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Article |
Correspondence to Staffan Strömblad: staffan.stromblad{at}labmed.ki.se; or Wenjie Bao: wenjie.bao{at}labmed.ki.se
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Abstract |
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Introduction |
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Integrin-mediated cellmatrix interactions trigger a variety of signaling pathways (Giancotti and Ruoslahti, 1999). Signal transduction in cells within 3D-matrices appears to be markedly different from signaling events in cells attached onto two-dimensional (2D) substrates (Cukierman et al., 2002). For example, tyrosine phosphorylation of FAK and EGF-receptor signaling was different in response to cell adhesion within 3D-matrices as compared with attachment to 2D substrates coated onto tissue culture plates (Wang et al., 1998; Cukierman et al., 2001). The integrin-induced MAPK kinase (MEK)-extracellular signal-regulated kinase (ERK) MAPK cascades are key signaling pathways involved in the regulation of adhesion-dependent cell growth and survival (Howe et al., 2002). In melanoma cells, MEK and ERK1/2 can be activated by active mutations of BRAF in 2D cultures (Satyamoorthy et al., 2003). Given that BRAF is mutated in most melanomas, BRAF-dependent MEK activation might be associated with oncogenic behavior of melanoma (Smalley, 2003). However, the role of the RafMEK1ERK1/2 pathway in the regulation of melanoma growth and cell survival is not well characterized. Furthermore, although cell anchorage is needed for activating ERK1/2 in melanocytes (Conner et al., 2003), it is unclear if integrin v may regulate melanoma cell MEK1ERK1/2 activity within 3D environments and if this may play a role for the control of melanoma cell survival.
p53-induced apoptotic cell death plays a central role for suppression of tumor growth (Schmitt et al., 2002). Upon activation by various types of stress stimuli, p53 transcriptionally regulates target genes, including PUMA, Apaf 1, Bax, and Bcl-2, which critically regulate mitochondrial apoptotic cascades (Vousden and Lu, 2002). p53 may also induce apoptosis by directly affecting mitochondria (Mihara et al., 2003). In addition, p53 has been associated with death receptors and activation of caspase-8 (Ashkenazi and Dixit, 1998). In angiogenesis, ligation of integrin vß3 inactivated vascular cell p53, whereas p53 null mice were refractory to an integrin
v-antagonist that blocked angiogenesis in wild-type (wt) mice (Strömblad et al., 1996, 2002). Interestingly, the p53 gene is rarely mutated in melanoma, although p53 is mutated in most human cancers (Geara and Ang, 1996; Jenrette, 1996). Therefore, melanoma cells typically express wt p53 protein and would because of this be expected to be sensitive to DNA-damaging agents. However, most melanoma cells are extremely radio resistant and irradiation of melanoma cells expressing wt p53 leads to accumulation of p53 but not to apoptosis (Satyamoorthy et al., 2000). Similarly, overexpression of wt p53 by adenovirus in melanoma cells did not induce apoptosis (Satyamoorthy et al., 2000). However, it is unclear why melanoma cells harboring wt p53 can still form tumors and survive. Based on our previous finding that integrin
vß3 inhibits endothelial cell p53 activity, and that dermal malignant melanoma express high levels of integrin
vß3, we hypothesized that melanoma cell integrin
vß3 might suppress p53 activity and thereby obviate the need for p53 mutations.
To elucidate molecular mechanisms for how integrin vß3 may promote melanoma cell survival, we used a 3D dermal collagen type I gel model because 3D cell culture systems are capable to provide distinct advantages as compared with 2D systems for studying cellular behavior in a 3D tissuelike context (Zahir and Weaver, 2004). For example, a 3D laminin-rich basement membrane model has been effectively used to examine mammary epithelial cell morphogenesis, polarity, differentiation, proliferation, and survival (Bissell et al., 2002; Zahir and Weaver, 2004). The model with a dermal 3D collagen type I gel that we used mimics the physiological dermal microenvironment where
90% of the protein is collagen type I (Montgomery et al., 1994). We found that M21 and M0-
v melanoma cells depended on integrin
v for survival in 3D-collagen. Furthermore, integrin
v functionally promoted melanoma cell survival by suppression of p53 activity and by activation of a MEK1 signaling pathway. Importantly, our results indicate that p53 functionally acts upstream of MEK1 in an integrin
v-dependent pathway that regulates melanoma cell survival in 3D environments, but that melanoma cells are independent of this pathway under 2D culture conditions. This also emphasizes the importance of studying functional intracellular pathways within a 3D environment that mimics the tissue composition.
