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Multiple murineBRafV600E melanomacell lines withsensitivity toPLX4032 Molly H. JenkinsShannon M. SteinbergMatthew P. Alexander2014 год

Multiple murine BRafV600E melanoma cell lines with sensitivity to PLX4032
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Аннотация: Rates of melanoma are steadily on the rise, with a current lifetime incidence of 1 in 50 individuals (Balch et al., 2004; Smalley et al., 2005). In nearly 50% of human melanomas, the BRAF oncogene encodes BRAFV600E (Lee et al., 2011; Long et al., 2011; Menzies et al., 2012; Rubinstein et al., 2010), a protein whose expression increases melanoma progression via activation of signaling cascades and downstream genes (Chapman et al., 2011; Joseph et al., 2010; Koya et al., 2012; Vultur et al., 2011; Yang et al., 2010). Vemurafenib, also known as PLX4032, is a small molecule inhibitor that binds to the ATP-binding site of mutated BRAF kinase, inhibiting ERK signaling only in tumor cells expressing BRAFV600E (Joseph et al., 2010). Clinical use of PLX4032, to treat BRAFV600E tumors, induces rapid tumor regression and has revolutionized targeted therapy for melanoma (Boni et al., 2010; Khalili et al., 2012; Koya et al., 2012; Straussman et al., 2012). However, resistance to PLX4032 typically develops within one yr (Joseph et al., 2010; Straussman et al., 2012; Vultur et al., 2011), underscoring the need for increased understanding of molecular mechanisms underlying BRAFV600E and for developing additional therapeutic strategies. The use of human melanoma cell lines is well established for in vitro and in vivo investigations of BRAFV600E melanoma (Herlyn and Fukunaga-Kalabis, 2010). However, human cell lines rely on xenografts in immune-compromised mice, which limit studies of host-tumor cell interactions in a genetically identical (syngeneic) background. Recently developed mouse models of BRafV600E melanoma provide researchers with the ability to investigate in vivo tumor responses to PLX4032 treatment (Dankort et al., 2009; Dhomen et al., 2009). Backcrossing these mice onto a pure background has allowed for in vivo testing of immunologic therapies in a syngeneic system (Hooijkaas et al., 2012b; A. Hooijkaas et al., 2012a). However, for in vitro analysis of the molecular mechanisms underlying BRafV600E melanoma, stable cell lines that recapitulate the human disease are needed. These lines could be used to identify critical mechanisms: signal transduction pathways, patterns of gene expression, and interactions with cells within the tumor microenvironment (Herlyn and Fukunaga-Kalabis, 2010). To date, two groups have reported the isolation of murine BRafV600E cell lines from mouse models of BRafV600E melanoma (Hooijkaas et al., 2012b; Koya et al., 2012). However, given the heterogeneity of human BRAFV600E cell lines, the establishment of only two murine cell lines may not adequately represent the true biology of BRafV600E cells in vitro. For example, both of the reported murine BRafV600E cell lines are only partially sensitive to PLX4032 and PLX4720 (Hooijkaas et al., 2012b; Knight et al., 2013; Koya et al., 2012), with respect to decreased pERK signaling and cellular proliferation. These in vitro results differ from in vivo results seen in a corresponding mouse model (Hooijkaas et al., 2012b) and with human BRAFV600E cell lines (Joseph et al., 2010; Straussman et al., 2012; Yang et al., 2010). Similar to the work of Hooijkaas et al., 2012b; our group backcrossed the transgenic mouse model, Tyr::CreER;BrafCA;Ptenlox/lox (Dankort et al., 2009), a gift from Marcus Bosenberg (Yale), to a C57BL/6 (B6) background to achieve >98% B6 DNA; these mice are hereafter referred to as Braf/Pten mice. In our studies, the establishment of stable cell lines from this model was difficult, perhaps due to the heterogeneous population of cells in the primary tumor and the slow growth of the tumor cells in culture. However, we developed a technique which we used to establish several stable cell lines that represent the in vivo biology of tumors in Braf/Pten mice. We found that the establishment of stable cell lines from Braf/Pten mice required a protocol of sequential in vitro and in vivo growth. Induced tumors (10 mm in diameter) were resected from Braf/Pten mice and