Nck2 promotes human melanoma cell proliferation, migration and invasion in vitro and primary melanoma-derived tumor growth in vivo
© Labelle-Côté et al; licensee BioMed Central Ltd. 2011
Received: 27 July 2011
Accepted: 12 October 2011
Published: 12 October 2011
Nck1 and Nck2 adaptor proteins are involved in signaling pathways mediating proliferation, cytoskeleton organization and integrated stress response. Overexpression of Nck1 in fibroblasts has been shown to be oncogenic. Through the years this concept has been challenged and the consensus is now that overexpression of either Nck cooperates with strong oncogenes to transform cells. Therefore, variations in Nck expression levels in transformed cells could endorse cancer progression.
Expression of Nck1 and Nck2 proteins in various cancer cell lines at different stages of progression were analyzed by western blots. We created human primary melanoma cell lines overexpressing GFP-Nck2 and investigated their ability to proliferate along with metastatic characteristics such as migration and invasion. By western blot analysis, we compared levels of proteins phosphorylated on tyrosine as well as cadherins and integrins in human melanoma cells overexpressing or not Nck2. Finally, in mice we assessed tumor growth rate of human melanoma cells expressing increasing levels of Nck2.
We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts. Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes. We demonstrated the involvement of Nck2 in proliferation, migration and invasion in human melanoma cells. Moreover, we discovered that Nck2 overexpression in human primary melanoma cells correlates with higher levels of proteins phosphorylated on tyrosine residues, assembly of Nck2-dependent pY-proteins-containing molecular complexes and downregulation of cadherins and integrins. Importantly, we uncovered that injection of Nck2-overexpressing human primary melanoma cells into mice increases melanoma-derived tumor growth rate.
Collectively, our data indicate that Nck2 effectively influences human melanoma phenotype progression. At the molecular level, we propose that Nck2 in human primary melanoma promotes the formation of molecular complexes regulating proliferation and actin cytoskeleton dynamics by modulating kinases or phosphatases activities that results in increased levels of proteins phosphorylated on tyrosine residues. This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.
Melanoma skin cancer is one of the most devastating types of cancer, extremely aggressive with high metastatic potential. Melanoma metastasis to distant organs is the primary cause of human cancer-related deaths. Worldwide, the incidence of cutaneous malignant melanoma is increasing faster than any other type of cancer. Cutaneous melanoma originates from pigment-producing melanocytes localized at the epidermal-dermal junction in human skin and develops through different steps . Among various hypotheses, it is proposed that these involve radial (RGP) and vertical (VGP) aberrant growth phases of preexisting nevi or at new site. Then to metastasize at distant sites, melanoma detach from a primary lesion, acquire motility and proteolytic activities to reach lymphatic and blood circulation and undergo growth to distinct organs, all this according to stepwise molecular changes involving defined genetic events [2, 3]. However, the exact mechanisms underlying this devastating process are complex and somehow still poorly understood. From a molecular point of view, oncogenic activation of the mitogen-activated protein kinase (MAPK) pathway, due to somatic mutations in B-RAF (V600E), is frequently observed in melanoma (70%) .
In mammals, the family of Nck (non-catalytic region of tyrosine kinase) proteins is represented by two highly conserved members, Nck1 and Nck2, composed of three N-terminal SH3 (Src homology 3) domains followed by a unique C-terminal SH2 (Src homology 2) domain and devoid of any catalytic activity [5, 6]. Like other SH2/SH3 domain-containing proteins, Nck1 and Nck2 behave as adaptor proteins by physically coupling activated membrane receptors to specific downstream effectors . In mice, individual Nck knockout resulted in no phenotype, confirming redundancy of Nck proteins, while early embryonic lethality of the double Nck knockout mice revealed their crucial role in embryonic development . However, regardless that Nck1 and Nck2 share high amino acid identity, and common cellular functions and binding partners, increasing evidence support specific roles and proteins interactions, as well as tissue expression patterns for these adaptors [7, 9–15]. Previous studies have reported that overexpression of Nck1 in fibroblasts induces cellular transformation and that these cells form tumors in mice [16, 17]. Furthermore, either Nck has been shown to cooperate with potent oncogenes (v-Abl and Ras) to transform cells, influence cell morphology and anchorage-independent growth . Although, these studies strongly suggest a role for Nck in cancer development, the mechanism by which Nck oncogenic potential is achieved still remains to be established.
Originally the Nck1 cDNA was isolated from a human melanoma cDNA expression library using a monoclonal antibody produced against the human melanoma-associated antigen , which has no similarity with Nck1. This suggests that the Nck1 mRNA might be abundant in human melanoma. Most recently, the Nck2 gene was found as being overexpressed in human metastatic melanoma compared with non-metastatic melanoma lesions . In agreement, the cancer microarray database Oncomine (https://www.oncomine.org/) reports Nck2 as a gene upregulated in several human cancer cell lines, including human melanoma. Therefore, the concept that deregulated expression of Nck adaptor proteins could contribute to promote melanoma development and/or progression deserves further investigation. In the present study, using human melanoma cell lines harboring the activating B-RAF (V600E) mutation, that are well defined for stage of cancer progression [19, 20], we demonstrate that Nck2 protein and mRNA levels are increased in human metastatic melanoma cells compared with human primary melanoma cells that rarely metastasis. We show that Nck2 promotes cell proliferation, migration and invasion in human melanoma cells. In addition, using an in vivo xenograft model, we provide evidence that increased Nck2 expression in human primary melanoma cells promotes melanoma-derived tumor growth rate. Collectively, our findings indicate that Nck2 plays a role in human melanoma progression.
