ABSTRACT An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and

selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug

resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of

melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is

reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF

inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-_κ_B pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway,

particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma. SIMILAR CONTENT BEING VIEWED BY OTHERS TARGETING MTOR SIGNALING OVERCOMES

ACQUIRED RESISTANCE TO COMBINED BRAF AND MEK INHIBITION IN _BRAF_-MUTANT MELANOMA Article Open access 24 July 2021 PERK MEDIATES RESISTANCE TO BRAF INHIBITION IN MELANOMA WITH IMPAIRED PTEN

Article Open access 19 July 2021 COMBATING ACQUIRED RESISTANCE TO MAPK INHIBITORS IN MELANOMA BY TARGETING ABL1/2-MEDIATED REACTIVATION OF MEK/ERK/MYC SIGNALING Article Open access 29

October 2020 MAIN Mutant-v-raf murine sarcoma viral oncogene homolog B1 (BRAF) is found in approximately 50% of melanomas1 and induces constituent MEK/extracellular signal regulated kinase

(ERK) signaling in melanoma cells.2 The most common mutation in BRAF is a substitution of valine with glutamic acid at amino-acid position 600 (V600E) in exon 15.1, 3 The potent and

selective BRAF (V600E) inhibitors,4 vemurafenib (PLX 4032) and dabrafenib (GSK2118436), have significantly improved survival for patients with V600 BRAF-mutant metastatic melanoma.5, 6, 7, 8

Eventually, the majority of patients relapsed with a median progression-free survival period of 5–7 months, indicating the acquisition of resistance to BRAF inhibition in these patients.5,

7, 8 Over the past 2 years, tremendous efforts have been directed toward understanding the molecular mechanisms of acquired BRAF inhibitor resistance. Emerging evidence suggests that NRAS

mutations, amplification of _BRAF (V600E)_ and overexpression of v-raf-leukemia viral oncogene 1 or COT lead to reactivation of MEK that counteracts the inhibition of BRAF.9, 10, 11, 12 In

addition, re-activated IGF-1R/PI3K and FGFR3/RAS/MAPK signaling pathways were shown to have an important role in the development of resistance to BRAF inhibitors.13, 14 More recently,

aberrant expression of splicing isoforms of BRAF (V600E) that dimerize in a RAS-independent manner has been shown to mediate acquired BRAF inhibitor resistance.15 Upregulated expression of

receptor tyrosine kinase and FOXD3 is also associated with BRAF inhibitor resistance.11, 16, 17 Given the diversity and complexity of these identified signaling pathways associated with BRAF

inhibitor resistance, fully defining the underlying mechanisms of resistance is still a priority to develop rational strategies to secure long-term responses of mutant BRAF melanomas to

specific inhibitors. To identify novel mechanism(s) of resistance to BRAF inhibition, we generated PLX4032-resistant cell lines by chronic exposure of a BRAF (V600E)-positive melanoma cell

line to PLX4032. Here we report _AEBP1_ as a novel PLX4032 resistance-associated gene. AEBP1 (adipocyte enhancer-binding protein 1) is highly upregulated in PLX4032-resistant melanoma cells

because of hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. Increased expression of AEBP1 is shown to confer acquired PLX4032 resistance by

facilitating activation of NF-_κ_B. We also show that AEBP1 is indeed upregulated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Collectively, these data

suggest that the PI3K/Akt-CREB-AEBP1-NF-_κ_B pathway has an important role in the development of BRAF inhibitor resistance in mutant BRAF melanomas. RESULTS AEBP1 CONFERS ACQUIRED RESISTANCE

TO BRAF (V600E) INHIBITION IN MELANOMA To identify novel molecular mechanism(s) of resistance, we generated PLX4032-resistant cell lines by chronic exposure of the BRAF (V600E)-positive

parental melanoma cell line Mel-CV to PLX4032. After approximately 6 months of continuous selection, 24 independent PLX4032-resistant clones (Mel-CVR1-Mel-CVR24) were generated. The

PLX4032-resistance phenotype of Mel-CVR18 and Mel-CVR21 clones was confirmed by MTT and long-term colony formation assays (Supplementary Figures 1A and B). These two clones were then

subjected to cDNA microarray analysis to identify potential PLX4032 resistance-associated genes. Through the cDNA microarray analysis, AEBP1 was revealed to be most dramatically upregulated

in both Mel-CVR18 and Mel-CVR21 compared with Mel-CV cells (Figure 1a). Real-time RT-PCR analysis showed that most of the PLX4032-resistant Mel-CVR sub-lines (18 of 24) exhibited a more than

twofold increase in AEBP1 mRNA expression compared with the Mel-CV cell line (Figure 1b). The subsequent western blot analysis also confirmed the upregulation of AEBP1 protein expression in

