RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

Abstract

NRAS and its effector BRAF are frequently mutated in melanoma. Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma. Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance. We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma. After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases. Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation. These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.

Figure 1. Effect of early deletion of BRAF and/or CRAF on hyperpigmentation and melanoma formation induced by NRAS^Q61K.

(a) Representative pictures of depilated back skin from 6-month-old mice with the indicated genotypes compared to control NRAS (Tyr::NRAS^Q61K/^o;Ink4a^+/−) and wild-type mice showing the effect of CRAF, BRAF or BRAF/CRAF loss on hyperpigmented phenotype. (b) High-magnification pictures of bottom part of depilated back skin. Skin lesions are indicated by white arrows. (c) Haematoxylin and eosin (H&E) staining of representative back skin histological sections from 6-month-old control, mutant and wild-type mice. Typical melanin deposits and melanocyte clusters are shown in the papillary dermis (black arrowheads) and in the subcutaneous fat layer (arrows). Scale bar, 100 μm. (d) X-gal staining of representative skin sections from 10-day-old mice. Melanocytes are observed in papillary dermis (arrow) and in the shaft and the bulb of the hair follicule (black and open arrowheads, respectively). Scale bar, 100 μm. (e) Number of pigmented spots per cm² on bottom back skin of 6-month-old mice (b). (f) Number of dermal melanocytes per microscopic field (× 10 objective) in skin sections from 10-day-old mice (d). (g) Quantification of pERK-positive melanocytes in the skin from RAS control (Tyr::NRAS^Q61K/^o; Ink4a^+/−; Tyr::Cre/^o; Dct::LacZ/^o), CRAF KO (Braf^+/+; Craf^f/f; Tyr::NRAS^Q61K/^o; Ink4a^+/−; Tyr::Cre/^o; Dct::LacZ/^o) or BRAF KO (Braf^f/f; Craf^+/+; Tyr::NRAS^Q61K/^o; Ink4a^+/−; Tyr::Cre/^o; Dct::LacZ/^o) mice at P10. (h) Kaplan–Meier curves of melanoma incidence. In all, 21% of CRAF-deficient mice (7 of 33) develop melanoma with a latency of 15.7±1.8 months compared to 53.5% of control mice (31 of 58) in 10.8±2.7 months. No melanoma was observed in BRAF-deficient or BRAF/CRAF-deficient mice (17 and 21 mice, respectively, per cohort). **P value<0.01, ***P value<0.001 compared by Student's t-test. ns, not significant. All data are represented as mean±s.d. Overall, 123 males on a SV129/C57Bl6 mixed genetic background were used for a–c,e,h at the indicated age. Forty males and females on a SV129/C57Bl6 mixed genetic background were used for d,f,g at the p10.

Figure 2. RAF signalling is required for cell proliferation and tumour growth in NRAS^Q61K-induced murine melanoma.

(a) A melanoma from an untreated Braf^f/f;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o mouse was cut into small pieces and subcutaneously grafted into two groups of nude mice that were treated either with tamoxifen or vehicle for 2 weeks. The effect on tumour growth was assessed by measuring tumour volume over a 6-week period. Tumour volumes are plotted relative to the initial volume at the start of treatment. This experiment is representative of three independent experiments requiring 48 Swiss Nu/Nu females (6-week-old) for one primary tumour from a 1-year-old female on a SV129/C57Bl6 mixed genetic background. (b) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels (pMEK and pERK, respectively) in protein lysates from culture in c on days 4 and 7 of 4OHT treatment compared to DMSO-treated culture. Total MEK, total ERK and β-actin are shown as a loading control. (c) Growth curve analysis of melanoma cell culture established from an untreated Braf^f/f;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o primary mouse tumour in response to 4OHT or DMSO for 9 days. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. (d) Cell cycle analysis by FACS from culture in c on day 6 of 4OHT treatment compared to DMSO-treated culture. Data are the mean value of three independent experiments. *P value <0.05 and **P value <0.01 compared by Student's t-test. ns, not significant. All data are represented as mean±s.d.

Figure 3. Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS^Q61K-induced murine melanoma.

(a,e) A melanoma from an untreated Braf^f/f;Craf^+/+;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o or Braf^+/+;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o mouse (a,e, respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a. These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. (b,f) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a,e, respectively. β-actin is used as a loading control. (c,g) Growth curve analysis of melanoma cell culture established from an untreated Braf^f/f;Craf^+/+;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o or Braf^+/+;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o primary mouse tumour (c,g, respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c. (d,h) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c,g respectively, as in Fig. 2b. All data are represented as mean±s.d.

Figure 4. NRAS-binding and kinase activity of BRAF and CRAF in NRAS-induced melanoma.

