furin is a direct transcription target of notch1 notch1 auto-activation

Furin is a direct transcription target of Notch1

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Notch1 auto-activation via transcriptional regulation of Furin, which sustains Notch1 signaling by 1

processing Notch1-activating proteases ADAM10 and MT1-MMP* 2

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Hong Qiu, Xiaoying Tang, Jun Ma, Khvaramze Shaverdashvili, Keman Zhang, Barbara Bedogni 4

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Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106 6

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*Running title: furin is a direct transcription target of Notch1 8

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To whom correspondence should be addressed: Barbara Bedogni, Dept. Of Biochemistry, Case Western 10

Reserve University School of Medicine, 10900 Euclid Ave, Cleveland, OH 44106, USA; Tel: 216-368-7602; 11

Fax: 216-368-3419; email: [email protected] 12

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Keywords: Notch1, MT1-MMP, Furin, ADAM10 14

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MCB Accepted Manuscript Posted Online 17 August 2015Mol. Cell. Biol. doi:10.1128/MCB.00116-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 31

Notch1 is an evolutionarily conserved transmembrane receptor involved in melanoma growth. Notch1 is 32

first cleaved by Furin in the Golgi to produce the biologically active heterodimer. Following ligand binding, 33

Notch1 is cleaved at the cell membrane by proteases such as ADAM10 and 17 and MT1-MMP, the latter of 34

which we recently identified as a novel protease involved in Notch1 processing. The final cleavage is 35

γ-secretase-dependent and releases the active intracellular domain (NIC). We now demonstrate that Notch1 36

directly regulates Furin expression. Aside from activating Notch1, Furin cleaves and activates several 37

proteases including MT1-MMP, ADAM10 and 17. By chromatin immunoprecipitation and reporter assay, 38

we demonstrate that Notch1 binds at position -1236 of the Furin promoter and drives Furin expression. The 39

Notch1-dependent enhancement in Furin increases the activity of MT1-MMP and ADAM10 but not 40

ADAM17, as demonstrated by shRNA knockdown of Furin. And promotes the cleavage of Notch1 itself. 41

These data highlight a novel positive feedback loop whereby Notch1-dependent Furin expression can induce 42

Notch1 signaling by increasing Notch1 processing and by potentiating the activity of the proteases 43

responsible of its activation. This leads to Notch1 signal amplification that can promote melanoma tumor 44

growth and progression as demonstrated by the inhibition of cell migration and invasion upon furin 45

inhibition downstream of Notch1. Disruption of such feedback signaling might represent an avenue to treat 46

melanoma. 47

48

INTRODUCTION 49

Melanoma is the deadliest form of skin cancer, causing approximately fifty thousand deaths a year, 50

despite new promising therapeutic treatments (1-3). It is imperative to understand melanoma biology in 51

order to find novel therapeutic targets. 52

Notch proteins are transmembrane receptors of approximately 300 kD. In humans, there are four Notch 53

receptors, Notch1-4, which share the same basic structure: 1) an extracellular domain containing 54

EGF-repeats (epidermal growth factor-like repeat domain), an LNR domain (LIN12-Notch-repeat domain) 55

and a HD domain (heterodimerization domain); and 2) an intracellular domain containing a RAM domain 56

(RBP-JK-associated module, an ANK domain (ankyrin repeat), a TAD domain (transactivation) and a PEST 57

domain (proline-glutamate-serine-threonine) (4). The full length Notch is first cleaved by Furin in the trans 58

Golgi. Such cleavage is necessary to properly position Notch1 at the plasma membrane (5, 6). Once at the 59

membrane and following ligand binding, a second cleavage occurs that is canonically driven by ADAM 60

proteases (ADAM10 and 17) (7-10). This produces an unstable fragment that is immediately cleaved by 61


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γ-secretase to release the intracellular domain (NIC) (11). NIC translocates into the nucleus, and binds with 62

transcription factors CSL (C-promoter binding factor-1) and MAML (Mastermind-like) to regulate the 63

transcription of multiple downstream targets, including HES family members, c-Myc, and Cyclin D3 64

(12-20). 65

The Notch pathway is an evolutionarily conserved signaling cascade that plays essential roles in 66

embryogenesis and in cell renewal in the adult by participating in the maintenance of stem cell pluripotency 67

in a variety of tissues (21-23). Previous studies have shown that Notch1 plays essential roles in melanocyte 68

stem and precursor cell homeostasis (24-27). Furthermore, Notch1 is highly expressed in melanomas. 69

Overexpression of active Notch1 (NIC1) transforms primary human melanocytes in vitro and confers 70

metastatic properties to primary melanoma cells (28-31). Notch1 was also shown to be required for 71

melanoma cell growth and survival (31). However, no Notch1 activating mutations have been observed in 72

melanoma so far, suggesting other mechanisms are responsible for the high activity of Notch1 in melanoma 73

cells. 74

One potential mechanism is an increase in processing. We recently demonstrated that MT1-MMP, a 75

membrane-tethered zinc dependent matrix metalloproteinase, acts as a protease involved in the second 76

cleavage of Notch1 (32). MT1-MMP is very abundant in melanomas and associates with disease progression 77

and poor patient outcome (32, 33). MT1-MMP is synthesized as a pre-pro-enzyme of 64kDa that is also 78

cleaved by Furin prior to going to the plasma membrane as an active 55kDa enzyme (34). MT1-MMP is one 79

of the most important MMPs that promote cell migration and invasion in cancer. In melanoma, we 80