PLX4032-resistant Mel-CVR cells (Supplementary Figure 2). As Mel-CVR18 and Mel-CVR21 cells showed similar responses to PLX4032 treatment (Supplementary Figures 1A and B), in the subsequent

functional studies only Mel-CVR18 was used. For convenience, Mel-CVR18 was hereafter referred to as Mel-CVR. To exclude the possibility that AEBP1 upregulation only occurs in Mel-CVR cell

type, we also generated other two PLX4032-resistant cell lines from different melanoma cell types Mel-RMu and SK-Mel-28 by chronic exposure to PLX4032. As was expected, both mRNA and protein

levels of AEBP1 were upregulated in these PLX4032-resistant cell lines (Supplementary Figure 3). We next determined whether upregulation of AEBP1 expression confers acquired PLX4032

resistance to melanoma cells. We first knocked down AEBP1 in Mel-CVR cells. Compared with control cells, AEBP1 knockdown Mel-CVR cells showed a dramatic decrease in cell viability in

response to PLX4032 treatment (Figure 1c). Accordingly, AEBP1 knockdown Mel-CVR cells exhibited high levels of caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage compared

with control cells after PLX4032 treatment (Figure 1d). Similar results were obtained when AEBP1 was knocked down in Mel-RMuR and SK-Mel-28R cells (Supplementary Figure 4). We next evaluated

the effect of AEBP1 on PLX4032-induced inhibition on cell viability in Mel-CV cells. As shown in Figure 1e, the inhibitory effect of PLX-4032 on cell viability was greatly reversed by

ectopic expression of AEBP1 in Mel-CV cells. Correlated with this, AEBP1 overexpression resulted in a great decrease in PLX4032-induced caspase-3 activation and PARP cleavage (Figure 1f).

Furthermore, long-term colony formation assay also revealed that when AEBP1 was knocked down in Mel-CVR-, Mel-RMuR- and SK-Mel-28R-resistant cells, these cells became sensitive to PLX4032

treatment (Figure 1g and Supplementary Figure 5). Taken together, these results strongly suggest the important role of AEBP1 in the development of acquired resistance to BRAF inhibition.

AEBP1 IS REGULATED BY THE PI3K/AKT-CREB PATHWAY To investigate the molecular mechanism whereby AEBP1 is upregulated in Mel-CVR cells, we first inspected the genomic sequence upstream of

AEBP1 coding region by using the JASPAR software.18 One putative CREB-binding site located 761–753 bp upstream of AEBP1 translational start site was identified (Figure 2a). To verify this

potential CREB-binding site is indeed responsive to CREB, a series of pGL3-based luciferase reporter plasmids containing the wild-type CREB-binding region (BR), a mutant CREB-BR or deletion

of CREB-BR were generated (Figure 2b). These plasmids were individually transfected into Mel-CV and Mel-CVR cells with or without CREB knockdown, followed by the measurement of their

transcriptional activities using luciferase assays. As shown in Figure 2c, pGL3 luciferase reporter plasmids containing the wild-type CREB-BR (WT and m4), but not the mutant plasmids (m1,

m2, m3 and m5), showed a CREB-responsive transcriptional activity in both Mel-CV and Mel-CVR cells. Of note, the transcriptional activities of pGL3-AEBP-WT and pGL3-AEBP-m4 were much higher

in Mel-CVR cells than those in Mel-CV cells (Figure 2c), indicating that the basal CREB transcriptional activity may be higher in Mel-CVR cells than that in Mel-CV cells. Given that the

transcriptional activity of CREB is induced by its phosphorylation at S133,19, 20 we sought to evaluate the effects of wild-type CREB and its mutant CREB S133A on the AEBP1 promoter

activity. As shown in Figure 2d, wild-type CREB, but not CREB S133A, increased the transcriptional activity of the AEBP1 promoter in a dose-dependent manner. The subsequent chromatin

immunoprecipitation (ChIP) assays showed that the chromatin fragments, corresponding to the putative CREB BR, were specifically present in anti-CREB immunoprecipitates from both Mel-CV and

Mel-CVR cells (Figure 2e), which was, however, diminished by CREB knockdown (Figure 2e). In addition, knockdown of CREB resulted in a dramatic decrease in AEBP1 mRNA levels in Mel-CVR cells

