BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma Lin Feng a, 1, Jun Li a, 1, Xin Bu b, 1, Yan Zuo c, Liangliang Shen b, **, Xuan Qu a, * a

Shaanxi University of Chinese Medicine, Xianyang, 712046, China The State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi’an, 710032, China c Affiliated Hospital of Xizang Minzu University, Xianyang, 712082, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2019 Accepted 19 December 2019 Available online xxx

BRAFV600E mutation is frequently observed in melanoma, and contributes to tumor malignancy. Despite inhibition of BRAF causes a profound cell growth inhibition and a strong clinical benefit in BRAFV600E melanoma, acquired drug resistance is still the major hurdle. In this study, we demonstrate that BRAFV600E drives cell growth and glycolysis in melanoma cells but does so by a previously unappreciated mechanism that involves direct induction of Skp2. Skp2 is highly expressed in melanoma tissues and particularly in tissues with BRAFV600E mutation. The inhibition of BRAFV600E by either siRNA or inhibitor vemurafenib suppressed Skp2 expression and cell growth. Mechanistic study shows that BRAFV600E suppression of Skp2 is dependent on c-Myc transcription factor via specifically bounding to the E-box region on SKP2 promoter. Further, the overexpression of Skp2 resulted in a markedly increase in cell growth, cell cycle progression and glycolysis which were repressed by BRAFV600E inhibition. Supporting the biological significance, Skp2 is specifically correlated with poor patient outcome in BRAFV600E but did not in BRAFWT melanomas. Thus, as a downstream target of BRAFV600E, Skp2 is critical for responses to BRAF inhibition, indicating targeting Skp2 might be a promising strategy for the treatment of BRAFi resistant melanomas. © 2020 Elsevier Inc. All rights reserved.

Keywords: BRAFV600E c-Myc Skp2 Melanoma Transcriptional regulation

1. Introduction Melanoma is an aggressive disease with a rapidly increasing incidence and mortality rate globally [1,2]. The increased glycolysis, which supports the increased energetic and biosynthetic demands of tumor cells, is observed in various cancers, including melanoma [3]. The specific molecular changes that involved in driving the metabolic reprogramming and tumor progression have been fully addressed in melanoma cells, such as the mutation of protooncogene BRAF [4].

Abbreviations: Skp2, S-phase kinase-associated protein 2; WT, wild type; Vem, vemurafenib; BRAFi, BRAF inhibitor; TCGA, the cancer genome atlas. * Corresponding author. ** Corresponding author. The State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Shaanxi, Xi’an, 710032, China. E-mail addresses: [email protected] (L. Shen), [email protected] (X. Qu). 1 These authors contributed equally to this work.

As a member of the serine kinase family, BRAF functions as an immediate downstream effector of RAS, and further activates MEKERK signaling, which contributes to the cell survival/proliferation [5]. The mutation of BRAF is particularly prevalent in melanoma where they occur at a frequency of ~60% [6]. At present, more than 50 mutation sites have been identified in BRAF gene, in which V600E amino acid substitution is the most common form, accounting for more than 90% of BRAF mutations [6,7]. BRAFV600E mutation results in the constitutive activation of BRAF-MEK-ERK signaling and eventually drives tumor formation and progression [8]. Numerous studies show that targeting oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAFV600E-driven tumors, potentially through inhibiting a network of BRAF-regulated transcription factors, including c-Myc and HIF-1a [4,9]. Especially, the BRAF inhibitor (BRAFi) vemurafenib and dabrafenib were shown striking effects on the treatment of BRAFV600E melanoma, accompanied with the suppression of tumor glucose uptake and glycolysis [10,11]. However, the acquired BRAFi resistance have been identified within a matter of a few

