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Functional impairment of p16INK4A due to CDKN2A p.Gly23Asp missense mutation
Mutation Research 671 (2009) 26–32
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Functional impairment of p16INK4A due to CDKN2A p.Gly23Asp missense mutation Maria Chiara Scaini a,∗ , Elisabetta Rossi a , Paula Lobao Antunes de Siqueira Torres g , Daniela Zullato b , Monia Callegaro a , Cinzia Casella a , Monica Quaggio a , Simona Agata b , Sandro Malacrida f , Vanna Chiarion-Sileni c , Antonella Vecchiato d , Mauro Alaibac e , Marco Montagna b , Graham J. Mann g , Chiara Menin b , Emma D’Andrea a,b a
Section of Oncology, Department of Oncology and Surgical Sciences, University of Padova, via Gattamelata, 64, I-35128 Padova, Italy Molecular Diagnostic Immunology and Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy c Medical Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy d Surgical Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy e Unit of Dermatology, University of Padova, Italy f Department of Biology, University of Padova, Italy g Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute and Melanoma Institute Australia, Westmead, NSW, Australia b
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Article history: Received 6 March 2009 Received in revised form 11 August 2009 Accepted 12 August 2009 Available online 25 August 2009 Keywords: Familial melanoma CDKN2A locus Functional assays p16INK4A unclassified variant
a b s t r a c t The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16INK4A and p14ARF , involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16INK4A properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16wt , p1623Asp , or the p1632Pro pathogenic variant. We found that p1623Asp was less efficient than p16wt in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p1623Asp in familial melanoma. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The CDKN2A locus maps to chromosome 9p21 and codes, by alternative splicing of different first exons (1␣ and 1), for two oncosuppressors: p16INK4A involved in cell cycle regulation as a cyclin dependent kinase (CDK4 and CDK6) inhibitor, and p14ARF that acts in p53 stabilization by binding and sequestering MDM2 [1,2]. CDKN2A is the most common, high penetrance, susceptibility gene identified to date in melanoma families [3,4]. The CDKN2A mutation detection rate in Italian high risk melanoma families is about 20–40%, but it might vary from 5% to 60% according to population characteristics and family selection criteria. A high number
∗ Corresponding author. Tel.: +39 0498215855; fax: +39 0498072854. E-mail address: [email protected] (M.C. Scaini). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.08.007
of melanoma patients within a family, earlier age at onset, and the presence of multiple primary melanomas (MPM) are all factors that strongly influence mutation detection rates regardless of the technical approach employed. CDKN2A mutations can affect both p16INK4A and p14ARF functions because the two proteins share exon 2 and part of exon 3, even though they are read in different frames. To date, p14ARF -only mutations are limited to a few melanoma families. A GenoMEL study on 466 families with at least three melanoma patients reported a frequency of p14ARF mutations of 1–2%, while 38% of the families had mutations which involved p16INK4A ; overall, 70% were missense or nonsense mutations, 23% were insertions or deletions, 5% were splicing, and 2% were regulatory mutations [5]. The GenoMEL study group used computational algorithms, such as the Grantham scale or BLOSUM62 matrix, to evaluate the severity of the missense mutations. In general, functionally impaired variants had larger chemical changes, suggesting