(Figure 2f). These results suggest that transcriptional regulation of AEBP1 is mediated by CREB. The importance of the CREB-AEBP1 axis in acquired PLX4032 resistance was also confirmed by

the finding that knockdown of either CREB or AEBP1 increased PLX4032-induced inhibition of viability in Mel-CVR cells (Figure 2g). Intriguingly, in contrast to Mel-CVR cells, CREB knockdown

only led to a subtle decrease in AEBP1 mRNA levels in Mel-CV cells (Figure 2f). This discrepancy further suggests that Mel-CVR cells possess higher CREB transcriptional activity than Mel-CV

cells. In support of this conclusion, we also found that levels of phosphorylated CREB (S133) were much higher in Mel-CVR cells than those in Mel-CV cells, although total CREB protein showed

similar levels in these two cells (Figure 2h). Furthermore, Mel-CVR cells were shown to exhibit higher levels of phosphorylated Akt (S473) (p-Akt) than Mel-CV cells (Figure 2h), indicating

that PI3K/Akt signaling is enhanced in PLX4032-resistant melanoma cells. The similar results were also obtained in other PLX4032-resistant Mel-CVR, Mel-RMuR and SK-Mel-28R clones

(Supplementary Figure 6). To confirm this, Mel-CV and Mel-CVR cells were treated with PLX4032 for prolonged periods. The remaining live cells were assayed for Akt activation by evaluating

protein levels of p-AKT. As shown in Supplementary Figure 7A, PLX4032 treatment resulted in progressive activation of Akt and CREB in Mel-CV cells, which was correlated well with the gradual

upregulation of AEBP1 expression by PLX4032 treatment (Supplementary Figure 7B), indicating that activation of Akt is associated with acquired PLX4032 resistance. In contrast to Mel-CV

cells, the enhancing effect of PLX4032 on activation of the PI3K/Akt-CREB pathway and upregulation of AEBP1 expression was minimal in Mel-CVR cells (Supplementary Figures 7A and B). This

suggests that the PI3K/Akt-CREB-AEBP1 pathway may have already been fully activated in Mel-CVR cells. The PI3K/Akt pathway has previously been shown to induce CREB activation by promoting

its phosphorylation at S133.19, 20 To determine the specific role of the PI3K/Akt pathway in mediating CREB activation and AEBP1 upregulation, Mel-CV and Mel-CVR cells were treated with

LY294002 or U0126, which inhibits the PI3K/Akt and MEK/ERK pathways, respectively. As shown in Figure 2i, LY294002 treatment, but not U0126 treatment, led to a dramatic decrease in both

p-CREB and AEBP1 protein levels in Mel-CVR cells with or without PLX4032 treatment. More importantly, pre-treatment of Mel-CVR with LY294002 led to a strong increase in PARP cleavage and

caspase-3 activation (Figure 2i) and a dramatic decrease in cell viability in response to PLX4032 treatment (Supplementary Figure 8). Similar results were also obtained from

PLX4032-resistant Mel-RMuR and SK-Mel-28R cells that were pre-treated with LY294002 (Supplementary Figure 9). However, when expression levels of p-CREB and AEBP were induced by ectopic

expression of constitutively active form of AKT (Myr-AKT) in Mel-CV and Mel-RMu cells (Supplementary Figure 10A), these cells became resistant to PLX4032 treatment (Supplementary Figures 10B

and C). We also found that the LY294002 pretreatment did not further enhance PLX4032-mediated cell viability inhibition in AEBP1 knockdown Mel-CVR cells (Supplementary Figure 8), indicating

that AEBP1 functions in full alignment with PI3K/Akt activity in the development of acquired resistance to PLX4032. Taken together, these data suggest that AEBP1 is upregulated by the

PI3K/Akt-CREB pathway, through which AEBP1 contributes to the acquired resistance of mutant BRAF melanoma cells to BRAF inhibition. AEBP1 MEDIATES ACTIVATION OF NF-_Κ_B AND THEREBY

CONTRIBUTES TO ACQUIRED RESISTANCE TO BRAF INHIBITION We next investigated how AEBP1 contributes to acquired resistance to BRAF inhibition. AEBP1 has recently been recognized as a positive

regulator of NF-_κ_B activation through accelerating degradation of I_κ_B_α_.21 It has also been shown that NF-_κ_B activation protects cells from BRAF inhibition-induced cell death.22 We

therefore asked whether AEBP1 could activate NF-_κ_B and thus confer resistance to BRAF inhibition. Consistent with the previous report, with the increasing expression of AEBP1, levels of