demonstrated that MT1-MMP mediates melanoma growth and metastasis via various mechanisms including 81

activation of Notch1 (32). 82

ADAM10 and ADAM17 belong to the ADAM (A disintegrin and metalloproteinase) family of zinc 83

dependent metalloproteinases and are considered the canonical proteases involved in the cleavage of Notch1 84

at the S2 site (7). Structurally similar to MT1-MMP, they also are activated in the Golgi by Furin, to then 85

arrive at the membrane as active enzymes (34). The dysregulated expression of ADAM proteins has been 86

reported in multiple tumors. They are implicated as positive regulators in tumor cell proliferation, 87

angiogenesis and metastasis (35). Importantly, both ADAM10 and ADAM17 have been shown to be 88

overexpressed in melanoma (36-39). 89

Furin cleaves many substrates, including MT1-MMP, Notch1, ADAM10, and ADAM17 (6, 34, 40-42). 90

Furin is a calcium-dependent serine protease that belongs to the proprotein convertases (PCs) family. The 91

expression levels of Furin vary from different cell types and cell differentiation degree (43-47). Furin has 92


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been reported to regulate tumor growth and malignant tumor phenotypes (48-50). Importantly, Furin 93

expression has been shown to coordinate with that of its substrates, such as TGF, BMP and insulin like 94

growth factor (51-53). 95

Our present data establish that Furin is regulated by its own substrate Notch1. In turn, Notch1 induced 96

Furin further activates MT1-MMP and ADAM10, which can then enhance the cleavage of Notch1. Hence, 97

we have identified a novel positive feedback loop whereby Notch1 signaling self-activates by regulating not 98

only its own processing but that of its regulatory proteases. This novel feedback signaling might account for 99

the high activity of Notch1 in melanoma cells. 100

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MATERIALS AND METHODS 102

Cell lines - Primary and metastatic melanoma cells were in part purchased from ATCC (American Type 103

Culture Collection, Manassas, VA) or were gifts from Dr. Marianne Broome Powell (Stanford University) 104

(54). The use of the cells was approved by the Case Cancer Institutional Review Board (IRB). The cell lines 105

used in this study are: V2387, WM115, WM266-4 and SKMEL2. These are human melanoma cells derived 106

from primary melanoma (WM115), metastases to the skin (WM266-4 and SKMEL2) and lymph nodes 107

(V2387). All cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 108

10% FCS (fetal calf serum), 1% glutamine, and 1% penicillin-streptomycin. 109

shRNAs and Expression Plasmids - shRNAs against human Notch1 (TRCN0000003359) and Furin 110

(TRCN000075238 and TRCN000075239) were purchased from Sigma . The Notch1-NIC lentiviral 111

expression vector was constructed by inserting the cDNA sequence corresponding to human Notch1-NIC 112

(base pairs 5278-7668 of full length human Notch1) into the pLM-CMV-Ha-puro-PL3 lentiviral plasmid 113

(55), between XbaI-XhoI. The lentiviral iDuet101a-DN-MAML plasmid (dominant negative MAML) was 114

kindly provided by Dr. Eli Bar (Case Western Reserve University). Viral particles were produced in 293-FT 115

cells using the X-tremegene 9 reagent (Roche, Mannheim, Germany) according to manufacturer's 116

instructions. The packaging plasmids used were pMD2.G and psPAX2 that were purchased from Addgene 117

(Cambridge, MA). 118

Western Blot Analysis - Total protein for all assays was extracted with urea lysis buffer (9 M urea; 75 mM 119

Tris-HCl, pH 7.5, and 100 mM 2-mercaptoethanol), and 20μg/sample was separated by 8–10% SDS-PAGE 120

and transferred onto PVDF membranes. Membranes were probed with the following antibodies: anti-Notch1 121

(C20, Santa Cruz Biotechnology, Santa Cruz, CA); anti Notch1mN1A (full length and cleaved – Novus 122

Biologicals LLC, Littleton , CO); anti-Notch1NIC (cleaved Val-1744 - Cell Signaling Technologies, 123


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Beverly, MA); anti-MT1-MMP (clone LEM-2/15.8, Millipore, Billerica, MA); anti-ADAM10 (Abcam, 124

Cambridge, MA); anti-TACE (tumor necrosis factor- α- converting enzyme, ADAM17) (eBioscience, San 125

Diego, CA) and anti-Furin (Abcam, Cambridge, MA). Bands were detected using Super Signal detection 126

reagent (Thermo Scientific). Loading was normalized with anti-β-actin (Santa Cruz Biotechnology). 127

Densitometric quantifications (by ImageJ) of band intensity were normalized to band intensity of the 128

respective β-actin. 129

Real-time PCR Analysis - cDNA was synthesized from total RNA using SuperScript first-strand synthesis 130

system for RT-PCR (Invitrogen), then used for PCR amplification with SYBR Green PCR master mix 131

(Roche). The following primer sets were used to amplify specific target genes: human ADAM10 forward: 132

