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Oncotarget. 2016 December 13; 7(50): 81995–82012.
Published online 2016 November 9. doi:  10.18632/oncotarget.13226
PMCID: PMC5347669

Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

Abstract

Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.

Keywords: melanoma, BRAF, vemurafenib, PLX-4720, TGF-beta

INTRODUCTION

Malignant melanoma is the most aggressive form of skin cancer with around 55,500 deaths worldwide in 2012 [1]. While primary localised melanoma may be cured by surgical removal alone, metastatic melanoma is associated with poor long-term prognosis. Somatic mutations that constitutively activate the RAS-RAF-mitogen activated protein kinase-extracellular signal-regulated kinase (RAS-RAF-MEK-ERK) signalling pathway are frequently detected in melanoma; mutations in BRAF and NRAS have been detected in approximately 50% and 20% of melanomas, respectively [2]. The identification of genetic drivers of melanoma [2] has led to the development of small-molecule inhibitors inhibitors (e.g. vemurafenib, dabrafenib) (BRAFi), which selectively target mutant BRAF. Their use in the clinic has significantly increased survival of metastatic melanoma patients [35]. However, the development of drug resistance remains a significant problem with the vast majority of patients with advanced melanoma dying of drug-resistant disease.

Numerous mechanisms of resistance to BRAF inhibitors have been described, many involving reactivation of the MAPK pathway (reviewed in [6]). As a result, the combined use of BRAF inhibitors with MEK inhibitors (e.g. cobimetinib, trametinib) has been proposed as a way to overcome the development of resistance [79]. While this approach significantly improves patient survival (resulting in a median expected survival of approximately 25 months for eligible patients), the efficacy of combinatorial therapies which target the same signalling pathway ultimately may be limited because of augmented BRAF inhibitor drug resistance mechanisms or secondary mutations [10, 11].

Additionally, secondary epigenetic events that do not necessarily affect MEK/ERK activity can occur to limit the tumour cells' dependence on the MAPK pathway, or restrict tumour immune surveillance. These resistance mechanisms include changes in the methylome affecting tumour cell apoptosis [12], increases in PI3K/AKT activity [1315] and/or increases in receptor tyrosine kinase (RTK) signalling. For instance, loss of microphthalmia-associated transcription factor (MITF) expression correlates with increased RTK expression and resistance [16]. Vemurafenib-resistance induced increases in EGFR signalling have been shown to activate an EGFR-SRC-STAT3 signalling cascade in melanoma, and targeting this pathway using inhibitors of SRC inhibits growth of vemurafenib-resistant xenografts [17, 18].

As well as cell autonomous effects, drug-induced stimulation of melanoma-associated fibroblasts stimulates matrix remodelling and, in this case, signals via integrins to increase SRC and FAK activity. This change in the microenvironment promotes melanoma cell survival and provides a “safe haven” to enable emergence of drug-resistant tumour cells [19]. Clearly, stromal remodelling and SRC activation have emerged as contributors to BRAF inhibitor resistance, and it is apparent that the therapy-induced secretome is key in driving resistance. Increased transforming growth factor-beta (TGFβ) secretion may be part of the therapy-induced secretome, and has been implicated in both in vitro derived drug resistance [20] and in vemurafenib-resistant patient material [21]. Increased TGFβ signalling can result in an upregulation of EGFR and PDGFR [21], positioning TGFβ signalling upstream of well described vemurafenib-resistance associated RTK pathways. Despite this, the potential for TGFβ pathway inhibitors in combating BRAF kinase inhibitor resistance has not been studied to date.

TGFβ ligand binds to the constitutively active high affinity type 2 serine/threonine kinase receptor TGFBR2 which trans-phosphorylates and activates TGFBR1. As part of the canonical signalling pathway, TGFBR1 phosphorylates and activates the intracellular signalling transcription factors SMAD2 and SMAD3, and following binding to SMAD4, the SMAD complex accumulates in the nucleus where it regulates target gene transcription. Additionally, TGFβ can signal via numerous non-canonical pathways including RHO/ROCK, MAPK, and PI3-Kinase (reviewed in [22]). In normal melanocytes, TGFβ inhibits proliferation and DNA synthesis and induces melanocyte stem cell quiescence, however, melanoma cells are able to evade the tumour suppressive effects of TGFβ. TGFβ levels are elevated in the plasma of melanoma patients (regardless of their exposure to BRAF inhibitors), and increases in expression are associated with progressive disease [23]. The mechanisms of growth arrest and their evasion by melanoma cells, however, have not been fully characterised and are likely to be multi-factorial (reviewed in [24]).

There is little evidence of mutation of TGFβ receptors in melanoma [25], so, it appears that with functional receptors and apparently intact SMAD function [26, 27], melanoma cells are able to evade growth suppressive effects of TGFβ while simultaneously utilising pro-tumourigenic functions of TGFβ. TGFβ signalling promotes migration of BRAF-transformed melanocytes in in vitro organotypic skin cultures [28] and is involved in metastasis of mouse melanoma cells to the bone through expression of tissue-specific genes known to promote bone osteolysis [26, 29]. In addition, melanoma cells engineered to over-express TGFβ exert paracrine effects on stromal fibroblasts whereby they secrete matrix components (including fibronectin, collagens, and tenascin) to promote melanoma tumour formation [30]. These observations are reminiscent of the vemurafenib-induced activation of melanoma-associated fibroblasts providing a “safe haven” for melanoma tumour cells, however, no link has been formally established between vemurafenib-induced fibroblast activation and TGFβ signalling.

