Supplementary Figures 1-2 from In Melanoma, RAS Mutations Are Accompanied by Switching Signaling from BRAF to CRAF and Disrupted Cyclic AMP Signaling

Supplementary Figures 1-2 from Tumor Necrosis Factor-α Blocks Apoptosis in Melanoma Cells when BRAF Signaling Is Inhibited

Supplementary Methods, Figures 1-9 from BRAF Inactivation Drives Aneuploidy by Deregulating CRAF

Supplementary Figures 1-4 from A Mouse Model of Melanoma Driven by Oncogenic KRAS

Supplementary Tables 1-2 from A Mouse Model of Melanoma Driven by Oncogenic KRAS

The mitogen-activated proteinkinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.Activation of transforming growth factor (TGF-) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF- signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitorinduced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval 38.65% to 155.50%, P .005).Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

cutaneous squamous cell carcinomas 7,12-dimethylbenz-(a)anthracene keratoacanthomas mitogen-activated protein kinase 12-O-tetradecanoyl-phorbol-13-acetate 5-fluorouracil TO THE EDITOR The protein kinase BRAF regulates cell growth through the mitogen-activated protein kinase (MAPK) pathway. In about half of the melanomas, BRAF is mutated and acts as a driver oncogene that stimulates cell proliferation, survival, and tumor progression (Davies et al., 2002Davies H. Bignell G.R. Cox C. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8271) Google Scholar; Gray-Schopfer et al., 2007Gray-Schopfer V. Wellbrock C. Marais R. Melanoma biology and new targeted therapy.Nature. 2007; 445: 851-857Crossref PubMed Scopus (1042) Google Scholar. The anti-BRAF drugs vemurafenib (PLX4032/RG7204) and dabrafenib (GSK2118436) achieve objective clinical responses in about 60% of melanoma patients whose tumors express mutant BRAF (Flaherty et al., 2010Flaherty K.T. Puzanov I. Kim K.B. et al.Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2953) Google Scholar; Chapman et al., 2011Chapman P.B. Hauschild A. Robert C. et al.Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6115) Google Scholar; Sosman et al., 2012Sosman J.A. Kim K.B. Schuchter L. et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1769) Google Scholar, validating these drugs as a therapeutic option for BRAF-mutant melanoma patients. An unexpected side effect of vemurafenib is that it induces keratoacanthomas (KA) and well-differentiated cutaneous squamous cell carcinomas (cuSCCs) in ∼26% of patients (Flaherty et al., 2010Flaherty K.T. Puzanov I. Kim K.B. et al.Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2953) Google Scholar; Sosman et al., 2012Sosman J.A. Kim K.B. Schuchter L. et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1769) Google Scholar. This is because, although BRAF inhibitors block MAPK signaling in cells harboring BRAF mutations, they activate the pathway in cells expressing mutant RAS (Rat sarcoma) or when RAS is activated by receptor tyrosine kinases (Hatzivassiliou et al., 2010Hatzivassiliou G. Song K. Yen I. et al.RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.Nature. 2010; 464: 431-435Crossref PubMed Scopus (1280) Google Scholar; Heidorn et al., 2010Heidorn S.J. Milagre C. Whittaker S. et al.Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.Cell. 2010; 140: 209-221Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar; Poulikakos et al., 2010Poulikakos P.I. Zhang C. Bollag G. et al.RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010; 464: 427-430Crossref PubMed Scopus (1396) Google Scholar. Critically, 21–57% of the nonmelanoma skin lesions that develop in vemurafenib-treated patients carry somatic mutations in HRAS or KRAS (Oberholzer et al., 2011Oberholzer P.A. Kee D. Dziunycz P. et al.RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors.J Clin Oncol. 2011; 30: 316-321Crossref PubMed Scopus (345) Google Scholar; Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We recently used a mouse two-stage skin carcinogenesis model to investigate how BRAF inhibitors drive the development of these nonmelanoma skin lesions (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. FVB/N mice were treated with a single topical application of the carcinogen 7,12-dimethylbenz-(a)anthracene (DMBA), which induces HRAS Q61L mutations in keratinocytes. The mice were then treated weekly with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and daily with oral doses of PLX4270 (see Supplementary Experimental Procedures online), a BRAF inhibitor that is preferred for mouse studies because of its superior oral bioavailability (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We demonstrated that PLX4720 accelerated the growth of squamoproliferative lesions in DMBA/TPA-treated mice but did not induce lesions in mice treated with DMBA alone (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. These data demonstrated that vermurafenib is not a tumor promoter per se; rather, it acts by accelerating the progression of preexisting, premalignant lesions in susceptible individuals. Download .pdf (.05 MB) Help with pdf files Supplementary Information It is not known whether the cuSCCs that develop in vemurafenib-treated patients have metastatic potential different from cuSCCs that develop in the absence of BRAF inhibition, because in both the clinical settings these lesions are treated by surgical excision. However, advanced-stage melanoma patients on BRAF inhibitors can develop multiple eruptive lesions early during the course of BRAF inhibitor therapy and these can be challenging to manage surgically. We therefore sought to develop a noninvasive approach to treat these particular patients. Previous studies have shown that BRAF inhibitors accelerate the proliferation of RAS-transformed cells (Hatzivassiliou et al., 2010Hatzivassiliou G. Song K. Yen I. et al.RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.Nature. 