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Results |
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Inactivation of p53 compensates for lack of integrin v in melanoma cell survival and tumor growth
We then examined whether the regulation of p53 activity by integrin v could functionally mediate the role of integrin
v in melanoma cell survival in 3D-collagen and tumor growth in vivo. To this end, we blocked integrin
v-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 (
v) and M21 (
v+) cells were cultured in 3D-collagen and analyzed for apoptosis. In contrast to the parental M21L (
v) cells, all four M21L-p53His175 clones survived to a similar degree as the integrin
v-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
v in melanoma cell survival. Furthermore, M21 (
v+) and M21L (
v) 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
v in melanoma tumor growth in vivo. Consistent with previous studies (Felding-Habermann et al., 1992), M21 (
v+) cells formed large tumors, whereas M21L (
v) only formed small or no tumors at all (Fig. 4, BD). Importantly, M21L (
v) cells overexpressing dn p53-His175 formed tumors to a similar extent and with a similar growth rate as the integrin
v-positive M21 cells (Fig. 4, BD). This indicates that inhibition of p53 is essential for melanoma tumor growth.
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Integrin v-dependent MEK1 activity is required for melanoma cell survival in 3D-collagen
Integrin vß3 ligation promotes sustained activation of the MEK1ERK1/2 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
vß3 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
v-positive M21 and integrin
v-negative M21L cells cultured in 3D-collagen. We detected no differences in MEK1 or ERK1/2 activities between M21 (
v+) and M21L (
v) cells under 2D culture conditions (Fig. 5 A, d 0). However, both MEK1 and ERK1/2 activities were markedly reduced in
v-negative M21L cells in contrast to
v-positive M21 cells after 37 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
v 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
v-mediated melanoma cell survival, we blocked MEK1 activity by treatment of integrin
v-positive M21 cells with two specific MEK1 inhibitors, PD98059 and U0126. Both inhibitors strongly induced apoptosis in M21 (
v+) cells within 3D-collagen (Fig. 5, B and C), but with no changes in integrin
vß3 cell surface expression levels (not depicted). This shows that integrin
v-dependent MEK1 signaling is required for melanoma cell survival within a 3D environment.
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Suppression of p53 rescues MEK1 activity in integrin v-negative melanoma cells
Given that MEK1 signaling did not act as a mediator of integrin v-dependent regulation of p53, we instead tested if suppression of p53 might affect MEK1ERK1/2 activity within 3D-collagen. We used the M21L (
v) 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
v-negative M21L cells to similar levels as in integrin
v-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
v 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 (
v) clones that normally survived to the same extent as M21 (
v+) 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
v-mediated melanoma cell survival. However, overexpression of ca MEK1 (S218D/S222D) did not rescue M21L (
v) cell survival in 3D-collagen (unpublished data). Given that block of p53 rescued the same M21L (
v) cells, this suggests that p53 may act not only through MEK1 regulation, but also through additional downstream pathways to induce melanoma cell apoptosis.
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Discussion |
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Acetylations are known to be important modulators of p53 activity (Brooks and Gu, 2003). We found that p53 activation caused by lack of integrin v correlated to p53-K382 acetylation as well as to regulation of p300. Acetylation of p53-K382 by p300 is known to be an important regulatory mechanism for p53 activity (Sakaguchi et al., 1998) and p300 levels are mainly regulated by the rate of proteasomal degradation (Poizat et al., 2000). Given our recent finding that ligation of integrins induces specific proteasomal proteolysis (Bao et al., 2002), it is possible that melanoma cell integrin
vß3 may specifically regulate the proteasomal turnover of p300 and thereby modulate acetylation of p53-K382 and p53 activity. However, it remains to be elucidated if p300-mediated p53-acetylation is functional in integrin
v-mediated regulation of p53 activity.