In this study, we now provide evidence that melanoma cells are “hard-wired” to depend on autocrine TGFβ signalling through TGFBR1 for tumour establishment and clonogenicity. We show that the fundamental addiction of melanoma cells to TGFβ is: induced by the presence of mutant BRAF; mediated by a SMAD4-independent pathway; and correlates with TGFβ regulation of RHOA activity, thus providing support for the notion that non-canonical signalling pathways are key mediators of pro-tumourigenic TGFβ function in melanoma. Importantly, we also provide evidence that vemurafenib resistant patient-derived cells retain sensitivity to inhibitors of TGFBR1. TGFBR1 inhibitors block the enhanced proliferation of paradoxically activated PLX-4720 treated melanoma cells, and can be used to effectively inhibit metastatic melanoma in a zebrafish xenograft model.

RESULTS

Mutant BRAF confers TGFβ addiction

We demonstrated previously that autocrine signalling through TGFBR1, is required for transformation of rodent fibroblasts by oncogenic BRAF [31], but did not investigate this dependence in human models of activated RAS/RAF-driven cancer. Since mutational activation of BRAF is frequently observed in melanoma [2], we tested the susceptibility of immortalised mouse melanocytes stably transfected with either wild-type or mutant BRAF to inhibition by the TGFBR1 kinase inhibitor SB-431542. Unlike parental or wildtype BRAF transfected cells, melanocytes transfected with oncogenic V600E BRAF required TGFBR1 kinase activity for their proliferation since SB-431542 decreased cell numbers (Figure (Figure1a).1a). These data suggest that the presence of mutant BRAF in melanocytes confers a dependence (or addiction) on the TGFβ/TGFBR1 signalling pathway for cell proliferation. Similar results were observed in soft agar assays measuring anchorage independent growth (Figure (Figure1b).1b). We determined the amount of autocrine TGFβ produced by the transfected melanocytes, using a bioassay of NIH3T3 cells stably transfected with a CAGA12-luciferase reporter construct (Supplementary Figure 1a). The dependence on TGFBR1 activity for colony formation did not correlate simply with an increase in latent autocrine TGFβ production following transfection with mutant BRAF (no active TGFβ was detectable without medium acidification) (Supplementary Figure 1b). There was also no elevated signalling via the TGFβ receptor-regulated intracellular signalling transcription factor, SMAD2 in SB-431542 sensitive cells (Supplementary Figure 1c).

Figure 1
BRAFV600E confers sensitivity to TGFBR1 inhibition

We tested whether human cancer cells with activating mutations in MAPK pathway components were also dependent on TGFBR1 for growth. A panel of human tumour cell lines carrying wild type RAS/BRAF or mutations in BRAF, HRAS, KRAS or NRAS (details of all cell lines are given in Supplementary Table 1) were tested for sensitivity to SB-431542 (Figure (Figure1c).1c). Inhibition of TGFBR1 resulted in a range of cellular responses in the wildtype, H-, K-, and N-RAS mutant groups, such that any dependence on TGFBR1 for colony formation could not be predicted in cells carrying these mutations. However, consistent with data obtained in mouse melanocytes (Figure (Figure1b),1b), colony formation in all seven human cell lines carrying mutant BRAF was significantly inhibited (Figure (Figure1c).1c). Again, sensitivity to the TGFBR1 inhibitor did not correlate with levels of autocrine TGFβ production (Supplementary Table S2). The effect of SB-431542 was dose-dependent in low density 2D-culture assay conditions established to assess more accurately clonogenic potential, reaching statistically significant inhibitory concentrations at 10μM (Figure (Figure1d1d and and1e).1e). Similar dose dependent effects were seen in anchorage-independent soft agar assays (Figure (Figure1f).1f). We attempted to select out TGFBR1 inhibitor-resistant cells following repeated rounds of treatment for over a month, but saw no evidence of outgrowth of refractory subpopulations or acquired-resistance during this time frame (Figure (Figure1g).1g). Taken together these data suggest that cells with mutational activation of BRAF, require TGFBR1 for efficient colony formation and that TGFβ would predictably function as a tumour promoter.