2010; 464: 431-435Crossref PubMed Scopus (1280) Google Scholar; Heidorn et al., 2010Heidorn S.J. Milagre C. Whittaker S. et al.Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.Cell. 2010; 140: 209-221Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar; Poulikakos et al., 2010Poulikakos P.I. Zhang C. Bollag G. et al.RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010; 464: 427-430Crossref PubMed Scopus (1396) Google Scholar; Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We confirmed that the nonmelanoma skin lesions in DMBA/TPA/PLX4720-treated mice have a high proliferative index by showing that they have a high mitotic index (5 × 10 high-power fields) and strong positive staining for Ki-67 (Figure 1a), and thus we investigated whether antiproliferative drugs could induce their regression. 5-Fluorouracil (5-FU) is an antimetabolite that blocks DNA synthesis and inhibits cell proliferation. Although topical application of 5-FU should be restricted to an area no greater than 500cm2 (approximately the area of an extended hand) (MedaPharmaceuticals, 2012MedaPharmaceuticals Summary of Product Characteristics. 2012http://www.medicines.org.uk/EMC/medicine/6219/SPC/Efudix+Cream/Google Scholar, 5-FU is inexpensive and achieves objective clinical responses in 46–87% of cases of solar keratosis, lesions that are the precursor of invasive cuSCC (Gray and Meland, 2000Gray R.J. Meland N.B. Topical 5-fluorouracil as primary therapy for keratoacanthoma.Ann Plast Surg. 2000; 44: 82-85Crossref PubMed Scopus (28) Google Scholar; Shimizu et al., 2011Shimizu I. Cruz A. Chang K.H. et al.Treatment of squamous cell carcinoma in situ: a review.Dermatol Surg. 2011; 37: 1394-1411Crossref PubMed Scopus (40) Google Scholar. We confirm that 5-FU inhibited the growth of PDV cells, an HRAS mutant keratinocyte line, with IC50 (half maximal inhibitory concentration 50) values of ∼4.43μM in the absence of PLX4720 and ∼1.67μM in its presence (Figure 1b). To test 5-FU in vivo, FVB/N mice (six animals per group) were treated with DMBA, TPA, and PLX4720 as described (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. As reported (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar, palpable lesions first appeared after 30 days, increasing in number to approximately 11 lesions per mouse in 55 days (Figure 1c). We then treated the individual lesions with topical 5% 5-FU cream twice a week. Left untreated, we have shown that the tumors continue to grow and reach tumor burden limits after approximately 90 days (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar; however, with 5-FU, we observed an immediate response, with tumor regression leading to complete remission within 25 days (Figure 1c and d). On the basis of these observations, we tested the efficacy of 5-FU in two melanoma patients who were being treated with the BRAF inhibitor dabrafenib (GSK2118436). Both patients rapidly developed multiple eruptive lesions consistent with actinic keratosis and/or cuSCC (Figure 2a and b). Individual lesions were treated with 5% 5-FU cream twice a day, respecting recommended safety guidelines for total skin treatment area. Photo-documented lesions presenting the characteristics of KA and cuSCCs at various stages responded to 5-FU, whereas untreated concurrent lesions progressed and after biopsy were confirmed to be bona fide cuSCCs (Figure 2b and c). During follow-up periods of 11 and 18 months, respectively, none of the treated lesions in patients 1 and 2 recurred. As noted in these anecdotal cases, nascent lesions tended to respond more quickly compared with the established hyperkeratotic lesions. Secondary malignancies induced by conventional chemotherapies generally take many years to develop (Curtis et al., 1992Curtis R.E. Boice Jr., J.D. Stovall M. et al.Risk of leukemia after chemotherapy and radiation treatment for breast cancer.N Engl J Med. 1992; 326: 1745-1751Crossref PubMed Scopus (383) Google Scholar, but the nonmelanoma skin lesions induced by BRAF inhibitors develop within weeks of initiating treatment (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. Our previous studies show that BRAF inhibitors are not carcinogens per se; rather, they act to accelerate the development of preexisting subclinical lesions in susceptible patients. The approval of vemurafenib raises a pressing clinical need to provide treatment guidelines to manage these secondary lesions in patients for whom surgery is an undesirable option. The observation that BRAF inhibitors drive proliferation of RAS-mutant keratinocytes led us to test the efficacy of antiproliferative agents. We show that 5-FU provides a safe, noninvasive, inexpensive, and effective alternative to surgical intervention in clinical cases in which multiple surgeries are difficult or in cases in which lesions are incipient. Details concerning the ethics of animal and human studies are provided in the Supplementary Materials online. We thank Mr Eric Ward, Dr Kay Savage (ICR), and Ms Annette Lane (ICR) for their technical assistance with immunohistochemical preparations. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Correction to: Journal of Investigative Dermatology (2013) 133, 274–276; doi:10.1038/jid.2012.268; published online 16 August 2012Journal of Investigative DermatologyVol. 133Issue 6PreviewTopical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell Carcinoma Full-Text PDF Open Archive

