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. 2010 Apr 22;29(16):2449-56.
doi: 10.1038/onc.2009.521. Epub 2010 Feb 1.

Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation

P J Mishra  1 , L Ha, J Rieker, E V Sviderskaya, D C Bennett, M D Oberst, K Kelly, G Merlino
Affiliations

Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation

P J Mishra et al. Oncogene. .

Erratum in

  • Oncogene. 2011 Apr 14;30(15):1850

Abstract

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression and effects of NRas and its downstream effectors on melanocyte morphology and pigmentation. (a) Highly simplified schematic of NRas downstream pathway members RalGEF, BRaf and PI3K, showing the activating mutants used in this study. (b) Panels show evidence for expression (+) of BrafV600E RNA (RT–PCR), and p110α-CAAX and RLF-CAAX (HA tagged) protein (western blot). Arf-deficient mouse melanocytes were used in all studies to avoid the senescence-prone phenotype (Ha et al., 2007). (c) RalGEF activity in vector control and RLF-CAAX-transfected melanocytes, assayed by measuring the quantity of RalA-GTP bound to RALBP-1 in a pull-down assay (Yin et al., 2007). (d) PI3K pathway activation in p110α-CAAX melanocytes assayed by visualizing phospho-AKP (Ser473) and total AKT levels by western blotting. (e) Activation of BRaf pathway in BrafV600E melanocytes assayed by visualizing phospho-MEK levels by western blotting. (f) Morphological alterations resulting from constitutive activation of: control (vector), BRaf (expressing BrafV600E), PI3K (expressing p110α-CAAX), RalGEF (expressing RLF-CAAX) or NRas (expressing NRasQ61K). Bright field images are shown (20×). NRas, PI3K and BRaf mouse melanocytes were generated using LZRS retroviruses, as described earlier (Chudnovsky et al., 2005; Ha et al., 2007). Ral and RalBN28 melanocytes were generated using pBABE retroviruses (Yin et al., 2007). For pBABE infections, EP293 RLF-CAAX cells were grown overnight in DMEM with 10% FBS, L-glutamine (no puromycin selection). A measure of 8 μg/ml polybreene was added to the media and used to infect Arf-deficient melanocytes for 8 h with pBABE retroviruses. Melanocytes were grown in CMGM overnight and the infection was repeated the next day. Then, 24 h after the second pBABE infection, melanocytes were thereafter grown in CMGM plus 1.5 μg/ml puromycin. Vec, control vector. Note that melanocyte cell lines were taken through ~15 passages after transfection before conducting experiments, under selection where appropriate. (g) Measurement of melanin content in genetically engineered melanocytes showing the pathway activated (see Figure 1 for expressed mutant proteins). Cells were lysed in 1N NaOH at 37 °C overnight and the melanin content was measured by optical density at 475nm (Virador et al., 1999). Error bars represent standard deviation (s.d.). (h) The protein expression levels of key mouse pigmentation genes, tyrosinase-related protein 1 (TYRP1) and DCT were determined by western blotting using the rabbit antibodies PEP7 and PEP8H, respectively (Recio et al., 2002). β-actin was used as a loading control. Western blots were performed using standard procedures.
Figure 2
Figure 2
Consequences of expression of activated NRas and downstream effectors on anchorage-dependent growth, anchorage-independent growth and invasiveness. (a and b) The growth rates in 2D culture of melanocytes genetically modified to bear constitutively activated NRas or its individual effectors. Proliferation was quantified in triplicate using a hemocytometer to count trypsin-suspended cells, which were replated at 3×104 cells/ml, 2 ml/dish. Error bars represent s.d. (c) The anchorage-independent growth ability of melanocytes expressing activated NRas, its individual effectors, or combinations of these targets, as assayed by growth of the cells in 3% semi-solid agar medium as previously described (Ha et al., 2007). Briefly, 5×104 cells/well were seeded in triplicate in six-well plates in growth medium containing 3% agar. After 2 weeks of inoculation, colonies that developed in soft agar were counted. (d) Quantification of the soft agar colonies are shown in (c). Melanocyte cell lines were taken through about 15 passages after transfection before conducting experiments. (e) Invasive potential of the melanocytes was assayed by matrigel invasion assay using previously published methods (Albini et al., 1987). Unlike control and BRafV600E-expressing melanocytes, PI3K and RalGEF activation in melanocytes conferred invasiveness.
Figure 3
Figure 3
Comparative effects of activated CRaf and BRaf on melanocyte growth, pigmentation, transformation and senescence. (a) The activity of CRaf in melanocytes transfected with the pEF-CAAX-Raf-1 vector, using Lipofectamine 2000 (Invitrogen) transfection reagent as per the manufacturer’s protocol, was confirmed by measuring and quantifying phospho-MEK levels. (b) Anchorage-dependent growth was quantified in triplicate by counting trypsin-suspended cells using an automated cell counter Countess (Invitrogen); cells were replated in six-well plates, 1×105 cells/well. Error bars represent s.d. (c) Melanin content was assayed by lysing the cells in NaOH and measuring the optical density at 475 nm. (d and e) The anchorage-independent growth ability of melanocytes expressing activated BRaf, CRaf and Ral was assayed by growing melanocytes in 3% semi-solid agar as previously described (Ha et al., 2007). Briefly, 2×104 cells/well were seeded in triplicate in six-well plates in growth medium containing 3% agar. After 2 weeks of inoculation, colonies that developed in soft agar were counted. Individual soft agar colonies are shown in (d) and quantitation is shown in (e). Error bars represent s.d. (f) The ability of melanocytes to induce senescence was quantified using senescence-associated β-galactosidase (SA-β-gal) staining and senescent morphology, using previously published methods (Michaloglou et al., 2005; Ha et al., 2007). Error bars represent s.d.
Figure 4
Figure 4
Consequences of expression of an Ral-dominant negative protein (RalBN38) on growth, and summary of all phenotypes associated with constitutive pathway activation. (a) Western blot showing a higher level of RalB owing to expression of the dominant-negative RalBN38, which binds to and inhibits RalGEF (Yin et al., 2007). (b) RalBN38 reduced the growth rate of the NRasQ61K-expressing melanocytes in 2D culture. (c) RalBN38 reduced the anchorage-independent growth of the NRasQ61K-expressing melanocytes in soft agar (P<0.05; error bars show s.d.). Briefly, 2×104 cells/well were seeded in triplicate in six-well plates in growth medium containing 3% agar. After 2 weeks of inoculation, colonies that developed in soft agar were counted. (d) RalBN38 had no significant effect on pigmentation in NRasQ61K-expressing melanocytes. (e) Summary of phenotypes associated with constitutive activation of NRas and its downstream effectors. The dedifferentiation phenotype included loss of melanin and decrease in expression of DCT and TYRP1. The BRaf, CRaf, PI3K and Ral pathways were constitutively activated by BRafV600E, CAAX-RIF-1, p110a-CAAX and RLF-CAAX, respectively. NRas pathways were activated by expression of NRasQ61K. All melanocyte lines were passaged ~15 times before analysis. (−) No phenotype; (+) mild phenotype; (+ +) moderate phenotype; (+ + +) strong phenotype.
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