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Research Articles: Targets
Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases
1 The Wistar Institute, and 2 Abramson Cancer Center of the University of Pennsylvania, Philadelphia, Pennsylvania
Requests for reprints: Keiran S.M. Smalley, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3951. E-mail: ksmalley{at}wistar.org
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Introduction |
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Although much of recent melanoma therapy research has focused on the BRAF/MEK/ERK pathway, a number of other pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt, nuclear factor 
B, Janus-activated kinase/signal transducers and activators of transcription, and ß-catenin are also known to be active in melanoma (7). Of these, the PI3K/Akt pathway plays a critical role in the oncogenic behavior of melanoma through its ability to suppress apoptosis (8) and control cell cycle entry via the regulation of both cyclin D1 and myc (9, 10). In the present study, we have investigated whether targeting either the MEK/ERK pathway or the PI3K/Akt pathway is a viable approach to melanoma therapy. As preclinical studies in vitro are often poorly predictive of the outcome of clinical studies, we have developed a novel cell culture model wherein human tumor cells are grown as three-dimensional spheroids and then implanted into collagen gels to mimic the tumor architecture and microenvironment. Most other preclinical cell culture models fail to account for the fact that tumor cells exist embedded within a three-dimensional stromal matrix that includes other cell types. The demonstration that anti-ß1-integrin antibodies reverse the malignant phenotype of breast cancer cells in three-dimensional, but not two-dimensional, culture is a pertinent example of marked differences seen under the different experimental conditions (11). In the current study, we have developed a novel model of melanoma whereby melanoma cells are grown under nonadherent conditions, which permits the formation of three-dimensional aggregations or spheroids. Once formed, the spheroids were harvested and implanted into a matrix of collagen I, which mimics the microenvironment of human skin. Using this three-dimensional spheroid model, it was found that cell lines derived from melanoma metastases were highly resistant to both PI3K and MEK inhibitors. This was in marked contrast to the responses seen to the same cell lines grown under standard two-dimensional cell culture conditions. Further studies revealed that only the combination of PI3K and MEK inhibitors had any antitumor activity in three-dimensional culture and led us to conclude that targeting only one signaling pathway, such as MEK, may not be a viable strategy for treating the most aggressive of melanomas.
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Materials and Methods |
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Adherent Cell Proliferation Analysis
Cells were plated into a 96-well plate at a density of 2.5 x 104/mL and left to grow overnight. Cells were treated with increasing concentrations of LY294002 (0.01–30 µmol/L; Calbiochem, Darmstadt, Germany) or U0126 (0.01–30 µmol/L; Calbiochem) in triplicate. In each instance, cells were left to grow for 72 hours before being treated with 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 3 hours (Sigma, St. Louis, MO). After this time, the medium was rapidly removed and the MTT crystals were solubilized using DMSO. The resulting absorbance was read in a plate reader at 560 nm. Absorbance readings were subtracted from the value of blank wells; the reduction in cell growth was calculated as a percentage of control absorbance in the absence of any drug. Data show the mean of at least three independent experiments ±SE.
Western Blot Analysis
Proteins were extracted and blotted as described in ref. 12. After analysis, Western blots were stripped once and reprobed for ß-actin to show even protein loading. Antibodies to phospho-glycogen synthase kinase 3ß (GSK-3ß), cyclin D, and phospho-ERK were from Cell Signaling Technology (Beverly, MA). The antibody for p27Kip-1 was from BD PharMingen (San Jose, CA) and the monoclonal antibody to ß-actin was from Sigma.
Three-Dimensional Spheroid Growth
Melanoma spheroids were prepared using the liquid overlay method. Briefly, 200 µL of melanoma cells (25,000/mL) were added to a 96-well plate coated with 1.5% agar (Difco, Sparks, MD). Plates were left to incubate for 72 hours, by which time cells had organized into three-dimensional spheroids. Spheroids were then harvested using a P1000 pipette. The medium was removed and the spheroids were implanted into a gel of bovine collagen I containing EMEM, L-glutamine, and 2% fetal bovine serum. Normal 2% melanoma medium was overlaid on top of the solidified collagen. Spheroids were treated with either LY294002 (1–50 µmol/L), U0126 (1–50 µmol/L), or LY294002 and U0126 in combination before being left to grow for 72 hours. Spheroids were then washed twice in PBS before being treated with calcein-AM and ethidium bromide (Molecular Probes, Eugene, OR) for 1 hour at 37°C according to the instruction of the manufacturer. After this time, pictures of the invading spheroids were taken using a Nikon-300 inverted fluorescence microscope.
