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Originally published as JCO Early Release 10.1200/JCO.2004.11.070 on May 3 2004 © 2004 American Society of Clinical Oncology. Exposure of Melanoma Cells to Dacarbazine Results in Enhanced Tumor Growth and Metastasis In VivoFrom the Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Menashe Bar-Eli, PhD, Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Unit 0173, 7777 Knight Rd, Houston, TX 77054; e-mail: mbareli{at}mdanderson.org
PURPOSE: In recent years, the incidence of cutaneous melanoma has increased more than that of any other cancer. Dacarbazine is considered the gold standard for treatment, having a response rate of 15% to 20%, but most responses are not sustained. Previously, we have shown that short exposure of primary cutaneous melanoma cells to dacarbazine resulted in the upregulation of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The purpose of the present study was to determine how long-term exposure of melanoma cells to dacarbazine would affect their tumorigenic and metastatic potential in vivo. MATERIALS AND METHODS: The primary cutaneous melanoma cell lines SB2 and MeWo were repeatedly exposed in vitro to increasing concentrations of dacarbazine, and dacarbazine-resistant cell lines SB2-D and MeWo-D were selected and examined for their ability to grow and metastasize in nude mice. RESULTS: The dacarbazine-resistant cell lines SB2-D and MeWo-D exhibited increased tumor growth and metastatic behavior in vivo. This increase could be explained by the activation of RAF, MEK, and ERK, which led to the upregulation of IL-8 and VEGF. More IL-8, VEGF, matrix metalloproteinase-2 (MMP-2), and microvessel density (CD-31) were found in tumors produced by SB2-D and MeWo-D in vivo than in those produced by their parental counterparts. No mutations were observed in BRAF. CONCLUSION: Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.
While the incidence of many solid tumors is gradually decreasing, the incidence of cutaneous melanoma is still on the rise, making this malignancy a significant clinical problem. Overall, melanoma accounts for 1% to 3% of all malignant tumors and is increasing in incidence by 6% to 7% each year.1,2 In the early phase of its natural history, when diagnosed as a thin lesion, cure with surgical resection is possible in a high percentage of cases, with a 5-year survival rate of more than 80%. Once the metastatic phase develops, it is almost always fatal, with an estimated median survival range of 6 to 9 months and a 5-year survival rate of less than 5%.3-5 The standard treatment for patients with metastatic melanoma has not been defined. Various therapeutic approaches have been evaluated, including chemotherapy and biologic therapies, both as single treatments and in combination.3,6-8 To date, however, none of these treatment options has shown a significant survival effect. Most patients with advanced-stage melanoma will receive systemic chemotherapy, which is still considered the mainstay of treatment for stage IV melanoma, and is employed largely with palliative intent.6,8 Numerous chemotherapeutic agents have shown some activity in the treatment of malignant melanoma, with dacarbazine being the most widely used. Dacarbazine is a nonclassical alkylating agent, generally considered the most active agent for treating malignant melanoma, and has been approved by the US Food and Drug Administration.8,9 It exerts its antitumor activities by methylation of nucleic acids or direct DNA damage, and results in arrest of cell growth or cell death. However, response rates for single-agent dacarbazine are disappointing, ranging from 10% to 25%, with complete responses seen in less than 5% of patients. In addition, the response duration is often as brief as 5 to 6 months.10 Moreover, it is unclear whether combination therapies are superior to single-agent dacarbazine.10,11 The second clinical setup in which chemotherapy has been suggested for the treatment of melanoma is the adjuvant setting for high-risk patients.12,13 Patients presenting with thick primary melanomas or those with regional nodal metastases have a high risk of recurrence after surgery. However, as in metastatic disease, chemotherapy has only a minimal effect in these patients. Melanoma is known for its notorious resistance to chemotherapy, a major obstacle to successful treatment. The basis for drug resistance in melanoma is not well understood, and various mechanisms have been postulated, including dysregulation of apoptotic pathways, defects in drug transport, detoxification, and enhanced DNA repair. Defects at multiple levels and in both major apoptotic pathways have been previously described in melanoma cases.14-19 Previously, we have shown that a single exposure of nonaggressive melanoma cells in culture to dacarbazine can result in overexpression of angiogenic factors such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), leading to increased resistance to further chemotherapy.20 In the present study, we show that repeated treatment of melanoma cells with dacarbazine can result in the selection of more chemoresistant cell lines, with enhanced tumorigenic and metastatic potential in vivo. These results have significant clinical implications. It is possible that the implementation of dacarbazine for the treatment of melanoma is not only ineffective, but can be hazardous and counterproductive if not combined with other modalities that mitigate the promalignant, prometastatic effects induced by the drug.
