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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 activity is required for NRasQ61K-induced melanoma growth and lymph node spread. (a) Response of allografts to drug treatment (n⩾4) on day 3 of NSC23766 or EHT1864 treatment (P<0.05). (b) Drug-treated allografts stained with H&E, anti-dopachrome tautomerase (DCT), and anti-BrdU. (c) BrdU-positive cells per field (⩾3 tumors). (d) Western blot of vehicle/tamoxifen (Tam)-treated allografts. (e) Rac1 activity and western blot of vehicle-, NSC23766-, or EHT1864-treated allografts. (f) Left inguinal lymph node and (g) average lymph node mass for drug-treated mice (n⩾4). (h) Lymph nodes with anti-DCT or with anti-Ki67. L=lymph node tissue; M=melanocytes. (i) Percentage of mice with DCT-positive lymph nodes. Error bars show mean±SEM, **P<0.01 and *P<0.05 by t-test. Bars=50 μm. H&E, hematoxylin and eosin.
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fig6: Rac1 activity is required for NRasQ61K-induced melanoma growth and lymph node spread. (a) Response of allografts to drug treatment (n⩾4) on day 3 of NSC23766 or EHT1864 treatment (P<0.05). (b) Drug-treated allografts stained with H&E, anti-dopachrome tautomerase (DCT), and anti-BrdU. (c) BrdU-positive cells per field (⩾3 tumors). (d) Western blot of vehicle/tamoxifen (Tam)-treated allografts. (e) Rac1 activity and western blot of vehicle-, NSC23766-, or EHT1864-treated allografts. (f) Left inguinal lymph node and (g) average lymph node mass for drug-treated mice (n⩾4). (h) Lymph nodes with anti-DCT or with anti-Ki67. L=lymph node tissue; M=melanocytes. (i) Percentage of mice with DCT-positive lymph nodes. Error bars show mean±SEM, **P<0.01 and *P<0.05 by t-test. Bars=50 μm. H&E, hematoxylin and eosin.

Mentions: Although Rac function is required for KRasG12D-induced primary squamous cell skin and lung tumor initiation (Kissil et al., 2007; Samuel et al., 2011), the role of Rac1 in NRasQ61K-induced tumor growth and invasiveness in vivo has not been addressed. We transplanted #11 NRasQ61K Rac1 f/f melanocytes subcutaneously into the left flank of nude mice (CD-1). The growth of Rac1-deleted allografts on day 1–3 of tamoxifen treatment was similar to vehicle, likely because it requires 3 days to deplete endogenous Rac1 (Li et al., 2011). However, allograft growth halted at day 5 after tamoxifen treatment (Figure 6a). BrdU incorporation was reduced by 60% in Rac1-deleted allografts (Figure 6b and c). Pak2 (Mouse melanocytes do not express Pak1/3 (Li et al., 2011).), Erk1/2, and AKT activation were reduced in Rac1-deleted allografts. Although the other Rac1-dependent signaling pathways (JNK, p38, and NF-κB; Jaffe and Hall, 2005) remained unchanged (Figure 6d). Rac-specific inhibitors (NSC23766 or EHT1864) showed strong inhibition of Rac1 activity in vivo (Figure 6e) and significant inhibition of allografts growth (Figure 6a) without inducing body-weight loss (Supplementary Figure S5A online). Proliferation was decreased by about 50% (Figure 6b). Consistent with Rac1 deletion results, both NSC23766- and EHT1864-treated allografts showed a strong decrease in Pak2, Erk1/2, and AKT activation (Figure 6e). Interestingly, analysis of vehicle-treated mice systematically revealed pigmentation of left axillary and inguinal skin-draining lymph nodes (Figure 6f and Supplementary Figure S5B online), a characteristic of the metastatic process. Tumor-draining lymph nodes were enlarged (Figure 6f and g and Supplementary Figure S5B online), and contained large numbers of dopachrome tautomerase-positive pigmented proliferating melanocytic cells (Figure 6h) and disrupted architecture (Supplementary Figure S5C online). However, in tamoxifen, NSC23766- or EHT1864-treated mice, lymph nodes retained their normal architecture and weight (Figure 6f and g and Supplementary Figure S5B, C online). All vehicle-treated mice (6/6) had melanocytes in lymph nodes (Figure 6h and i, Supplementary Figure S5C online). Whereas, none of mice (0/6) treated with tamoxifen and only 1 in 4 mice treated with either NSC23766 or EHT1864 showed melanocytes in lymph nodes (Figure 6h and i, Supplementary Figure S5C online). Together, these data indicate that Rac1 activity is required for NRasQ61K-induced tumor growth and lymph node spread in vivo, which suggests that Rac1 may be a valid therapeutic target against NRasQ61K-induced metastatic melanoma.