I_κ_B_α_ were concomitantly decreased in Mel-CV cells (Figure 3a), thereby leading to the gradual activation of NF-_κ_B (Figure 3b). Similar to AEBP1, CREB also showed a similar decrease in

I_κ_B_α_ protein levels and an increase in NF-_κ_B activation (Figure 3a, lanes 4–6 and Figure 3b). This effect of CREB appeared to require its transcriptional activity, as revealed by the

finding that wild-type CREB, but not CREB S133A, promoted NF-_κ_B activation (Figures 3c and d). The failure of CREB to induce NF-_κ_B activation in AEBP1 knockdown Mel-CV cells (Figure 3e)

suggests that AEBP1 is the downstream mediator of CREB to promote NF-_κ_B activation. Given the upregulation of AEBP1 expression in Mel-CVR, Mel-RMuR and SK-Mel-28R cells, we sought to

determine whether this resulted in NF-_κ_B activation in these cells. As shown in Figure 3f and Supplementary Figure 11A, Mel-CVR, Mel-RMuR and SK-Mel-28R cells indeed showed higher NF-_κ_B

activity and lower I_κ_B_α_ expression compared with their corresponding sensitive parental cells. Correlated with the lower levels of I_κ_B_α_ in Mel-CVR cells, the half-life of I_κ_B_α_ in

Mel-CVR cells was much shorter than that in Mel-CV cells (Figure 3g). However, when AEBP1 was knocked down in Mel-CVR cells, the half-life of I_κ_B_α_ was significantly prolonged (Figure

3g). In addition, knockdown of either AEBP1 or CREB showed strong inhibitory effect on the NF-_κ_B transcriptional activity in Mel-CVR, Mel-RMuR and SK-Mel-28R cells (Figure 3h and

Supplementary Figure 11B). Treatment with LY294002, but not U0126, greatly inhibited the NF-_κ_B transcriptional activity in Mel-CVR, Mel-RMuR and SK-Mel-28R cells (Figure 3i and

Supplementary Figure 11C). Induction of constitutively active form of AKT (Myr-AKT) in Mel-CV and Mel-RMu cells strongly enhanced NF-_κ_B activity (Supplementary Figure 11D). These results

indicate that the hyperactivated PI3K/Akt-CREB-AEBP1 pathway is responsible for the increased NF-_κ_B activity in PLX4032-resistant melanoma cells. To further determine the physiological

significance of AEBP1-mediated NF-_κ_B activation in relation to the acquired resistance to PLX4032, we used PDTC, BAY-11-7082 and ALLN, which were specific inhibitors of NF-_κ_B activation.

When NF-_κ_B activation was inhibited by PDTC, BAY-11-7082 or ALLN, Mel-CVR cells exhibited a dramatic decrease in cell viability (Figure 3j and Supplementary Figure 12A) and a strong

increase in PARP cleavage and caspase-3 activation in response to PLX4032 treatment (Figure 3k and Supplementary Figure 12B). Similarly, when NF-_κ_B activation was inhibited by ectopic

expression of a non-degradable mutant of I_κ_B_α_ (I_κ_B_α_S32AS36A) in Mel-CVR cells, the cells became sensitive to PLX4032 treatment (Supplementary Figures 13A and B). Conversely, when

NF-_κ_B activity was induced by I_κ_B knockdown in Mel-CV and Mel-RMu cells (Supplementary Figure 13C), these cells became resistant to PLX4032 treatment without causing any change in AEBP1

expression (Supplementary Figures 13D–F). Taken together, these results suggest that NF-_κ_B is activated by the PI3K/Akt-CREB-AEBP1 pathway, and thus confers BRAF inhibitor resistance to

melanoma cells. BIOLOGICAL SIGNIFICANCE OF INCREASED EXPRESSION OF AEBP1 IN ACQUIRED RESISTANCE TO BRAF INHIBITION To validate the functional significance of AEBP1 upregulation in acquired

BRAF inhibitor resistance _in vivo_, we used a xenograft mouse model. Mel-CV and Mel-CVR cells with and without stable knockdown of either CREB or AEBP1 were individually injected

subcutaneously into the right flank of nude mice. As shown in Figure 4a, all these melanoma cells developed into tumors of comparable volumes at 15 days after injection, indicating that both

CREB and AEBP1 have no effects on melanomagenesis in this xenograft model. After tumor establishment (about 50 mm3), mice were treated with PLX4032 (50 mg/kg) through intraperitoneal (i.p.)