Autocrine TGFβ is required for in vivo melanoma xenograft tumour formation

Melanoma cells engineered to over-express TGFβ1 have increased tumour forming ability [30]. To discover whether endogenous autocrine TGFβ expression is required for tumour formation, we generated ligand knockdown clones of A375(M2) cells using stably transfected shRNA constructs targeting TGFβ1. Knockdown of TGFβ1 to levels below 20pg per 1x105 cells/hr (Figure (Figure2a)2a) was sufficient to decrease the ability of A375(M2) cells to form colonies in vitro (Figure (Figure2b2b and Supplementary Figure 2a). In xenograft assays, ligand knockdown reduced the percentage of mice with palpable tumours at all recorded time-points (Figure (Figure2c),2c), and significantly reduced tumour growth (Figure (Figure2d).2d). Immunohistochemical (IHC) staining of xenograft sections revealed increased expression of the cyclin-dependent kinase inhibitor CDKN1A (p21CIP1) in tumours generated by TGFβ knockdown cells (Figure (Figure2e2e and and2f).2f). Elevated CDKN1ACIP1 expression was also observed following SB-431542 treatment of A375(M2) cells (Supplementary Figure 2b).

Figure 2
Colony and in vivo tumour formation require the autocrine production of TGFβ1

So far, our data implicate autocrine TGFβ signalling through TGFBR1 as a critical factor in melanoma clonogenicity and tumour formation, however, it was important to rule out off-target effects of the inhibitor. We therefore assessed colony formation following transient transfection with two independent siRNAs targeting TGFBR1. TGFBR1 knockdown (Supplementary Figure 3a) reduced TGFBR1 protein expression and phosphorylation of SMAD2 in response to exogenous TGFβ (Figure (Figure3a3a and and3b),3b), and recapitulated the effect of chemical inhibition of the receptor. Colony formation and cell proliferation decreased following TGFBR1 knockdown (Figure 3c, 3d, and Supplementary Figure 3b and 3c), confirming that TGFBR1 is required for melanoma colony formation.

Figure 3
TGFBR1, but not SMAD4, is required for clonogenicity of mutant BRAFV600E melanoma cells

To discover whether the canonical SMAD pathway is required for either melanoma cell colony formation or for the inhibitory response to SB-431542, transient knockdown of the co-SMAD, SMAD4 was performed. Knockdown of SMAD4 (Figure (Figure3e)3e) had no significant effect on A375(M2) colony formation (Figure (Figure3f)3f) and significantly increased Colo829 cell proliferation (Figure (Figure3g).3g). These data suggest that SMAD4-dependent signalling is not necessary for colony outgrowth and may, in fact, repress colony growth in Colo829 cells. SMAD4 knockdown also did not block the inhibitory effect of SB-431542 (Figure (Figure3h),3h), indicating that the mechanism of inhibition is SMAD4 independent. In case the levels of knockdown were not sufficient to accurately assess the contribution of SMAD4, we tested a mutant BRAF/SMAD4 null cell line (HT29). These cells were also sensitive to TGFBR1 inhibition (Supplementary Figure 4a and 4b) thus supporting our conclusion that functional SMAD4 is not necessary for inhibition of colony formation by SB-431542.

We next considered any non-canonical signalling pathways that might be affected by TGFβ signalling in melanoma cells. We previously reported that a non-canonical TGFβ/TGFBR1/RHOA signalling pathway is necessary for initiation and maintenance of rodent fibroblast BRAFV600E transformed cultures [31]. Thus, in mutant BRAF human melanoma cells, it seemed plausible that this pathway could be involved in regulating melanoma cell clonogenicity. SB-431542 treatment reduced levels of active-GTP bound RHOA (Supplementary Figure 4c), while transfection of melanoma cells with the exoenzyme C3 transferase to inhibit RHOA [32] mimicked the effect SB-431542 (Supplementary Figure 4d). In addition, overexpression of either constitutively active RHOA, or the constitutively active RHOA specific guanine nucleotide exchange factors Δ558LARG or onco-LBC (to activate endogenous RHOA) [33], blocked the effect of the TGFBR1 inhibitor on colony formation (Supplementary Figure 4e). These data are consistent with our previous findings in rodent fibroblasts.

To gain further insight into the cellular pathways involved in the inhibition of colony formation, we analysed cells by microscopy for division and apoptosis, using BRDU incorporation or fluorogenic apoptosis reagents respectively. Initial experiments revealed that SB-431542 treatment significantly reduced BRDU incorporation but induced little apoptosis (Supplementary Figure 5a and data not shown). We questioned whether an apparently modest reduction in BRDU incorporation was sufficient to account for the dramatic reduction in colony formation and cell proliferation. To investigate in more detail, we generated A375(M2) cell lines stably transfected with an H2B-red fluorescent protein (RFP) fusion protein expression construct to enable kinetic single cell tracking using IncuCyte imaging. Imaging between days four and six of treatment (Supplementary Figure 5b) showed a reduction in their number, and a significant increase in the length of time cells remained in interphase (Supplementary Figure 5c). There were slight increases in the mean number of cells that failed to enter into mitosis or detached upon treatment, but these differences did not reach statistical significance (Supplementary Figure 5d and 5e). TGFBR1 inhibition, therefore, predominantly affects the proportion of cells in S-phase, and significantly affects the clonogenic potential of BRAF mutant cells through effects on the cell cycle.

