Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

Oncogene volume 30, pages 4590–4600 (2011)Cite this article

Subjects

Abstract

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  • Ackermann J, Frutschi M, Kaloulis K, McKee T, Trumpp A, Beermann F . (2005). Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background. Cancer Res 65: 4005–4011.

    Article  CAS  PubMed  Google Scholar 

  • Aiba Y, Oh-hora M, Kiyonaka S, Kimura Y, Hijikata A, Mori Y et al. (2004). Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation. Proc Natl Acad Sci USA 101: 16612–16617.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ball NJ, Yohn JJ, Morelli JG, Norris DA, Golitz LE, Hoeffler JP . (1994). Ras mutations in human melanoma: a marker of malignant progression. J Invest Dermatol 102: 285–290.

    Article  CAS  PubMed  Google Scholar 

  • Bastian BC, Olshen AB, LeBoit PE, Pinkel D . (2003). Classifying melanocytic tumors based on DNA copy number changes. Am J Pathol 163: 1765–1770.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bauer J, Bastian BC . (2006). Distinguishing melanocytic nevi from melanoma by DNA copy number changes: comparative genomic hybridization as a research and diagnostic tool. Dermatol Ther 19: 40–49.

    Article  PubMed  Google Scholar 

  • Brodie C, Steinhart R, Kazimirsky G, Rubinfeld H, Hyman T, Ayres JN et al. (2004). PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters. Mol Pharmacol 66: 76–84.

    Article  CAS  PubMed  Google Scholar 

  • Broome Powell M, Gause PR, Hyman P, Gregus J, Lluria-Prevatt M, Nagle R et al. (1999). Induction of melanoma in TPras transgenic mice. Carcinogenesis 20: 1747–1753.

    Article  CAS  PubMed  Google Scholar 

  • Coughlin JJ, Stang SL, Dower NA, Stone JC . (2005). RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling. J Immunol 175: 7179–7184.

    Article  CAS  PubMed  Google Scholar 

  • Cully M, You H, Levine AJ, Mak TW . (2006). Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6: 184–192.

    Article  CAS  PubMed  Google Scholar 

  • Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky Jr WE et al. (2009). Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat Genet 41: 544–552.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eckhart L, Bach J, Ban J, Tschachler E . (2000). Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. Biochem Biophys Res Commun 271: 726–730.

    Article  CAS  PubMed  Google Scholar 

  • Furge KA, Kiewlich D, Le P, Vo MN, Faure M, Howlett AR et al. (2001). Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. Proc Natl Acad Sci USA 98: 10722–10727.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao L, Feng Y, Bowers R, Becker-Hapak M, Gardner J, Council L et al. (2006). Ras-associated protein-1 regulates extracellular signal-regulated kinase activation and migration in melanoma cells: two processes important to melanoma tumorigenesis and metastasis. Cancer Res 66: 7880–7888.

    Article  CAS  PubMed  Google Scholar 

  • Gawecka JE, Griffiths GS, Ek-Rylander B, Ramos JW, Matter ML . (2010). R-Ras regulates migration through an interaction with filamin A in melanoma cells. PLoS One 5: e11269.

    Article  PubMed  PubMed Central  Google Scholar 

  • Guo FF, Kumahara E, Saffen D . (2001). A CalDAG-GEFI/Rap1/B-Raf cassette couples M(1) muscarinic acetylcholine receptors to the activation of ERK1/2. J Biol Chem 276: 25568–25581.

    Article  CAS  PubMed  Google Scholar 

  • Haluska FG, Ibrahim N . (2006). Therapeutic targets in melanoma: map kinase pathway. Curr Oncol Rep 8: 400–405.

    Article  CAS  PubMed  Google Scholar 

  • Hingorani SR, Jacobetz MA, Robertson GP, Herlyn M, Tuveson DA . (2003). Suppression of BRAF(V599E) in human melanoma abrogates transformation. Cancer Res 63: 5198–5202.

    CAS  PubMed  Google Scholar 

  • Houben R, Becker JC, Kappel A, Terheyden P, Brocker EB, Goetz R et al. (2004). Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 3: 6.