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that the biochemical nature of the amino acid change may be a useful predictor of functional loss [5,6]. Evolutionarily conserved amino acid may serve also as an indicator of functionally critical mutations that may be cancer-related. Nonetheless, the most important criterion for the pathogenic role of a CDKN2A variant remains the evaluation of its effect on cell cycle regulation. Structurally, p16INK4A is comprised of four ankyrin-type motifs: these domains are composed of two antiparallel helices and a loop, creating a cleft that binds to and blocks the function of CDK4 and CDK6. When associated with D-type cyclins, CDK4/6 promote cell cycle progression through the G1 phase by contributing to the phosphorylation and functional inactivation of the retinoblastoma gene product, pRb (reviewed in Refs. [7,8]). Different assays have been applied to investigate the clinical relevance of CDKN2A variants. Most of these tests, as the CDK4/6 binding and kinase activity inhibition assays, have been performed by in vitro translation of mutant proteins [9], as well as by yeast [10,11] or mammalian [12] two-hybrid binding assays. Alternatively, cell cycle-inhibition or colony-forming efficiency has been evaluated by ectopic expression of the wild-type and variant p16 into various cell lines [13–15]. We identified the CDKN2A p.Gly23Asp (c.68G>A) variant in a melanoma-prone family with three melanoma cases; only one of the two tested melanoma patients was a p.Gly23Asp carrier, likewise a melanoma-free first degree relative, affected by B-cell Non-Hodgkin Lymphoma (B-NHL). This variant has previously been reported as a somatic mutation in pancreatic carcinoma [16] and it was also detected in a French [17] and in an Italian melanomaprone family [18]. While cosegregation of p.Gly23Asp with disease was present in the Italian family, only one out of three melanoma cases carried the variant in the French family, thus mining its pathogenic relevance. The p.Gly23Asp mutation affects one of the amino acids belonging to the ankyrin consensus sequence and, as recently shown by Kannengiesser et al. [19], reduces the p16 binding to CDK4, thus likely affecting its cell cycle inhibitory activity. Other amino acid changes have been reported in codon 23 [5,20,21] and some evidence indicates that they might be involved in melanoma predisposition. Therefore, the functional effect of this variant deserves further investigation, and, above all, a reliable and clear cut functional assessment of this variant should explore all p16 properties, namely its ability to inhibit pRb phosphorylation, to evenly localize in the cell, and to cause cell cycle arrest. Here we report that p1623Asp is functionally impaired in all of its abilities, as assessed by different experimental approaches. 2. Materials and methods 2.1. Sample collection and CDKN2A mutational analysis The fourth pedigree, identified while recruiting melanoma-prone families from North Eastern Italy, was named “kindred 4”, and included in the screening for CDKN2A (GenBank AF527803.1; MIM# 600160) constitutive mutations. Written informed consent was obtained from the subjects prior to participation. DNA was isolated from blood and/or from lymphoblastoid cell line (LCL) using standard phenol–chloroform extraction procedure. The entire coding sequence of CDKN2A (exons 1␣, 1, 2, and 3), plus the 5 and 3 UTR were analyzed. All regions were amplified by PCR from genomic DNA using primers complementary to the flanking intron sequences and overlapping their 5 and 3 UTR; primer sequences were derived from “primer 3”, http://biotools.umassmed.edu/bioapps/primer3 www.cgi, and they are available upon request. Amplified products were sequenced using Big Dye terminator v1.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and analyzed on the ABI Prism 3130 sequencer (Applied Biosystems). As control population, we collected 186 blood samples from geographically matched healthy donors. After DNA extraction (NucleoSpin Blood Kit, Macherey-Nagel, GmbH & Co., Duren, Germany), DHPLC analysis (Denaturing High Performance Liquid Chromatography) of the amplified exon 1␣ was performed using p.Gly23Asp and wild-type samples, as controls. Data analysis was based on visual inspection of the chromatograms and comparison with normal and mutant controls included in each run.
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2.2. Cell culture Three different cell lines were used in this study. The NM-39, a human melanoma cell line, and the U2-OS, a human osteosarcoma cell line, both with no detectable p16INK4A or p14ARF ; and the SaOS 2, a p53 and pRb-null human osteosarcoma cell line. The cells were maintained at 37 ◦ C and 5% CO2 atmosphere in Dulbecco’s modified Eagle’s Medium (SAFC Biosciences, MO, USA), and/or Mc Coy’s 5A Medium Modified (Sigma–Aldrich, MO, USA) for U2-OS, supplemented with 10% foetal bovine serum (FBS, GIBCO-Invitrogen Corporation) and 1.5 mM Ultraglutamine (Cambrex Bio-Science, Wokingham, UK; CAS registry number: 56859) was used.
