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Activated mutant NRas(Q61K) drives aberrant melanocyte signaling, survival, and invasiveness via a Rac1-dependent mechanism.

Li A, Ma Y, Jin M, Mason S, Mort RL, Blyth K, Larue L, Sansom OJ, Machesky LM - J. Invest. Dermatol. (2012)

Bottom Line: The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling.We tested the effects of activated NRas(Q61K) on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model.We also show that genetic deletion or pharmacological inhibition of Rac1 in NRas(Q61K) induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK.

ABSTRACT
Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation. NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal-regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation. The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling. We tested the effects of activated NRas(Q61K) on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model. We find an important role for Rac1 downstream of NRas(Q61K) in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRas(Q61K) does not appear to affect melanoblast motility or proliferation during mouse embryogenesis. We also show that genetic deletion or pharmacological inhibition of Rac1 in NRas(Q61K) induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

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Rac1 is required for NRasQ61K-induced invadopodia formation and invasion in melanocytes. (a) #3, #4, #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) on cross-linked gelatin and stained with rhodamine phalloidin. (b) #10 or #11 melanocyte cells on cross-linked gelatin stained with anti-p34 (Arp2/3 complex), anti-cortactin, and phalloidin. Arrows indicate invadopodia. (c) Percentage of cells degrading matrix. (d) Relative matrix degradation to DMSO control #5 (e) or #10 or #11. All error bars show mean±SEM from 30 cells per experiment, n=3 independent experiments. (f) Organotypic invasion assays and quantification (g) for invasion of #10 or #11 melanocytes treated with DMSO or OHT. Melanocytes are indicated by red arrows. **P<0.01 and *P<0.05 by t-test. Bars=10 μm (a, b) and 20 μm (f). Insets show invadopodia.
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fig5: Rac1 is required for NRasQ61K-induced invadopodia formation and invasion in melanocytes. (a) #3, #4, #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) on cross-linked gelatin and stained with rhodamine phalloidin. (b) #10 or #11 melanocyte cells on cross-linked gelatin stained with anti-p34 (Arp2/3 complex), anti-cortactin, and phalloidin. Arrows indicate invadopodia. (c) Percentage of cells degrading matrix. (d) Relative matrix degradation to DMSO control #5 (e) or #10 or #11. All error bars show mean±SEM from 30 cells per experiment, n=3 independent experiments. (f) Organotypic invasion assays and quantification (g) for invasion of #10 or #11 melanocytes treated with DMSO or OHT. Melanocytes are indicated by red arrows. **P<0.01 and *P<0.05 by t-test. Bars=10 μm (a, b) and 20 μm (f). Insets show invadopodia.

Mentions: We asked whether expression of NRasQ61K could enhance the ability of melanocytes to degrade extracellular matrix and whether matrix-remodeling capacity was Rac1 dependent. Invadopodia are actin-based membrane protrusions formed in invasive cancer cells that have a matrix degradation activity (Buccione et al., 2004). They are rich in filamentous actin, cortactin, and Arp2/3 complex (Linder, 2007). To examine whether NRasQ61K could induce invadopodia formation, wild-type melanocyte cell lines (#3 and #4) and NRasQ61K melanocyte cell lines (#10 and #11) were cultured on cross-linked gelatin matrix. Wild-type melanocytes (#3 and #4) showed similar morphology compared with NRasQ61K-expressing melanocyte cell lines, but did not display invadopodia (Figure 5a). In contrast, NRasQ61K-expressing melanocyte cell lines (#5, #10 and #11) formed copious invadopodia (Figure 5b). We found ∼45% of NRasQ61K-expressing melanocytes and only 4% of normal melanocytes contained invadopodia (Figure 5c). Furthermore, the matrix degradation for individual cells was negligible in controls (#3 and #4) as compared with NRasQ61K-expressing melanocytes (Figure 5d).


Activated mutant NRas(Q61K) drives aberrant melanocyte signaling, survival, and invasiveness via a Rac1-dependent mechanism.

Li A, Ma Y, Jin M, Mason S, Mort RL, Blyth K, Larue L, Sansom OJ, Machesky LM - J. Invest. Dermatol. (2012)

Rac1 is required for NRasQ61K-induced invadopodia formation and invasion in melanocytes. (a) #3, #4, #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) on cross-linked gelatin and stained with rhodamine phalloidin. (b) #10 or #11 melanocyte cells on cross-linked gelatin stained with anti-p34 (Arp2/3 complex), anti-cortactin, and phalloidin. Arrows indicate invadopodia. (c) Percentage of cells degrading matrix. (d) Relative matrix degradation to DMSO control #5 (e) or #10 or #11. All error bars show mean±SEM from 30 cells per experiment, n=3 independent experiments. (f) Organotypic invasion assays and quantification (g) for invasion of #10 or #11 melanocytes treated with DMSO or OHT. Melanocytes are indicated by red arrows. **P<0.01 and *P<0.05 by t-test. Bars=10 μm (a, b) and 20 μm (f). Insets show invadopodia.
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fig5: Rac1 is required for NRasQ61K-induced invadopodia formation and invasion in melanocytes. (a) #3, #4, #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) on cross-linked gelatin and stained with rhodamine phalloidin. (b) #10 or #11 melanocyte cells on cross-linked gelatin stained with anti-p34 (Arp2/3 complex), anti-cortactin, and phalloidin. Arrows indicate invadopodia. (c) Percentage of cells degrading matrix. (d) Relative matrix degradation to DMSO control #5 (e) or #10 or #11. All error bars show mean±SEM from 30 cells per experiment, n=3 independent experiments. (f) Organotypic invasion assays and quantification (g) for invasion of #10 or #11 melanocytes treated with DMSO or OHT. Melanocytes are indicated by red arrows. **P<0.01 and *P<0.05 by t-test. Bars=10 μm (a, b) and 20 μm (f). Insets show invadopodia.
Mentions: We asked whether expression of NRasQ61K could enhance the ability of melanocytes to degrade extracellular matrix and whether matrix-remodeling capacity was Rac1 dependent. Invadopodia are actin-based membrane protrusions formed in invasive cancer cells that have a matrix degradation activity (Buccione et al., 2004). They are rich in filamentous actin, cortactin, and Arp2/3 complex (Linder, 2007). To examine whether NRasQ61K could induce invadopodia formation, wild-type melanocyte cell lines (#3 and #4) and NRasQ61K melanocyte cell lines (#10 and #11) were cultured on cross-linked gelatin matrix. Wild-type melanocytes (#3 and #4) showed similar morphology compared with NRasQ61K-expressing melanocyte cell lines, but did not display invadopodia (Figure 5a). In contrast, NRasQ61K-expressing melanocyte cell lines (#5, #10 and #11) formed copious invadopodia (Figure 5b). We found ∼45% of NRasQ61K-expressing melanocytes and only 4% of normal melanocytes contained invadopodia (Figure 5c). Furthermore, the matrix degradation for individual cells was negligible in controls (#3 and #4) as compared with NRasQ61K-expressing melanocytes (Figure 5d).

Bottom Line: The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling.We tested the effects of activated NRas(Q61K) on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model.We also show that genetic deletion or pharmacological inhibition of Rac1 in NRas(Q61K) induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK.

ABSTRACT
Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation. NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal-regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation. The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling. We tested the effects of activated NRas(Q61K) on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model. We find an important role for Rac1 downstream of NRas(Q61K) in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRas(Q61K) does not appear to affect melanoblast motility or proliferation during mouse embryogenesis. We also show that genetic deletion or pharmacological inhibition of Rac1 in NRas(Q61K) induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

Show MeSH
Related in: MedlinePlus