Cell Lines Two human melanoma cell lines, MeWo and SB2, were used for this study, and they were previously described at length.21,22
Establishment of Dacarbazine-Resistant Melanoma Cell Lines
Animals
In Vivo Tumor Growth and Metastasis Assays
Immunohistochemistry and Quantitation of Microvessel Density Sections of paraffin-embedded tumors were used to identify proliferating cell nuclear antigen (PCNA) -positive cells (ie, proliferating cells), IL-8, VEGF, and MMP-2. For all these reactions, the average measurements of the intensity of the staining quantitated in 10 areas of each sample were calculated with an image analyzer and the Optimas Image Analysis software (Bioscan, Edmonds, WA).
Cell Proliferation Assay
Enzyme-Linked Immunosorbent Assays (ELISA)
Western Blotting
Quantitative Real-Time Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction and Nucleotide Sequencing for N-, H-, and K-RAS, and BRAF
Statistical Analysis
Establishment of Dacarbazine-Resistant Melanoma Cell Lines Two melanoma cell lines were selected for this study: SB2, a low tumorigenic, nonmetastatic, and MeWo, which does produce tumors in immunocompromised mice and a low rate of metastasis when injected intravenously. We used these relatively unaggressive cell lines to evaluate their growth and metastatic potential after exposure to the drug. To investigate the effect of chronic (prolonged) exposure of melanoma cells to dacarbazine, we repetitively treated the cells in culture with increasing doses of the drug. In this manner, we sought to simulate the clinical setup in which chemotherapy is usually implemented in cycles, leaving the patient, but also the tumor, to resuscitate from the effects of the drug until the next cycle. After a total of three cycles of treatment with 500 µg/mL and one final treatment with 1,000 µg/mL, two new cell lines (SB2-D and MeWo-D) were established and were used for further experiments. First, we investigated the growth rate of the selected cell lines in vitro. No difference in the doubling time compared with that of the untreated parental cells was observed in repeated assays (Table 1). However, the selection did result in cells that had a higher resistance to the toxic effect of the chemotherapy IC50 of 750 µg/mL and 890 µg/mL, versus 220 µg/mL and 380 µg/mL in SB2-D and MeWo-D compared with the parent cells, respectively (Table 1).
Enhanced Tumorigenicity and Metastatic Potential of Dacarbazine-Resistant Cells In Vivo The tumorigenicity was evaluated by comparing the local growth rate of tumors in mice injected subcutaneously with the selected cell lines in comparison with the parent cells. The metastatic potential was evaluated after intravenous injections. Table 2 summarizes the tumorigenic and metastatic capabilities of the selected cell lines. There was a trend toward a higher tumor incidence for the selected cell line SB2-D than for SB2, though it did not achieve statistical significance (P = .0567). However, tumor weight was significantly higher in the SB2-D tumors (608.6 v 13.67 mg; P < .0123). Metastasis development was a rare event for the SB2 parental counterpart (one of 10 mice) for both macroscopic and microscopic tumors. In contrast, metastases developed in all the mice in the SB2-D group: lung metastases in all cases, deposits in the heart in five cases, and subcutaneous metastases in four cases. Similar results were found when we compared the growth of the MeWo-D cell line to its parental counterpart. Again, no major difference was found in tumor incidence, with 60% of the mice injected with the MeWo cell line developing local tumors. However, the tumors produced by the MeWo-D cells were twice the weight of the tumors produced by the parental MeWo cells. The rate of metastasis was also significantly higher in the MeWo-D group, in which they developed in the lungs of all the mice, in gastrointestinal tracts of six mice, in the livers of three mice, and in the adrenal glands of two mice. Figure 1A shows the typical macroscopic and microscopic appearance of the lung metastasis developing after intravenous injection of MeWo cells, with small nodules surrounded by the normal lung parenchyma. The number of lung colonies and the metastatic burden was much higher in the mice bearing tumors of the MeWo-D cell line, with tumor deposits replacing much of the normal lung (Fig 1B). Figure 1C shows an example of MeWo-D liver metastasis. No liver metastases were observed in the control group injected with the parental MeWo cells.