The Wistar melanoma cell lines (WM278, WM1232, WM115, 1205Lu, WM164, WM1617 and 451Lu) were obtained from Dr Meenhard Herlyn (PA, USA). Human Epidermal Melanocytes (HEM) cell line was purchased from Cell Applications Inc. Murine colon carcinoma cell (CT26, CT36 and CT51) were obtained from Dr. Nicole Beauchemin (McGill University, Montreal, QC). Breast cancer cell lines (MCF10, MCF7, T47D, MDA-MB-231) were kindly provided by Dr. Morag Park (McGill University, Montreal, Qc).
Unless specified, all chemicals used in this study are from regular commercial sources. Cells were maintained at 37°C in 5% CO2-95%O2 atmosphere. HEK293, colon and breast cancer cell lines were grown in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS and supplemented with 100 Units/ml of penicillin, 100 μg/ml of streptomycin and 0.25 μg/ml of Amphotericin B. Melanoma cell lines were grown in RPMI 1640 supplemented with 2 mg/ml NaHCO3 and 0.3 mg/ml glutamine. MCF10 cells were grown in DMEM containing 5% Horse Serum (Invitrogen), 20 μg/ml of mouse epidermal growth factor (mEGF, Collaborative Biomedical Products), 10 μg/ml of insulin and 0.1 μg/ml of cholera toxin. To induce MCF10 cell differentiation, cells were grown in media in absence of mEGF but supplemented with 0.5 μg/ml of hydrocortisone for two days. HEM (human epidermal melanocyte) cells were grown in HEM media (Cell Applications Inc.) and cultured according to the manufacturer's instructions.
To analyze phospho-tyrosine proteins, cells were exposed to protein phosphotyrosine phosphatase inhibitor (pervanadate (Na3VO4) or bpVPhen, 100 μM, 15 min at 37°C) prior to be harvested and total cell lysates processed for anti-phosphotyrosine western blot as reported below. Alternatively, total cell lysates (2 mg protein) were incubated with indicated antibodies (4 μg) for 2 hours at 4°C and 40 μl of 50% slurry solution of Protein-A immobilized on Sepharose beads (Santa Cruz Biotech.) were added for an additional 2 hours of incubation at 4°C. Immunoprecipitated samples were washed 3X with lysis buffer before to be finally recovered in Laemmli buffer and processed for anti-phosphotyrosine western blot as reposted below.
Nck polyclonal antibodies were raised by immunizing rabbits with GST-Nck fusion proteins as antigens. Crude serum samples were Protein-A-purified (ProChem, MA) and further tested for Nck specificity as described below. A pan-Nck antibody (1793), which recognizes both Nck isoforms was raised against residues 1-251 containing the three SH3 domains of human Nck1 as previously reported . Nck1 antibody (2383) and Nck2 antibody (3313) were raised against isoform specific amino acid sequence in between the last SH3 and the SH2 domain of each Nck as reported earlier . Other antibodies used are: CrkII (C-18), Integrin β3 (H-96), phospho-tyrosine (clone PY99), HA (Y-11) and GFP (B-2) from Santa Cruz Biotech. Antibodies against Integrin β1 (anti-CD29, clone 18), E-Cadherin (clone 34) and N-Cadherin (clone 32) were purchased from BD (ON, Canada). Antibodies to detect vinculin (clone h-VIN-1) and Tubulin (TUB2.1) were from Sigma-Aldrich, USA. Secondary antibodies coupled to HRP were from Bio-Rad Inc. Rhodamine-coupled to mouse anti-IgG was bought at Jackson ImmunoResearch Inc. Phalloidin-coupled to AlexaFluor®555 and 488 were purchased from Molecular Probes (Invitrogen, CA, USA)
Cell lysis and western blots
Cell lysates were prepared in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Sodium Pyrophosphate, 100 mM Sodium Fluoride, supplemented with the protease inhibitors Aprotinin and Leupeptin at 1 μg/ml and PMSF at 1 mM. Lysates normalized for protein content (Bradford protein assay, Bio-Rad) were prepared in Laemmli buffer, heated, subjected to SDS-PAGE on 10% acrylamide gels and transferred onto nitrocellulose membranes. For western blot analyses, membranes were blocked in TBS (Tris-buffered saline) containing 10% dry milk and 0.1% Tween-20, and then incubated overnight at 4°C with indicated primary antibodies appropriately diluted in the blocking solution. For pY western blot, blocking and primary antibody solution was TBS containing 5% bovine serum albumin (BSA, Sigma) and 0.1% Tween-20. Next morning, the membranes were washed twice with TBS for 5 minutes followed by two 5 minutes washes using TBS-T (TBS-0.1% Tween-20) and two 5 minutes washes with TBS. The membranes were then incubated with secondary antibody appropriately diluted in milk-blocking solution for 1 hour and washed as above. Finally, signal was detected using ECL Plus Western Blotting Detection System (GE Healthcare, UK) and XR film exposure.