dissociated into single cells by collagenase digestion (see Data S1). We discovered that DMEM/F-12 advanced/5% FBS media was optimal for survival of cells isolated from the primary tumor. However, these cells often grew slowing in culture, making them unsuitable for in vitro studies. To verify the tumorigenicity of dissociated and cultured primary tumor cells, we injected them into NOD/SCID/γ chainnull (NSG) mice, an immune-compromised, permissive host (Figure 1A). When secondary tumors from the host mice reached 10–12 mm in diameter, they were surgically excised and cultured using the same dissociation protocol and culture media. Of the seven tumors that were processed with this sequence, we successfully developed five stable D4M (Dartmouth Murine Mutant Malignant Melanoma) cell lines from both male and female Braf/Pten mice, demonstrating that we can routinely establish lines with this protocol. As both human BRAFV600E and mouse BRafV600E tumors have constitutive activation of MAPK signaling cascades, we evaluated levels of downstream pERK in three D4M cell lines, 3A, 5A, and 7A (Figure 1B). In keeping with a mutational activation of BRaf (Dankort et al., 2009), D4M cell lines have high constitutive levels of pERK compared to B16F1 cells, which are Brafwt. Two D4M cell lines, D4M.3A (from a male donor) and D4M.7A (from a female donor), were used for subsequent studies. Although these cell lines were generated using different modifications of our protocol (3A - with culture before re-injection, 7A - without culture before re-injection), they were morphologically similar in culture (Figure 1A). All D4M cell lines generated were unpigmented, which is commonly seen in human melanoma cell lines (Eberle et al., 1998), such as VMM5 and VMM12 cells (Blackburn et al., 2007, 2009; Croteau et al., 2012; Huntington et al., 2004; Yamshchikov et al., 2005) (Figure S1). Both D4M.3A and D4M.7A grow in advanced DMEM/F-12 media with or without serum (Figure 1C). Survival and growth in serum-free conditions, though significantly less than in serum-containing conditions (P < 0.05), will allow for analysis of secreted factors released from these cell lines in vitro. PLX4032 binds V600E mutant BRAF with high affinity and inhibits ERK signaling output. We tested the sensitivity of cultured D4M cells to this inhibitor by first evaluating cell signaling after treatment with PLX4032, using DMSO-treated cells as negative controls and U0126-treated cells as positive controls (Figure 2A). U0126 is a selective inhibitor of MEK1 and MEK2, blocking downstream signals including phosphorylation of ERK. Treatment of D4M.3A, D4M.5A, and D4M.7A cells with 3 μM PLX4032 for 45 or 90 min, led to decreased pERK, with no change in levels of pAKT (Figure 2A and not shown). Treatment with U0126 decreased pERK levels after 10 min. VMM5 and VMM12 cells, human BRAFV600E cell lines of melanoma (Huntington et al., 2004), showed similar responses to PLX4032 and U0126, with decreased pERK levels at 45 and 10 min, respectively, and no change in pAKT (Figure 2B and not shown). D4M cell lines are, thus, comparable to human BRAFV600E cell lines in their cell signaling response to PLX4032 treatment (Joseph et al., 2010; Straussman et al., 2012; Yang et al., 2010). We conclude that D4M cell lines have characteristics similar to human BRAFV600E melanoma and BRafV600E mouse models. We next evaluated cellular proliferation of D4M cell lines in response to PLX4032. Treatment with 3 μM PLX4032 significantly (P < 0.005) arrested in vitro growth of D4M.3A and D4M.7A cells 2–3 days post-treatment (Figure 2C). Human tumors with BRAFV600E are dependent on constitutive activation of ERK signaling for cellular proliferation; thus, addition of PLX4032 causes growth arrest of BRAFV600E tumor cells in vitro (Joseph et al., 2010). In keeping with reports of human BRAFV600E cell lines (Joseph et al., 2010; Yang et al., 2010), the response of the D4M cells to PLX4032 appeared to be primarily cytostatic. The previously published murine BRafV600E cell lines are relatively resistant to PLX4032 and PLX4720 treatment compared to human BRAFV600E cell lines, with respect to decreased pERK signaling and cellular proliferation (Hooijkaas et al., 2012b; Knight et al., 2013; Koya et al., 2012). To assess the tumorigenicity of D4M melanoma cells, we