injection. After 25 days of the commencement of treatment, the excised tumors were weighed and analyzed for expression of AEBP1 and CREB and activation of caspase-3/7. As shown in Figures

4a and b, Mel-CV xenografts were largely abolished by treatment of PLX4032, whereas Mel-CVR xenografts showed little, if any, responses to PLX4032 treatment. However, when AEBP1 or CREB was

knocked down in Mel-CVR cells, the xenografts showed the similar shrinkage rates as Mel-CV tumors in response to PLX4032 (Figures 4a and b). This was also confirmed by a significant decrease

in xenograft weights (Figure 4c). The knockdown efficiency of AEBP1 or CREB was confirmed by western blot analysis of xenograft samples post treatment with PLX4032 (Figure 4d). Also,

knockdown of either AEBP1 or CREB resulted in a dramatic increase in caspase-3/7 activity in Mel-CVR xenografts in response to treatment with PLX4032 (Figure 4e). These results point to an

important role of the CREB-AEBP1 axis in antagonizing PLX4032-mediated inhibition of melanoma growth _in vivo_. To test whether AEBP1 is involved in development of acquired resistance to

BRAF inhibition in melanoma patients, we first took advantage of primary melanoma cell cultures of paired BRAF (V600E) melanoma biopsy samples from four patients pre- and progressed

post-treatment with PLX4032.23 These melanomas responded initially to PLX4032 but relapsed after various progression-free durations,23 indicative of development of acquired resistance. We

found that two of four post-treatment cultures expressed lower levels of I_κ_B_α_ and higher levels of AEBP1 and p-Akt than corresponding pre-treatment cells (Figure 4f), consistent with the

findings in melanoma cell lines that increased AEBP1 expression, Akt phosphorylation and NF-_κ_B activation were associated with resistance to PLX4032. Similarly, examination of paired BRAF

(V600E) melanoma biopsy samples taken from five patients pre- and progressed post-treatment with the BRAF inhibitor dabrafenib by immunohistochemistry revealed that two of five melanomas

progression samples displayed increased expression of AEBP1 in comparison with the corresponding samples before treatment (Supplementary Figure 14). Together, these findings suggest that the

development of BRAF inhibitor resistance in a sub-group of BRAF (V600E) melanomas is closely associated with upregulation of AEBP1. DISCUSSION Approximately 80% of patients with mutant BRAF

melanomas respond initially to vemurafenib and dabrafenib treatment, but acquired resistance develops in the majority of patients commonly within 1 year.5, 6, 7, 8 In this study, we have

provided evidence that increased expression of AEBP1 contributes to the development of acquired resistance of mutant BRAF melanomas to specific inhibitors. AEBP1 was originally identified as

a transcriptional repressor with both carboxypeptidase and DNA-binding activities,24, 25 and has been implicated in the regulation of various biological processes.26, 27, 28 Our data

showing that AEBP1 is upregulated in at least a proportion of BRAF (V600E) melanomas that had acquired resistance to PLX4032, and that inhibition of AEBP1 restores its sensitivity to PLX4032

in resistant cells uncover a previously unidentified function of AEBP1 in association with BRAF inhibitor resistance. The increase of AEBP1 in BRAF (V600E) melanoma cells with PLX4032

resistance appears to be due to transcriptional upregulation mediated by the transcription factor CREB. It has been well known that the transcriptional activity of CREB is activated via its

phosporylation at S133 by various signaling pathways, such as Akt, MAPK and ERK.19, 20, 29, 30 Our finding that Akt inhibition, but not ERK inhibition, results in marked decreases in protein

levels of both phosphorylated CREB and AEBP1, pointing to a specific role of PI3K/Akt signaling in activating CREB and subsequent upregulation of AEBP1 in the context of PLX4032 resistance.

Along with upregulation of AEBP1, PI3K/Akt-CREB signaling is also highly activated in PLX4032-resistant melanoma cells, the detailed mechanism underlying this phenomenon still remains to be

further determined. Although we did not find the mutations in PTEN or PI3K in Mel-CVR cells (data not shown), it is still possible that the hyper-activation of the PI3K/Akt-CREB pathway in

PLX4032-resistant melanoma cells is due to the activation of upstream signaling cascade, such as HGF-MET pathway.31, 32 In this study, we also found that inhibition of the PI3K/Akt-CREB

pathway, but not of the MEK/ERK pathway, leads to a dramatic increase in cell death of PLX4032-resistant cells. These results indicate that AEBP1 is specifically upregulated by PI3K/Akt-CREB

signaling and contributes to the acquired resistance to BRAF inhibition. In agreement with our data, recent studies have also suggested that increased activation of PI3K/Akt signaling has

an important role in the development of BRAF inhibitors resistance.13, 33, 34 AEBP1 has been reported to interact with PTEN and promote its degradation.35 Consistently, AEBP1-deficient mice

exhibit increased PTEN levels and suppressed Akt activation.35 Therefore, it is conceivable that AEBP1 may be part of a positive autoregulatory feedback loop that reinforces the

PI3K/Akt-CREB signaling axis in BRAF inhibitor-resistant melanoma cells. Numerous studies have suggested the existence of a crosstalk between PI3K/Akt and NF-_κ_B signaling pathways.