Immunofluorescence Microscopy
Melanoma cells were seeded onto glass coverslips in six-well plates and incubated overnight. Cells were then fixed in 4% formaldehyde solution (Electron Microscopy Systems, Hatfield, PA) and permeabilized with Triton X-100 (0.2% v/v) before being blocked in PBS containing 1% bovine serum albumin. Primary antibody incubations (1:200) were done at 37°C in a humidified atmosphere for 1 hour. Coverslips were then washed thrice in PBS before being incubated with secondary antibodies for 1 hour under similar conditions to the primary antibody (dilution factor, 1:250). The monoclonal antibody to the p85
PI3K subunit was from BD PharMingen and the antimouse Texas red–conjugated secondary antibody was from Vector Laboratories (Burlingame, CA). Coverslips were then further washed in PBS and sterile water before being treated with VectorShield (Vector Laboratories) antifade and analyzed using either immunofluorescence or confocal microscopy.
Cell Cycle Analysis
Cell cycle analysis was done after treatment with kinase inhibitors for 24 hours (U0126, 10 µmol/L; LY294002, 20 µmol/L) and after incubation with kinase inhibitors for 24 hours followed by a further 24, 48, or 72 hours without inhibitors. Cells (1 x 106–2 x 106) grown adherently on a culture dish were harvested, washed in cold PBS, and resuspended in 200 µL cold PBS. Cells were fixed by adding 200 µL of above-mentioned cell solution to 4 mL of 70% ethanol and incubated on ice for at least 1 hour (typically overnight). Intracellular DNA was labeled with 200 µL propidium iodide solution containing 40 µg/mL propidium iodide and 100 µg/mL RNase in PBS and incubated at 37°C for 30 minutes in the darkness. Samples were analyzed using EPICS XL (Beckman-Coulter, Inc., Miami, FL). The cell cycle profile was obtained by analyzing 15,000 cells.
Analysis
Unless otherwise stated, all data show the mean of at least three independent experiments and, where appropriate, ±SE.
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Results |
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Inhibition of MEK Blocks the Growth of Aggressive Melanoma Cells in Two-Dimensional but not Three-Dimensional Culture
One of the best characterized signaling pathways in melanoma is the MAPK pathway (1), which is activated by BRAF V600E mutations as well as autocrine growth factor loops (1). With the exception of the C8161 cell line (14), all of the melanoma cell lines used in this study harbored the V600E BRAF mutation. Increasing concentrations of the MAPK inhibitor U0126 reduced the growth of the four melanoma cell lines in monolayer culture (Fig. 1A
). When grown adherently, the radial growth phase melanoma WM35 was especially susceptible to the growth inhibitory effects of U0126 whereas WM793, 1205Lu, and C8161 were less sensitive but were still inhibited with a similar potency (Fig. 1A). However, phospho-ERK levels were reduced in 1205Lu cells at concentrations much lower than those needed to block growth in adherent culture (Fig. 1B). U0126 reduced cell growth and viability only in the radial growth phase and vertical growth phase primary melanoma cell lines in three-dimensional spheroid culture (Fig. 1C). In this instance, the effects were very striking with virtually no viable cells remaining after 72 hours of treatment (Fig. 1C). In contrast, U0126 had no effect on the growth and invasion of three of four (1205Lu, C8161, and 451Lu) melanoma lines derived from metastases (Fig. 1C). Treatment of the WM164 cell line, which was also derived from a metastasis, resulted in reduced growth and survival (data not shown). In all cases, similar results were seen with another MEK inhibitor, PD 98059 (data not shown).
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40% as shown by MTT assays. Simultaneous treatment with both inhibitors reduced growth by 60%. This growth inhibition was readily reversible, as cells incubated without inhibitors for a further 72 hours showed the same extent of growth as untreated cells (Fig. 3A
). Under adherent culture conditions, the MEK inhibitor U0126 blocked growth through the up-regulation of the cyclin-dependent kinase (CDK) inhibitor p27Kip-1 (Fig. 3B) whereas the PI3K inhibitor LY294002 did not induce an up-regulation of p27. Cell cycle analysis revealed that this reduction in growth was associated with a G1-phase cell cycle arrest but not apoptosis (Fig. 3C and D). The MEK inhibition–induced cell cycle arrest was readily reversible with the melanoma cells reentering the cell cycle 24 hours after removal of the drug (Fig. 3E and F).