RNA isolation and RT-PCR
Total RNA was isolated from melanoma cells using the TRIZOL (Invitrogen) according to the manufacturer's protocol. Briefly, cells from 100-mm dishes (1 × 106 cells) were suspended in 7.8 ml of TRIZOL. The aqueous and organic phases were separated after addition of chloroform. Precipitated RNA by isopropyl alcohol addition was washed in 70% ethanol and dissolved in RNase-free water. RNA concentration and purity (OD260/280) was measured using an Ultrospec 2100 Pro UV/visible Spectrophotometer (Fisher Scientific, ON). First-strand cDNA synthesis was generated by reverse transcriptase reaction in a final volume of 50 μl. For this, 2.0 μg of total RNA were mixed in a total reaction volume of 20 μl of RNAse free water containing 1 μM Oligo d(T)20 for Nck amplifications or 6 μg of Random Primers for 18S amplification. The reactions were incubated at 65°C for 5 min and quenched on ice. Then, the RT reaction was assembled by adding 10 μl of the 5X 1st strand buffer (Invitrogen), 5 μl of 0.1 M DTT (Invitrogen), 2.5 μl of RNase Inhibitor (40 U/μl) (Invitrogen), 2.5 μl of 10 mM dNTPs, 5 μl of 50 mM MgSO4 and 2.5 μl of Superscript III (200 U/μl) (Invitrogen). Samples were incubated at 37°C for 50 min and deactivated at 70°C for 15 min. PCR amplification was performed using 0.5 μl of cDNA template in a final volume of 50 μl containing 5 μl of 10X PCR Enhancer buffer (Invitrogen), 1.5 mM MgCl2, 0.2 mM dNTPs, 50 pM of specific forward and reverse primers, 10 μl of Amplification buffer (Invitrogen) and 1 U of Taq DNA polymerase (Invitrogen) and DEPC water. Primers used were: Nck1 forward 5'-GCCAGATTCTGCATCTCCTG-3', Nck1 reverse 5'-ACACTTGCCCAGTATTTAGG-3', Nck2 forward 5'-CGAGTACCCCGCCAATGG-3' and Nck2 reverse 5'-CCCGTCACTGAGGACCACC-3'. Reactions were carried out on PTC-100 Programmable Thermal Controller (MJ Research Inc.) according to the following program conditions: initial denaturation at 94°C for 1 min, followed by 1 min at 94°C, 30 seconds of annealing (47°C for Nck 1, 53°C for Nck 2 and 55°C for 18S) and 1 min at 72°C. The final elongation step was 10 minutes at 72°C and the samples were kept at 4°C until analysis. PCR products were separated on a 1% agarose gels and imaged using an NIH Image J 1.30 system. Fifteen, 20, 25 and 30 cycles of PCR amplification products were analyzed to confirm that the amplification was in the linear range for each gene. Ratios of Nck1 and Nck2 over 18S were calculated from similar assays performed in triplicate and repeated three times.
Human HA-tagged Nck2 cDNA generously provided by Dr. Wei Li (University of Southern California, LA, CA) was subcloned into the retroviral vector pLXSN (Clonetech Laboratories Inc., CA, USA) and the viral particles produced using the GP2-293 cell line according to the manufacturers' instructions. Human Nck2 cDNA was also subcloned into the pEGFP-C1 plasmid (BD, NJ, USA). To establish stable clones of human WM278 primary melanoma overexpressing GFP or GFP-Nck2, cells plated in 100-mm dishes (1 × 106 cells) were transiently transfected with 10 μg of plasmid DNA (pEGFP or pEGFP-Nck2) using calcium phosphate and following selection with neomycin, clones were isolated, amplified and analyzed for GFP or GFP-Nck2 by western blot. 451Lu cells plated at 40-60% confluence were transfected with either 100 nM Nck2 or control siRNA 13379 (Ambion, Austin, TX) using Lipofectamine Plus reagent according to the manufacturer's protocol and analyzed for protein expression after 24 or 48 h.
Briefly, cells (4 × 103) were seeded in 96-well plates and 24, 48, 72 or 96 h after, cells were fixed by adding glutaraldehyde (20 min, final concentration 1%). Then, fixed cells were washed twice with deionised water and stained with Crystal Violet (20 min, 0.4% in 10% ethanol, Sigma). The excess of Crystal Violet was removed by washing the cells three times with water and finally, incorporated Crystal Violet was dissolved in 10% acetic acid and read at 570 nm using a spectrophotometer (Beckman Coulter). Wells without cells, but containing medium were used as blank value that was subtracted from all values. Data were expresses a raw OD at 570 nm or as ratio of OD at specific time point over initial OD at day 1.
Wound healing assays
Cell migration was assessed in classical wound healing assays. Confluent monolayer cells in a 6-well plate were wounded using a plastic pipette tip (P200) and rinsed with PBS before to add back culture medium. The bottoms of the wells were marked to indicate where the initial pictures of the wound area were taken. After 8 h incubation at 37°C, pictures (10X) of the same areas were recorded using an Axiovert 200 M microscope (Zeiss) equipped with a CoolSnap™ES camera (Photometric®, Roper Scientific) and closure of the wound evaluated using Metamorph® (V6.3, Molecular Devices Corp.).
Cell invasion assays using Transwells
Melanoma cells (1 × 105) resuspended in 10% serum containing medium were added to the top chamber of a Transwell (8 μm, DD Biosciences, NJ, USA) pre-coated with matrigel™ (BD Biosciences, NJ, USA) diluted in ice-cold PBS (175 μg/ml) at a total of 35 μg per well and allowed to migrate for 24 h. To evaluate the amount of cells that had invaded through each transwell, excess of media was discarded and the transwells were washed once with PBS and then placed in trypsin solution (0.025%) to release the invaded cells underside of the transwells and in the bottom chamber. Total invaded cells were estimated using Calcein AM (BD Biosciences, NJ, USA) as recommended by the manufacturer. Data were normalized according to the respective total amount of cells for each line plated at the same time in adjacent wells devoid of transwells to take into account variations in cell number between cell lines.
Spheroid formation assays
Spheroid formation and culture in 3D were performed according to the hanging drop method . Briefly, 2 × 104 cells in 20 μl of culture medium were suspended on the lid of tissue culture dishes containing 10 ml of culture medium for 48 h to form spheroids. Then spheroids were transferred in culture dishes containing culture medium and on 2% agar (Agar Select, Invitrogen, CA, USA) at the bottom. After 72 h of growth in suspension, individual spheroid has been transferred in 4-well plate containing 80% collagen type IV (PureCol®, Advanced BioMatrix) in RPMI without FBS. Following 30 min at 37°C to allow collagen polymerization, 500 μl of RPMI containing 10% FBS was added to each well. Images were recorded initially and at 12-24 hr intervals as reported above for wound healing assays. Spheroid invasion was determined qualitatively as either positive or negative comparing sequential images.