5’-CAAAGTCTGAGAAGTGTCGGG-3’; reverse: 5’-CTGCACATTGCCCATTAATG-3’; human 133

ADAM17 forward: 5’-ACCTGAAGAGCTTGTTCATCGAG-3’; reverse: 134

5’-CCATGAAGTGTTCCGATAGATGTC-3’; human GAPDH forward: 135

5’-CGCTCTCTGCTCCTCCTGTT-3’; reverse: 5’-CCATGGTGTCTGAGCGATGT-3’; human MT1-MMP 136

forward: 5’-CTCCCTCGGCTCGGCCCAAA-3’; reverse: 5’-CGCCTCATGGCCTTCATGGTGTCT-3’; 137

human Furin forward: 5’-GAAGCAGCAGCGGCCAGGAT-3’; reverse: 138

5’-CGAAGATCTGGCCCAGGTTGAGG-3’; human PC5 forward: 139

5’-TGCCAGGGACCAACCCAGGA-3’; reverse: 5’-TGCTCTCGGCCATAGTTGTCTGC-3’; human PC7 140

forward: 5’-CACCAGCACGGTTTCGGCCT-3’; reverse: 5’-AGCCGTTGGGATCCGAGTCCAT-3’. 141

Relative quantification of mRNA expression levels was normalized by GAPDH. 142

Chromatin immunoprecipitation assay - Chromatin immunoprecipitation (ChIP) was performed using ChIP 143

assay kits (Upstate Biotechnology) following the manufacturer's recommendations. The following primer 144

sequences were used: Furin promoter at B1 (-3556bp): forward: 145

5’-ATCAGGAGGGTCACCTATGGTGC-3’; reverse: 5’-CAGAGGTGCTGGGATTACAGGC-3’; Furin 146

promoter at B2 (-1236bp): forward: 5’-CTCAGAGCTTAGTTCCCAGCAGAC-3’; reverse: 147

5’-GATCTCCAGGTATTGCCAAATGTC-3’; Furin promoter at B3 (+960bp): forward: 5’- 148

ATGCAGATTGAAGAGGCAAGCTG-3’; reverse: 5’- GCAATACTTGCTGCTTCAGGTGC-3’; HES1 149

promoter: forward: 5’-CGTGTCTCCTCCTCCCATTG-3’; reverse: 150

5’-CCAGGACCAAGGAGAGAGGT-3’. The antibody employed for the immunoprecipitation was 151

previously described (56). 152

Luciferase Assays - The Hes1-reporter plasmid was a kind gift from Dr. Ryoichiro Kageyama (Kyoto 153

University, Kyoto, Japan) (57). The human Furin promoter-luciferase constructs pGL2-Basic, pGL2-SacI, 154


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was generously provided by Dr. Claire M. Dubois (University of Sherbrooke, Canada) (43). The mutations 155

within the pGL2-SacI reporter were generated by PCR using the following primers: 156

5’-cctgtgacgtcacagctcctcattctgcgacagtgg-3’ and 5’-gaatgaggagctgtgacgtcacaggatggtggttag-3’ which replaces 157

the sequence 5’-TTCCCAC-3’ with 5’-TCACAGC-3’. V2387, WM115 or SKMEL2 cells (5 ×104/well, in 158

24-well plates) were transfected using X-tremegene HP reagent (Roche Applied Science) as per the 159

manufacturer’s instructions. After 48 h, cells were lysed in 100 μl of lysis buffer (Promega, Madison, WI). 160

A Renilla luciferase reporter plasmid driven by a CMV promoter was co-transfected with the reporter 161

constructs at a 1:20 ratio to assess transfection efficiency. Activities of firefly and Renilla were assessed by 162

the Dual-Luciferase assay system (Promega), and light production was measured for 10 s in a Monolight 163

2010 luminometer (Molecular Devices, Sunnyvale, CA). 164

MT1-MMP and ADAM10 activity assay - Membrane proteins were extracted from 106 cells by three cycles 165

of freeze-thaw in dry ice/ethanol/37°C baths. The lysates were sonicated for 3 s, and the membranes were 166

pelleted by centrifugation (30 min, 13 000 g, 4°C) and resuspended in PBS. Equal protein amounts per 167

sample were incubated with a fluorogenic MT1-MMP substrate (Mca-PLGL-Dap (Dnp)-AR-NH2) or 168

5-FAM /QXLTM 520 ADAM10 substrate provided by the manufacturer (Anaspec, Fremont, CA, USA). 169

Fluorescence intensity was measured at Ex/Em = 490 + 20 nm/520 + 20 nm using a SpectraMax M2 Elisa 170

reader (Molecular Devices, Sunnyvale, CA, USA). 171

Notch ligand stimulation assay - Notch signaling was induced by plating cells (32,000/cm2) on dishes 172

displaying immobilized FC- or FC-JAGGED1 ligand anchored to protein-A. Plasmids expressing FC- and 173

FC-JAGGED1 (58, 59) were kindly provided by Dr. Aaron Proweller (Case Western Reserve University, 174

Cleveland). 175

Migration and invasion assays – SKMel2 cells were plated in a confluent monolayer in duplicate. A scratch 176

using a pipette tip was produced and the detached cells were gently washed away with PBS. Plates were 177

incubated under a time-lapse microscope for 24 hours. Pictures were taken every hour for the duration of the 178

experiment. Frames were aligned and distance between one of the edges of the wound in the first frame 179

(considered as time 0) to the migration front was calculated for the time points indicated. For the trans-well 180

invasion assay, 25 x104 cells suspension was added to an 8μ pore insert either uncoated (control) or coated 181

with growth factor reduced matrigel (BD Biosciences, MA). 5% FBS containing DMEM media was added 182

to the lower chamber (24 well plate) as chemo-attractant. After a 24 hours incubation cells that migrated 183

through the control inserts or matrigel and collected onto the bottom membrane, were fixed with 4% 184

Formaldehyde and stained with Coomassie blue. Each treatment was done in triplicate. Membranes were 185