BRAF inhibitor resistance

The addiction of mutant BRAF melanoma cells to signalling through TGFBR1 suggests a potential novel therapeutic approach for mutant BRAF-driven cancers. Ideally, not only would a novel treatment be effective as a single agent without evidence of refractory disease (Figure (Figure1),1), but the novel therapeutic drug would act in combination with existing therapies to enhance their efficacy or prevent the development of resistance. The current therapeutic modality for mutant BRAF metastatic melanoma is treatment with BRAF inhibitors vemurafenib or dabrafenib in combination with MEK inhibitors for suitable patients. However, the development of BRAF inhibitor-resistant disease through a variety of different mechanisms, including the paradoxical activation of the MAPK pathway, remains a significant clinical problem.

To assess the potential for a combination therapy targeting both BRAF and TGFBR1, we first tested the sensitivity of previously drug naïve A375(M2) and Colo829 to the mutant BRAF kinase inhibitor PLX-4720 in our clonogenic, low density assay conditions. As expected, at doses exceeding 250nM, growth of both cell lines was inhibited, however, at lower doses, we noted an unexpected significant increase in cell proliferation (Figure 4a, 4b and Supplementary Figure 6a). Suboptimal doses of PLX-4720 induced phosphorylation of ERK (Supplementary Figure 6b), and the enhanced proliferation of Colo829 (Figure (Figure4c)4c) and A375(M2) cells (data not shown) were abrogated by co-treatment with the MEK inhibitor PD184352 (Figure (Figure4c).4c). These data are consistent with low dose PLX-4720 paradoxically activating the RAS-MAPK pathway. Since both cell lines carry BRAFV600E and are wild type for RAS, the most likely interpretation is that low dose PLX-4720 relieves an inhibitory autophosphorylation [34]. Consistent with this hypothesis, low dose PLX-4720 did not promote the proliferation of three melanoma cell lines carrying wild type RAF/RAS (Supplementary Figure 7). Importantly co-treatment of PLX-4720 treated mutant BRAF cells with SB-431542 (10μM) abolished the increase in cell growth caused by low dose PLX-4720 (Figure (Figure4d4d and and4e).4e). We quantified the effect on clonogenicity [35] and showed that not only did SB-431542 significantly reduce clonogenicity as a single agent, but that the significant increase in clonogenicity induced by PLX-4720 alone was reversed by SB-431542 (Figure (Figure4f4f and Supplementary Figure 6c). This result was recapitulated by siRNA knockdown of TGFBR1 (Figure (Figure4g4g).

Figure 4
Low dose BRAF inhibitor (PLX-4720) enhances proliferation of drug naïve melanoma cells

To investigate further the potential for TGFBR1 inhibitors to prevent vemurafenib resistance, we tested both in vitro derived resistant lines (A375R), and patient derived vemurafenib-resistant recurrent tumour cells for sensitivity to SB-431542 (10μM). Patients #2 and #35 (stage IV) achieved a partial response having received vemurafenib for 3 months. Patient #5 (stage IV) had progressive disease and received vemurafenib for 2 months (Supplementary Table 1). The growth of vemurafenib-naïve A375 (Figure (Figure5a)5a) and patient tumour derived cells (Patient#1) (Figure (Figure5b)5b) [18] were both inhibited by SB-431542. Importantly, in vitro derived PLX-4720 resistant A375R cells (cultured in the presence of 1μM PLX-4720) were growth inhibited by SB-431542 (Figure (Figure5a).5a). The patient-derived vemurafenib resistant cells had readily detectable levels of phosphorylated SMAD2 that were reduced on SB-431542 treatment, indicating that they all had active autocrine TGFβ signalling (Figure (Figure5c).5c). In addition, all vemurafenib-resistant lines derived from patients were growth inhibited by SB-431542 in proliferation assays (Figure (Figure5d)5d) and in longer term colony formation assays (Figure (Figure5e).5e). Vemurafenib resistant cells therefore retain their sensitivity to inhibitors of the TGFβ signalling pathway.

Figure 5
Patient-derived BRAF inhibitor resistant tumour cells are sensitive to TGFBR1 inhibition

Several reports indicate that the development of resistance to BRAF kinase inhibitors may be associated with signalling through SRC-family kinases, and that resistance can be overcome by inhibition of SRC activity [1719, 36]. We therefore tested whether TGFβ signalling was associated with SRC phosphorylation in A375(M2) and Colo829 cells (Figure (Figure6).6). We noted that phosphorylated-SRC levels increased during the five day incubation period in cells initially plated at low density. SB-431542 (10μM) (Figure (Figure6a)6a) and TGFBR1 siRNA (Figure (Figure6b)6b) prevented any accumulation of phosphorylated SRC during the time course, and SB-431542 blocked an increase in phospho-SRC levels induced by low-dose PLX-4720 in A375(M2) cells (Figure (Figure6a).6a). An important implication of these data is that inhibition of TGFBR1 signalling may restrict signalling through a known mediator of vemurafenib resistance.