Actin and focal adhesions
Cells were plated at 3 × 104 cells/well on glass coverslips pre-coated or not with various extracellular matrices and incubated in culture medium for 24 h. All steps were carried at room temperature and coverslips were rinsed with PBS between each step. Cells were fixed in freshly prepared 3.7% formaldehyde for 10 min, permeabilized in 0.2% Triton-X-100 for 5 min and blocked in 0.1% BSA for 30 min. For vinculin staining, cells were incubated with primary monoclonal anti-vinculin antibody (1:400) for 1 h and with a mixture of secondary tetramethylrhodamine isothiocyanate-conjugated phalloidin-conjugated goat mouse antibody (TRITC-GAM, Sigma) for 30 min. Actin staining was performed by incubating the coverslips for 30 min with Phalloidin-AlexaFluor®555. Coverslips were mounted by inverting them on glass slides using Prolong anti-fade mounting media (Molecular Probes). Coverslips were examined on a Zeiss Axiovert 200 M microscope (Zeiss) using 40X or oil immersion 63× objective lens. Fluorescent images were captured using a CoolSnap™ES camera (Photometric®, Roper Scientific) and analyzed using Metamorph® (V6.3, Molecular Devices Corp.).
Tumor growth in vivo
WM278 primary melanoma cells either parental, overexpressing GFP (C2) or GFP-Nck2 at low (N7) or high (N14) levels were grown in RPMI medium supplemented with 10% FBS to 80% confluency. Cells (5 × 106) resuspended in 500 μl at 50% Matrigel™ were injected subcutaneously in the right flank of 6-week-old CD-1 Nude mice (Charles River, Qc, Canada) (n = 5 for each group). Tumors development was followed for 20 weeks. Tumor size was measured every week with calipers to assess tumor volume ([πlength × width2]/6). Mice were housed in McGill University Animal facilities at the Genome building. Mice experiments were conducted under a McGill University-approved animal use protocol (Dr. P.M. Siegel) in accordance with guidelines established by the Canadian Council on Animal Care.
Data analysis and statistics
Densitometry analysis results are expressed as means ± S.E.M. Student's t test was used to evaluate the statistical significance of the results. A p ≤value 0.05 is assumed to be significant.
Nck2 protein and mRNA levels are increased in human metastatic melanoma cell lines
To investigate the potential involvement of Nck proteins in human melanoma development and progression, we first analyzed total Nck protein levels in human melanoma cell lines at different stages of cancer progression and compared with normal human melanocytes. The human melanoma cell lines used in this study were provided by the laboratory of Dr. Meenhard Herlyn at the Wistar Institute (PA, USA) and already used in vivo for tumorigenicity and experimental metastasis [23, 24]. Mainly, these include the WM278, a melanoma cell line derived from a human primary tumor in vertical growth phase that rarely metastasis; WM1617, a WM278 sister melanoma cell line derived from lymph nodes metastasis in the same patient few years later; 451Lu, a melanoma cell line isolated from lung metastasis in mice injected with the WM164 cell line, which is a human melanoma cell line isolated from lymph nodes metastasis similar to WM1617, but from a different patient.
Nck2 promotes human melanoma cell proliferation
Nck2 modulates migration and invasion of human melanoma cells
Nck2 modulates focal adhesions in human melanoma cells
Nck2 promotes phosphorylation of proteins on tyrosine and downregulation of cell surface adhesion proteins in human primary melanoma cells
Nck2 promotes primary melanoma-derived tumor growth in vivo
Nck2 expression is upregulated in invasive colon and breast cancer cell lines
Nck1 and Nck2 SH2-SH3 domain-containing proteins have been reported to be differently expressed in numerous mouse tissues [8, 15]. In agreement with the ability of both Nck to collaborate with strong oncogenes to transform cells , Nck1 and Nck2 genes were found upregulated in several human cancer cell lines, including melanoma (https://www.oncomine.org/). However, Nck proteins expression levels in cancer tissues and possible mechanism(s) by which these adaptors contribute to cancer development have been poorly investigated to date. In this study, we provide evidence that Nck2 plays a role in promoting proliferation, migration and invasion of human melanoma cells in vitro and growth of melanoma-derived tumors in vivo, while its expression is upregulated in metastatic cancer cells, including colon, breast and melanoma.