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incubated in 10% acetic acid to extract the Coomassie stain and color intensity was quantified at 295nm. % 186

invasion is calculated as follows: (mean reads of matrigel/mean reads of insert)x100. 187

188

RESULTS 189

Notch1 modulates MT1-MMP activity - Our previous data showed that MT1-MMP is involved in the 190

cleavage of Notch1, and that it is a critical modulator of melanoma metastasis and melanoma cell growth (32, 191

33, 60). Thus, we decided to explore the mechanisms of MT1-MMP regulation. Interestingly, we observed 192

that Notch1 promotes MT1-MMP activity. Expression of an active Notch1 (NIC) in melanoma cells 193

increases the 55 kDa fraction of MT1-MMP (Fig 1A), the catalytically active enzyme, as assessed by two 194

different antibodies, one of which recognizes the active MT1-MMP peptide specifically. This protein 195

fraction is indeed the active protein as it leads to an increase in MT1-MMP activity as measured by an in 196

vitro activity assay (Fig 1B) (61, 62). Furthermore, expression of active Notch1 leads to an increase in 197

MMP2 processing (Fig. 1C), a known target of MT1-MMP (63). Given that MT1-MMP activation requires 198

the cleavage of the inhibitory propeptide by Furin or Furin-like convertases, and given that expression of 199

Notch1 did not change the levels of MT1-MMP transcript in the cells (Fig. 1D), we investigated if Notch1 200

could regulate the expression of the convertases. Indeed, we found that Notch1-NIC increased both mRNA 201

and protein levels of Furin (Fig 1D, E). The transcripts of the Furin-like convertases PC5 and PC7, also 202

involved in MT1-MMP activation, were increased as well (Fig 1D). Conversely, inhibition of Notch1 by a 203

specific shRNA, resulted in a decrease of Furin mRNA and protein. In a parallel experiment, cleavage of 204

endogenous Notch1 by stimulation with recombinant JAGGED1 ligand, led to an increase in Furin and a 205

correspondent increase in MT1-MMP processing (Fig. 2), further suggesting Notch1 activates MT1-MMP 206

likely via furin expression. 207

Notch1 affects Furin expression through binding to the Furin promoter - Based on the previous data, we 208

hypothesized that Notch1 affects the activity of its protease MT1-MMP by increasing the levels of Furin 209

available to cleave the inhibitory propeptide. We therefore sought to determine whether Notch1 might 210

regulate Furin by directly driving its expression. Active Notch1, although not capable of directly binding to 211

DNA sequences, functions in a transcription complex together with CBF-1 and Master Mind-Like (MAML). 212

Sequence analysis of the Furin promoter through Motif Search (http://www.genome.jp/tools/motif/) revealed 213

three putative CBF1/Notch-binding sequences (TTCCCAC) within a 5 kb sequence encompassing 4 kb 214

upstream and 1 kb downstream the Furin transcription start site: one at position -3556bp, one at -1236bp, 215

and one at +960bp (Fig. 3A). The sites were then tested for Notch1 occupancy in V2387 and WM266-4 cells 216


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expressing either a control shRNA (shGFP) or shNotch1 (Fig. 3B), by chromatin immunoprecipitation 217

(ChIP) assay. We observed enrichment at the B2 site (position -1236) in shGFP cells that was inhibited in 218

shNotch1 expressing cells, but not at positions -3556 and +960 (Fig 3C). Binding of Notch1 to the HES1 219

promoter was performed as a positive control, which also showed enrichment in shGFP cells compared to 220

shNotch1 cells (Fig 3D). 221

To further confirm that the B2 site is indeed where Notch1 binds and drives transcription of Furin, we 222

performed a series of luciferase reporter assays (fig. 4A). In V2387 cells, the reporter SacI that contains the 223

WT B2 binding site, showed a 10 fold induction of luciferase activity in cells expressing the active form of 224

Notch1 (NIC) compared to control (pLM) expressing cells (Fig 4B). The HES1 reporter construct was used 225

as a positive control. Mutation of the B2 binding site form TTCCCAC to TCACAGC, significantly reduced 226

the induction of luciferase activity in NIC expressing cells, compared to the wild type (WT) in three 227

different melanoma cell lines (Fig 4C). The upper panel shows the expression levels of Notch1-NIC in each 228

line tested. These data identify Furin as a novel Notch1 direct target. 229

Notch1 affects MT1-MMP activity through Furin - Once we established that Notch1 modulates Furin 230

expression, and given that MT1-MMP activity is dependent on the cleavage of the inhibitory propeptide by 231

Furin, we next evaluated whether Notch1 plays a role in the conversion of MT1-MMP from inactive (64Kd) 232

to active (55Kd) through Furin. We tested whether inhibition of Furin by specific shRNA sequences would 233

impair MT1-MMP activation downstream of Notch1. Since Furin also processes Notch1, we used cell lines 234

expressing an exogenous, active Notch1-NIC that is independent of Furin cleavage. Western blot analysis 235

showed that the ratio of active MT1-MMP increases in NIC expressing cells, but decreases in Furin knock 236

down cells (Fig 5A, 4B). Importantly, inhibition of Furin expression abolishes the ability of active Notch1 to 237

increase the active MT1-MMP fraction (Fig 5A, 4B), indicating that Notch1 affects MT1-MMP activity 238

through Furin. 239

Notch1 affects ADAM10 activity - ADAM10 and ADAM17 are generally accepted as proteases responsible 240

for Notch1 cleavage following ligand binding. Similar to MT1-MMP, ADAM 10 and 17 are also activated 241

by Furin prior to reaching the plasma membrane as fully active enzymes (7, 34, 40). Previous data from our 242

group showed that melanoma tumors and cells do express these proteases, although not at the same levels as 243