Topical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell Carcinoma Following the publication of this article, the authors noted that the second affiliation for Dr Amaya Viros is incomplete. The work was performed at Seccio Dermatologia, Departament de Medicina, Hospital Universitari Vall d’Hebron, Universitat Autonoma de Barcelona, Spain. The authors regret the error. Topical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell CarcinomaJournal of Investigative DermatologyVol. 133Issue 1Previewcutaneous squamous cell carcinomas Full-Text PDF Open Archive

BACKGROUND Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

ethanol KIT ligand metabotropic glutamate receptor-1 TO THE EDITOR Transgenic mouse models of melanoma driven by melanocyte-specific expression of oncogenes have been developed to study gene–gene and gene–environment interactions (Damsky and Bosenberg, 2010Damsky Jr., W.E. Bosenberg M. Mouse melanoma models and cell lines.Pigment Cell Melanoma Res. 2010; 23: 853-859Crossref PubMed Scopus (22) Google Scholar). Oncogene expression can be constitutive or inducible, but is generally insufficient for melanomagenesis unless tumor-suppressor genes are deleted (Pten, Tp53, Cdkn2a), or the mice are exposed to mutagens such as UV light. Critically, in mice melanoma is largely restricted to the dermal layers of the skin and the epidermis is rarely involved, whereas human cutaneous malignant melanoma generally develops in the epidermis and then invades the deeper layers of skin. This difference presumably occurs because mouse melanocytes are predominantly located in the follicles and dermis and provide pelt pigmentation, whereas human melanocytes are predominantly epidermal and provide protection from UV light. Melanocyte location in the skin is controlled by the KIT ligand (KITL). Human keratinocytes express KITL throughout life and melanocytes persist in the epidermis. In postnatal mouse skin, KITL expression is restricted to the hair matrix (Yoshida et al., 2001Yoshida H. Kunisada T. Grimm T. et al.Review: melanocyte migration and survival controlled by SCF/c-kit expression.J Investig Dermatol Symp Proc. 2001; 6: 1-5Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and the melanocytes are largely located in the follicles. However, if mouse keratinocytes are engineered to express KITL using the keratin-14 gene promoter (K14-Kitl), a proportion of the melanocytes remain in the epidermis throughout life (Kunisada et al., 1998Kunisada T. Lu S.Z. Yoshida H. et al.Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor.J Exp Med. 1998; 187: 1565-1573Crossref PubMed Scopus (162) Google Scholar). Using this approach, melanoma driven by tyrosinase-expressed Q61KNras (Tyr-Q61KNras), p19Arf deletion, and neonatal UV light exposure can be translocated to the mouse epidermis (Walker et al., 2011Walker G.J. Soyer H.P. Handoko H.Y. et al.Superficial spreading-like melanoma in Arf(-/-)::Tyr-Nras(Q61K)::K14-Kitl mice: keratinocyte kit ligand expression sufficient to “translocate” melanomas from dermis to epidermis.J Invest Dermatol. 2011; 131: 1384-1387Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The protein kinase BRAF is mutated in about half of human melanomas and we developed a V600EBraf-driven mouse melanoma model using the tamoxifen-activated Cre-recombinase/loxP technology (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). V600EBraf-driven melanomas are exclusively dermal (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar); therefore we tested whether a membrane-bound version of KITL under the control of the K14 promoter could translocate these tumors to the epidermis. All animal procedures were approved by the local animal ethics committee (ICR) in accordance with the Home Office regulations under the Animals (Scientific Procedures) Act of 1986 (Workman et al., 2010Workman P. Aboagye E.O. Balkwill F. Committee of the National Cancer Research InstituteGuidelines for the welfare and use of animals in cancer research.Br J Cancer. 2010; 102: 1555-1577Crossref PubMed Scopus (1038) Google Scholar). When LSL-V600EBraf::Tyr-CreERT2 (henceforth V600EBraf) mice were crossed with K14-Kitl mice, we observed S100-positive melanocytes in the basal layers of the interfollicular epidermis (Figure 1a) and melanin capping of the nuclei in the keratinocytes (Figure 1b). These features were absent in V600EBraf mice (Figures 1a and b). As previously described (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar), V600EBraf induced mild hyperpigmentation of the ears and feet (Figure 1c). Notably, KITL also induced increased pigmentation of the feet and ears and V600EBraf further enhanced this effect (Figure 1c). V600EBraf also induced nevi comprising pigmented epithelioid and dendritic melanocytes in the deep reticular dermis and hypodermis of the skin (Figure 1d; see Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). However, in V600EBraf::K14-Kitl mice, V600EBraf induced highly cellular nevi presenting large populations of pigment-laden epithelioid and dendritic melanocytes that occasionally formed ill-defined nests (Figure 1d; Supplementary Figure S1 online). Nevi in V600EBraf::K14-Kitl mice were located in the papillary and reticular dermis and were separated from the epidermis by a narrow Grenz zone. They occasionally reached the hypodermis surrounding the adnexae, but did not present an epidermal component (Figure 1d; Supplementary Figure S1 online). Download .pdf (2.8 MB) Help with pdf files