could compensate for lack of integrin αv in melanoma tumor growth in vivo. Consistent with previous studies (Felding-Habermann et al., 1992), M21 (αv+) cells formed large tumors, whereas M21L (αv−) only formed small or no tumors at all (Fig. 4, B–D). Importantly, M21L (αv−) cells overexpressing dn p53-His175 formed tumors to a similar extent and with a similar growth rate as the integrin αv-positive M21 cells (Fig. 4, B–D). This indicates that inhibition of p53 is essential for melanoma tumor growth.


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)

Suppression of p53 compensates for the lack of integrin αv for melanoma cell survival and tumor growth. (A) Apoptosis was analyzed by Annexin-V staining in M21L-p53His175 clones (Lp53His175), M21L (αv−) and M21 (αv+) cells, cultured under 2D conditions (day 0) and within 3D-collagen for the indicated times. The bar graph (top) shows the quantifications of Annexin-V staining of one representative among three independent experiments. p53 activities were detected by EMSA in the indicated cells cultured in 3D-collagen for 5 d (bottom) to control for suppression of p53 activity by p53-His175 within the 3D collagen model used. (B–D) Melanoma cells were injected s.c. into the back of C57/BL mice as described in Materials and methods. (B) Photographs show representative mice from each group 25 d after tumor cell injections. (C) The graph shows the mean tumor volumes for each group of mice over time (n = 7). (D) The wet weights were measured in dissected tumors after 25 d. The graph shows mean weight ± S.D. for each group (n = 7; P < 0.05, unpaired two-tailed t test). (E) M21 (αv+), M21L (αv−), and five individual M21L-p53-siRNA clones (Lp53siRNA) were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Apoptosis was detected by Annexin-V staining. The Western blots for p53 and actin protein levels are shown in the vertical display for the cells indicated immediately to the left of each lane. The displayed results are representative among three independent experiments. (F) The protein levels of PUMA, Bax, and Apaf1 were detected by Western blotting in M21L-p53-siRNA clones, M21L (αv−) and M21 (αv+) cells, cultured in 3D-collagen for the indicated times, controlled by p53 and actin levels.
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fig4: Suppression of p53 compensates for the lack of integrin αv for melanoma cell survival and tumor growth. (A) Apoptosis was analyzed by Annexin-V staining in M21L-p53His175 clones (Lp53His175), M21L (αv−) and M21 (αv+) cells, cultured under 2D conditions (day 0) and within 3D-collagen for the indicated times. The bar graph (top) shows the quantifications of Annexin-V staining of one representative among three independent experiments. p53 activities were detected by EMSA in the indicated cells cultured in 3D-collagen for 5 d (bottom) to control for suppression of p53 activity by p53-His175 within the 3D collagen model used. (B–D) Melanoma cells were injected s.c. into the back of C57/BL mice as described in Materials and methods. (B) Photographs show representative mice from each group 25 d after tumor cell injections. (C) The graph shows the mean tumor volumes for each group of mice over time (n = 7). (D) The wet weights were measured in dissected tumors after 25 d. The graph shows mean weight ± S.D. for each group (n = 7; P < 0.05, unpaired two-tailed t test). (E) M21 (αv+), M21L (αv−), and five individual M21L-p53-siRNA clones (Lp53siRNA) were cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Apoptosis was detected by Annexin-V staining. The Western blots for p53 and actin protein levels are shown in the vertical display for the cells indicated immediately to the left of each lane. The displayed results are representative among three independent experiments. (F) The protein levels of PUMA, Bax, and Apaf1 were detected by Western blotting in M21L-p53-siRNA clones, M21L (αv−) and M21 (αv+) cells, cultured in 3D-collagen for the indicated times, controlled by p53 and actin levels.
Mentions: We then examined whether the regulation of p53 activity by integrin αv could functionally mediate the role of integrin αv in melanoma cell survival in 3D-collagen and tumor growth in vivo. To this end, we blocked integrin αv-negative M21L cell p53 activity by stably transfecting a dominant negative (dn) p53-His175 cDNA. A number of p53-His175 expressing clones were identified using Western blotting (unpublished data). The M21L-p53-His175 clones, M21L (αv−) and M21 (αv+) cells were cultured in 3D-collagen and analyzed for apoptosis. In contrast to the parental M21L (αv−) cells, all four M21L-p53His175 clones survived to a similar degree as the integrin αv-positive M21 cells (Fig. 4 A, top). To control for the inhibitory effect of p53-His175 in these clones within 3D-collagen, p53 DNA-binding activities were analyzed after 5 d in 3D-collagen using EMSA (Fig. 4 A, bottom). These results indicate that block of p53 function by p53-His175 compensates for the lack of integrin αv in melanoma cell survival. Furthermore, M21 (αv+) and M21L (αv−) cells as well as two of the M21L-p53-His175 clones were injected subcutaneously (s.c.) into the back of C57/BL nude mice, to test if block of p53 function could compensate for lack of integrin αv in melanoma tumor growth in vivo. Consistent with previous studies (Felding-Habermann et al., 1992), M21 (αv+) cells formed large tumors, whereas M21L (αv−) only formed small or no tumors at all (Fig. 4, B–D). Importantly, M21L (αv−) cells overexpressing dn p53-His175 formed tumors to a similar extent and with a similar growth rate as the integrin αv-positive M21 cells (Fig. 4, B–D). This indicates that inhibition of p53 is essential for melanoma tumor growth.

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