PI3K/Akt signaling can activate NF-_κ_B in response to a variety of stimuli by phosphorylating I_κ_K_α_ (I_κ_B kinase _α_).36 In addition, Akt also phosphorylates the NF-_κ_B subunit p65 and

increase binding of the NF-_κ_B complex to DNA.37 Our results showing that PI3K/Akt-CREB-mediated upregulation of AEBP1 leads to NF-_κ_B activation through accelerating I_κ_B_α_ degradation

in BRAF inhibitor-resistant melanoma cells, together with the finding from another study,21 demonstrate that AEBP1 has an important role in the activation of NF-_κ_B. Given that AEBP1 is

subjected to transcriptional regulation by PI3K/Akt pathway, these data identify AEBP1 as a novel critical node that connects the PI3K/Akt and NF-_κ_B signaling pathways. Although activation

of NF-_κ_B is critical for melanoma cell growth,38, 39 whether it is associated with acquired BRAF inhibitors resistance remains uncertain. Our findings that NF-_κ_B is highly activated

because of the AEBP1 upregulation in PLX4032-resistant melanoma cells, and that inhibition of NF-_κ_B reverses the resistance phenotype of the cells, suggest that NF-_κ_B activation has an

important role in the acquisition of BRAF inhibitor resistance in melanoma cells. The increased expression of AEBP1 in relapsed melanomas from patients post treatment with BRAF inhibitor

suggests that upregulation of AEBP1 is, at least in a proportion of mutant BRAF melanomas, associated with resistance to BRAF inhibition, and provides novel insights into potential

therapeutic strategies for the treatment of mutant BRAF melanoma cells that have developed resistance to the inhibitors. In support, an _in vivo_ xenograft mouse model shows that inhibition

of AEBP1 greatly enhances anti-tumor effect of PLX4032 on BRAF inhibitor-resistant melanomas. As mentioned earlier, several mechanisms of BRAF inhibitor resistance have been proposed. In

this study, we also examined whether Mel-CVR-, SK-Mel-28R- and Mel-RMuR-resistant cells harbor mutations in NRAS and MEK, and we failed to detect any mutations in either NRAS or MEK in these

three resistant cell lines (Supplementary Figure 15). In addition, the previously reported BRAF splice variants were not detected in Mel-CVR cells either (Supplementary Figure 16). These

data indicate that activation of AEBP1 represents a unique mechanism of acquired resistance to BRAF inhibition. In summary, this study presents a novel PI3K/Akt-CREB-AEBP1-NF-_κ_B signaling

pathway whose activation contributes to acquired resistance to PLX4032 in BRAF-mutant melanoma cells, and identifies AEBP1 as a potential therapeutic target for the treatment of BRAF

inhibitor-resistant melanomas (Figure 4g). MATERIALS AND METHODS CELL CULTURE AND REAGENTS Human melanoma cell lines described previously were cultured in DMEM containing 5% FCS.40 Mel-CV

and Mel-RMu were from lymph node metastases, and SK-Mel-28 was from a primary melanoma.40 Primary melanoma cultures of paired BRAF (V600E) melanoma biopsy samples from patients pre- and

post-treatment with vemurafenib were established and described previously.22 PLX4032 was purchased from Selleck Inc. (Houston, TX, USA). It was dissolved in DMSO to make up stock solutions

of 10 _μ_M. The PI3K inhibitor LY294002 and the MEK inhibitor UO126 were obtained from Sigma (St. Louis, MO, USA). The following antibodies were used in this study: antibodies against AKT

(Cell Signaling, Danvers, MA, USA; 2920S), I_κ_B_α_ (Cell Signaling, 4812S), phospho-ERK (Cell Signaling, 4370S), CREB (Cell Signaling, 9197S) and phospho-CREB (Ser133) (Cell Signaling,