MT1-MMP (32). We therefore wanted to determine whether ADAM10 and 17 were also affected by Notch1 244

through Furin induction. As shown in Fig 6A, the active band of ADAM10 (60kDa, the bottom band) 245

increases in the NIC expressing cells, indicating an increase in processing. Similarly, in cells stimulated by 246

recombinant JAGGED1, ADAM10 processing is increased (Fig. 2). The same was not true for ADAM17, 247


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however. We further measured the activity of ADAM10 in vitro using a commercially available activity 248

assay (ANASPEC), and found that overexpression of NIC significantly increased ADAM10 activity in both 249

SKMel2 and V2387 cells (Fig 6C, D). Of note, Notch1 did not affect ADAM10 gene expression (Fig 6B). 250

We next evaluated whether Notch1 played a role in the conversion of ADAM10 through Furin. As Fig 7 251

shows, the active band of ADAM10 increases in the NIC expressing cells, but decreases in the Furin knock 252

down cells. Again, inhibition of Furin abolished the ability of Notch1-NIC to process ADAM10, as 253

previously seen for MT1-MMP. The in vitro activity assay mirrored the western blot data, showing 254

induction of activation in the presence of NIC and lack of increase by Notch1 when Furin is inhibited (fig. 255

7C, D). 256

Inhibition of furin diminishes Notch1 processing – given that Furin is promotes the first cleavage of 257

Notch1 and such cleavage is required for proper Notch positioning at the plasma memnbrane and access to 258

the ligand (5, 6), we first wanted to determine whether interruption of Notch1 signaling at the DNA binding 259

levels would affect furin and consequently Notch1-NIC release. To do so, cells were transduced with the 260

dominant negative MAML. Expression of MAML-DN inhibited the expression of furin and HEY1, a known 261

direct Notch transcription target, and resulted in diminished processing of full length to cleaved Notch1 as 262

shown by an antibody that recognized both full length and processed Notch1 (Fig. 8A upper panel and graph) 263

and by one that specifically recognizes Notch1 cleaved by γ-secretase at valin 1744 (Fig 8A, NIC specific 264

panel). Inhibition of furin by a specific shRNA reduced Notch processing in a similar manner (Fig. 8B). 265

These data indicate Notch1 can promote its own activation through a signaling loop involving furin and the 266

proteases ADAM10 and MT1-MMP. 267

Inhibition of furin affects melanoma cell migration and invasion – We have previously shown that 268

MT1-MMP is required for melanoma cell migration and invasion (64). Given that Notch1 dependent furin 269

expression promotes MT1-MMP activation we next examined whether furin inhibition could affect 270

migration and invasion of cells downstream of Notch1. Furin was downregulated in SKMel2 cells 271

expressing constitutive Notch1-NIC (fig. 9A). Then a scratch assay and a matrigel invasion assay were 272

performed. Cells expressing Notch1-NIC migrated faster than controls, however, a 30% reduction in 273

migration (on average) was observed when furin was inhibited in both control (pLM) and NIC expressing 274

cells (fig. 9B and suppl. Fig 1). Similarly, cells expressing active Notch1 were more invasive than control 275

cells, but inhibition of furin reduced these invasion promoting effects (fig, 9C). 276

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DISCUSSION 279

Notch processing is an unexplored and potentially therapeutically targetable mechanism of increased 280

Notch signaling in cancer. We have established that elevated Notch plays a critical role in the pathogenesis 281

of melanoma, and describe here a novel circuit by which Notch drives an autoregulatory positive feedback 282

loop initiating with the elevated expression of Furin. The full length precursor of Notch1 is cleaved by Furin 283

in the trans-Golgi network, prior to arriving to the plasma membrane as a heterodimer where it can be 284

activated by interaction with Delta-like and Jagged ligands expressed on the surface of adjacent cells (6, 9, 285

65, 66). The cleavage by Furin is required for the proper surface presentation of Notch1 and proper ligand 286

binding (65). In fact, studies show that Furin-resistant Notch1 receptors exhibit decreased surface expression 287

and ligand-mediated receptor activation (5). Although Furin can be regulated by a variety of factors 288

including Sox9 (67), TGFβ (68) and BMPs (69), here we find that Furin is a novel direct transcriptional 289

target of its own substrate Notch1. Furthermore, we show that the activity of two major proteases involved 290

in Notch1 cleavage following ligand binding, are induced by active Notch1 through the modulation of Furin. 291

Both MT1-MMP and ADAM10 are synthesized as inactive zymogens whose activation requires cleavage of 292

an inhibitory prodomain sequence (40, 70). Furin is an essential activator of pro-MT1-MMP that controls 293

the level of active MT1-MMP on the cell surface (34). On the other hand, previous studies demonstrated that 294

overexpression of Furin and protein convertase PC7 increased the levels of active ADAM10, and mutation 295

of the convertase dependent cleavage sites blocked the processing of ADAM10 to its active form (40). 296

Indeed, here we demonstrate that Notch1 promotes the processing of MT1-MMP and ADAM10 to their 297

active forms through Furin, as down regulation of Furin expression is sufficient to abolish the ability of 298