A notable fact is that most malignant melanoma harbor wt p53, but display strong resistance to chemotherapy and radiation, suggesting that p53 may be dysfunctional (Satyamoorthy et al., 2001). A number of ways have been suggested to result in the dysfunction of wt p53 in melanoma, including defective downstream effectors. For example, Apaf1, an essential downstream component in p53-induced apoptosis, has been suggested to be lost in malignant melanoma leading to melanoma cells escaping from apoptosis and thereby exhibiting strong chemotherapy resistance (Soengas et al., 2001). However, our results displayed no loss of Apaf1 protein in M21 melanoma cells. Moreover, caspase-9 cleavage was still regulated by integrin v, suggesting no defect in Apaf1 activity because Apaf1 is needed for caspase-9 cleavage (Zou et al., 1999). However, in preliminary experiments, we tested the integrin
vß3 cell surface expression in 13 different malignant melanoma cell lines carrying wt p53 and found that eleven of these cell lines expressed high levels of integrin
vß3, whereas four out of five melanoma cell lines tested with mutant p53 had low or no integrin
vß3 expression (unpublished data). This indicates a correlation between the p53 mutational status and integrin
vß3 expression in malignant melanoma cells. Moreover, integrin
vß3 is known to be highly expressed in most dermal malignant melanoma in patients (Albelda et al., 1990; Seftor et al., 1999; Van Belle et al., 1999). Together, with the role for integrin
v in suppressing wt p53 activity presented here, integrin
v-mediated inactivation of wt p53 might help to explain the lack of need for malignant melanoma p53 mutations in vivo.
Concomitant with enhanced p53 activity, PUMA levels were increased upon lack of integrin v, but were reduced when also p53 was suppressed, implicating that melanoma cell PUMA might be a downstream target of p53 (Jeffers et al., 2003). Although the levels of Bax protein were not regulated in response to loss of integrin
v, a transcription-independent regulation of bax by p53 might induce apoptosis (Chipuk et al., 2004). Considering that caspase-9 is activated by loss of integrin
v, and also that caspase-9 is known to be essential for p53-induced apoptosis (Soengas et al., 1999), loss of integrin
v might trigger a mitochondrial apoptotic pathway, which is commonly associated with increased PUMA and active caspase-9 (Wang, 2001). However, unlike the suggested caspase-8dependent apoptotic pathway that may be caused by unligated integrin
vß3 in epithelial cells (Stupack et al., 2001), our results rule out activation of caspase-8 in the induction of melanoma cell apoptosis caused by lack of integrin
v.
Integrin-activated RafMEK1ERK1/2 signaling regulates various cellular functions, including cell survival (Howe et al., 2002) and melanocytes need anchorage to activate ERK1/2 signaling (Conner et al., 2003). However, although most melanoma cells cultured under 2D conditions display active ERK1/2 induced by a ca BRAF V599E mutation (Satyamoorthy et al., 2003), we found that melanoma cell MEK1ERK1/2 signaling depended on integrin v within 3D-collagen. Importantly, this integrin
v-mediated signaling pathway was necessary for melanoma cell survival in 3D-collagen because block of MEK1 activity induced melanoma cell apoptosis, whereas a previous study found no such effect by blocking MEK1 under 2D culture conditions (Smalley and Eisen, 2002). This is another example of how our results display differences in signal transduction and cell survival triggered by cell to 3D matrix interactions as compared with 2D culture conditions. Furthermore, our results are also consistent with an in vivo need for integrin
vß3 for sustained vascular cell ERK1/2 activation during angiogenesis (Eliceiri et al., 1998). Recently, integrin
vß3 was found to trigger FAK as well as PAK1-mediated c-Raf-S338 phosphorylation in angiogenic endothelial cells leading to activation of MEK and ERK (Hood et al., 2003). However, we found neither FAK-Y397 or c-Raf-S338 phosphorylation, nor PAK1 kinase activity to be regulated by integrin
v in melanoma cells in 3D-collagen (unpublished data). Because we did not observe any integrin
v-dependent regulation of FAK activity within 3D-collagen, our results do not favor the possibility that integrin-activated FAK may suppress p53 in the regulation of apoptosis (Ilic et al., 1998), although we used a different cell type and a different model compared with previous studies. However, surprisingly, we found that inactivation of p53 functions upstream of MEK1 in regulating integrin
v-mediated melanoma cell survival. p53 can transcriptionally activate MAPK phosphatases, including PAC1, which may play a role in p53-dependent apoptosis by dephosphorylating and inactivating ERK (Yin et al., 2003). However, we found no regulation of PAC1 levels by p53 within our system (unpublished data).