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The role of the collagen gel in modulating drug resistance was investigated by treating the spheroids on top of agar with the PI3K and MAPK inhibitors. In this instance, both LY294002 and U0126 reduced cell viability. The combination of the two drugs was synergistic and completely blocked cell survival (Fig. 5C). In the absence of the collagen matrix, the spheroids were no longer resistant to the signal transduction inhibitors, suggesting that critical survival signals involved in drug resistance are received from the stromal microenvironment (15).
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Discussion |
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The role of constitutive MEK activity in melanoma is more well defined and includes increased cell proliferation, enhanced matrix metalloprotease secretion, and invasion (reviewed in ref. 1). A recent study has claimed that the presence of BRAF mutations in melanoma confers selectivity to MEK inhibitors and that MEK inhibition offers a rational therapeutic approach to melanomas with BRAF mutations (3). However, there is little evidence to suggest that MEK inhibition induces apoptosis in tumor cells and it seems more likely that these drugs would instead only induce cell cycle arrest. The current study has investigated whether targeting MEK is a viable strategy for treating melanoma cell lines derived from radial growth phase, vertical growth phase, and metastatic lesions. With the exception of the C8161 cell line, all of the melanoma cell lines tested harbor the V600E mutation in BRAF.
In agreement with other work, it was noted that increasing concentrations of the MEK inhibitor U0126 reduced the growth of all of the melanoma cell lines tested under normal adherent culture conditions (3). Cell cycle analysis revealed that this reduction in growth was associated with loss of cells in S phase and up-regulation of the CDK inhibitor p27Kip-1 and G1-phase cell cycle arrest. Cell proliferation is regulated during the first pause in the cell cycle at the G1 checkpoint. Under normal conditions, cell growth is tightly controlled by the growth-promoting cyclins and CDKs of which the activity is opposed by the CDK inhibitors. The progression through the G1-phase checkpoint is driven by CDK4, CDK6, and their interaction with the cyclin D family of proteins (20). Activation of the MAPK pathway via ERK-induced regulation of cyclin D1 expression is a key regulator of the G1 transition (21). The ability of U0126 to arrest melanoma cells in the G1 phase of the cell cycle is consistent with a requirement for constitutive MAPK activity in the progression of melanoma cells through this checkpoint. The effects of U0126 on G1 cell cycle arrest were readily reversible with the melanoma cells reentering the cell cycle 24 hours after removal of the drug. Consistent with these results, it was also noted that U0126 did not induce apoptosis in any of the melanoma cells grown in two-dimensional culture, as assessed by propidium iodide staining and the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay (data not shown).
Interestingly, there was no correlation between the concentrations of U0126 required to block phospho-ERK activity and to inhibit cell growth. Indeed, for the 1205Lu cell line, the concentrations of U0126 required to inhibit cell growth were nearly 100-fold higher than those required to block kinase activity. In other studies, it was shown that sensitivity to MEK inhibition was correlated with BRAF mutational status (3) and that cell lines in which MEK inhibition and growth arrest were disconnected were wild-type for both BRAF and N-Ras (3). However, in our study, there was only a close correlation between MEK inhibition and attenuation of cell growth in the early-stage WM35 cells, which harbored the V600E BRAF mutation. Therefore, in our cell lines, it seemed unlikely that the correlation between the U0126 concentrations required for MEK inhibition and those for growth inhibition was due to BRAF mutational status.
When the same panel of melanoma cell lines were grown under three-dimensional collagen-implanted spheroid culture conditions, the results were strikingly different. It was found that U0126 treatment completely inhibited the growth and survival of the cells derived from earlier melanoma lesions (radial growth phase and vertical growth phase) but had little effect on any of the metastatic lesions. It seems that under three-dimensional culture conditions, the metastatic melanoma lines were very resistant to MEK inhibition. Further experiments using U0126 concentrations up to 50 µmol/L also showed a lack of inhibitory effects on cell growth or invasion. These results were in marked contrast to those from the recent study of Solit et al. (3), which showed that melanoma cells with BRAF mutations were susceptible to MEK inhibition. One possible explanation for this discrepancy is the choice of melanoma lines. In our study, three of four of the most aggressive melanoma lines (1205Lu, 451Lu, and C8161) were resistant to MEK inhibition whereas one (WM164) was not, suggesting that not all metastatic melanoma lines are equally resistant. Of these lines, two of the three resistant lines were BRAF mutant (1205Lu and 451Lu) and one was BRAF wild-type (C8161). As even the nonresistant line (WM164) harbored the V600E BRAF mutation, this seemed unlikely to provide the explanation for the difference in sensitivity observed. It is also pertinent to note that the resistance was only seen under three-dimensional culture conditions and was not predicted by any of the assays done using two-dimensional cell culture techniques.