Figure 6
SB-431542 treatment and TGFBR1 knockdown inhibit phosphorylation of SRC

Zebrafish embryo xenograft metastasis model

So far, our murine xenograft assays, and inhibition of clonogenic potential of melanoma cells in low cell density 3D and 2D culture systems, suggest that TGFBR1 inhibitors would be effective in preventing establishment of disease. To further examine whether TGFBR1 inhibitors could effectively treat established cell cultures, we seeded cells at low cell density and progressively delayed addition of drug throughout the lag phase of cell growth. SB-431542 was effective if administered during the lag phase, but delaying treatment until the cells start to exit the lag phase considerably reduced its efficacy (Supplementary Figure 8a). Similarly, seeding cells at higher cell numbers also reduced the efficacy of SB-431542 in both vemurafenib naïve and resistant cells (Supplementary Figure 8b). Given these observations, we posit that cell:cell contact and/or allowing the secretion of growth factors or matrix components has a protective effect against TGFBR1 inhibitors; the implication is that TGFBR1 inhibitors might not be useful as first line, single agents or as debulking therapeutic agents in established solid tumours. Nevertheless, the dependence of melanoma cells on TGFBR1 for clonogenicity suggests that TGFBR1 inhibitors could be effective in preventing spread or outgrowth of micrometastases. To test this hypothesis we used a zebrafish embryo metastasis model [37, 38] to visualise and quantify numbers of invasive melanoma cells. This model has been used successfully to examine the effect of SB-431542 on breast cancer cell invasion [39]. We generated stable TGFBR1 knockdown A375(M2) cell lines using LMP-TGFBR1 shRNA plasmids, which had reduced TGFBR1 protein expression and reduced capacity to signal (Figure (Figure7a)7a) as well as reduced ability to form colonies on plastic (Figure (Figure7b).7b). The stable lines were labelled with the mCherry fluorophore, injected into the Duct of Cuvier (DoC) of zebrafish embryos and the numbers of invasive cells in the avascular tail fins were analysed. Representative con-focal images of metastatic spread into the tail fin are shown in Figure Figure7c.7c. Control non-silencing (NS) A375(M2) cells were capable of metastatic spread into the fish fin (arrows indicate micro-metastases). Using doses of 1μM SB-431542 (SBi), we observed a significant decrease in the ability of SB-431542 treated NS cells to invade. Stable TGFBR1 knockdown also reduced colonisation of zebrafish tissue (Figure (Figure7d).7d). Our data overall suggest that TGFBR1 inhibitors would reduce the metastatic burden in BRAF mutant melanoma by preventing invasion and/or outgrowth of metastatic colonies.

Figure 7
TGFBR1 is required for tumour cell metastasis in xenografted zebrafish

DISCUSSION

The outcome for patients with advanced melanoma has improved dramatically in recent years. The development of targeted therapy of the MAPK pathway, and advances in immunotherapy have resulted in improvements in median survival from 9 months to 25-31 months. However, long-term prognosis remains uncertain. For targeted therapy using BRAFi, drug resistance mechanisms identified to date are numerous, and there is no established effective second-line targeted therapy for patients progressing on combination BRAFi + MEKi. Often, drug-resistance mechanisms involve induction of either an autocrine, or a paracrine, drug-induced secretome which helps to promote expansion and dissemination of the drug-resistant cells [15] and/or protect potentially sensitive tumour cells from the inhibitory effects of the chemotherapeutic agent [15, 19, 21]. Phosphorylated ERK and SRC are frequently elevated in resistant tumours [18] suggesting that paradoxical activation of the MAPK pathway and growth factor signalling are involved in resistance development. In this study we investigated the potential of targeting TGFβ1 as second-line therapy for advanced melanoma.

We found that autocrine TGFβ signalling through TGFBR1 is an intrinsic requirement for the clonogenic potential of mutant BRAF transformed cells, indicating that mutation of BRAF may be useful as a biomarker for TGFβ tumour promoting activity in melanoma. Although TGFβ levels are elevated post-vemurafenib treatment [20, 21], our data suggest that a TGFβ/TGFBR1-dependent state is an adaptation to the presence of mutant BRAF. Thus, with pro-tumourigenic autocrine TGFβ signalling pathways having already been established, elevated levels of signalling during therapy are perhaps more readily selected for than would otherwise be the case. How the initial switch from tumour suppressor to tumour promoter function of TGFβ in melanocytes is mediated by mutant BRAF remains to be determined.

Our in vitro assays were specifically designed to mimic conditions of cellular stress (i.e. low density 2D and anchorage independent colony formation assays) to more accurately assess the clonogenic potential of melanoma cells and their cancer stem-cell like properties. TGFBR1 inhibition was highly effective in inhibiting growth of both naïve and vemurafenib-resistant cells when administered during the lag phase of growth; less so when cell seeding numbers were increased. The implication of these data is that targeting TGFβ/TGFBR1 may not be an effective therapeutic strategy in established tumours. A secondary consideration is that TGFBR1 inhibition affected proliferation of the tumour cells without inducing apoptosis, and so may not result in significant tumour shrinkage. As a consequence, we predicted that inhibiting TGFβ/TGFBR1 signalling would more likely be effective in preventing tumour metastasis and outgrowth of micrometastasis, rather than reducing established tumour burden. Indeed, our murine and zebrafish xenograft models show that targeting autocrine TGFβ secretion and TGFBR1 kinase activity inhibits xenograft tumour establishment in mice and prevents metastatic spread in zebrafish tissues. TGFBR1 inhibitors therefore may have potential as an adjuvant therapy in high risk, resected disease, or a maintenance therapy in patients responding to BRAF inhibitors. The inhibition of glioblastoma cancer initiating (stem) cells by TGFBR1 inhibitors [40, 41] is consistent with our data, and provides support for our conclusion that TGFBR1 activity is required for melanoma stem-cell like properties.