https://doi.org/10.1016/j.bbrc.2019.12.085 0006-291X/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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months after BRAF kinase targeted therapy [12,13]. Although several mechanisms of acquired BRAFi resistance have been identified, which is broadly categorized as either dependent or independent of MEK-ERK signaling, the basis for acquired resistance remains unknown in over 40% of melanomas [14,15]. S-phase kinase-associated protein 2 (Skp2) is an F-box protein as a subunit in SCF (SKP1- CUL1-F-box protein) ubiquitin E3 ligase complex. The crucial role of Skp2 has been validated in the regulation of cell survival through either promoting the ubiquitination and degradation of CDK inhibitors or increasing the oncogenic signals by activating Akt or c-Myc [16e19]. Aberrant Skp2 expression has been observed in multiple cancers and contributes to tumor progression [16e19]. We and others previously reported that elevated Skp2 is required for c-Myc or Akt dependent induction of epithelial-mesenchymal transition (EMT) in melanoma cells [18,20]. Here we investigate how Skp2 is involved in BRAF signaling, and reports that the constitutive activation of BRAF stimulates cell cycle progression and glycolysis to support cell growth through c-Myc dependent induction of Skp2 in BRAFV600E melanoma cells. 2. Materials and methods Immunohistochemistry staining Tissue microarrays were purchased from Servicebio (Wuhan, China) and additional human samples were collected at the Second Affiliated Hospital of Xi’an Jiaotong University. The immunohistochemistry staining assay was performed and scored as previously described [20]. The primary antibodies for anti-Skp2 (#ab68455, Abcam) and anti-c-Myc (#ab32072, Abcam) were applied. The protein levels were statistically analyzed by Student’s t-test. Linear regression and Pearson’s correlation significance were used to analyze Skp2 and c-Myc correlation. Survival analysis of TCGA patient data The Cancer Genome Atlas cBioPortal was used to determine overall patient survival in the dataset for Skin Cutaneous Melanoma (TCGA, Provisional). cMyc and Skp2 were entered as the query genes. The raw data were downloaded and survival analysis was determined with patient progression-free survival data. Patient data with Skp2 mRNA levels were used to estimate medians and bounds for upper and lower means. KaplaneMeier survival graphs were plotted, and log-rank tests were performed. Cell culture and reagents A375, A2058 and WM3211 cells were purchased from ATCC and cultured in DMEM with (Hyclone) with 10% FBS. SK-MEL-31 cells were purchased from Cobioer (Nanjing, China) and cultured in MEM (Hyclone) with 1% non-essential amino acids (Hyclone), 2 mM glutamine (Hyclone) and 10% FBS. BRAF inhibitor vemurafenib was obtained from MedCemExpress (Shanghai, China). Plasmid, siRNA and virus The human Skp2 promoter pGL4SKP2-1.2 and pGL4-SKP2-1.2 with E-box mutation were created in the previous study [20]. BRAF siRNA were purchased from Thermo Fisher Scientific (n319368). Silencer® Select Negative Control #1 siRNA was used as a negative control (4390843). Lentivirus BRAFV600E and Skp2 were packaged in Hanbio Biotechnology (China) and further stable expressed in the indicated melanoma cells. Cell based assays MTT assay, cell cycle assay, and glycolysis relevant assays were performed as described previously [21]. Skp2 promoter related assays Luciferase reporter assay and Chromatin immunoprecipitation were performed as described previously [20]. c-Myc enrichment at the Skp2 locus (forward 50 GCCTTCTCCCCGACGGAGGAAG-30 , reverse 50 -GGCTGCTGAGTGGTGACAGTAG-30 ) was determined by normalizing qPCR to the c-Myc off-target loci PFKFB3 (forward 50 -CAGGAGTGGAGTGGGACTC-30 ,