Several ECM proteins can be recognized by v-integrins, including vitronectin, denatured collagen type I, and osteopontin (Strömblad and Cheresh, 1996). Although integrin
2ß1 is a major receptor for collagen type I, M21 melanoma cells are capable of degrading native collagen type I to expose cryptic RGD sites for integrin
vß3 ligation, suggesting that degraded collagen type I could be a functional ligand for melanoma cell integrin
v-mediated cell survival (Montgomery et al., 1994). Alternatively, given that osteopontin may be involved in melanoma progression from the radial growth phase to the vertical growth phase (Sturm et al., 2002), and that overexpression of osteopontin may promote melanocyte survival in 3D-collagen (Geissinger et al., 2002), endogenously produced osteopontin is another potential ligand for integrin
v within 3D-collagen. It should be noted that although the dermis contains
90% collagen type I, it also contains other types of collagen as well as fibronectin and additional ECM components. Furthermore, other cell types are present in the dermis in vivo and the collagen density and assembly in vivo might also differ from the used in vitro 3D-collagen model. Therefore, the used in vitro model is not an exact replica of the dermis environment. However, importantly, our results obtained in the 3D collagen model were consistent with our results obtained in vivo in mouse skin, indicating that the in vitro 3D-collagen model is valid for studies of molecular pathways involved in integrin
v regulation of melanoma cell survival.
In conclusion, we have identified an integrin v-dependent melanoma cell survival pathway within 3D dermal collagen. We found that integrin
v caused inactivation of wt p53, which functionally promoted melanoma cell survival and tumor growth in vivo. Simultaneously, integrin
v-mediated activation of MEK1 signaling was also required for melanoma cell survival. Surprisingly, we found that whereas p53 acts downstream of integrin
v, it also regulates MEK1 activity in the same functional melanoma cell survival pathway. Although the melanoma cells depended on this integrin
v-mediated pathway for survival in 3D environments, they were independent of this pathway under 2D culture conditions, stressing the importance of studying the role of cell to matrix interactions and their functional signaling pathways within 3D contexts.
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Materials and methods |
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Flow cytometric determination of integrin expression and apoptosis
1 x 106 cells were incubated with 1 µg/µl of anti-vß3 mAb LM609 (CHEMICON International, Inc.), 5 µg/µl of anti-ß3 mAb AP3 (GTI) or 10 µg/µl anti-
2ß1 mAb 12F1 (CHEMICON International, Inc.) for 1 h at RT. After washing, cells were incubated with FITC-conjugated goat antimouse IgG (1:50; Jackson ImmunoResearch Laboratories) for 30 min and analyzed by flow cytometry with CellQuest Software (Becton Dickinson). The fraction of apoptotic cells was detected by flow cytometry after staining with Annexin-V FITC as described by the manufacturer (Biosource International), or by TUNEL using an in situ Cell Death Detection kit (Boehringer-Mannheim).