intradermally injected a range (300–300 000) of cells into B6 and NSG host mice. Each concentration of cells was injected into four B6 and four NSG mice. Of the eight mice injected with 300 cells, no tumors developed in any of the mice after 27 weeks. In contrast, it took only two weeks for tumors to be observed in mice that received ≥3000 cells, and only one week for tumors to be observed in mice that received 300 000 cells (Table 1). Tumors arose with similar kinetics in B6 and NSG host mice; there was no significant difference in the average tumor size between NSG and B6 mice injected with 3000 D4M.3A cells, at any time point (Figure 3A). Histologic examination revealed no gross morphologic differences between tumors in B6 or NSG hosts (data not shown). We conclude that the D4M cells are equally tumorigenic in both immune-compromised and syngeneic hosts. We next tested the efficacy of PLX4720 (a sister compound of PLX4032) (Tsai et al., 2008) to inhibit tumor progression and phosphorylated ERK signaling in vivo. D4M.3A cells were injected intradermally into B6 mice (300 000 cells, per mouse). When tumors reached 5 mm in diameter (8 days post-injections), mice were either given drug-amixed chow containing PLX4720 (Plexxikon, Berkeley, CA) or control chow. Tumors were measured 4 and 7 days after the start of treatment and tumor volumes were calculated. After 7 days of treatment, mice that were fed the PLX4720 chow had significantly smaller tumors than mice that were fed the control chow (Figure 3B, P < 0.05). Mice injected with D4M.7A cells showed comparable results, with smaller tumors after 7 days in mice fed the PLX4720 chow compared with those fed the control chow (data not shown). In addition, tumors in mice fed the PLX4720 chow for 7 days had decreased pERK levels compared to tumors in mice fed the control chow (Figure 3C). To further verify that D4M cell lines have melanoma characteristics, we examined expression of the murine melanoma marker, Pmel. Several melanocyte differentiation antigens (MDAs), such as PMEL (also referred to as gp100) are widely expressed in human melanoma and recognized by tumor-infiltrating lymphocytes (Kawakami, 1994), making it a potentially useful biomarker. We saw relatively low levels of Pmel expression in D4M.3A cultured cells; however, expression increased in tumors that arose when D4M.3A cells were injected into either NSG or B6 mice (Figure 4A). Further, when these tumors were excised and re-cultured once or twice (Figure 4B), Pmel levels fell in culture, but significantly increased again upon implantation into mice (Figure 4C, P < 0.0005). This trend was also observed with Mart-1, Tryrosinase, and Tryp-1 expression (Figure S2 and not shown). This indicates that D4M cells maintain the ability to express MDAs, in vivo and in vitro. Further, the in vivo upregulation of MDAs suggests that D4M cells are responding to host factors within the tumor microenvironment. The fact that Pmel levels equally increase upon transplantation of the D4M cells into either NSG or B6 mice suggests that non-immune cells may be mediating most of this increase. Indeed, the stromal compartment of adjacent fibroblasts has been implicated as one prominent modulator of tumor cell behavior in vivo (Bhowmick et al., 2004; Eck et al., 2009; Marsh et al., 2013; Smalley et al., 2005). In addition, we have found discrepancies in the levels of gene expression between cultured human melanoma cells and those excised from nude mice (Croteau et al., 2012), underscoring the importance of utilizing both in vitro and in vivo model systems. Investigations with human melanoma cell lines and clinical tissue samples demonstrate that PLX4720 (a precursor of PLX4032) (Tsai et al., 2008) treatment leads to increased expression of MDAs, such as PMEL (Boni et al., 2010; Frederick et al., 2013). As D4M cells are sensitive to PLX4032, we treated cells with PLX4032 and analyzed mRNA levels of Pmel. In D4M.3A and D4M.7A cell lines, basal levels of Pmel were relatively low and comparable to levels in 3T3 cells. However, we observed an approximate 4-fold increase (P < 0.005) in Pmel expression 48 hrs after the addition of PLX4032 in these two D4M cell lines, but not in Brafwt 3T3 cells (D4M.3A - Figure 5A and D4M.7A - data not shown). Similarly, when the two human melanoma