Notch1 to promote the processing of the proteases and to increase their activity. Based on these data we 299

suggest that Notch1, by regulating the expression of Furin, establishes a positive feedback loop, which not 300

only can promote Notch1 processing but also its activation by modulating the activity of MT1-MMP and 301

ADAM10. 302

MT1-MMP belongs to a family of membrane-tethered matrix metalloproteinase and it is considered one 303

of the most important MMPs in promoting cancer cell migration and invasion (33). Recently, we 304

demonstrated that MT1-MMP operates as a protease involved in Notch1 activation, and that MT1-MMP 305

dependent Notch1 activation mediates part of the pro-tumorigernic properties of MT1-MMP (32). 306

Melanomas express high levels of active Notch1 and its expression correlated with that of MT1-MMP (32, 307

54, 71, 72). Hence, considering the results presented here, it is likely that in tumors where the two proteins 308

are present, they functionally influence each other by promoting their reciprocal activation. The decrease in 309


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cell migration and invasion downstream of active Notch1 upon furin inhibition would substantiate this 310

notion. 311

On the other hand, although ADAM10 and ADAM17 are both capable of processing Notch1 at the S2 312

site (10, 73), more and more evidence point to ADAM10 as the main enzyme responsible of such activity. 313

Firstly, the defects found in ADAM10 deficient mice phenocopy those of Notch1 deficient mice (74-76); 314

and secondly, Van Tetering et al. (10) demonstrated that only in ADAM10 knock out fibroblasts Notch1 315

cleavage is severely diminished, but not so in ADAM17 knock out cells. In melanoma, expression of 316

ADAM10 has been shown to be significantly increased in melanoma metastases compared to primary 317

tumors (37, 38). Knock down of ADAM10 inhibits melanoma cell growth and migration, while 318

overexpression of ADAM10 increases migration of melanoma cells (38). Our study shows that 319

overexpression of Notch1 increases the activity of ADAM10 through the modulation of Furin. In contrast, 320

Notch1 did not promote the processing of ADAM17. These data suggest that ADAM17, at least in 321

melanoma cells, is not under the influence of Notch1 signaling. 322

Together, the data presented here highlight a novel positive feedback signaling whereby Notch1, by 323

regulating Furin expression, promotes its own cleavage in the Golgi, a process that is required for the proper 324

localization of Notch1 on the plasma membrane; and stimulates the activation of ADAM10 and MT1-MMP, 325

which not only are involved in the cleavage and activation of Notch1, but that contribute to tumorigenesis by 326

modulating the processing of a variety of substrates including extracellular matrix and surface receptors. 327

These findings add further complexity to Notch signaling deregulation in cancer. Notch not only promotes 328

tumorigenesis by directly regulating genes involved in tumor development and progression (77), but can 329

also indirectly do so by influencing the activity of protumorigenic enzymes. We therefore propose that the 330

positive feedback loop between Notch1 and its proteases promotes the amplification of Notch1 signaling and 331

may represent a novel therapeutic target to treat melanoma. 332

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51. Blanchette F, Day R, Dong W, Laprise MH and Dubois CM: TGF beta 1 regulates gene 479 expression of its own converting enzyme furin. Journal Of Clinical Investigation 99: 1974-1983, 480 1997. 481 52. Dickson MC, Slager HG, Duffie E, Mummery CL and Akhurst RJ: Rna And Protein 482 Localizations Of Tgf-Beta-2 In the Early Mouse Embryo Suggest an Involvement In Cardiac 483 Development. Development 117: 625-639, 1993. 484 53. Lee JE, Pintar J and Efstratiadis A: Pattern Of the Insulin-Like Growth Factor-Ii 485 Gene-Expression during Early Mouse Embryogenesis. Development 110: 151-&, 1990. 486 54. Bedogni B, Warneke JA, Nickoloff BJ, Giaccia AJ and Powell MB: Notch1 is an effector of Akt 487 and hypoxia in melanoma development. Journal Of Clinical Investigation 118: 3660-3670, 2008. 488 55. Razorenova OV, Agapova LS, Budanov AV, Ivanov AV, Strunina SM and Chumakov PM: 489 Retroviral reporter systems for assessing the activity of stress-inducible signal transduction pathways 490 controlled by the p53, HIF-1, and HSF-1 transcription factors. Molecular Biology 39: 253-259, 2005. 491 56. Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, Barnes KC, O'Neil J, 492 Neuberg D, Weng AP, Aster JC, Sigaux F, Soulier J, Look AT, Young RA, Califano A and Ferrando 493 AA: NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network 494 promoting leukemic cell growth. Proceedings Of the National Academy Of Sciences Of the United 495 States Of America 103: 18261-18266, 2006. 496 57. Nishimura M, Isaka F, Ishibashi M, Tomita K, Tsuda H, Nakanishi S and Kageyama R: 497 Structure, chromosomal locus, and promoter of mouse Hes2 gene, a homologue of Drosophila hairy 498 and Enhancer of split. Genomics 49: 69-75, 1998. 499 58. Proweller A, Pear WS and Parmacek MS: Notch signaling represses myocardin-induced smooth 500 muscle cell differentiation. J Biol Chem 280: 8994-9004, 2005. 501 59. Buas MF, Kabak S and Kadesch T: Inhibition of myogenesis by Notch: evidence for multiple 502 pathways. J Cell Physiol 218: 84-93, 2009. 503 60. Shaverdashvili K and Bedogni B: Matrix Metalloproteinase MT1-MMP Regulates Melanoma 504 Metastasis by Activation RAC1. Journal Der Deutschen Dermatologischen Gesellschaft 11: 44-44, 505 2013. 506 61. Knauper V, Will H, Lopez-Otin C, Smith B, Atkinson SJ, Stanton H, Hembry RM and Murphy 507 G: Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that 508 MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem 509 271: 17124-31, 1996. 510 62. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E and Seiki M: A matrix 511 metalloproteinase expressed on the surface of invasive tumour cells. Nature 370: 61-5, 1994. 512 63. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA and Goldberg GI: Mechanism of 513 cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the 514 membrane metalloprotease. J Biol Chem 270: 5331-8, 1995. 515 64. Shaverdashvili k. WP, Ma J., Zhang K., Osman I., Bedogni B.: MT1-MMP modulates 516 melanoma cell dissemination and metastasis through activation of MMP2 and RAC1. Pigment 517 Cell and melanoma Res 27: 287-96, 2014. 518 65. Nichols JT, Miyamoto A, Olsen SL, D'Souza B, Yao C and Weinmaster G: DSL ligand 519 endocytosis physically dissociates Notch 1 heterodimers before activating proteolysis can occur. 520 Journal Of Cell Biology 176: 445-458, 2007. 521 66. van Tetering G and Vooijs M: Proteolytic Cleavage of Notch: "HIT and RUN". Current 522 Molecular Medicine 11: 255-269, 2011. 523 67. Guimont P GF, Dubois CM.: Sox9-dependent transcriptional regulation of the proprotein 524 convertase furin. Am J Physiol Cell Physiol 293: C172-83, 2007. 525