Supplementary Information V600EBraf also induced rapidly growing skin tumors in V600EBraf and V600EBraf::K14-Kitl mice (Figure 2a). The tumors from the two strains of mice were macroscopically similar, but developed with a significantly lower latency in the V600EBraf::K14-Kitl mice (4.8 compared with 12.5 months; log-rank test P<0.0001; Figure 2b). V600EBraf::K14-Kitl mice also showed greater tumor penetrance (100% in 7 months compared with 70% in 24 months; Figure 2b) and developed a greater number of tumors (average 3 compared with 0.78; Figure 2c). Using PCR, we showed that LSL-V600EBraf was recombined in tumors from V600EBraf::K14-Kitl mice (Figure 2d), and using RT-PCR we confirmed the expression of the melanocytic markers tyrosinase, silver (gp100), Mitf-M, Dct (Trp2), Sox10, Pax3, and Pou3f2 (Brn2; Figure 2e). S100 expression was confirmed by immunostaining (Figure 2f). These data are consistent with a diagnosis of malignant melanoma. The smaller, less advanced tumors presented an intact epidermis and pigmented melanocytes in the reticular dermis, suggestive of residual intradermal nevi (Figure 2g). These lesions were generally amelanotic, paucicellular, and composed of spindle/dendritic melanocytes occupying the deeper layers of skin (Figure 2h). The larger lesions were generally asymmetric, poorly circumscribed, and within the superficial dermis (Figure 2i). Prominent ulceration and effacement of the epidermis were common (Figure 2j), as was locally destructive invasion into the subcutaneous layers (Figure 2i and k). These tumors were also generally amelanotic, with rare pigmented melanocytes occupying the superficial aspects. The deeper aspects were composed of atypical spindle melanocytes occasionally arranged in storiform patterns (Figure 2l). Mitotic figures were abundant (Figure 2m). Crucially, as in V600EBraf mice (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar), the tumors in V600EBraf::K14-Kitl mice were exclusively dermal; neither epidermal invasion nor a junctional component was observed (Figures 2g, i, j, and l), and we note that K14-Kitl also failed to translocate melanoma driven by the metabotropic glutamate receptor-1 (mGluR1) to the epidermis (Abdel-Daim et al., 2010Abdel-Daim M. Funasaka Y. Komoto M. et al.Pharmacogenomics of metabotropic glutamate receptor subtype 1 and in vivo malignant melanoma formation.J Dermatol. 2010; 37: 635-646Crossref PubMed Scopus (23) Google Scholar). We set out to translocate V600EBraf tumors to the epidermis using K14-Kitl. We cannot discount the possibility that rare melanoma cells did colonize the epidermis, but overall the V600EBraf::K14-Kitl melanomas appeared to be exclusively dermal. This may be because, although melanocytes do occupy the epidermis of V600EBraf::K14-Kitl mice, ∼90% of the melanocytes remain dermal (Supplementary Figure S2 online); therefore, the probability of developing dermal melanoma may simply be higher. However, we examined 30 tumors and ∼50 nevi and found no evidence of epidermal lesions, suggesting that these are not chance events and that even when expressing KITL, the mouse epidermis does not provide the microenvironment necessary for V600EBraf-driven melanomagenesis. There are several plausible explanations as to why epidermal melanoma developed in the Tyr-Q61KNras::p19Arf-/−::K14-Kitl, but not in the V600EBraf::K14-Kitl or mGluR1::K14-Kitl mice. UV light is critical for efficient melanomagenesis in Tyr-Q61KNras::p19Arf−/− mice (Ferguson et al., 2010Ferguson B. Konrad Muller H. Handoko H.Y. et al.Differential roles of the pRb and Arf/p53 pathways in murine naevus and melanoma genesis.Pigment Cell Melanoma Res. 2010; 23: 771-780Crossref PubMed Scopus (36) Google Scholar) and has been shown to recruit melanocytes to the epidermis of neonatal mice (Walker et al., 2009Walker G.J. Kimlin M.G. Hacker E. et al.Murine neonatal melanocytes exhibit a heightened proliferative response to ultraviolet radiation and migrate to the epidermal basal layer.J Invest Dermatol. 2009; 129: 184-193Crossref PubMed Scopus (41) Google Scholar). Perhaps the exogenous KITL traps these newly recruited melanocytes and simply increases the chance that epidermal melanoma will develop, or perhaps it provides a microenvironment that supports melanomagenesis in this population in this location. Alternatively, differences in the timing of oncogene expression (prenatal in Tyr-Q61KNras::p19Arf−/− mice, induced in juveniles in V600EBraf::K14-Kitl and mGluR1::K14-Kitl mice), whether p19Arf is deleted or not, or the genetic background of the mice (pure C57BL6 compared with mixed FVB/C57BL6) could account for the differences. Clearly, these are testable hypotheses and, although the KITL did not translocate V600EBraf melanoma to the epidermis, it increased tumor penetrance and reduced tumor latency and does therefore provide a considerably more robust and versatile mouse model of melanoma. This work was supported by Cancer Research UK (refs: C107/A10433) and the Institute of Cancer Research. JSR-F is in part funded by the Breakthrough Breast Cancer Research Centre and is a recipient of the 2010 CRUK Future Leaders Prize. We acknowledge NHS funding to the NIHR Biomedical Research Centre. We thank Dr B Jack Longley (University of Wisconsin, Madison) for providing the K14-Kitl mice and also thank Mr Eric Ward (ICR), Dr Kay Savage, and Ms Annette Lane (ICR) for their technical assistance with histological preparations. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