Our investigation revealing that Nck2 overexpression in human primary melanoma cells induces metastatic characteristics point towards Nck2 sufficiency to promote metastasis phenotype. In this study, we did not address whether Nck2 is necessary for melanoma metastasis. However, we provided some insights suggesting that Nck2 could play such function. In fact, we found higher levels of Nck2 expression in metastatic compared to non metastatic cell lines in three different types of cancer. In addition, we demonstrate that depletion of Nck2 in metastatic melanoma reduces cell proliferation. This does not exclude that other yet identified players could be required to fully promote metastasis in melanoma overexpressing Nck2. None the less, our findings clearly demonstrate that overexpression of Nck2 in human primary melanoma correlates with upregulation of the total phospho-tyrosine proteins content, assembly of novel Nck2-dependent pY-protein complexes and downregulation of E- and N-cadherins, and β-1 and -3 integrins. E-cadherin, found at adherens junctions, is the principal effector of cell-cell adhesion . Loss of E-cadherin expression in cancer cells weakens cell-cell adhesion and is associated with cancer progression, invasion and metastasis [41–43]. At the present time, there is no evidence for a direct link between E-cadherin and Nck2. Further investigation is required to elucidate the molecular events responsible for E-cadherin downregulation associated with overexpression of Nck2 in human primary melanoma cells and whether downregulation of Nck2 in metastatic human melanoma cells would restore E-cadherin expression remains to be determined. On the other hand, the degree of cancer cells cohesion in primary tumor also depends on the strength of cell-ECM contacts mediated by integrins . Alteration in integrins expression has been also implicated in cancer progression, invasion and metastasis [45, 46]. Integrins signaling associated with regulation of the actin cytoskeleton leading to adhesive attachment involves the activation of the focal adhesion kinase (FAK) and the integrin-like kinase (ILK) (reviewed in ). Interestingly, Nck2 has been shown to affect cell motility through its direct interaction with FAK . Moreover, increasing evidence support a close relationship between integrins and growth factor receptor tyrosine kinases to activate signaling pathways that promote proliferation and metastatic activity (reviewed in ). Nck2 has been reported to function as a molecular link connecting integrins and growth factor receptor tyrosine kinases signaling pathways. In fact, Nck2 associates with numerous receptor tyrosine kinases [17, 50–54] through its SH2 domain and using its third SH3 domain, it binds to a LIM domain in PINCH (Particularly Interesting Cys-His-rich Protein) . PINCH, a binding protein for ILK, plays an important role in mediating integrins-induced cell-ECM interaction by directing ILK to focal adhesions . It is recognized that the ILK-PINCH complex participates to signaling pathways regulating fundamental cellular processes (reviewed in , including cell shape and migration . A crucial role for Nck in regulating these processes was particularly illustrated by the findings showing that fibroblasts derived from Nck double knockout mice embryos display major defects in cell attachment, cell motility and actin remodeling . Therefore, increased expression of Nck2 in human primary melanoma cells may elicit protein interactions that re-wire signaling pathways in a fashion that alters focal adhesions and promotes cell motility by interacting with FAK and PINCH. Alternatively, increased expression of Nck2 could passively destroy proper stoichiometry of molecular complexes and in this manner, indirectly contributes to cancer progression by altering signaling pathways regulating the actin cytoskeleton network supporting cell migration.
Nck proteins are known to couple activated receptor tyrosine kinases, as well as non receptor tyrosine kinases, to effectors involved in signaling pathways regulating proliferation and actin cytoskeleton dynamics [7, 14, 59–61]. Non-receptor protein kinases of the Src and Abl families are often overexpressed or aberrantly activated in a wide variety of human cancers and their roles in cancer progression, including proliferation, survival, motility, invasiveness, metastasis and angiogenesis, is significant. Of note, Nck directly binds to and promotes Abl activation and signaling [62, 63], and associates with p60v-src in vitro . c-Src has been recently reported to be overexpressed in human metastatic melanoma tumors . Interestingly, Src-dependent phosphorylation of Tks5 and cortactin recruits Nck to invadopodia, where it regulates actin assembly and ECM degradation [65–67]. Invadopodia, exclusive invasive cancer cell membrane actin-based protrusions enriched in signaling and proteolytic activities, are used by invasive cancer cells to degrade the ECM and invade surrounding tissues [68–71]. It is then possible that upregulation of tyrosine phosphorylated proteins and downregulation of cadherins and integrins in human primary melanoma cells that overexpress Nck2 may endow melanoma cells with altered adhesive properties and spatial relationships that favor uncontrolled proliferation, migration and invasion.
Nck1 and Nck2 proteins are highly identical, but despite high homology, redundant functions and common binding partners, increasing evidence suggest specific roles and protein interactions [7, 9–12, 14], as well as specific tissue expression patterns for Nck proteins [8, 15]. In this study, the effect of Nck1 overexpression on melanoma phenotype was not addressed. However, our results demonstrate that increased endogenous expression of Nck2 in human metastatic melanoma cells relative to primary melanoma cells and melanocytes results from increased Nck2 transcription, suggesting that Nck1 and Nck2 promoters are under different regulatory controls.
In conclusion, in this study we provide evidence for a role of the adaptor protein Nck2 in melanoma proliferation, migration and invasion in vitro and melanoma-derived tumor growth in vivo. Collectively, our data support Nck2 as a cornerstone governing the aspects that promote melanoma progression. Given other common metastatic cancer cell lines also overexpress Nck2, a general paradigm could make Nck2 a potential molecular marker of cancer progression and a novel target for anti-cancer drug therapy.
This work was supported by a grant to LL from the Canadian Health Research Institutes (CIHR). MLC was supported by a studentship from the CIHR. JD is postdoctoral fellow supported by the Fond de la Recherche en Santé du Québec (FRSQ). SI received support from AstraZeneca. LL is FRSQ Chercheur National. We thank Caterina Russo and Dr. Josée-France Villemure for expert experimental assistance. We also thank Lama Yamani and Dr. Nathalie Lamarche-Vane for critical reading of the manuscript.