50 -CCTCTCAGAGCCCCTGTTC-30 ). Western blotting Western blotting assay was performed as previously described [20]. Primary antibodies were used at dilutions of 1:1000 for anti-BRAF (Abcam, #ab33899), anti-c-Myc (#ab32072, Abcam), anti-Skp2 (#ab68455, Abcam), 1:500 for antib-actin (A5441, Sigma). Quantitative PCR (qPCR) Extract the RNA and generate complementary DNA through GoScript Reverse Transcription System (Promega). The qPCR assay was performed as described previously [20]. Primer sequences are available on request. Statistical analysis Data are expressed as mean ± SD. Statistical analysis was performed with the SPSS10.0 software package by using Student’s t-test for independent groups. Statistical significance was based on a value of P  0.05. 3. Results 3.1. High Skp2 expression in BRAFV600E melanoma tissues predicts poor patient outcome Our group previously demonstrated that Skp2 is high expressed in metastatic melanomas and associated with EMT [20]. To further evaluate the clinical significance of Skp2 in melanoma patients, we firstly determined its expression in 84 cases of normal skin and melanoma tissues. Immunohistochemical and statistical analysis showed that Skp2 is broadly stained in melanoma tissues (Fig. 1A). A positive association of Skp2 with c-Myc has been observed (Fig. 1B), which is consistent with our previous work [20]. Intriguingly, Skp2 and c-Myc were more highly expressed in melanoma tissues with BRAFV600E mutation than BRAFWT tissues (Fig. 1A, 1C and 1D), indicating Skp2 and c-Myc might be involved in the mutant BRAF pathway. Since BRAFV600E-dependent c-Myc induction has been fully addressed [4,22], we thus sought to investigate how Skp2 plays a role in BRAFV600E-driven melanomas. In silico analysis of 262 melanoma tissues of the multidimensional data set from TCGA showed that, BRAFV600E mutation had no significant power to correlate with clinical outcome (Fig. 1E), which might be attribute to targeted drugs for the treatment of patients with BRAFV600E melanoma. However, subdividing the METABRIC data into the melanoma subtypes revealed that Skp2 correlated with poor patient outcome in BRAFV600E melanomas but did not correlate with clinical outcome in BRAFWT melanomas (Fig. 1F and 1G). Therefore, Skp2 probably be involved in BRAFV600E induction of melanoma malignancy and related to prognosis. 3.2. BRAFV600E dictates Skp2 expression To further determine the involvement of Skp2 in BRAFV600E pathway during melanoma progression, a panel of BRAFWT (WM3211 and SK-MEL-31) and BRAFV600E (A375 and A2058) human melanoma cells were introduced. In BRAFV600E cells, Skp2 protein levels were dramatically decreased in response to the siRNA-mediated knockdown of BRAF (Fig. 2A). This change was associated with significant reduction in Skp2 mRNA expression (Fig. 2B). Thus the high expression of Skp2 in melanoma cells is dependent on BRAF activity. Consistently, the overexpression of BRAFV600E in BRAFWT cells increased Skp2 protein and mRNA levels (Fig. 2C and 2D), which indicates that BRAFV600E regulates Skp2 expression in transcriptional level. To further confirm this point, melanoma cells with or without BRAFV600E mutation were treated with the BRAF inhibitor vemurafenib. Importantly, vemurafenib treatment suppressed Skp2 expression in BRAFV600E but not BRAFWT melanoma cells (Fig. 2E and 2F). Moreover, dual luciferase reporter assay indicated that Skp2 promoter activity was increased in BRAFWT cells after BRAFV600E overexpression and decreased in

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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Fig. 1. High Skp2 expression in BRAFV600E melanoma tissues predicts poor patient outcome (A) Immunohistochemical staining of c-Myc and SKP2 in human normal skin and melanoma tissues with or without BRAFV600E mutation. (B) Positive correlation between c-Myc and Skp2 expression levels with linear regression and Pearson’s correlation significance (P < 0.0001, ANOVA test). (C and D) c-Myc (C) and Skp2 (D) expression levels in normal skin and melanoma tissues with or without BRAFV600E mutation. (EeG) Silico analysis of 262 cases of melanoma tissues of the multidimensional data set from TCGA cbioportal data set. KaplaneMeier plots indicate the clinical outcomes for BRAFV600E mutation (E) or Skp2 levels (F and G) in the indicated melanoma tissues. n indicates the number of patient samples evaluated in each analysis. P-values were calculated using the ManteleCox log-rank test. ***P < 0.001.

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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Fig. 2. BRAFV600E dictates Skp2 expression (A and B) A375 or A2058 were transfected BRAFespecific siRNA to reduce BRAFV600E levels. 48 h after the transfection, cells were harvested to determine indicated protein expressions by Western blotting (A) or mRNA levels by qPCR assay (B). (C and D) We overexpressed BRAFV600E in BRAFWT cell line SK-MEL31 or WM3211. The indicated protein (A) or mRNA (B) levels were determined respectively. (E and F) Effect of vemurafenib (Vem) on protein or mRNA expressions in melanoma cells were determined by Western blotting (E) or qPCR (F) (control vs. 3 mM Vem; 20 h). (G and H) Skp2 promoter was transfected into BRAFWT cells with or without BRAFV600E overexpression (G), or BRAFV600E with or without BRAF siRNA transfection (H). 48 h after transfection, cells were harvest for luciferase reporter assay. Data are expressed as means ± SD (n ¼ 3).