Abstract The antidiabetic drug metformin has antitumor activity in a variety of cancers because it blocks cell growth by inhibiting TORC1. Here, we show that melanoma cells that are driven by oncogenic BRAF are resistant to the growth-inhibitory effects of metformin because RSK sustains TORC1 activity even when AMP-activated protein kinase (AMPK) is activated. We further show that AMPK targets the dual-specificity protein phosphatase DUSP6 for degradation and this increases ERK activity, which then upregulates the VEGF-A protein. Critically, this drives angiogenesis and accelerates the growth of BRAF-driven tumors in mice. Unexpectedly, however, when VEGF signaling is inhibited, instead of accelerating tumor growth, metformin inhibits tumor growth. Thus, we show that BRAF-driven melanoma cells are resistant to the antigrowth effects of AMPK and that AMPK mediates cell-autonomous and cell-nonautonomous effects that accelerate the growth of these cells in vivo. Significance: Metformin inhibits the growth of most tumor cells, but BRAF-mutant melanoma cells are resistant to metformin in vitro, and metformin accelerates their growth in vivo. Unexpectedly, VEGF inhibitors and metformin synergize to suppress the growth of BRAF-mutant tumors, revealing a combination of drugs that may be effective in these patients. Cancer Discov; 2(4); 344–55. ©2012 AACR. This article is highlighted in the In This Issue feature, 288

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca2+, stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

We recently demonstrated that expression of (V600E)Braf in mature mouse melanocytes induces melanoma. Here, we show that expression of (V600E)Braf using the tyrosinase promoter leads to an unexpected embryonic lethality, with the animals dying before, at, or shortly after birth. The mice suffer from a range of developmental defects in the skin, the brain, the eyes and the heart, tissues that are normally colonized by melanocytes. We show that the (V600E)Braf expressing cells are potential melanocytic precursors that are fully transformed, suggesting that (V600E)Braf stimulates proliferation and blocks differentiation of these cells. Our data suggests that the presence of these cells in the organs that are normally occupied by melanocytes leads to severe developmental disruption, resulting in catastrophic defects and leading to death of the individual.