cell lines (VMM5 and VMM12) were treated with PLX4032 for 48 hrs, PMEL levels significantly increased, while no effect was observed in human dermal fibroblasts (HDFs) (Figure S3). For functional analysis of Pmel antigen recognition, we used a cytotoxicity assay to assess melanoma cell sensitivity to lysis by Pmel TCR transgenic T cells (Overwijk et al., 2003). Activated Pmel T cells were added to DMSO or PLX4032-treated D4M.3A cells at different E:T ratios. PLX4032-treatment increased lysis of D4M.3A cells by Pmel T cells compared to DMSO-treatment (Figure 5B, P < 0.005). EL-4 (antigen-negative) cells either pulsed or unpulsed with Pmel peptide were used as a control target, and Pmel T cells specifically recognized and lysed peptide-pulsed, but not unpulsed, EL-4 cells (data not shown). Taken together, these data are consistent with increased MDA expression in human melanoma cell lines in response to BRAFV600E inhibition (Boni et al., 2010), and further validate the D4M cell lines as bone fide melanoma cells and an appropriate murine model system. Although D4M cell lines do not have high endogenous levels of MDAs in vitro, our data illustrate that D4M.3A cells express Pmel in a functionally relevant manner. D4M cell lines recapitulate human BRAFV600E melanoma in vitro. However, unlike human cell line xenografts, D4M cell lines are readily transplantable into syngeneic host mice (Figure 3A), which will allow for immunological studies. The fact that there was no significant difference between tumorigenicity and tumor volume in NSG and B6 hosts could suggest that D4M cell lines are relatively non-immunogenic, like murine B16 cells (Ashley and Kotlarski, 1986; Leveson et al., 1979). This is consistent with the expression of low levels of MDAs in culture and the relative paucity of immune cells in the tumor tissue (as seen by histology; data not shown). Given the heterogeneity of human BRAFV600E melanoma cell lines, having multiple murine BRafV600E cell lines will be an important resource for the scientific community. The established BRafV600E SM1 cell line has been a useful model in supporting the therapeutic potential of combining BRAF inhibitors with immunotherapy (Knight et al., 2013). Future studies with D4M cells should be similarly useful in developing models of new therapeutic modalities. In addition, the fact that, at 3 μM PLX4032, our D4M cells show substantial decreases in pERK and decrease in cell proliferation, indicates that our cell lines are distinct from those already published and will be a valuable contribution to the scientific community. In conclusion, our study describes a useful model of murine BRafV600E melanoma for in vitro and in vivo experiments. D4M cell lines grow readily in culture, are sensitive to PLX4032, and are transplantable in syngeneic hosts. These cell lines are stable and reproducible, allowing for biological replicates for in vitro and in vivo experiments, and may be of use to many investigators. The authors thank Dr. Marcus Bosenberg for supplying Tyr::CreER;BrafCA;Ptenlox4-5/lox4-5 mice. We thank Dr. Shaofeng Yan, MD, for pathology analysis. We thank Diane Mellinger and Jennifer Fields for technical support. We thank Dr. Gideon Bollag and Plexxicon Inc. (Berkeley, CA) for use of control and PLX4720 mouse chow. This research was funded, in part by: NIH P30 - Center for Molecular, Cellular and Translational Research (P30RR032136); NIH T32 - Cancer Center Training Grant CA009658 (MHJ/CEB); NIH T32 – Immunology Training Grant AI007363 (MHJ/DWM); NIH T32 – Molecular and Cellular Biology Training Grant GM00874 (SMS/MJT); NIH R01 AR-26599 and CA-77267 (CEB); NIH R01 CA120777 (MJT); The American Cancer Society RSG LIB-121864 (MJT); the Melanoma Research Alliance Development Award (MJT); Hitchcock Foundation Pilot Studies Award (DWM and CEB); and NIH R01 CA134799 (DWM). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Год издания: 2014
Авторы: Molly H. Jenkins, Shannon M. Steinberg, Matthew P. Alexander, Jan L. Fisher, Marc S. Ernstoff, Mary Jo Turk, David W. Mullins, Constance Brinckerhoff
Издательство: Wiley
Источник: Pigment Cell & Melanoma Research
Ключевые слова: Melanoma and MAPK Pathways, Cell Adhesion Molecules Research, Cytokine Signaling Pathways and Interactions
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