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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BRAFV600E cells upon vemurafenib treatment (Fig. 2G and 2H). Thus Skp2 is transcriptionally regulated by BRAFV600E, potentially associated with BRAFV600E-driven melanoma progression. 3.3. c-Myc is essential for BRAFV600E induction of Skp2 We next sought to investigate the molecular mechanisms underlying BRAFV600E regulation of Skp2 in melanoma cells. Since cMyc was shown positively associated with Skp2 in response to BRAF inhibition or BRAFV600E expression in both melanoma tissues and cell lines (Fig. 1A, 1B, 2A-2F), we suppose c-Myc might be required for BRAFV600E-dependent Skp2 expression. Especially, a much higher enrichment of c-Myc was found in Skp2 promoter in BRAFV600E cells (Fig. 3A). Thus, we overexpressed c-Myc in BRAFV600E melanoma cell A2058 and then treated the cells with vemurafenib. Expectedly, c-Myc overexpression blocked vemurafenib effects on the repression of Skp2 protein and mRNA levels (Fig. 3B and 3C). Furthermore, Skp2 promoter activity, repressed by vemurafenib, was restored in the presence of c-Myc (Fig. 3D). The mutation of c-Myc-binding E-box abolished Skp2 promoter activity in BRAFV600E cells with either c-Myc overexpression or

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vemurafenib treatment (Fig. 3D), suggesting BRAFV600E induction of Skp2 via c-Myc depends strongly on an intact E-box on Skp2 promoter. Accordingly, in BRAFWT cells, BRAFV600E overexpression increased Skp2 promoter activity, which was decreased after the mutation of c-Myc-binding E-box (Fig. 3E). The enrichment of cMyc on Skp2 promoter was decreased in BRAFV600E cells with vemurafenib treatment, and increased in BRAFWT cells after BRAFV600E overexpression. Therefore, BRAFV600E induction of Skp2 transcription is dependent on c-Myc transcriptional activity in melanoma cells. 3.4. BRAFV600E promotes cell cycle and glycolysis via the regulation of Skp2 Given the broad spectrum of oncogenic functions of Skp2, we then determined whether gain of Skp2 function in response to BRAF inhibition is functionally linked to BRAFV600E-induced melanoma progression. Thus we further overexpressed Skp2 in BRAFV600E cells (Fig. 4A). The repression of BRAF by siRNA diminished cell growth rate in both A375 and A2058 cells (Fig. 4B and 4C). Whereas Skp2 expression partially reversed BRAF siRNA-mediated

Figure 3. c-Myc is essential for BRAFV600E induction of Skp2 (A) The amount of c-Myc bound to the Skp2 promoter was determined by ChIP in BRAFWT or BRAFV600E cells. (B and C) Levels of indicated proteins (B) or Skp2 mRNA (C) were determined in A2058 cells with or without c-Myc overexpression following 3 mM Vem treatment for 20 h. (D) Wild-type or E-box-mutant Skp2 promoter was transfected into A2058 cells with or without c-Myc overexpression. 24 h after transfection, cells were then treated with 3 mM Vem for additional 16 h and harvested for luciferase reporter assay. (E) Skp2 promoter activity was determined in SK-MEL-31 cells with or without BRAFV600E overexpression. (F and G) The amount of c-Myc bound to the Skp2 promoter was determined by ChIP in A2058 cells following Vem treatment (control vs. 3 mM Vem; 20 h) (F) or SK-MEL-31 cells with or without BRAFV600E overexpression (G). Data are expressed as means ± SD (n ¼ 3).