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 activity is required for NRasQ61K-induced melanoma growth and lymph node spread. (a) Response of allografts to drug treatment (n⩾4) on day 3 of NSC23766 or EHT1864 treatment (P<0.05). (b) Drug-treated allografts stained with H&E, anti-dopachrome tautomerase (DCT), and anti-BrdU. (c) BrdU-positive cells per field (⩾3 tumors). (d) Western blot of vehicle/tamoxifen (Tam)-treated allografts. (e) Rac1 activity and western blot of vehicle-, NSC23766-, or EHT1864-treated allografts. (f) Left inguinal lymph node and (g) average lymph node mass for drug-treated mice (n⩾4). (h) Lymph nodes with anti-DCT or with anti-Ki67. L=lymph node tissue; M=melanocytes. (i) Percentage of mice with DCT-positive lymph nodes. Error bars show mean±SEM, **P<0.01 and *P<0.05 by t-test. Bars=50 μm. H&E, hematoxylin and eosin.
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fig6: Rac1 activity is required for NRasQ61K-induced melanoma growth and lymph node spread. (a) Response of allografts to drug treatment (n⩾4) on day 3 of NSC23766 or EHT1864 treatment (P<0.05). (b) Drug-treated allografts stained with H&E, anti-dopachrome tautomerase (DCT), and anti-BrdU. (c) BrdU-positive cells per field (⩾3 tumors). (d) Western blot of vehicle/tamoxifen (Tam)-treated allografts. (e) Rac1 activity and western blot of vehicle-, NSC23766-, or EHT1864-treated allografts. (f) Left inguinal lymph node and (g) average lymph node mass for drug-treated mice (n⩾4). (h) Lymph nodes with anti-DCT or with anti-Ki67. L=lymph node tissue; M=melanocytes. (i) Percentage of mice with DCT-positive lymph nodes. Error bars show mean±SEM, **P<0.01 and *P<0.05 by t-test. Bars=50 μm. H&E, hematoxylin and eosin.
Mentions: Although Rac function is required for KRasG12D-induced primary squamous cell skin and lung tumor initiation (Kissil et al., 2007; Samuel et al., 2011), the role of Rac1 in NRasQ61K-induced tumor growth and invasiveness in vivo has not been addressed. We transplanted #11 NRasQ61K Rac1 f/f melanocytes subcutaneously into the left flank of nude mice (CD-1). The growth of Rac1-deleted allografts on day 1–3 of tamoxifen treatment was similar to vehicle, likely because it requires 3 days to deplete endogenous Rac1 (Li et al., 2011). However, allograft growth halted at day 5 after tamoxifen treatment (Figure 6a). BrdU incorporation was reduced by 60% in Rac1-deleted allografts (Figure 6b and c). Pak2 (Mouse melanocytes do not express Pak1/3 (Li et al., 2011).), Erk1/2, and AKT activation were reduced in Rac1-deleted allografts. Although the other Rac1-dependent signaling pathways (JNK, p38, and NF-κB; Jaffe and Hall, 2005) remained unchanged (Figure 6d). Rac-specific inhibitors (NSC23766 or EHT1864) showed strong inhibition of Rac1 activity in vivo (Figure 6e) and significant inhibition of allografts growth (Figure 6a) without inducing body-weight loss (Supplementary Figure S5A online). Proliferation was decreased by about 50% (Figure 6b). Consistent with Rac1 deletion results, both NSC23766- and EHT1864-treated allografts showed a strong decrease in Pak2, Erk1/2, and AKT activation (Figure 6e). Interestingly, analysis of vehicle-treated mice systematically revealed pigmentation of left axillary and inguinal skin-draining lymph nodes (Figure 6f and Supplementary Figure S5B online), a characteristic of the metastatic process. Tumor-draining lymph nodes were enlarged (Figure 6f and g and Supplementary Figure S5B online), and contained large numbers of dopachrome tautomerase-positive pigmented proliferating melanocytic cells (Figure 6h) and disrupted architecture (Supplementary Figure S5C online). However, in tamoxifen, NSC23766- or EHT1864-treated mice, lymph nodes retained their normal architecture and weight (Figure 6f and g and Supplementary Figure S5B, C online). All vehicle-treated mice (6/6) had melanocytes in lymph nodes (Figure 6h and i, Supplementary Figure S5C online). Whereas, none of mice (0/6) treated with tamoxifen and only 1 in 4 mice treated with either NSC23766 or EHT1864 showed melanocytes in lymph nodes (Figure 6h and i, Supplementary Figure S5C online). Together, these data indicate that Rac1 activity is required for NRasQ61K-induced tumor growth and lymph node spread in vivo, which suggests that Rac1 may be a valid therapeutic target against NRasQ61K-induced metastatic melanoma.

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