The Dacarbazine-Resistant Cell Lines Express Significantly Higher Levels of Angiogenic Factors Two angiogenic cytokines, IL-8 and VEGF, are thought to be important for melanoma progression. Based on our previous observation that a short single exposure of melanoma cells to dacarbazine resulted in the upregulation of IL-8 and VEGF mRNA and protein,20 we investigated the expression of these cytokines in the dacarbazine-selected cell lines. IL-8 protein levels were found to be 10 and 6.8 times higher in the SB2-D and MeWo-D cell lines, respectively (Fig 2A), than in their parental cells. Similarly, 3.7- and 5.6-fold increases in VEGF protein levels were observed in the dacarbazine-resistant cells (Fig 2B). Using quantitative real-time PCR, we found that the elevated protein levels could be explained by mRNA overexpression. IL-8 mRNA levels were 35.5- and 15.5-fold higher in the SB2-D and MeWo-D cell lines, respectively, than in their parental counterparts (Figs 2C and 2D). VEGF mRNA levels were elevated by 2.7- and six-fold (Fig 2C and D). This overexpression was seen long after the drug was withdrawn and after many cycles of freezing and thawing of the cells in culture.
The Activity of the RAF-MEK-ERK Pathway Is Constitutively Upregulated in Dacarbazine-Resistant Cell Lines Recently, the ERK pathway was found to play a major role in the progression and metastatic phenotype of cutaneous melanoma.27 Because the dacarbazine-resistant cell lines present a more aggressive phenotype than their parental counterparts, we looked for the expression of pERK in these cells, since the upregulation of this pathway could partially be responsible for the observed overexpression of IL-8 and VEGF. Furthermore, previously we have shown that pERK was activated after a short exposure to dacarbazine.20 Using immunobloting (Fig 3), we found significantly higher expression of pERK in both SB2-D and MeWo-D than in SB2 and MeWo (by 58- and 78-fold, respectively, as determined by densitometry). This overexpression can be explained by the overexpression of both pMEK (98- and 30-fold higher) and its upstream activator pRAF (5.6- and 5.5-fold higher) in both dacarbazine-resistant cell lines, as shown in Figure 3. No major difference in the expression of pAKT was noticed in the studied cell lines (data not shown).
Inhibition of pMEK in SB2-D and MeWo-D Enhances Their Sensitivity to Dacarbazine To study whether the induction of activated ERK in these cell lines could explain their increased resistance to dacarbazine in vitro, we exposed the cells to dacarbazine (100 µg/mL) with and without UO126 (a specific pMEK inhibitor) for 48 hours, and examined their proliferation rate using an MTT assay (Fig 4) and found that the effect of dacarbazine at this concentration was only minor in both SB2-D and MeWo-D cells. Exposing the cells to UO126 alone resulted in a minor reduction in cell proliferation. In contrast, combination treatment of dacarbazine plus UO126 caused the most significant effect, suggesting that the inhibition of ERK activation renders these cells more responsive to dacarbazine.
Exposure to Dacarbazine Did Not Lead to Acquired BRAF and RAS Mutations According to previous reports, one of the suggested mechanisms for the increased phosphorylation of ERK in melanoma is activating mutations in the BRAF or RAS oncogenes.28-31 To evaluate whether exposure to dacarbzine could lead to acquired mutations in these genes, genomic DNA from SB2, SB2-D, MeWo, and MeWo-D cells was analyzed for mutations in exons 11 and 15 of the BRAF and exons 1 and 2 of the H-, K-, and N-RAS genes. PCR amplification followed by nucleotide sequencing revealed that the SB2 and SB2-D cells both had an activating mutation in both alleles of N-RAS at codon 61 (CAAAAA, GlnLys; Fig 5), and polymorphism in H-RAS at codon 27 (CATCAC, HisHis). None of the cell lines had mutations in BRAF exons 11 or 15. No mutations at codon 599 (V599E) were observed after treatment with dacarbazine in either cell line. Dacarbazine treatment did not induce any additional mutations in BRAF or RAS (data not shown).
The Upregulation of pERK, IL-8, and VEGF Is Sustained In Vivo To investigate the characteristics of the tumors established after injection of MeWo and MeWo-D cells to nude mice, we performed a series of immunohistochemical studies for the expression of IL-8, VEGF, MMP-2, and CD-31 (Fig 6). Using antibodies against PCNA, we found that although the selected cell lines do not proliferate faster in vitro (Table 1), they do show a higher proliferation rate in vivo. This might be explained by the more robust angiogenesis seen in these tumors, as shown by the CD-31 staining (Fig 6). In accordance with our previous results,20 the dacarbazine-resistant cell line tumors expressed higher levels of IL-8 and VEGF than the tumors produced by the parental cell line. Similarly, higher levels of MMP-2 were found in the dacarbazine-resistant cell lines. The elevated MMP-2 expression could be a direct result of a direct effect of the prolonged exposure to dacarbazine, or a secondary effect of elevated IL-8 expression as we have previously demonstrated.24
Staining for pERK demonstrated that the overexpression seen in protein lysates from the resistant cells in vitro was also sustained in the established tumors in vivo (Fig 7). No difference in the expression of pAKT was observed between tumors produced by the MeWo parental cells and those produced by MeWo-D cells in vivo (Fig 7), confirming the results obtained in vitro.