- Larue L, Beermann F: Cutaneous melanoma in genetically modified animals. Pigment cell research/sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society. 2007, 20 (6): 485-497.View ArticleGoogle Scholar
- Gupta PB, Kuperwasser C, Brunet JP, Ramaswamy S, Kuo WL, Gray JW, Naber SP, Weinberg RA: The melanocyte differentiation program predisposes to metastasis after neoplastic transformation. Nature genetics. 2005, 37 (10): 1047-1054. 10.1038/ng1634.View ArticlePubMedPubMed CentralGoogle Scholar
- Miller AJ, Mihm MC: Melanoma. The New England journal of medicine. 2006, 355 (1): 51-65. 10.1056/NEJMra052166.View ArticlePubMedGoogle Scholar
- Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka J, et al: High frequency of BRAF mutations in nevi. Nature genetics. 2003, 33 (1): 19-20. 10.1038/ng1054.View ArticlePubMedGoogle Scholar
- Lehmann JM, Riethmuller G, Johnson JP: Nck, a melanoma cDNA encoding a cytoplasmic protein consisting of the src homology units SH2 and SH3. Nucleic Acids Res. 1990, 18 (4): 1048-10.1093/nar/18.4.1048.View ArticlePubMedPubMed CentralGoogle Scholar
- Braverman LE, Quilliam LA: Identification of Grb4/Nck beta, a src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck. J Biol Chem. 1999, 274: 5542-5549. 10.1074/jbc.274.9.5542.View ArticlePubMedGoogle Scholar
- McCarty JH: The Nck SH2/SH3 adaptor protein: a regulator of multiple intracellular signal transduction events. BioEssays. 1998, 20: 913-921. 10.1002/(SICI)1521-1878(199811)20:11<913::AID-BIES6>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
- Bladt F, Aippersbach E, Gelkop S, Strasser GA, Nash P, Tafuri A, Gertler FB, Pawson T: The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Molecular and cellular biology. 2003, 23 (13): 4586-4597. 10.1128/MCB.23.13.4586-4597.2003.View ArticlePubMedPubMed CentralGoogle Scholar
- Buday L: Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins. Biochem Biophys Acta. 1999, 1422: 187-204.PubMedGoogle Scholar
- Buday L, Wunderlich L, Tamas P: The Nck family of adapter proteins. Regulators of actin cytoskeleton. Cellular signalling. 2002, 14 (9): 723-731. 10.1016/S0898-6568(02)00027-X.View ArticlePubMedGoogle Scholar
- Cowan CA, Henkemeyer M: The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals. Nature. 2001, 413 (6852): 174-179. 10.1038/35093123.View ArticlePubMedGoogle Scholar
- Lawe DC, Hahn C, Wong AJ: The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68. Oncogene. 1997, 14: 223-231. 10.1038/sj.onc.1200821.View ArticlePubMedGoogle Scholar
- Oser M, Dovas A, Cox D, Condeelis J: Nck1 and Grb2 localization patterns can distinguish invadopodia from podosomes. Eur J Cell Biol. 2010Google Scholar
- Li W, She H: The SH2 and SH3 adapter Nck: a two-gene family and a linker between tyrosine kinases and multiple signaling networks. Histol Histopathol. 2000, 15: 947-955.PubMedGoogle Scholar
- Latreille M, Laberge MK, Bourret G, Yamani L, Larose L: Deletion of Nck1 attenuates hepatic ER stress signaling, improves glucose tolerance and insulin signaling in liver of obese mice. American journal of physiology. 2011, E423-E434.Google Scholar
- Chou MM, Fajardo JE, Hanafusa H: The SH2- and SH3-Containing Nck Protein Transforms Mammalian Fibroblasts in the Absence of Elevated Phosphotyrosine Levels. Mol Cell Biol. 1992, 12 (12): 5834-5842.View ArticlePubMedPubMed CentralGoogle Scholar
- Li W, Hu P, Skolnik EY, Ullrich A, Schlessinger J: The SH2 and SH3 Domain-Containing Nck Protein Oncogenic and a Common Target for Phosphorylation by Different Surface Receptors. Mol Cell Biol. 1992, 12 (12): 5824-5833.View ArticlePubMedPubMed CentralGoogle Scholar
- de Wit NJ, Rijntjes J, Diepstra JH, van Kuppevelt TH, Weidle UH, Ruiter DJ, van Muijen GN: Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays. British journal of cancer. 2005, 92 (12): 2249-2261. 10.1038/sj.bjc.6602612.View ArticlePubMedPubMed CentralGoogle Scholar
- The Wistar melanoma Cell Lines. [http://www.wistar.org/lab/meenhard-herlyn-dvm-dsc/page/melanoma-cell-lines-metastatic-melanoma-cell-lines-the-primary-le-0]
- Smalley KS, Contractor R, Haass NK, Lee JT, Nathanson KL, Medina CA, Flaherty KT, Herlyn M: Ki67 expression levels are a better marker of reduced melanoma growth following MEK inhibitor treatment than phospho-ERK levels. British journal of cancer. 2007, 96 (3): 445-449. 10.1038/sj.bjc.6603596.View ArticlePubMedPubMed CentralGoogle Scholar
- Lussier G, Larose L: A Casein Kinase I activity is constitutively associated with Nck. J Biol Chem. 1997, 272: 2688-2694. 10.1074/jbc.272.5.2688.View ArticlePubMedGoogle Scholar
- Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK: Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng. 2003, 83 (2): 173-180. 10.1002/bit.10655.View ArticlePubMedGoogle Scholar
- Graeven U, Rodeck U, Karpinski S, Jost M, Philippou S, Schmiegel W: Modulation of Angiogenesis and Tumorigenicity of Human Melanocytic Cells by Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor. Cancer research. 2001, 61 (19): 7282-7290.PubMedGoogle Scholar
- Iliopoulos D, Ernst C, Steplewski Z, Jambrosic JA, Rodeck U, Herlyn M, Clark WH, Koprowski H, Herlyn D: Inhibition of Metastases of a Human Melanoma Xenograft by Monoclonal Antibody to the GD2/GD3 Gangliosides. J Natl Cancer Inst. 1989, 81 (6): 440-444. 10.1093/jnci/81.6.440.View ArticlePubMedGoogle Scholar