Given the vast number of context specific genes regulated by TGFβ, it is likely that a number of different downstream effectors will mediate the autocrine TGFβ-induced promotion of melanoma cell growth and drug-resistance. We showed that although melanoma cells rely on TGFBR1 kinase activity, they do not require SMAD4 for either colony formation, or for the response to TGFBR1 inhibition. Signalling via RHOA, however, rescued the effect of SB-431542 which is both consistent with our previous analysis of rodent fibroblast transformation [31], and with a role for TGFβ-activated non-canonical signalling pathways in this response. The establishment of an adaptive autocrine TGFβ/TGFBR1 signalling pathway through RHOA following BRAF mutation may be necessary to overcome CDKN1A expression and growth arrest induced as a response to oncogenic stress [31, 42]. Consistent with this are our data showing CDKN1A induction by disrupting TGFβ signalling both in vitro and in vivo (Figure (Figure22 and Supplementary Figure S2). Interestingly, inhibition of ROCK (using Y27632), a downstream target of RHOA signalling, also induces CDKN1A in melanoma [43], and ROCK1 has been identified as a potential candidate for combinatorial therapy with BRAF inhibitors. In these studies inhibition of ROCK1 sensitises melanoma cells to PLX-4720 [44]. Our data now suggest that the involvement of ROCK1 in resistance mechanisms is potentially a result of TGFβ/TGFBR1/RHOA signalling. Although the SMAD-dependent induction of RHO GEFs has been described in other studies, [45, 46] how TGFBR1 directly activates RHOA in melanoma cells in a SMAD-independent manner is unclear at present.

Several other TGFβ target genes have been implicated in melanoma biological responses. Melanoma cells exposed to the high levels of exogenous TGFβ present in bone, upregulate osteolytic genes (including IL-11, PTHrP, and CTGF) which may aid more effective colonisation of this metastatic niche. Blocking receptor function by over-expression of the natural inhibitor SMAD7 extended survival of mice xenografted with SMAD7 expressing melanoma cells. However, a causal role for the TGFβ-regulated osteolytic genes in bone metastasis was not directly demonstrated [26]. Similarly, the balance between the TGFβ target gene GLI2 and the melanocyte specific isoform of MITF (M-MITF) appears important for invasion through matrigel in vitro, with high GLI-2/low M-MITF correlating with invasion. However, these expression profiles were independent of BRAF mutation status and did not correlate with either proliferation in vitro or with subcutaneous xenograft tumour establishment [47]. We suspect that TGFβ target genes induced by exogenous TGFβ exposure may be quite different from those genes regulated by non-canonical signalling as a result of autocrine TGFβ ‘addiction’ established following BRAF mutation. Further work to identify which are the key TGFβ target genes involved in both promoting these stem-cell properties, and in driving drug-resistance, is underway and we expect that these studies will suggest novel, selective therapeutic targets.

We show that drug-naïve melanoma cells are growth promoted by low-dose PLX-4720, likely by paradoxical activation of the MAPK pathway. This may have important implications clinically, since low doses of bioavailable BRAFi reaching some tumour tissue could actually potentiate tumour growth. Importantly, we show that both paradoxically activated, previously drug naïve cells, as well as vemurafenib resistant cells, retain sensitivity to TGFBR1 inhibitors. In addition, SB-431542 prevented phosphorylation of SRC which is frequently associated with vemurafenib resistance, suggesting that TGFBR1 inhibitors would prevent relapse with vemurafenib-resistant metastases. How SB-431542 regulates SRC activation is currently under investigation in our laboratory. It will be important to test the sensitivity of BRAFi/MEKi resistant cells derived from patients treated with combination therapy when established. Nevertheless, we predict that targeting an independent signalling pathway may have some advantages over combination therapies which target different components of the same signalling pathway. In addition, blocking the immunosuppressive effects of TGFβ could potentiate the efficacy of immune based therapeutics. Since dependence on TGFβ signalling appears to be universal in mutant BRAF melanoma cells, targeting TGFβ or downstream effectors may also provide useful therapeutic options for blocking metastatic outgrowth of vemurafenib refractory disease which occurs in approximately 20% of patients receiving treatment. There are currently a number of TGFβ pathway inhibitors progressing through Phase 1-3 clinical trials [48]. The small molecule TGFBR1 inhibitor Galunisertib is being evaluated in cancer patients with unmet need. This inhibitor is deemed tolerable, with an acceptable margin of safety when administered using intermittent dosing regimens [49], demonstrating that TGFBR1 inhibitors are suitable for clinical use and may provide new opportunities for therapy of BRAF-inhibitor resistant cancer.