(a,b) PLA showing NRAS/BRAF and NRAS/CRAF complexes in BRAF^Δ/Δ and CRAF^Δ/Δ murine melanoma cultures compared to the parental cultures (a,b, respectively). NRAS/BRAF and NRAS/CRAF interactions in cultures established from Fig. 3c,g were visualized as red dots by using a fluorescent microscope. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Box plots represent the average number of dots per cell. Lower, median and upper quartiles are shown, with whiskers extending to the lowest and highest values. Scale bar, 100 μm. (c,d) BRAF and CRAF in vitro kinase assays in BRAF^Δ/Δ or CRAF^Δ/Δ cultures compared to parental control cultures (c,d, respectively). BRAF or CRAF were immunoprecipitated and their intrinsic kinase activity against kinase-inactive MEK was determined by western blotting using anti-pMEK antibody. Immune complexes and total extracts were immunoblotted with anti-BRAF and anti-CRAF antibodies. β-actin was used as a loading control. BRAF^Δ/Δ and CRAF^Δ/Δ refer to Braf^Δ/Δ;Craf^+/+;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o and Braf^+/+;Craf^Δ/Δ;Tyr::NRAS^Q61K/^o; Ink4a^+/−; Tyr::CreERT2/^o, respectively and control to the corresponding parental cultures.

Figure 5. NRAS-mutated human melanoma cells require both BRAF and CRAF for ERK activation and proliferation.

(a) Western blot analysis of BRAF and CRAF protein expression and pERK activation in NRAS-mutated human melanoma cell lines (WM1361, WM852 and Sbcl2) transfected with the scrambled control (−) or short interfering RNA to BRAF and/or CRAF (siBRAF1 and siCRAF1). Total ERK and β-actin are used as a loading control. (b) Proliferation rate in WM1361, WM852 and Sbcl2 cells transfected with scrambled control (scr), BRAF siRNA (B1), CRAF siRNA (C1) or BRAFsiRNA/CRAFsiRNA (B1C1) was measured after BrdU incorporation during 3 hours. **P-value<0.01 and ***P-value<0.001 compared by Student's t-test. All data are represented as mean±s.d. (c) PLA showing NRAS/BRAF and NRAS/CRAF complexes in WM1361, WM852 and Sbcl2 cells. Numbers in white indicate the average number of dots per cell. Numbers between brackets represent s.d.'s. Scale bar, 100 μm.

Figure 6. ARAF is required for the survival NRAS^Q61K-induced murine melanoma cell lines in absence of BRAF and CRAF.

(a) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF^Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. (b) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF^Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. (c) qRT–PCR analysis of ARAF expression in BRAF/CRAF^Δ/Δ and parental control cultures. ***P value<0.001 compared by Student's t-test. (d,f) Parental control and BRAF/CRAF^Δ/Δ cultures (d,f, respectively) were infected by lentiviruses encoding control shRNA or targeting ARAF (shARAF.1; shARAF.2; shARAF.3). After puromycin selection, cells were stained with crystal violet. (e,g) Western blot analysis of ARAF, BRAF and CRAF protein expression levels and pERK activation in parental control and BRAF/CRAF^Δ/Δ cultures (e,g, respectively) after infection by control or ARAF shRNA encoding viruses and selection. Total ERK and β-actin are used as a loading control. Control refers to Braf^f/f;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o parental culture and BRAF/CRAF^Δ/Δ refers to Braf^Δ/Δ;Craf^Δ/Δ;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o double knockout culture. (h) Cell counting of parental control and BRAF/CRAF^Δ/Δ cultures infected with lentiviruses encoding control or ARAF shRNA on days 1, 6 and 10 after puromycin selection. (i) Proliferation rate in parental control and BRAF/CRAF^Δ/Δ cultures infected by lentiviruses encoding control or ARAF shRNA was measured after BrdU incorporation during 9 h. ***P value<0.001 compared by Student's t-test. ns, not significant. All data are represented as mean±s.d.

Figure 7. Vemurafenib induces ERK paradoxical activation in BRAF- and CRAF-decifient NRAS-induced melanoma by increasing ARAF kinase activity.

(a) Western blot analysis of ERK activation (pERK) and ARAF, BRAF and CRAF protein expression in parental control and BRAF/CRAF^Δ/Δ cultures after treatment with 1 μM Vemurafenib (Vemu) or DMSO during 1 h. Total ERK and β-actin are used as loading controls. (b) ARAF in vitro kinase assays in BRAF/CRAF^Δ/Δ cultures after treatment with 1 μM Vemurafenib or DMSO during 1 h. ARAF was immunoprecipitated and its intrinsic kinase activity was measured on kinase-inactive MEK as substrate by western blotting using anti-pMEK antibody. Immune complexes and total cell extracts were immunoblotted with anti-ARAF, pMEK, MEK, pERK and ERK antibodies. β-actin was used as a loading control.