- Mayer BJ, Hamaguchi M, Hanafusa H: Characterization of p47gag-crk, a novel oncogene product with sequence similarity to a putative modulatory domain of protein-tyrosine kinases and phospholipase C. Cold Spring Harb Symp Quant Biol. 1988, 53 (Pt 2): 907-914.View ArticlePubMedGoogle Scholar
- Watanabe T, Tsuda M, Tanaka S, Ohba Y, Kawaguchi H, Majima T, Sawa H, Minami A: Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation. Molecular Cancer Research. 2009, 7 (9): 1582-1592. 10.1158/1541-7786.MCR-09-0064.View ArticlePubMedGoogle Scholar
- Crowley E, Horwitz AF: Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions. The Journal of cell biology. 1995, 131 (2): 525-537. 10.1083/jcb.131.2.525.View ArticlePubMedGoogle Scholar
- Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B, Kam Z: Molecular diversity of cell-matrix adhesions. Journal of cell science. 1999, 112 (Pt 11): 1655-1669.PubMedGoogle Scholar
- Chan KT, Cortesio CL, Huttenlocher A: FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion. The Journal of cell biology. 2009, 185 (2): 357-370. 10.1083/jcb.200809110.View ArticlePubMedPubMed CentralGoogle Scholar
- Mader CC, Oser M, Magalhaes MAO, Bravo-Cordero JJ, Condeelis J, Koleske AJ, Gil-Henn H: An EGFR-Src-Arg-Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion. Cancer research. 2011, 71 (5): 1730-1741. 10.1158/0008-5472.CAN-10-1432.View ArticlePubMedPubMed CentralGoogle Scholar
- Smith-Pearson PS, Greuber EK, Yogalingam G, Pendergast AM: Abl Kinases Are Required for Invadopodia Formation and Chemokine-induced Invasion. Journal of Biological Chemistry. 2010, 285 (51): 40201-40211. 10.1074/jbc.M110.147330.View ArticlePubMedPubMed CentralGoogle Scholar
- Balzer EM, Whipple RA, Thompson K, Boggs AE, Slovic J, Cho EH, Matrone MA, Yoneda T, Mueller SC, Martin SS: c-Src differentially regulates the functions of microtentacles and invadopodia. Oncogene. 2010, 29 (48): 6402-6408. 10.1038/onc.2010.360.View ArticlePubMedPubMed CentralGoogle Scholar
- Kelley LC, Ammer AG, Hayes KE, Martin KH, Machida K, Jia L, Mayer BJ, Weed SA: Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation. Journal of cell science. 2010, 123 (22): 3923-3932. 10.1242/jcs.075200.View ArticlePubMedPubMed CentralGoogle Scholar
- Fantus IG, Kadota S, Deragon G, Foster B, Posner BI: Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. Biochemistry. 1989, 28 (22): 8864-8871. 10.1021/bi00448a027.View ArticlePubMedGoogle Scholar
- Kadota S, Fantus IG, Deragon G, Guyda HJ, Hersh B, Posner BI: Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase. Biochemical and biophysical research communications. 1987, 147 (1): 259-266. 10.1016/S0006-291X(87)80115-8.View ArticlePubMedGoogle Scholar
- Posner BI, Faure R, Burgess JW, Bevan AP, Lachance D, Zhang-Sun G, Fantus IG, Ng JB, Hall DA, Lum BS: Peroxovanadium compounds. A new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetics. Journal of Biological Chemistry. 1994, 269 (6): 4596-4604.PubMedGoogle Scholar
- Brattain MG, Strobel-Stevens J, Fine D, Webb M, Sarrif AM: Establishment of mouse colonic carcinoma cell lines with different metastatic properties. Cancer research. 1980, 40 (7): 2142-2146.PubMedGoogle Scholar
- Lacroix M, Leclercq G: Relevance of breast cancer cell lines as models for breast tumours: an update. Breast cancer research and treatment. 2004, 83 (3): 249-289. 10.1023/B:BREA.0000014042.54925.cc.View ArticlePubMedGoogle Scholar
- Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, et al: A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer cell. 2006, 10 (6): 515-527. 10.1016/j.ccr.2006.10.008.View ArticlePubMedPubMed CentralGoogle Scholar
- Takeichi M: Morphogenetic roles of classic cadherins. Curr Opin Cell Biol. 1995, 7 (5): 619-627. 10.1016/0955-0674(95)80102-2.View ArticlePubMedGoogle Scholar
- Perl AK, Wilgenbus P, Dahl U, Semb H, Christofori G: A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature. 1998, 392 (6672): 190-193. 10.1038/32433.View ArticlePubMedGoogle Scholar
- Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A, Lochner D, Birchmeier W: E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. The Journal of cell biology. 1991, 113 (1): 173-185. 10.1083/jcb.113.1.173.View ArticlePubMedGoogle Scholar
- Mbalaviele G, Dunstan CR, Sasaki A, Williams PJ, Mundy GR, Yoneda T: E-cadherin expression in human breast cancer cells suppresses the development of osteolytic bone metastases in an experimental metastasis model. Cancer research. 1996, 56 (17): 4063-4070.PubMedGoogle Scholar
- Hynes RO: Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992, 69 (1): 11-25. 10.1016/0092-8674(92)90115-S.View ArticlePubMedGoogle Scholar
- Giancotti FG, Mainiero F: Integrin-mediated adhesion and signaling in tumorigenesis. Biochim Biophys Acta. 1994, 1198 (1): 47-64.PubMedGoogle Scholar
- Juliano RL, Varner JA: Adhesion molecules in cancer: the role of integrins. Curr Opin Cell Biol. 1993, 5 (5): 812-818. 10.1016/0955-0674(93)90030-T.View ArticlePubMedGoogle Scholar
- Giancotti FG, Tarone G: Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol. 2003, 19: 173-206. 10.1146/annurev.cellbio.19.031103.133334.View ArticlePubMedGoogle Scholar
- Goicoechea SM, Tu Y, Hua Y, Chen K, Shen TL, Guan JL, Wu C: Nck-2 interacts with focal adhesion kinase and modulates cell motility. The international journal of biochemistry & cell biology. 2002, 34 (7): 791-805. 10.1016/S1357-2725(02)00002-X.View ArticleGoogle Scholar