4276S); antibodies against AEBP1 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-271374), GAPDH (Santa Cruz Biotechnology, SC-32233), PARP (Santa Cruz Biotechnology, SC-8007); anti-caspase-3

(Stressgene, CA-Brockville, ON, Canada; AAP-113) and anti-phospho-AKT (Ser473) (Epitomics, Burlingame, CA, USA; 2118-1). Primary melanoma cell cultures were established as described

previously.23 GENERATION OF PLX4032-RESISTANT MELANOMA CELL LINE Mel-CV cells were initially treated with 20 _μ_M PLX4032 for 4 days. The remaining live cells were switched into fresh medium

containing 5 _μ_M PLX4032 and cultured for about 2 weeks till the colonies became visible. Twenty-four independent resistant colonies (Mel-CVR1-24) were picked and expanded in the culture

medium containing 5 _μ_M PLX4032. Then the cells were passaged and cultured in the medium containing 5 _μ_M PLX4032 for at least 20 weeks before they were used for the subsequent studies.

The similar procedures were used to generate other two PLX4032-resistant cell lines Mel-RMuR and SK-Mel-28R. RETROVIRAL OVEREXPRESSION OF AEBP1 To produce retrovirus expressing AEBP1,

HEK293T cells were co-transfected with pBABE-Flag-AEBP1 and pCL-Ampho packaging vector. 24 h after transfection, cells were cultured with fresh medium for an additional 24 h. The culture

medium containing the retrovirus particles was then centrifuged at 12 000 × _g_ for 5 min, and virus in the supernatant was used for melanoma cells infection. LENTIVIRAL SMALL HARPIN RNA

(SHRNA)-MEDIATED KNOCKDOWN OF CREB AND AEBP1 To generate lentiviruses expressing CREB, AEBP1 or control shRNAs, HEK293T cells grown on a 6-cm dish were transfected with 2 _μ_g of CREB shRNA,

AEBP1 shRNA (cloned in PLKO.1) or control vector, 2 _μ_g of pREV, 2 _μ_g of pGag/Pol/PRE and 1 _μ_g of pVSVG. 24 h after transfection, cells were cultured with DMEM medium containing 20%

FBS for another 24 h. The culture medium containing lentivirus particles was cleaned by centrifugation to get rid of the cell debris at 12 000 × _g_ for 5 min, and used for the target cells

infection. The shRNA sequences targeting CREB and AEBP were 5′-TACAGCTGGCTAACAATGG-3′ and 5′-CGATGACATGGACTATTAC-3′, respectively. DUAL-LUCIFERASE REPORTER ASSAY Mel-CV cells were

transiently transfected with the indicated luciferase reporter plasmid, together with either Flag-CREB or control vector. _Renilla_ plasmid was also included in each transfection to

normalize the transfection efficiency. Firefly and _Renilla_ luciferase activities were analyzed by Dual-Luciferase Reporter Assay system according to the manufacturer’s instructions

(Promega, Madison, WI, USA). The relative luciferase activities were calculated by normalizing the firely luciferase activity to _Renilla_ luciferase activity. The represented data were

mean±S.D. of three independent experiments. REAL-TIME RT-PCR Total RNA was isolated using Trizol (Sangon Company, Shanghai, China). 1 _μ_g of RNA was used to synthesize cDNA using

PrimeScriptTM RT reagent kit (TaKaRa, Otsu, Shiga, Japan; DRR037A) according to the manufacturer’s instruction. Real-time PCR was performed using SYBR premix EX Taq (TaKaRa) and ROX and

analyzed with Stratagene Mx3000p (Agilent Technologies, Santa Clara, CA, USA). Real-time primer sequences for AEBP1 were as follows: 5′- AGGGCAGAGACTCCAGCAT -3′ and 5′- AGAGCACCCCAGCACCTC

-3′. COLONY FORMATION ASSAY Mel-CV and Mel-CVR cells with and without stable knockdown of CREB or AEBP1 were plated at a density of 20 000 cells per well on a six-well plate in the normal

medium. Next day, cells were incubated with culture medium in the presence or absence of 5 _μ_M PLX4032 for 3 weeks, with refreshment of medium twice a week. The cells were then fixed with

10% cold methanol for 5 min and stained with 0.005% (m/v) crystal violet for 30 min at room temperature. After extensive wash, the colonies were photographed. CHIP ASSAY Mel-CV and Mel-CVR

with or without CREB stably knockdown were crosslinked with 1% formaldehyde for 10 min at room temperature. ChIP assay was performed according to the manufacturer’s instructions by using

anti-CREB and the ChIP assay kit (Millipore, Merck KGaA, Darmstadt, Germany). Anti-rabbit IgG were used as controls. The bound DNA fragments were eluted and amplified by PCR using the

following primer pair: 5′-GCAACCAAACTTCTCTGTCC-3′ and 5′-TCCTGCCTCAGCCTCCCAA-3′. PCR products were separated on 2% agarose gel by gel electrophoresis. XENOGRAFT MOUSE MODEL Mel-CV and

Mel-CVR cells with and without stable knockdown of CREB or AEBP1 (about 1 × 107) were injected subcutaneously into the right flank of nude mice (Shanghai SLAC Laboratory, Shanghai, China).