    Article  PubMed  PubMed Central  Google Scholar 

  • Huang HP, Shih YW, Chang YC, Hung CN, Wang CJ . (2008). Chemoinhibitory effect of mulberry anthocyanins on melanoma metastasis involved in the Ras/PI3K pathway. J Agric Food Chem 56: 9286–9293.

    Article  CAS  PubMed  Google Scholar 

  • Ikediobi ON, Davies H, Bignell G, Edkins S, Stevens C, O'Meara S et al. (2006). Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther 5: 2606–2612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ikenoue T, Hikiba Y, Kanai F, Aragaki J, Tanaka Y, Imamura J et al. (2004). Different effects of point mutations within the B-Raf glycine-rich loop in colorectal tumors on mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase and nuclear factor kappaB pathway and cellular transformation. Cancer Res 64: 3428–3435.

    Article  CAS  PubMed  Google Scholar 

  • Ikenoue T, Hikiba Y, Kanai F, Tanaka Y, Imamura J, Imamura T et al. (2003). Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res 63: 8132–8137.

    CAS  PubMed  Google Scholar 

  • Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F et al. (2004). B-RAF is a therapeutic target in melanoma. Oncogene 23: 6292–6298.

    Article  CAS  PubMed  Google Scholar 

  • Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J . (1997). Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J 16: 2783–2793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lorenzo PS, Beheshti M, Pettit GR, Stone JC, Blumberg PM . (2000). The guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters. Mol Pharmacol 57: 840–846.

    CAS  PubMed  Google Scholar 

  • Lorenzo PS, Kung JW, Bottorff DA, Garfield SH, Stone JC, Blumberg PM . (2001). Phorbol esters modulate the Ras exchange factor RasGRP3. Cancer Res 61: 943–949.

    CAS  PubMed  Google Scholar 

  • Oh-hora M, Johmura S, Hashimoto A, Hikida M, Kurosaki T . (2003). Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-gamma2 to Ras in B cell receptor signaling. J Exp Med 198: 1841–1851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohba Y, Ikuta K, Ogura A, Matsuda J, Mochizuki N, Nagashima K et al. (2001). Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis. Embo J 20: 3333–3341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oki-Idouchi CE, Lorenzo PS . (2007). Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin. Cancer Res 67: 276–280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Orian-Rousseau V, Morrison H, Matzke A, Kastilan T, Pace G, Herrlich P et al. (2007). Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol Biol Cell 18: 76–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT, Walker EH et al. (2000). Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell 103: 931–943.

    Article  CAS  PubMed  Google Scholar 

  • Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM et al. (2003). High frequency of BRAF mutations in nevi. Nat Genet 33: 19–20.

    Article  CAS  PubMed  Google Scholar 

  • Pu Y, Perry NA, Yang D, Lewin NE, Kedei N, Braun DC et al. (2005). A novel diacylglycerol-lactone shows marked selectivity in vitro among C1 domains of protein kinase C (PKC) isoforms alpha and delta as well as selectivity for RasGRP compared with PKCalpha. J Biol Chem 280: 27329–27338.

    Article  CAS  PubMed  Google Scholar 

  • Roberts DM, Anderson AL, Hidaka M, Swetenburg RL, Patterson C, Stanford WL et al. (2004). A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters. Mol Cell Biol 24: 10515–10528.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ et al. (1994). Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527–532.

    Article  CAS  PubMed  Google Scholar 

  • Sarkisian CJ, Keister BA, Stairs DB, Boxer RB, Moody SE, Chodosh LA . (2007). Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis. Nat Cell Biol 9: 493–505.

    Article  CAS  PubMed  Google Scholar 

  • Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL et al. (2003). Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res 63: 756–759.

    CAS  PubMed  Google Scholar 

  • Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW . (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88: 593–602.

    Article  CAS  PubMed  Google Scholar 

  • Stark M, Hayward N . (2007). Genome-wide loss of heterozygosity and copy number analysis in melanoma using high-density single-nucleotide polymorphism arrays. Cancer Res 67: 2632–2642.

    Article  CAS  PubMed  Google Scholar 

  • Stone JC . (2006). Regulation of Ras in lymphocytes: get a GRP. Biochem Soc Trans 34: 858–861.

