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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 anchorage-independent growth. (a) Western blots of primary melanocyte cell lines with antibodies as indicated. (b) Western blot of #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) for 5 days were probed with antibodies as indicated. (c) Representative images of primary melanocyte cell lines treated with DMSO or OHT were plated in soft agar in the presence of 200 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks. (d) Relative number of colonies and (e) relative size of each colony in the presence of 200 nM TPA. All error bars show mean±SEM from three independent experiments. Bar=500 μm; **P<0.01 by t-test. DCT, dopachrome tautomerase; ERK, extracellular signal-–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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fig1: Rac1 is required for NRasQ61K-induced anchorage-independent growth. (a) Western blots of primary melanocyte cell lines with antibodies as indicated. (b) Western blot of #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) for 5 days were probed with antibodies as indicated. (c) Representative images of primary melanocyte cell lines treated with DMSO or OHT were plated in soft agar in the presence of 200 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks. (d) Relative number of colonies and (e) relative size of each colony in the presence of 200 nM TPA. All error bars show mean±SEM from three independent experiments. Bar=500 μm; **P<0.01 by t-test. DCT, dopachrome tautomerase; ERK, extracellular signal-–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mentions: In our recent study (Li et al., 2011), we generated two independent immortalized Rac1 f/f Ink4a−/− Tyr∷CreERt2+/o melanocyte cell lines #3 and #4, which were unable to grow in soft agar (Figure 1c–e). To test the importance of Rac1 downstream of NRasQ61K in oncogenic transformation for melanocytes, we generated melanocyte cell lines from 1-day-old Tyr∷NRasQ61K+/o Rac1 f/f Ink4−/− Tyr∷CreERt2+/o (#10 and #11) and Tyr∷NRasQ61K+/o Rac1 f/f Ink4a−/− Tyr∷CreERt2o/o (#5) littermate mice (also littermate of #3 and #4). As expected, NRasQ61K+/o-expressing melanocytes (#5, #10 and #11) have elevated activation of mitogen-activated protein kinase signaling compared with Rac1 f/f Ink4a−/− Tyr∷CreERt2+/o melanocyte cell lines #3 and #4 (Figure 1a; Ackermann et al., 2005; Whitwam et al., 2007; Li et al., 2011), but Rac1 expression level was not affected by expression of NRasQ61K (Figure 1a). Rac1 deletion was induced with tamoxifen analog 4-hydroxytamoxifen (OHT) in cell lines that express CreERt2 (#10 and #11), but not in cell line #5, which is CreERt2 negative (Figure 1b). Neither Rac2 nor Rac3 was detectable, regardless of Rac1 deletion (Figure 1b). All the NRasQ61K-expressing melanocyte cell lines (#5, #10 and #11) were able to induce rapid AIG in soft agar and form large colonies (Figure 1c–e). Rac1 deletion reduced colony number by about 80% and the average colony size by about 60% (Figure 1c–e). Rac1-deleted NRasQ61K-expressing melanocytes additionally showed reduced proliferation (Supplementary Figure S1A online). 12-O-Tetradecanoylphorbol 13-acetate acts as a growth factor for cultured melanocytes, but is not required for growth when melanocytes are transformed by constitutively active c-kit receptor (Larue et al., 1992). Similarly, 12-O-tetradecanoylphorbol 13-acetate is not required for NRasQ61K-expressing melanocytes growth on either 2D or in soft agar (Supplementary Figure S1 online). Thus, NRasQ61K confers Rac1-dependent AIG on primary melanocytes. This suggests an important, but previously undescribed, role for Rac1 in mediating oncogenic transformation induced by NRasQ61K in melanocytes.


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 anchorage-independent growth. (a) Western blots of primary melanocyte cell lines with antibodies as indicated. (b) Western blot of #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) for 5 days were probed with antibodies as indicated. (c) Representative images of primary melanocyte cell lines treated with DMSO or OHT were plated in soft agar in the presence of 200 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks. (d) Relative number of colonies and (e) relative size of each colony in the presence of 200 nM TPA. All error bars show mean±SEM from three independent experiments. Bar=500 μm; **P<0.01 by t-test. DCT, dopachrome tautomerase; ERK, extracellular signal-–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
© Copyright Policy - open-access
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fig1: Rac1 is required for NRasQ61K-induced anchorage-independent growth. (a) Western blots of primary melanocyte cell lines with antibodies as indicated. (b) Western blot of #5, #10, and #11 primary melanocyte cell lines treated with DMSO or 4-hydroxytamoxifen (OHT) for 5 days were probed with antibodies as indicated. (c) Representative images of primary melanocyte cell lines treated with DMSO or OHT were plated in soft agar in the presence of 200 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks. (d) Relative number of colonies and (e) relative size of each colony in the presence of 200 nM TPA. All error bars show mean±SEM from three independent experiments. Bar=500 μm; **P<0.01 by t-test. DCT, dopachrome tautomerase; ERK, extracellular signal-–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Mentions: In our recent study (Li et al., 2011), we generated two independent immortalized Rac1 f/f Ink4a−/− Tyr∷CreERt2+/o melanocyte cell lines #3 and #4, which were unable to grow in soft agar (Figure 1c–e). To test the importance of Rac1 downstream of NRasQ61K in oncogenic transformation for melanocytes, we generated melanocyte cell lines from 1-day-old Tyr∷NRasQ61K+/o Rac1 f/f Ink4−/− Tyr∷CreERt2+/o (#10 and #11) and Tyr∷NRasQ61K+/o Rac1 f/f Ink4a−/− Tyr∷CreERt2o/o (#5) littermate mice (also littermate of #3 and #4). As expected, NRasQ61K+/o-expressing melanocytes (#5, #10 and #11) have elevated activation of mitogen-activated protein kinase signaling compared with Rac1 f/f Ink4a−/− Tyr∷CreERt2+/o melanocyte cell lines #3 and #4 (Figure 1a; Ackermann et al., 2005; Whitwam et al., 2007; Li et al., 2011), but Rac1 expression level was not affected by expression of NRasQ61K (Figure 1a). Rac1 deletion was induced with tamoxifen analog 4-hydroxytamoxifen (OHT) in cell lines that express CreERt2 (#10 and #11), but not in cell line #5, which is CreERt2 negative (Figure 1b). Neither Rac2 nor Rac3 was detectable, regardless of Rac1 deletion (Figure 1b). All the NRasQ61K-expressing melanocyte cell lines (#5, #10 and #11) were able to induce rapid AIG in soft agar and form large colonies (Figure 1c–e). Rac1 deletion reduced colony number by about 80% and the average colony size by about 60% (Figure 1c–e). Rac1-deleted NRasQ61K-expressing melanocytes additionally showed reduced proliferation (Supplementary Figure S1A online). 12-O-Tetradecanoylphorbol 13-acetate acts as a growth factor for cultured melanocytes, but is not required for growth when melanocytes are transformed by constitutively active c-kit receptor (Larue et al., 1992). Similarly, 12-O-tetradecanoylphorbol 13-acetate is not required for NRasQ61K-expressing melanocytes growth on either 2D or in soft agar (Supplementary Figure S1 online). Thus, NRasQ61K confers Rac1-dependent AIG on primary melanocytes. This suggests an important, but previously undescribed, role for Rac1 in mediating oncogenic transformation induced by NRasQ61K in melanocytes.

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