2.3. Construction of expression vectors The cDNA of the appropriate lymphoblastoid cell line was amplified using primers F (5 -CACCGCGGGGAGCAGCATGGAG-3 ), and R (5 CCTGTAGGACCTTCGGTGAC-3 ) generating a 553 bp product encompassing the region from exon 1␣ ATG codon to exon 3 TGA codon. PCR products were suitable for cloning in the pcDNATM 3.1 D/V5-His-TOPO® expression vector (Invitrogen, CA, USA). For immunostaining, kinase activity assay and cell cycle tests, the p16-null human osteosarcoma cell line, U2-OS, was transfected with the appropriate plasmid DNA (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP, pcDNA3.1-lacZ) and then maintained in medium containing 300 g/ml G418 (G418, 50 mg/ml active Geneticin® ; GIBCO-Invitrogen Corporation; CAS registry number: 108321422), unless otherwise indicated. Alternatively, the constructs harbouring the desired nucleotide change (p.Gly23Asp and p.Leu32Pro) were engineered by polymerase chain reactionmediated mutagenesis (QuikChange® II XL Site-Directed Mutagenesis Kits, Stratagene, CA, USA, following manufacturer’s instructions). All mutants were ligated to the N terminus of the FLAG epitope encoded by the pFLAG-CMV-5b vector (Sigma–Aldrich) and fully sequenced. The constructs were also cloned in frame with the GAL4 nuclear localization signal in the pM vector (Clontech, CA, USA): after a PCR reaction using primers p16 ATG fwd (5 -GAATTCCCGCCACCATGGAGCCGGCGGCGGGGA-3 ) and p16 TGA rev (5 -GGATCCCGATCGGGGATGTC-3 ), plasmids were digested with the suitable restriction enzymes BamHI and EcoRI (New England Biolabs, MA, USA) and ligated to the destination plasmid.
2.4. CDK4 binding and kinase activity assay Two different approaches were used to determine impairment of p1623Asp ability for CDK4 binding. For the mammalian two-hybrid assay, p16wt and its variant constructs (p1632Pro and p1623Asp ) were each cloned in frame with the GAL4 nuclear localization signal plus the GAL4 binding domain in the pM vector (Clontech). The CDK4 cDNA was cloned in frame with the SV40 nuclear localization sequence plus the GAL4 activation domain in the pVP16 vector. SaOS 2 cells were then seeded in six-well plates (1 × 105 cells/well) and after 24 h co-transfected with either p16 wild-type or its variants constructs, CDK4 vector, a transfection efficiency control vector, -Gal, and the vector coding for the reporter gene, pG5luc (1.5 g pMBD DNA, 1.5 g CDK4-pVP16, 300 ng pG5luc and 200 ng -Gal, 1 l Lipofectamine 2000). Forty hours after transfection the interaction of p16INK4A and CDK4 was quantified with a luciferase assay using a microplate scintillation and luminescence counter (Top Count NXT, PerkinElmer Life Sciences, MA, USA). For immunoprecipitation assay, U2-OS transfectants were obtained with 1 l Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, added with 2.5 g of the appropriate plasmid DNA (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP) and then maintained in medium containing 300 g/ml G418 (50 mg/ml active Geneticin® ; GIBCO-Invitrogen Corporation) added 24 h after transfection. Seventy-two hours after transfection, U2-OS cells were lysed in NP-40 buffer (PBS, 1% NP-40, CAS registry number: 9036195, 2 mM EDTA, CAS registry number: 62339), plus proteases inhibitors (Complete Mini, Roche Life Sciences, Basel, Switzerland), scraped and centrifuged. The supernatant was mixed with 2 g of anti-p16 antibody (C-20: sc468; Santa Cruz Biotechnology, CA, USA) and the immunocomplexes absorbed to 100 l of a 3:1 slurry of protein G-Sepharose (Amersham Biosciences UK Ltd, Little Chalfont, Buckinghamshire, UK). The beads were re-suspended in sodium dodecyl sulphate (SDS, CAS registry number: 151213) sample buffer, boiled, electrophoresed on 12% SDS-Polyacrylamide (CAS registry number: 9003058) gel and subjected to Western Blotting analysis with antibodies anti-CDK4 (H-22:sc-601, Santa Cruz Biotechnology) to detect the presence of the co-immunoprecipitated CDK4, and anti-p16. For the pRb phosphorylation analysis, U2-OS cells transfected with the proper construct (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP) using the same conditions described for immunoprecipitation, were collected in SDS sample buffer supplemented with proteases (Roche Life Sciences) and phosphatases inhibitors (200 M Na3 VO4, CAS registry number: 13721-39-6; 50 mM NaF, CAS registry number: 7681-49-4) 72 h after transfection. Western Blot analysis (6% SDS-PAGE) was performed with monoclonal antibodies against total Rb (␣ pRb; 4H1, Cell Signaling Technology, MA, USA) and tubulin (␣ tubulin; Clone B-5-1-2; Sigma–Aldrich, MO, USA), and with a polyclonal antibody against hyperphosphorylated pRb (␣ ppRb; Ser807 and Ser811: 9308; Cell Signaling Technology).
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2.5. Immunostaining To perform subcellular distribution assays, U2-OS or NM-39 cells were seeded on coverslips in six-well plates (1 × 105 cells/well) and, after 24 h, transfected with 1 g of each p16 constructs, cloned in frame with the FLAG epitope, and 1 l Lipofectamine 2000 (Invitrogen). After 48 h the cells were fixed in 4% formaldehyde (CAS registry number: 50000) in phosphate-buffered saline (PBS) for 15 min and then permeabilised with 0.2% Triton-X (CAS registry number: 9002931) in PBS for 10 min at room temperature. The cells were immunostained for 50 min with rabbit IgG anti-FLAG antibodies (Sigma–Aldrich) diluted 1:300 in PBS/FCS, followed by another 50 min exposure to goat IgG anti-rabbit, conjugated with Alexa-488 (Invitrogen), secondary antibodies, diluted 1:1000 in PBS/FCS. Nuclei were visualized by Hoechst 33258 staining (2 g/ml, CAS registry number: 23491454). The coverslips were mounted in glycerol (CAS registry number: 56815):PBS (9:1) containing 0.2 N n-propyl-gallate (CAS registry number: 121799) to reduce bleaching. The slides were observed under a fluorescence microscope Olympus BX51 (Shinjuku Monolith, Tokyo, Japan), equipped with a RT-SE18 digital camera. Subcellular distribution was determined from a total of 600 fluorescent cells, in at least three independent transfection experiments. 2.6. Cell cycle inhibition assay U2-OS cells trasfected with p16 (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt ) and control (pcDNA3.1-EGFP) constructs were trypsinized 72 h after transfection, pelleted, re-suspended in 1 ml of GM solution (1.1 g/l glucose (CAS registry number: 50997), 8 g/l NaCl (CAS registry number: 7647145), 0.4 g/l KCl (CAS registry number: 744740-7), 0.2 g/l Na2 HPO4 .2H2 O (CAS registry number: 7558794), 0.15 g/l KH2 PO4 (CAS registry number: 7778770), 0.5 mM EDTA) and fixed in 3 ml of cold ethanol (CAS registry number: 64175). By using 600 g/ml of G418, almost all cells (>98%) were fluorescent. Before the flow cytometry analysis (FACScalibur, BD, NJ, USA; “Modfit” software), the pellet was treated with 5 U/ml of RNase A (Roche Diagnostics, Basel, Switzerland) and incubated for 2 h with 0.1 g/ml Propidium Iodide (CAS registry number: 25535164). 2.7. Cell proliferation assays G418-resistant U2-OS cells, transfected as in the previous experiment, were maintained under G418 selection (300 g/ml) and monitored for a period of 10 days. Using trypan blue dye (CAS registry number: 72571), manual cell counts (average of two replicates) were performed at different time points. Alternatively, U2-OS cells
expressing p1623Asp , p16wt , or control (lacZ) were analysed 2 weeks after transfection; 15000 cells were plated in duplicate and grown under 300 g/ml G418 selection. They were then washed twice with PBS and stained with 1.5% crystal violet solution (CAS registry number: 548629). Total colony number (±standard deviation) was determined from two replicates.