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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Fig. 4. BRAFV600E promotes cell cycle and glycolysis via the regulation of Skp2. (AeJ) We overexpressed Skp2 using Skp2 overexpressing lentivirus with Flag-tag in A375 or A2058 cells. (A) Identification of Skp2 protein overexpression in indicated cells. (B and C) Cell viabilities in each group were determined by MTT assay. (D and E) The glucose uptake rate (D) and internal ATP levels (E) were determined as indicated. (F) Cell viabilities were determined in melanoma cells with or without Skp2 overexpression following 0e10 mM Vem treatment for 72 h. (GeJ) A2058 cells with or without Skp2 overexpression were treated with 3 mM Vem for 20 h. The glucose uptake rate (G), internal ATP levels (H), cell cycle distribution (I) and glycolytic gene expressions (J) were determined as indicated. Data are expressed as means ± SD (n ¼ 3).

growth inhibition. Accumulated evidence showed that BRAFV600E drives glycolysis to contribute to the malignancy of melanoma cells [4,23]. In accordance, we observed the decreased glucose uptake and subsequent ATP production in A2058 cells after BRAF knockdown (Fig. 4D and 4E). Moreover, Skp2 expression resulted in a markedly increase in the glucose uptake and ATP levels which were repressed by BRAF siRNA, indicating Skp2 is critical for BRAFV600Einduced glycolysis and tumor growth. Further, we treated melanoma cells with vemurafenib to complement BRAF knockdown approaches. Vemurafenib-mediated suppressions of proliferation, glucose uptake and ATP production were restored in the presence of Skp2 in BRAFV600E melanoma cells (Fig. 4F-H). Interestingly, Skp2 overexpression resumed the cell cycle progression and increased the mRNA levels of the key glycolytic enzymes (GLUT1, HK2, PFK1, PKM2, LDHA) which were suppressed by vemurafenib (Fig. 4I and 4J). Thus, Skp2 regulates cell growth and glycolysis in response to BRAFV600E through both driving cell cycle entry and glycolytic enzyme expressions in melanoma. 4. Discussion BRAFV600E mutation accounts for a large proportion of

metastatic melanomas. Despite great breakthrough of related small molecular inhibitor developments revolutionized the treatment strategies, acquired drug resistance is still the major hurdle [12,13]. The related mechanism is commonly associated with the mutation of RAS or MEK, or the activation of PI3K/AKT pathway, which can overcome the BRAFi effect and result in the acquired drug resistance [14,15]. However, the basis for acquired resistance remains unknown in over 40% of melanomas [24]. In this study, we demonstrated that BRAFV600E regulates melanoma cell survival by a previously unappreciated mechanism that involves the activation of c-Myc/Skp2 axis. The suppression of Skp2 is a critical step for BRAFi effects on the inhibition of both glycolysis and cell cycle progression (Fig. 4FeI). Thus, targeting Skp2 might be a promising strategy for the treatment of BRAFi resistant melanomas. Since the significant role of Skp2 in regulation of TGF-bdependent EMT has been documented in our previous study [20], we thus further determined Skp2 association with tumor malignancy in melanoma tissues. The high expression of Skp2 specifically in BRAFV600E tissues indicates that Skp2 probably be involved in BRAF signaling during tumor progression. Especially, the high levels of Skp2 correlates with poor patient outcome in BRAFV600E melanomas but not in BRAFWT melanomas (Fig. 1F and 1G),

Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085

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suggesting Skp2 predominantly contributes to the aggressive behavior of this melanoma subclass. These data validate the clinical significance of the reciprocal relationship between Skp2 and BRAFV600E and suggest that the deleterious effects of BRAFV600E are exacerbated by high Skp2 expression. c-Myc is an oncogene downstream of BRAF-MEK-ERK pathway and essential for BRAFV600E induced tumor progression [22]. The constitutive activation of BRAF in melanomas increases c-Myc expression through both transcriptional and post-translational manners, resulting in the metabolic reprogramming, epigenetic silencing, or drug resistance [22,25]. Confirming this point, we observed c-Myc is predominantly expressed in BRAFV600E melanoma tissues, and transcriptionally induced by BRAFV600E. Intriguingly, Skp2 levels is positively correlated with c-Myc levels in both clinical samples and cells lines with the manipulation of BRAF, indicating the reciprocal relationship between c-Myc and Skp2 in BRAF pathway. Our previous work identified a c-Myc binding E-box region on Skp2 promoter which is critical for c-Myc transcriptionally activation of Skp2 [20]. In this study, BRAFV600E induced Skp2 promoter activity was abolished upon the E-box mutation. Thus we conclude that c-Myc is essential for BRAFV600E-dependent induction of Skp2 in melanomas. Given the central role of Skp2 E3 ligase activity, aberrant Skp2 activation drives tumor growth by targeting different substrates. Evidence obtained show that Skp2 promotes cell cycle through the ubiquitinations and degradations of CDK inhibitor p21 and p27, and induces aerobic glycolysis by the ubiquitination and activation of Akt [16,18]. Here, we demonstrate that Skp2 regulates cell survival and glycolysis in response to BRAFV600E through two ways. First, Skp2 is required for BRAFV600E-induced cell cycle progression. Since the abrogation of p27 is enough to suppress cell quiescence [26], the overexpression of Skp2 can drive cell cycle entry through degradation of p27 even when BRAF is inhibited, and subsequently increased the glucose uptake for energy supply. Secondly, our work also found Skp2 increased the key glycolytic enzyme expressions broadly, raising the possibility that Skp2 may cooperate with other transcription factors to mediate the transcriptional regulation. It is widely accepted that c-Myc transcriptionally activates glycolytic gene expressions [27], and Skp2 is a transcriptional coactivator for Myc [19]. Thus we suppose that Myc and Skp2 probably form a feedback loop responding to BRAFV600E-driven glycolytic gene expressions and glycolysis. Further studies are needed to fully address the mechanism. Recent reports have shown a link between BRAFV600E-driven glycolysis and BRAFi resistance [4]. BRAFi was found to repress glucose uptake in melanoma cells and tumors of patients independently of cell-cycle progression or cell death [4]. Moreover, inhibition of glucose uptake significantly increased vemurafenib sensitivity, suggesting a possible role for glycolysis in resistance of melanoma to BRAFV600E-targeted therapies [4]. Accordingly, our study confirmed that vemurafenib treatment decreased glucose uptake, ATP production and tumor growth in BRAFV600E melanoma cells. Moreover, the association of Skp2 with the resistances of various chemotherapeutic agents was documented broadly, such as tamoxifen [28], or Bortezomib [29]. Based on these evidence, we firstly point out that Skp2 contributes to BRAFi resistance in melanomas predominantly through the modulation of BRAFV600Edependent glycolysis. In summary, our study provides a mechanistic framework to understand BRAFV600E-driven glycolysis and BRAFi resistance in melanoma, involving c-Myc and Skp2. BRAFV600E induces c-Mycdependent transcription of Skp2, and the inhibition of Skp2 is critical for the suppression of cell growth in response to BRAFV600E inhibition. Thus, we significantly expand on the current understanding of BRAFV600E pathway and suggest a possible role for Skp2