MATERIALS AND METHODS

Western blotting

Cell lysates were analysed by SDS-PAGE using the following antibodies: PO4-SMAD2 (Ser465/467) (rabbit polyclonal, #3101, Cell Signalling Technology [CST]), SMAD2 (mouse monoclonal, C16D3, CST), SMAD2/3 (mouse monoclonal, Clone 18, BD transduction Laboratories), SMAD4 (mouse monoclonal, B-8, Santa Cruz Biotechnology), TGFBR1 (rabbit polyclonal, V-22, Santa Cruz Biotechnology), CDKN1A (rabbit polyclonal, C19, Santa Cruz Biotechnology), RHOA (mouse monoclonal, 26C4, Santa Cruz Biotechnology), PO4-SRC (Tyr416) (rabbit monoclonal, D49G4, CST), SRC (rabbit monoclonal, 36D10, CST), PO4-p44/p42 MAPK (ERK1/2) (Thr202/Tyr404) (rabbit polyclonal, #9101, CST), p44/p42 MAPK (ERK1/2) (rabbit polyclonal, #9102, CST), β-actin (mouse monoclonal, AC-74, Sigma). Secondary HRP-conjugated antibodies (Dako) and enhanced chemiluminescence (GE Healthcare) was used to detect bound antibody.

Cell culture

Details of the cell lines and media supplements used are shown in Supplementary Table S1. All cells lines were tested regularly for mycoplasma contamination by the Institute's mycoplasma testing service. Patient derived cell lines were passaged for approximately 1 month. Where indicated the cells were transfected with Lipofectamine or Lipofectamine 2000 (Invitrogen) using the following plasmids; pRK5 C3-transferase and pEF-Flag LARG Δ558 (kind gifts of R. Grosse), pRK5-RhoA V14 (kindly supplied by Alan Hall), pSR-Flag onco LBC (kindly supplied by Mike Olson), or pSuper-TGFβ1. LMP-scrambled non-silencing (NS) and LMP-TGFBR1 shRNA constructs were generated in house with the following hairpin sequences:

NS - 5′ CGAGAAGGTATATTGCTGTTGACAGTGAGCGACT CATAGCGATGTGAACTCAATAGTGAAGCCACAGA TGTATTGAGTTCACATCGCTATGAGCTGCCTACTG CCTCGG -3′;

TGFBR1#1 - 5′ TCGAGAAGGTA TATTGCTGTTGACAGTGAGCGACTCATAGAGAT TTGAAATCAATAGTGAAGCCACAGATGTATTGAT TTCAAATCTCTATGAGCTGCCTACTGCCTCGG -3′;

TGFBR1#2 - 5′ TCGAGAAGG TATATTGCTGTTGACAGTGAGCGACAGTGTAATA AAGTCAATTAATAGTGAAGCCACAGATGTATTAAT TGACTTTATTACACTGCTGCCTACTGCCTCGG. -3′.

Cells were transfected with either Oligofectamine or HiPerFect (Qiagen) to introduce, at a final concentration of 20 - -50nM, the following siRNA; allstars negative control, TGFBR1 [HS_TGFBR1_6 (TGFBR1#1) and HS_TGFBR1_7 (TGFBR1#2) (Qiagen)] or SMAD4 (Dharmacon smartpool). Mock transfections (no siRNA) were included in each experiment. A375(M2) pSuper or pSuper-TGFβ1 stable cell lines were selected and maintained in 0.6mg/mL puromycin. A375(M2) histone H2B-RFP stable cell lines were selected and maintained in 800μg/mL G418, and LMP-scrambled or LMP-TGFBR1 shRNA derivatives were maintained in 800μg/mL G418 plus 1μg/mL puromycin. Where indicated the cells were treated with SB-431542 (Tocris) [50], PLX-4720 (Selleck Chemicals) or PD184352 (Cell Signalling) (prepared in DMSO).

Soft agar assay

Soft agar assays were carried out essentially as previously described [31]. Briefly, six well plates were coated in 2mLs of media supplemented with 0.9% low melting point agar (Invitrogen). 2mL cells (1x104/mL) in media supplemented with 0.45% low melting point agar were overlaid with either SB-431542 or vehicle control. Wells were fed twice weekly for 2-4 weeks, and the number of colonies (> 80μm in diameter) in nine fields of view was scored using an Olympus CKX41 microscope, fitted with a 4X objective and an eyepiece graticule (250μm gradations). Statistical analyses were carried out by Students TTEST unless stated otherwise.

Proliferation assay

Cell proliferation kinetics were either monitored using an IncuCyte ZoomTM imaging system and software (percent confluence) (Essen Biosciences), or by trypsinisation and cell counts using a Casy cell-counter (model TT, Innovatis).

Colony formation and clonogenicity assays

Colony formation: Cells were seeded in 10cm dishes at an appropriate density to form approximately 250 discrete colonies after 2-3 weeks in culture. Colonies were fixed in methanol and stained with toluidine blue/borax solution for counting.