- Guo W, Giancotti FG: Integrin signalling during tumour progression. Nat Rev Mol Cell Biol. 2004, 5 (10): 816-826. 10.1038/nrm1490.View ArticlePubMedGoogle Scholar
- Holland SJ, Gale NV, Gish GD, Roth RA, Songyang Z, Cantley LC, Henkemeyer M, Yancopoulos GD, Pawson T: Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells. EMBO J. 1997, 16 (13): 3877-3888. 10.1093/emboj/16.13.3877.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee C-H, Li W, Zhou M, Batzer AG, Myers MG, White MF, Schlessinger J, Skolnik EY: Nck associates with the SH2 domain-docking protein IRS-1 in insulin-stimulated cells. Proc Natl Acad Sci USA. 1993, 90: 11713-11717. 10.1073/pnas.90.24.11713.View ArticlePubMedPubMed CentralGoogle Scholar
- Meisenhelder J, Hunter T: The SH2/SH3 Domain-Containing Protein Nck Is Recognized by Certain Anti-Phospholipase C-γ1 Monoclonal Antibodies, And Its Phosphorylation on Tyrosine Is Stimulated by Platelet-Derived Growth Factor and Epidermal Growth Factor Treatment. Mol Cell Biol. 1992, 12 (12): 5843-5856.View ArticlePubMedPubMed CentralGoogle Scholar
- Park D, Rhee SG: Phosphorylation of Nck in Response to a Variety of Receptors, Phorbol Myristate Acetate, and Cyclic AMP. Mol Cell Biol. 1992, 12 (12): 5816-5823.View ArticlePubMedPubMed CentralGoogle Scholar
- Stein E, Huynh-Do U, Lane AA, Ceretti DP, Daniel TO: Nck Recruitment to Eph Receptor, EphB1/ELK, Couples Ligand Activation to c-Jun Kinase. J Biol Chem. 1998, 273 (3): 1303-1308. 10.1074/jbc.273.3.1303.View ArticlePubMedGoogle Scholar
- Tu Y, Li F, Wu C: Nck-2, a Novel Src Homology 2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways. Mol Biol Cell. 1998, 9: 3367-3382.View ArticlePubMedPubMed CentralGoogle Scholar
- Tu Y, Li F, Goicoechea S, Wu C: The LIM-only protein PINCH directly interacts with the integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. M Cell Biol. 1999, 19: 2425-2434.View ArticleGoogle Scholar
- Wu C, Dedhar S: Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. The Journal of cell biology. 2001, 155 (4): 505-510. 10.1083/jcb.200108077.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Guo L, Chen K, Wu C: A critical role of the PINCH-integrin-linked kinase interaction in the regulation of cell shape change and migration. The Journal of biological chemistry. 2002, 277 (1): 318-326.View ArticlePubMedGoogle Scholar
- Blasutig IM, New LA, Thanabalasuriar A, Dayarathna TK, Goudreault M, Quaggin SE, Li SS, Gruenheid S, Jones N, Pawson T: Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization. Molecular and cellular biology. 2008, 28 (6): 2035-2046. 10.1128/MCB.01770-07.View ArticlePubMedPubMed CentralGoogle Scholar
- Li X, Meriane M, Triki I, Shekarabi M, Kennedy TE, Larose L, Lamarche-Vane N: The adaptor protein Nck-1 couples the netrin-1 receptor DCC (deleted in colorectal cancer) to the activation of the small GTPase Rac1 through an atypical mechanism. The Journal of biological chemistry. 2002, 277 (40): 37788-37797. 10.1074/jbc.M205428200.View ArticlePubMedGoogle Scholar
- Antoku S, Saksela K, Rivera GM, Mayer BJ: A crucial role in cell spreading for the interaction of Abl PxxP motifs with Crk and Nck adaptors. Journal of cell science. 2008, 121 (Pt 18): 3071-3082.View ArticlePubMedPubMed CentralGoogle Scholar
- Smith JM, Katz S, Mayer BJ: Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology2/Src homology 3 adaptor Nck. J Biol Chem. 1999, 274 (39): 27956-27962. 10.1074/jbc.274.39.27956.View ArticlePubMedGoogle Scholar
- Preisinger C, Kolch W: The Bcr-Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway. Cellular signalling. 2010, 22 (5): 848-856. 10.1016/j.cellsig.2009.12.012.View ArticlePubMedGoogle Scholar
- Lee JH, Pyon J-K, Kim DW, Lee SH, Nam HS, Kim CH, Kang SG, Lee YJ, Park MY, Jeong DJ, et al: Elevated c-Src and c-Yes expression in malignant skin cancers. Journal of Experimental & Clinical Cancer Research. 2010, 29 (1): 116-10.1186/1756-9966-29-116.View ArticleGoogle Scholar
- Stylli SS, Stacey TT, Verhagen AM, Xu SS, Pass I, Courtneidge SA, Lock P: Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation. Journal of cell science. 2009, 122 (Pt 15): 2727-2740.View ArticlePubMedPubMed CentralGoogle Scholar
- Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, et al: Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. The Journal of cell biology. 2005, 168 (3): 441-452. 10.1083/jcb.200407076.View ArticlePubMedPubMed CentralGoogle Scholar
- Ayala I, Baldassarre M, Giacchetti G, Caldieri G, Tete S, Luini A, Buccione R: Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation. Journal of cell science. 2008, 121 (Pt 3): 369-378.View ArticlePubMedGoogle Scholar
- Chen WT: Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J Exp Zool. 1989, 251 (2): 167-185. 10.1002/jez.1402510206.View ArticlePubMedGoogle Scholar
- Condeelis J, Segall JE: Intravital imaging of cell movement in tumours. Nature reviews. 2003, 3 (12): 921-930.PubMedGoogle Scholar
- Buccione R, Orth JD, McNiven MA: Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat Rev Mol Cell Biol. 2004, 5 (8): 647-657. 10.1038/nrm1436.View ArticlePubMedGoogle Scholar
- McNiven MA, Baldassarre M, Buccione R: The role of dynamin in the assembly and function of podosomes and invadopodia. Front Biosci. 2004, 9: 1944-1953. 10.2741/1348.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/443/prepub
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