3. Results 3.1. Identification of p.Gly23Asp mutation While screening for patients with familial predisposition for malignant melanoma, we identified a family (kindred 4, Fig. 1) with a history of melanoma as well as other cancers. Mutational analysis of CDKN2A exon 1␣ of the proband (indicated by arrow in Fig. 1) was uninformative, while analysis of the second melanoma patient (indicated by asterisk), and of her father with B-NHL disclosed the presence of the germline missense mutation p.Gly23Asp in both of them. This variant was not found in 186 healthy controls, thus excluding the possibility of a common benign polymorphism. The nucleotide change (c.68G>A) located in p16-specific exon 1␣, belongs to an evolutionary conserved domain located in the first ankyrin repeat that specifically binds to CDK4/6, thus inhibiting kinase function. To investigate if this variant impairs the activity of the p16 protein, different functional assays were performed. 3.2. Loss of CDK4 binding and pRb hyperphosphorylation CDK4 is the main known target of p16INK4A , and its kinase activity is inhibited by mutual binding. Therefore, p1623Asp variant was analyzed for loss of its binding ability with a subsequent accumulation of the hyperphosphorylated pRb form (ppRb). The in vivo binding activity of this variant was assessed using a mammalian two-hybrid assay. Each p16 construct (the pathogenic p1632Pro ,
Fig. 1. The high risk melanoma kindred 4. Mutational analysis of the proband (indicated by arrow) was uninformative, while analysis of the second melanoma patient (indicated by asterisk), and her father with B-NHL, disclosed the presence of the germ-line missense mutation p.Gly23Asp in both of them. Age at pedigree compilation, age of melanoma onset and type of tumor are shown under each symbol. CDKN2A mutation status, when determined, is also indicated.
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phosphorylated (ppRb) and total pRb (Fig. 2C). The specific ␣ ppRb antibody detects endogenous levels of pRb only when phosphorylated at serine 807/811, i.e. when cells progress from G1 to S phase. As expected, the expression of wild-type p16 caused a reduction in the amount of the hyperphosphorylated form of pRb (lane 3). Furthermore, the bands corresponding to pRb and ppRb are visible only in p16wt cells but not in p1623Asp cells (lane 2), where only the ppRb band is present, thus suggesting that p1623Asp is much less efficient than p16wt in blocking cell cycle progression. 3.3. Different subcellular distribution of p1623Asp Each of the p16 construct (p1623Asp and p16wt ) was cloned in frame with the FLAG epitope in the pFLAG-CMV-5b vector. The subcellular distribution of each construct was evaluated in transiently transfected U2-OS osteosarcoma cells (Fig. 3A) and NM-39 melanoma cells (Fig. 3B). As expected, p16wt localized evenly throughout the cell, in both cell lines; on the contrary, p1623Asp presented a different subcellular distribution when compared to the wild-type pattern (Fig. 3A and B). Cytoplasmic aggregates (large areas of intense fluorescence) could be detected in cells transfected with the variant, possibly indicating an incorrect folding of the mutant protein during post-translational processing in the ER and Golgi apparatus. Besides the cytoplasmic aggregates observed in the U2-OS cells (Fig. 3A), nuclear speckles (small discrete areas of intense fluorescence) could also be detected in NM-39 cells, transfected with the p1623Asp variant (Fig. 3B), thus suggesting that even though pleomorphic, aggregates of this variant are readily detectable in different cell lines. 3.4. Loss of G1 cell cycle arrest in p1623Asp cells
Fig. 2. Binding of p16 to CDK4 and inhibition of pRb phosphorylation. (A) Twohybrid system assay in p53 and pRb-null SaOS 2 cells. Binding of variant proteins was determined as a percentage relative to the binding activity of the wild-type (p16wt ) protein. At least four independent transfection experiments were performed in duplicate for each construct. (B) Western Blotting detection of CDK4 in U2-OS cell lysates immunoprecipitated with p16. Cells expressing p1623Asp , p16wt or a control EGFP plasmid (negative control), were lysed and immunoprecipitated with a polyclonal antibody anti-p16. Western Blotting was performed using a polyclonal antibody anti-CDK4. Hela cells lysates, expressing endogenous p16wt , were used as positive control. (C) Analysis of pRb phosphorylation inhibition by p16 constructs and control vector in U2-OS cells. Samples were immunoblotted with antibody against total Rb (␣ pRb), hyperphosphorylated pRb (␣ ppRb) and tubulin (␣ tubulin) as shown on the left; the positions of hyper- and hypophosphorylated forms of the protein are indicated by arrows on the right.