Clonogenicity: Cells were seeded overnight at 1 and 3 cells/well in 60 wells of a 96-well plate, prior to treatment. Wells were fed twice weekly, and wells examined by light microscopy. After approximately two weeks, media was removed, colonies fixed in methanol and stained with 0.4% (w/v) sulforhodamineB (SRB)/1% acetic acid. Colonies > 50 cells in size were counted, and the plating efficiency and surviving fractions after drug treatment determined according to Franken et al [35].

TGFβ1 ELISA

The TGFβ1 assay has been described previously [31]. Briefly, cells were cultured in media containing 0.1% FBS for 24 hours. Media was harvested and the cells trypsinized and counted. The media was acid treated to activate latent TGFβ, and TGFβ1 levels determined by ELISA using anti-TGFβ1 (MAB1835) (capture antibody) and biotinylated anti-TGFβ1 (BAF240) (detection antibody). Recombinant hTGFβ1 (Peprotech) was used as a standard. Results were expressed as TGFβ1 produced per 1x105 cells/hour.

Mouse xenografts

Nude mouse subcutaneous xenograft experiments were performed according to Home Office guidelines and were approved by the local research and ethics committee (BICRLREC). 1x106 cells were injected subcutaneously into the flank of CD1 nude mice (n = 8) (Charles Rivers). Palpable tumours were observed 8 days post-injection and tumour volumes were calculated using calliper measurement and the formula V = (E2xA)/2 where E = shortest and A = the longest diameter measurement.

Immunohistochemistry

Sections from formalin, paraffin embedded, pSuper (Control shRNA) or pSuper-TGFβ1 shRNA tumours were stained for CDKN1A (M19, Santa Cruz Biotechnology) using an Envision kit (Dako) according to the manufacturer's instructions. The sections were counter stained with Haemotoxylin and were scored for CDKN1A expression. One representative field of view (that contained a minimum of 350 cells) was scored (blind) for each tumour.

IncuCyte zoom and analysis

An IncuCyte Zoom live cell imaging microscope (Essen Biosciences) with 10x objective and data management software was used to monitor kinetic cell proliferation. The mean ± SEM percent confluence from four phase-contrast images/well, with a minimum of 3 replicate wells/treatment was determined according to software processing definitions as recommended by the manufacturer. Statistical analysis was carried out using Graphpad software and pairwise comparisons using the compareGrowthCurves function (statmod, R project, 10,000 permutations).

Embryo preparation and tumor cell implantation

Zebrafish and embryos were raised, staged and maintained according to standard procedures. The Institutional Committee for Animal Welfare of the Leiden University Medical Center (LUMC) approved this study. Tg(Fli1:GFP) zebrafish embryos were dechorionated at two days post-fertilisation (dpf). Single cell suspensions of melanoma cells were prepared in PBS and kept at 4°C before implantation. The cell suspension was loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus) and the injections were performed using a Pneumatic Picopump and a manipulator (World Precision Instruments, Stevenage, UK). Dechorionated embryos were anaesthetised with 0.003% 3-amino benzoic acid ethyl estertricaine, (Sigma)] and mounted on 10 cm Petridishes coated with 1% agarose. Approximately 200 cells were injected at the duct of Cuvier (DoC). Implanted zebrafish embryos were maintained at 33°C. Zebrafish in the Non-Silencing (NS) + SB431542 group were treated with 1μM SB-431542 added to the eggwater. All implantations were repeated at least three times with at least 30 embryos per group.

Microscopy and analysis of zebrafish

Zebrafish embryos were fixed with 4% paraformaldehyde for two hours at room temperature. Embryos were imaged in PBS/0.1% Tween-20 (Merck, Amsterdam, Netherlands) using a Leica SP5 STED confocal microscope (Leica, Rijswijk, Netherlands). Confocal stacks were processed for maximum intensity projections with Image J. Images were adjusted for brightness and contrast, and overlays created using Adobe Photoshop CS6. Statistical analysis was performed using Prism 4 software (GraphPad, La Jolla, USA). Results are expressed as the mean ± SD. One-way analysis of variance (ANOVA) were performed followed by the Tukey's method for multiple comparison. P < 0.05 was considered to be statistically significant (*0.01 < P < 0.05; **0.001 < P < 0.01; *** P < 0.001). In one experiment the results were scored blinded; all results were confirmed by an independent observer.

SUPPLEMENTARY VIDEOS

Acknowledgments

We are indebted to Norman Sharpless, Jeff Evans, Marene Landstrom, Kevin Ryan, Wolfgang Schultz, Simon Cook, Mina Bissel and Holger Kalthoff for providing cell lines.

Footnotes

GRANT SUPPORT

Financial support: GJI, LCS, GJF, GS, TH, OJS and DFV were supported by the CRUK Beatson Institute. LS was also funded by a Worldwide Cancer Research (formerly AICR) project grant to GJI (11-078). MRG and RM are supported by the CRUK Manchester Institute [C5759/A12328] and the Wellcome Trust [100282/Z/12/Z]. SL, CC and PtD are supported by the Cancer Genomics Centre, Netherlands.

CONFLICTS OF INTERESTS

We declare no conflict of interest

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