    Article  CAS  PubMed  Google Scholar 

  • Stope MB, Vom Dorp F, Szatkowski D, Bohm A, Keiper M, Nolte J et al. (2004). Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3. Mol Cell Biol 24: 4664–4676.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sumimoto H, Miyagishi M, Miyoshi H, Yamagata S, Shimizu A, Taira K et al. (2004). Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 23: 6031–6039.

    Article  CAS  PubMed  Google Scholar 

  • Sutlovic D, Definis Gojanovic M, Andelinovic S, Gugic D, Primorac D . (2005). Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid. Croat Med J 46: 556–562.

    PubMed  Google Scholar 

  • Suzuki T, Shen H, Akagi K, Morse HC, Malley JD, Naiman DQ et al. (2002). New genes involved in cancer identified by retroviral tagging. Nat Genet 32: 166–174.

    Article  CAS  PubMed  Google Scholar 

  • Takayama H, LaRochelle WJ, Sharp R, Otsuka T, Kriebel P, Anver M et al. (1997). Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proc Natl Acad Sci USA 94: 701–706.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Teixeira C, Stang SL, Zheng Y, Beswick NS, Stone JC . (2003). Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3. Blood 102: 1414–1420.

    Article  CAS  PubMed  Google Scholar 

  • Tuveson DA, Shaw AT, Willis NA, Silver DP, Jackson EL, Chang S et al. (2004). Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 5: 375–387.

    Article  CAS  PubMed  Google Scholar 

  • van Elsas A, Zerp S, van der Flier S, Kruse-Wolters M, Vacca A, Ruiter DJ et al. (1995). Analysis of N-ras mutations in human cutaneous melanoma: tumor heterogeneity detected by polymerase chain reaction/single-stranded conformation polymorphism analysis. Recent Results Cancer Res 139: 57–67.

    Article  CAS  PubMed  Google Scholar 

  • Wang D, Yang W, Du J, Devalaraja MN, Liang P, Matsumoto K et al. (2000). MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression. Oncogene 19: 4647–4659.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu H, Goel V, Haluska FG . (2003). PTEN signaling pathways in melanoma. Oncogene 22: 3113–3122.

    Article  CAS  PubMed  Google Scholar 

  • Yamashita S, Mochizuki N, Ohba Y, Tobiume M, Okada Y, Sawa H et al. (2000). CalDAG-GEFIII activation of Ras, R-ras, and Rap1. J Biol Chem 275: 25488–25493.

    Article  CAS  PubMed  Google Scholar 

  • Yang D, Kedei N, Li L, Tao J, Velasquez JF, Michalowski AM et al. (2010). RasGRP3 contributes to formation and maintenance of the prostate cancer phenotype. Cancer Res 70: 7905–7917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng H, Gao L, Feng Y, Yuan L, Zhao H, Cornelius LA . (2009). Down-regulation of Rap1GAP via promoter hypermethylation promotes melanoma cell proliferation, survival, and migration. Cancer Res 69: 449–457.

    Article  CAS  PubMed  Google Scholar 

  • Zheng Y, Liu H, Coughlin J, Zheng J, Li L, Stone JC . (2005). Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and RAS signaling systems in B cells. Blood 105: 3648–3654.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported in part by the intramural program of the NIH, Center for Cancer Research, National Cancer Institute (project number Z1A BC 005270) and in part by grant ETT 495/09. ZE Toth is supported by the Bolyai fellowship. We thank Glenn Merlino for helpful comments.

Author information

Author notes

  1. D Yang and J Tao: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    D Yang, J Tao, L Li, N Kedei, A Czap, J F Velasquez, D Mihova, A M Michalowski, S H Yuspa & P M Blumberg

  2. Department of Anatomy, Neuromorphological and Neuroendocrine Research Laboratory, Histology and Embryology, Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary

    Z E Tóth

Authors
  1. D Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N Kedei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Z E Tóth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Czap
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J F Velasquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D Mihova
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. A M Michalowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. S H Yuspa
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. P M Blumberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P M Blumberg.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, D., Tao, J., Li, L. et al. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma. Oncogene 30, 4590–4600 (2011). https://doi.org/10.1038/onc.2011.166

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.166

Keywords

This article is cited by

Search

Advanced search

Quick links