To ascertain whether this acquired resistance seen under three-dimensional culture conditions was specific to the MEK/ERK pathway, we did similar experiments targeting the PI3K/Akt pathway using LY294002. In these studies, LY294002 blocked the growth of all four of the melanoma cell lines with a similar potency in two-dimensional culture. Again, like U0126, the metastatic melanoma lines (three of four tested) were completely resistant to the inhibitor when grown in three-dimensional spheroid culture. To investigate the mechanism of this acquired resistance in the three-dimensional culture, the 1205Lu melanoma cell lines were infected with a dominant-negative form of the p85 subunit of PI3K and grown as spheroids. In this instance, we found that the melanoma cells were less invasive but still viable after 72 hours of growth, showing that the melanoma cells were able to survive in three-dimensional culture in the absence of PI3K activity. Similar results were also seen when the 1205Lu spheroids were treated with higher concentrations of LY294002 (50 µmol/L; data not shown). This suggested that resistance to LY294002 in three-dimensional culture was not through altered drug transport mechanisms. The underlying mechanisms of drug resistance of the metastatic cell lines are likely due to high activity in multiple prosurvival signaling pathways. The data presented in this study have shown that blocking one signaling pathway may not be a viable therapeutic strategy for the most aggressive melanomas and that multiple pathways may have to be targeted simultaneously. To further investigate this idea, we treated the aggressive melanoma lines with both LY294002 and U0126 simultaneously and found an additive effect in two-dimensional adherent culture. When the same drug treatment was applied to the aggressive melanoma lines, there was some synergism; the spheroids were smaller in size and were less invasive. However, some viable cells still remained, suggesting that this approach may not be curative.
It is possible that the observed resistance seen with the aggressive melanoma lines in three-dimensional culture is a result of the collagen matrix sequestering the drug or being somehow less permeable. We would argue that this is unlikely to be the case. The fact that spheroids derived from the radial growth phase and vertical growth phase lesions, which are the same size as those from the metastatic lines, are exquisitely sensitive to these inhibitors suggests that there are no issues with either permeability or drug absorbance onto the matrix. In addition, previous studies using fluorescent drug conjugates, such as BODIPY-taxol, have shown the rapid (<360 minutes) transport of drugs to the core of the spheroid (22).
In the final part of the study, we looked at the effects of modulating the microenvironment on melanoma sensitivity to MEK and PI3K inhibition and whether this played a role in the observed resistance. Removing the serum from the medium and the surrounding collagen completely reversed the resistance of 1205Lu spheroids to U0126 but not to LY294002. It therefore seemed that other extrinsic serum-derived survival signals were responsible for mediating resistance to MEK but not to PI3K/Akt inhibition and suggested that activity in the MEK/ERK pathway is more critical than that in PI3K/Akt for melanoma survival. The fact that serum removal reversed the resistance of 1205Lu cells to U0126 further adds to the argument that the drug is able to reach the spheroid.
We further showed a role for the collagen mediating resistance to both U0126 and LY294002. When spheroids on top of agar were treated with either of the inhibitors, the cells underwent substantial cell death, which was not seen when the spheroids were implanted into the collagen. It is likely that integrin-collagen engagement activates outside-in signaling, which is critical for cell survival. Recent work has shown that engagement of v integrin with a three-dimensional collagen matrix increases melanoma survival via suppression of p53 activity (23). The observation that modulating the microenvironment alters drug sensitivity suggests that a better understanding of the tumor-stromal interaction will allow the development of strategies to overcome drug resistance in melanoma.
Although the preliminary in vitro data look promising (5, 24), it is unclear whether BRAF or MAPK inhibition will translate into the clinical treatment of melanoma. This contention is supported by a limited number of clinical studies on the MEK inhibitor CI-1040, which has shown pharmacologic activity at the level of phospho-ERK inhibition but little clinical benefit above stable disease (25, 26). It seems that the responses to MEK inhibition in vivo are more complex than those seen under normal tissue culture conditions and that the microenvironment may modulate drug resistance in unforeseen ways. Clearly, these results have important implications for the future therapeutic use of MEK inhibitors and suggest that a greater knowledge of signaling within the tumor microenvironment is required to overcome therapeutic resistance.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: K.S.M. Smalley and N.K. Haass contributed equally to this work.
3 Supplementary materials for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). 
Received 2/14/06; accepted 3/13/06.
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v-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen. J Cell Biol 2004;167:745–56.This article has been cited by other articles:
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