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68. Blanchette F RP, Grondin F, Attisano L, Dubois CM: Involvement of Smads in 526 TGFbeta1-induced furin (fur) transcription. J Cell Physiol 188: 264-73, 2001. 527 69. Chang HM CJ, Klausen C, Leung PC.: Recombinant BMP4 and BMP7 increase activin A 528 production by up-regulating inhibin βA subunit and furin expression in human granulosa-lutein cells. 529 J Clin Endocrinol Metab 100: E375-86, 2015. 530 70. Remacle AG, Chekanov AV, Golubkov VS, Rozanov DV, Fugere M, Day R and Strongin AY: 531 Proprotein convertases and glycosylation regulate MT1-MMP activity. Matrix Biology 25: S49-S49, 532 2006. 533 71. Bedogni B and Powell MB: Unique transforming properties of Notch1 in human melanocytes. 534 Pigment Cell & Melanoma Research 22: 702-703, 2009. 535 72. Bedogni B and Powell MB: Hypoxia, melanocytes and melanoma - survival and tumor 536 development in the permissive microenvironment of the skin. Pigment Cell & Melanoma Research 537 22: 166-174, 2009. 538 73. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA 539 and Israel A: A novel proteolytic cleavage involved in Notch signaling: The role of the 540 disintegrin-metalloprotease TACE. Molecular Cell 5: 207-216, 2000. 541 74. Hartmann D, De Strooper B, Serneels L, Craessaerts K, Herreman A, Annaert W, Brabant V, 542 Luebke T, Illert AL, von Figura K and Saftig P: Deficiency for the disintegrin metalloprotease 543 ADAM10 causes disturbed alpha-secretase function and a notch deficiency-related phenotype in 544 mice. Neurobiology Of Aging 23: S183-S183, 2002. 545 75. Krebs LT, Xue YZ, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, 546 Kitajewski J, Callahan R, Smith GH, Stark KL and Gridley T: Notch signaling is essential for 547 vascular morphogenesis in mice. Genes & Development 14: 1343-1352, 2000. 548 76. Limbourg FP, Takeshita K, Radtke F, Bronson RT, Chin MT and Liao JK: Essential role of 549 endothelial Notch1 in angiogenesis. Circulation 111: 1826-1832, 2005. 550 77. Ntziachristos P LJ, Sage J, Aifantis I.: From fly wings to targeted cancer therapies: a centennial 551 for notch signaling. Cancer Cell 25: 318-34, 2014. 552 553

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This work was supported by a grant from the Harry Lloyd Charitable Trust and by a grant from the 559

Concern Foundation. 560

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FIGURE LEGENDS 566

Figure 1. Notch1 promotes MT1-MMP activity. A. The active MT1-MMP fraction (55kDa, bottom 567

band) is increased in Notch1-NIC expressing cells. B. Notch1-NIC increases MT1-MMP in vitro 568

activity. Neg=negative control (buffer); pos= positive control (0.5 μg/ml recombinant active 569

MT1-MMP). C. Zymography image of MMP2 processing in pLM or NIC expressing cells. 570

Numbers represent fold induction of active band (bottom band) over controls (pLM) ±SD. Results 571

are the average between three repeats. *p<0.05, Student’s T test. D. Notch1 increases both mRNA 572

and protein levels of Furin, but doesn’t affect the expression of MT1-MMP. qRT-PCR and Western 573

blot of the cells in A showing expression changes of Furin and Furin like convertases. Differences in 574

expression are significant (p<0.001, Student’s T test). E. Inhibition of Notch1 reduces both Furin 575

mRNA and protein. *p<0.001, Student’s T test. β-actin is used as loading control. 576

Figure 2. Stimulation of endogenous Notch1. JAGGED1 dependent activation of endogenous 577

Notch1 increases furin and promotes MT1-MMP and ADAM10 processing in two melanoma cell 578

lines. 579

Figure 3. Furin is a direct target of Notch1. A. Schematic representation of the Furin promoter. 580