Our findings provide evidence that repeated exposure of melanoma cells with a nonaggressive or mildly aggressive phenotype to dacarbazine resulted in the acquisition of a much more tumorigenic and metastatic phenotype in vivo. When injected to immuncompromised mice, these cells produced distinguishably larger tumors, suggesting a higher capacity for local growth. In human melanoma, the ability to grow locally, as estimated by depth of invasion of the skin, is the most important prognosticator for the aggressiveness of the tumor and the ability to develop metastatic disease.32 Furthermore, on intravenous injection of the dacarbazine-resistant cells, metastases developed in all the mice. In contrast, metastasis was a rare event when the parental cells were injected. There was a significant difference in the metastatic burden and in the pattern of spread, which was confined to the lungs in the parental tumors, but extended to various other viscera and tissues in the dacarbazine-resistant tumors, mimicking the clinical pattern in melanoma patients. The information presented in our study might also prove to be clinically valuable, especially nowadays when much emphasis is put on shifting therapy for melanoma to an adjuvant setup in an attempt to achieve higher cure rates. Using chemotherapeutics such as dacarbazine for the treatment of dormant circulating melanoma cells might lead to an unexpected result—the selection of more aggressive clones. We have previously shown that a brief exposure of melanoma cells to dacarbazine resulted in the induction of various angiogenic molecules such as IL-8 and VEGF that might render the cells to a more aggressive phenotype.20 Hypothetically, this was attributed to a stress response initiated by the chemotherapy. However, this cannot be a valid explanation for the present data, because the cells that survived the repeated exposure to dacarbazine sustained their aggressive and metastatic behavior long after the withdrawal of the chemotherapeutic agent and after repeated freeze/thaw cycles under culture conditions. It is more likely that long-term exposure to dacarbazine resulted in the selection and expansion of cells with higher tumorigenic or metastatic capabilities. Dacarbazine is also known to exert its antitumor activities by the methylation of nucleic acids,8,9 so it is possible that the dacarbazine-induced phenotype might be brought about by epigenetic changes, such as the activation of previously silent genes or the silencing of previously active genes. Our findings provide many testable hypotheses regarding the progression of melanoma. Interestingly, we were able to demonstrate increased pERK expression in the more aggressive cell lines. This finding possibly underscores the role of ERK phosphorylation in melanoma progression. The aberrant activation of MAP kinase pathways are implicated in the growth and pathologic behavior of various cancers.33-37 There is a growing body of evidence suggesting that activation of the Ras/Raf/MEK/ERK pathway may be involved in the aggressive behavior of melanoma.27,28 Erk1/2 has been found to be constitutively activated in human melanoma cell lines and tumors in a progression stage-dependent manner.28 This high basal ERK activity is implicated in the rapid melanoma cell growth, increased cell survival, and resistance to apoptosis. The activity of ERK may also be a major driving force behind the highly metastatic behavior through its ability to regulate the expression of various cytokines and angiogenic factors, extracellular membrane degrading enzymes, and invasion-promoting integrins.26 The possible role of ERK in the oncogenic activity of melanoma suggests that the MAP kinase pathway would be an ideal therapeutic target. Indeed, many groups have already started to explore the therapeutic potential of drugs that block the activity of Ras, such as the farnesyl transferase inhibitors, FTS, and the RAF inhibitors.38,39 The mechanisms leading to the upregulation of pERK in melanoma are not well established and are to be either secondary to an activating mutation in N-RAS or BRAF or a result of the upregulation of various cytokines and their receptors that function upstream of ERK.27 We therefore examined whether dacarbazine treatment induced RAS or BRAF mutations in SB2 and MeWo cells. Sequencing analysis revealed that the SB2 parental cell line had an activating mutation in N-RAS codon 61 (Fig 5). Dacarbazine treatment induced no additional mutations in RAS or BRAF (including at codon 599), indicating that the increase in MAPK activity in dacarbazine-treated clones did not result from newly acquired mutations. In conclusion, repeated exposure of melanoma cells to dacarbazine, which is considered the chemotherapeutic agent of choice for this disease, may lead to the selection of cells with a much more aggressive phenotype. We propose that combination treatment with novel targeted agents such as anti VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.
The authors indicated no potential conflicts of interest.
Supported in part by National Institutes of Health grant CA76098. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