Stable transfections
Expression vectors with human p53 dn mutant p53-His175 (pCMV-Neo-BAM-p53-His175) and p53-siRNA (pSUPER-p53) were gifts from K. Wiman (Karolinska Institutet) and R. Agami (The Netherlands Cancer Institute, Amsterdam, Netherlands), respectively. M21L (v) cells were transfected with 10 µg of p53-His175 cDNA or cotransfected with 10 µg of pSUPER-p53 and 1 µg of pCI-neo plasmid using Lipofectamine 2000 (Invitrogen). 72 h after transfections, cells were treated with 600 µg/ml of G-418 (Invitrogen). After 14 d, G418-resistant clones were picked, expanded and eventually selected based on p53 protein levels and p53 DNA binding activities. ca MEK1 S218D/S222D cDNA, a gift from P. Gerwins (Uppsala University, Uppsala, Sweden), was stably introduced into M21L (
v) cells by the same method. Positive clones were selected according to induced levels of phosphorylated ERK1/2.
EMSA
Nuclear extracts were prepared and subjected to EMSA as described previously (Selivanova et al., 1996). The protein concentrations of the crude nuclear extracts were determined using a BCA protein quantification kit (Pierce Chemical Co.) using BSA as a standard. 32P-endlabeled double stranded oligonucleotides containing the following human p53 recognition sequence was used: (5'-CAGGCATGTCTGCAGGCAAAGGCATGTCTG-3'). For specific or nonspecific competition, the corresponding unlabeled oligonucleotides or random oligonucleotides were added together in reactions. For supershift, anti-p53 mAb Pab421 (Oncogene) was used and performed as described previously (Selivanova et al., 1996). The analysis was finalized by autoradiography or by a phosphor-imager (Packard).
Western blotting
Cell lysates, protein determination, and Western blotting were performed as described previously (Bao and Strömblad, 2002). The following primary antibodies were used: anti-p53 mAb (DO1), anti-bax pab (N-20), anti-MEK1 pab (C-18), and anti-PAC1 pab (N-19) were purchased from Santa Cruz Biotechnology, Inc.; anti-p53 mAb (Pab421), anti-p53 pab (Ab-7), anti-acetylated p53 Lys382 pab (Ab-1), anti-p300 pab (N-15), anti-PUMA pab (Ab-1), and anticaspase-8 mAb (clone 13) were purchased from Oncogene Research Products; antiphospho-MEK1/2 pab (Ser 217/221), antiphospho-ERK1ERK2 pab (Thr202/Thr204), antiphospho-p53 pab (Ser 15), antiphospho-p53 pab (Ser 20), and anticaspase-9 pab were purchased from New England Biolabs, Inc.; anti-Apaf1 mAb (2E12) was purchased from Alexis; anti-actin mAb (JLA 20) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa.
In vivo tumor growth
Animal experiments were performed according to a permit from the Stockholm South ethical committee in Sweden. C57BL6 black nude female mice (68 w) were obtained from M&B. Melanoma cells (1 x 106) were injected s.c. at the back of the mice. The tumor size was measured every other day with calipers and tumor volumes were calculated by the formula 0.52 x length x width2. The wet weight of dissected tumors was determined at the end of experiments.
Statistical analyses
The following statistical analyses were performed for the results represented in Fig. 3. Although the integrin v-negative cells constantly displayed higher p53 DNA-binding activity as compared with the integrin
v-positive cells after exposure in 3D-collagen, the absolute values (magnitude) and the time of onset of p53 activity varied between experiments. Therefore, we calculated the ratio of p53 activity between
v-negative cells and the corresponding
v-positive cells at each time point (Fig. 3, B and D, closed bars) and performed a statistical analysis of these ratios compared with the corresponding ratios at the start of the experiments (d 0) by use of unpaired two-tailed t test. Likewise, the ratios between results obtained from integrin
v-negative and integrin
v-positive cells were used for the statistical analyses of Western blot results represented in Fig. 3 (HM) in the same manner. Statistical evaluations of experiments shown in other figures were performed as described in the figure legends.
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Acknowledgments |
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This work was supported by grants to SS from the Swedish Cancer Society, the Swedish Research Council, the Swedish Medical Association and the Cancer Society of Stockholm and to WB from the Robert Lundberg Memorial Fund. SS holds a Senior Scientist position from the Swedish Research Council.
Submitted: 4 April 2004
Accepted: 14 September 2004
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