There are three putative CSL binding sequences TTCCCAC, located at -3556 (B1), -1236 (B2), and 581

+960 (B3). B. Western blot analysis in V2387 and WM266-4 cells showing Notch1 protein levels in 582

control and shNotch1 expressing cells. C. ChIP analysis showing association of Notch1 to the 583

binding site -1236 (B2) both in V2387 and WM266-4 cells (**, *** p＜0.01, Student’s T test). D. 584

The HES1 promoter was used as a positive control for CSL/Notch-binding activity (**, *** p＜0.01, 585

Student’s T test). All data are representative of three independent experiments. 586

Figure 4. Notch1 affects Furin expression through binding to the Furin promoter. A. Schematic 587

representation of the Luciferase reporter constructs containing a WT sequence TTCCCAC or the 588

mutated sequence TCACAGC. The construct SacI contains the sequence from PstI at the TSS 589

(transcription start site) to SacI. B. Luciferase reporter assay in V2387 cells in control (PLM) or 590

Notch1-NIC expressing cells. Constructs: -Basic indicates pGL2 basic – luciferase construct without 591

promoter region and represents the negative control for the luciferase assay; HES1 contains the 592

promoter region of the Notch1 target HES1 (positive control; *p<0.01, Student’s T test). A 593

significant induction of luciferase activity was observed in NIC expressing cells when using the WT 594

construct SacI (**p<0.01, Student’s T test). C. Induction of luciferase in NIC expressing cells is seen 595

in WT SacI expressing cells (*p<0.01, Student’s T test), and it is significantly reduced when the SacI 596


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construct is mutated (**p<0.01, Student’s T test). Upper panel: Notch1-NIC protein levels in pLM 597

and NIC expressing cells. Experiments were done in three cell lines: WM115, Skmel2, V2387. Data 598

are the average of three independent experiments. 599

Figure 5. Notch1 induces MT1-MMP activation through Furin. A. Western blot analysis showing 600

the active fraction of MT1-MMP increases in NIC expressing cells, but decreases in Furin knockdown 601

cells. Experiments were done in three cell lines: SkMel2, V2387, and WM115, as shown. Values 602

reported below the blots are the average quantification of furin bands ±SD among three separate 603

experiments. B. Ratio between the active and inactive (pro-) MT1-MMP bands normalized to the 604

corresponding β-actin. Data are the average between three independent experiments (pSKMel2<0.01; 605

pV2387<0.01; pWM115<0.01, Student’s T test). 606

Figure 6. Notch1 promotes ADAM10 activity. A. In Skmel2 and V2387 cells, the active ADAM10 607

fraction (60kDa, bottom band) is increased in Notch1-NIC expressing cells. ADAM17 does not 608

change. Ratio between inactive (pro) and active ADAM10 normalized to their corresponding β-actin 609

is indicated. Values are the average ± standard deviation of three independent experiments. B. 610

qRT-PCR of ADAM10 in pLM or NIC expressing cells showing mRNA levels of ADAM10 611

normalized to GAPDH. C. ADAM10 in vitro activity assay in pLM or NIC expressing cells. Fold 612

change of RFU (relative fluorescence units) in SKMel2 and V2387 cells were detected. Statistical 613

significance is indicated (Student’s T test). 614

Figure 7. Notch1 increases the active fraction of ADAM10 through Furin. A. Western blot 615

analysis showing expression of pro and active ADAM10 and Notch1-NIC in NIC expressing cells 616

(SKMel2 – left panel; V2387 – right panel) in the presence of shGFP or shFurin (shF2 and shF3). B. 617

Ratio between active and inactive ADAM10 of the lanes (normalized to β-actin) in the blots in A. 618

*pSKMel2<0.01; pV2387<0.01, Student’s T test. Data are the average of four independent 619

experiments. C-D. ADAM10 in vitro activity assay in SKmel2 and V2387 expressing empty vector 620

(pLM) or active Notch1 (NIC) in the presence of shGFP or shFurin (shF2 and shF3). Statistical 621

significance is indicated (Student’s T test). Results are the average of four independent experiments. 622

Figure 8. Inhibition of Furin decreases Notch1 processing. A. Notch1 full length (FL) and 623

intracellular (NIC) (Ab: mN1A), Notch-NIC (Ab: Cleaved Val1744), MT1-MMP, ADAM10 and 624

Furin expression in cells stimulated with recombinant JAGGED1 and expressing either empty vector 625

(pLM) or dominant negative MAML (MAML-DN). B. Notch1 full length (FL) and intracellular 626

(NIC) (Ab: mN1A), Notch-NIC (Ab: Cleaved Val1744), MT1-MMP, ADAM10 and Furin expression 627


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in cells stimulated with recJAGGED1 and expressing a specific furin shRNA (shF2) or control 628

(shGFP). The graphs are the means±SD of the ratio between NIC and FL Notch1 among two 629

independent experiments. P values were calculated by the Student’s T test. 630

Figure 9. Furin inhibition reduces melanoma cell migration and invasion. A. Expression levels 631

of Notch1-NIC and furin in cells expressing active Notch1 and furin shRNA. B. Migration (scratch 632

assay) over 24 hours of the cells in A. C. Invasion through Matrigel over a 24 hours period of the 633

cells in A. Reduction of migration and invasion in shFurin expressing cells is statistically significant 634

(Student’s T test). 635


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furin is a direct transcription target of notch1 notch1 auto-activation

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