Fifteen days after injection, palpable tumors were present in all mice. The mice bearing tumor of similar size (about 50 mm3) from each group (_n_=6) were treated with PLX4032 (50 mg/kg)

through i.p. injection. Twenty-five days after PLX4032 treatment, mice were killed and tumors were excised and weighed. Studies on animals were conducted with approval from the Animal

Research Ethics Committee of University of Science and Technology of China. PATIENT TISSUES Ten tumor samples were collected from five metastatic melanoma patients treated with a BRAF

inhibitor as part of the phase I (_n_=2), and phase II-brain metastases (_n_=2) clinical trials of dabrafenib,7, 41 or the expanded access program for vemurafenib (_n_=1). Patients eligible

for these studies had unresectable AJCC stage III or stage IV BRAF mutant melanoma and were treated with either dabrafenib (150 mg, twice daily) or vemurafenib (960 mg twice daily). Biopsied

tumor specimens were collected from consenting patients before BRAF inhibitor treatment and at disease progression, as part of the Treat Excise Analyze for Melanoma Study at the Melanoma

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the brain (BREAK-MB): a multicentre, open-label, phase 2 trial. _Lancet Oncol_ 2012; 13: 1087–1095. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful

to Dr. Peter Heresy for establishment of the primary melanoma cell lines from paired melanoma samples pre- and post-treatment with vemurafenib; Professor Richard Kefford, Jessica Hyman,

James Wilmott, Helen Rizos and the MIA surgeons, specifically Julie Howle, Kerwin Shannon, Robyn Saw, John Thompson, Andrew Spillane and Jonathan Stretch for their assistance in procuring

melanoma specimens; and Dr. Hyo-Sung Ro for kindly providing us with pCDNA-AEBP1 plasmid. This work was supported by grants from the Ministry of Science and Technology of China (2010CB912804

and 2011CB966302); National Natural Science Foundation of China 31030046 and the Fundamental Research Funds for Central Universities (WK2060190018). The project was also partly supported by

Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University to Qiao Wu. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Hefei National Laboratory for Physical

Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Anhui, China W Hu, L Jin, Y Mei & M Wu * Department of Immunology, Anhui Medical

University, Anhui, China W Hu * School of Biomedical Science and Pharmacy, University of Newcastle, Newcastle, NSW, Australia C C Jiang & X D Zhang * Melanoma Institute Australia and

Westmead Institute for Cancer Research, University of Sydney, Sydney, NSW, Australia G V Long * Discipline of Pathology, University of Sydney; Tissue Pathology and Diagnostic Oncology, Royal

Prince Alfred Hospital; Melanoma Institute Australia, Sydney, NSW, Australia R A Scolyer * State Key Laboratory of Cellular Stress Biology, School of Life sciences, Xiamen University,

Xiamen, China Q Wu Authors * W Hu View author publications You can also search for this author inPubMed Google Scholar * L Jin View author publications You can also search for this author

inPubMed Google Scholar * C C Jiang View author publications You can also search for this author inPubMed Google Scholar * G V Long View author publications You can also search for this

author inPubMed Google Scholar * R A Scolyer View author publications You can also search for this author inPubMed Google Scholar * Q Wu View author publications You can also search for this

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author inPubMed Google Scholar * M Wu View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to X D Zhang, Y Mei or M Wu.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by G Raschellá Supplementary Information accompanies this paper on Cell

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CITE THIS ARTICLE Hu, W., Jin, L., Jiang, C. _et al._ AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma. _Cell Death Dis_ 4, e914 (2013).

https://doi.org/10.1038/cddis.2013.441 Download citation * Received: 17 July 2013 * Revised: 30 September 2013 * Accepted: 04 October 2013 * Published: 07 November 2013 * Issue Date:

November 2013 * DOI: https://doi.org/10.1038/cddis.2013.441 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a

shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * AEBP1 * BRAF * melanoma