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in responses and resistance of melanoma to BRAFV600E-targeted therapies. Acknowledgements This work was financially sponsored by grants from the National Natural Science Foundation of China (No.81502370, No.81702845), and the Natural Science Basic Research Plan in Shaanxi Province of China (2019JQ-879). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.085 References [1] A. Jemal, R. Siegel, J. Xu, E. Ward, Cancer statistics, 2010, CA A Cancer J. Clin. 60 (2010) 277e300. [2] K.M. Rubin, D.P. Lawrence, Your patient with melanoma: staging, prognosis, and treatment, Oncology 23 (2009) 13e21. [3] M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Science 324 (2009) 1029e1033. [4] T.J. Parmenter, M. Kleinschmidt, K.M. Kinross, S.T. Bond, J. Li, M.R. Kaadige, A. Rao, K.E. Sheppard, W. Hugo, G.M. Pupo, R.B. Pearson, S.L. McGee, G.V. Long, R.A. Scolyer, H. Rizos, R.S. Lo, C. Cullinane, D.E. Ayer, A. Ribas, R.W. Johnstone, R.J. Hicks, G.A. McArthur, Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis, Cancer Discov. 4 (2014) 423e433. [5] N. Dhomen, R. Marais, New insight into BRAF mutations in cancer, Curr. Opin. Genet. Dev. 17 (2007) 31e39. [6] B.G. Davies H, C. Cox, et al., Mutations of the BRAF gene in human cancer, Nature 417 (2002) 949e954. [7] M.R. Garnett MJ, Guilty as charged: B-RAF is a human oncogene, Cancer Cell 6 (2004) 313e319. [8] V.C. Gray-Schopfer, S. da Rocha Dias, R. Marais, The role of B-RAF in melanoma, Cancer Metastasis Rev. 24 (2005) 165e183. [9] J. Yun, C. Rago, I. Cheong, R. Pagliarini, P. Angenendt, H. Rajagopalan, K. Schmidt, J.K. Willson, S. Markowitz, S. Zhou, L.A. Diaz Jr., V.E. Velculescu, C. Lengauer, K.W. Kinzler, B. Vogelstein, N. Papadopoulos, Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells, Science 325 (2009) 1555e1559. [10] G. Bollag, P. Hirth, J. Tsai, J. Zhang, P.N. Ibrahim, H. Cho, W. Spevak, C. Zhang, Y. Zhang, G. Habets, E.A. Burton, B. Wong, G. Tsang, B.L. West, B. Powell, R. Shellooe, A. Marimuthu, H. Nguyen, K.Y. Zhang, D.R. Artis, J. Schlessinger, F. Su, B. Higgins, R. Iyer, K. D’Andrea, A. Koehler, M. Stumm, P.S. Lin, R.J. Lee, J. Grippo, I. Puzanov, K.B. Kim, A. Ribas, G.A. McArthur, J.A. Sosman, P.B. Chapman, K.T. Flaherty, X. Xu, K.L. Nathanson, K. Nolop, Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma, Nature 467 (2010) 596e599. [11] K.T. Flaherty, I. Puzanov, K.B. Kim, A. Ribas, G.A. McArthur, J.A. Sosman, P.J. O’Dwyer, R.J. Lee, J.F. Grippo, K. Nolop, P.B. Chapman, Inhibition of mutated, activated BRAF in metastatic melanoma, N. Engl. J. Med. 363 (2010) 809e819. [12] R. Nazarian, H. Shi, Q. Wang, X. Kong, R.C. Koya, H. Lee, Z. Chen, M.K. Lee, N. Attar, H. Sazegar, T. Chodon, S.F. Nelson, G. McArthur, J.A. Sosman, A. Ribas, R.S. Lo, Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation, Nature 468 (2010) 973e977. [13] D.J. Wong, A. Ribas, Targeted therapy for melanoma, Cancer Treat Res. 167 (2016) 251e262. [14] D.B. Johnson, A.M. Menzies, L. Zimmer, Z. Eroglu, F. Ye, S. Zhao, H. Rizos, A. Sucker, R.A. Scolyer, R. Gutzmer, H. Gogas, R.F. Kefford, J.F. Thompson, J.C. Becker, C. Berking, F. Egberts, C. Loquai, S.M. Goldinger, G.M. Pupo, W. Hugo, X. Kong, L.A. Garraway, J.A. Sosman, A. Ribas, R.S. Lo, G.V. Long, D. Schadendorf, Acquired BRAF inhibitor resistance: a multicenter metaanalysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms, Eur. J. Cancer 51 (2015) 2792e2799. [15] H. Rizos, A.M. Menzies, G.M. Pupo, M.S. Carlino, C. Fung, J. Hyman, L.E. Haydu, B. Mijatov, T.M. Becker, S.C. Boyd, J. Howle, R. Saw, J.F. Thompson, R.F. Kefford, R.A. Scolyer, G.V. Long, BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact, Clin. Cancer Res. 20 (2014) 1965e1977. [16] A.C. Carrano, E. Eytan, A. Hershko, M. Pagano, SKP2 is required for ubiquitinmediated degradation of the CDK inhibitor p27, Nat. Cell Biol. 1 (1999) 193e199. [17] H. Sutterluty, E. Chatelain, A. Marti, C. Wirbelauer, M. Senften, U. Muller, W. Krek, p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells, Nat. Cell Biol. 1 (1999) 207e214. [18] C.H. Chan, C.F. Li, W.L. Yang, Y. Gao, S.W. Lee, Z. Feng, H.Y. Huang, K.K. Tsai,

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Please cite this article as: L. Feng et al., BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.085