p16wt and p1623Asp ) was cloned in frame with the GAL4 nuclear localization sequence, and transiently co-transfected with a CDK4 expression plasmid, into SaOS 2 cells. As shown in Fig. 2A, the ability of p1623Asp to bind CDK4 is null, and comparable to that of p1632Pro . In a different approach, U2-OS cells expressing p1623Asp , p16wt or EGFP, as control plasmid, were lysed and immunoprecipitated with a polyclonal antibody anti-p16: the presence of co-precipitating CDK4 was then assessed by western blotting using a polyclonal antibody anti-CDK4. Contrary to p16wt , p1623Asp was unable to bind CDK4, as shown in Fig. 2B. These results were consistent with those obtained by the mammalian two-hybrid binding assay, and showed once more no affinity of p1623Asp for CDK4. A further proof of the p1623Asp loss of CDK4 binding activity was obtained evaluating its effect on pRb phosphorylation. Total protein samples from U2-OS cells transfected with pcDNA3.1p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP were fractionated in a 6% SDS-PAGE and immunoblotted with antibodies against hyper-
To further investigate the loss of p1623Asp proliferationinhibitory functions, we evaluated the cell cycle distribution in three experimental U2-OS cell samples (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP), using flow cytometry (Fig. 4A). Cell selection (98% of fluorescent cells) was obtained in 72 h using twice the standard G418 concentration (600 g/ml). The assay, in six replicates (as reported in Fig. 4A), confirmed the same conclusions reached previously, showing a higher percentage of G1 cells in p16wt (69.4%) than in p1623Asp and EGFP (∼50%) cultures; consequently, the percentage of S-phase cells was higher in p1623Asp and EGFP (∼35%) transfected cells than in control p16wt (19%) cultures. 3.5. Loss of cell proliferation arrest in p1623Asp cells G418-resistant U2-OS cells expressing p1623Asp , p16wt or EGFP plasmid were also monitored for a period of 10 days; their proliferation curves are reported in Fig. 4B, as an example of three independent experiments. Clearly, cells expressing p16wt expanded more slowly, due to the expression of the functional oncosuppressor. On the other hand, p1623Asp cells grew better than their wild-type counterpart p16wt cells; additionally, their growth curve precisely overlapped with that of the cells transfected with a control plasmid (EGFP), that has no effect on cell cycle regulation. Finally, we tested whether the higher proliferation rate of p1623Asp cells, when compared with p16wt cells, was also reflected by the ability of the cells to form colonies when seeded at low density. After 2 weeks under G418 selection, colony formation ability was much lower in p16wt cells than in p1623Asp cells (Fig. 4C). In fact, the number of p1623Asp colonies was similar (>200 colonies/plate) to the control clones (pcDNA3.1-lacZ) and much higher than that of p16wt cells (∼50 colonies/plate), thus confirming, once more, that the expression of p1623Asp was unable to arrest cell proliferation.
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Fig. 3. Cellular distribution of FLAG epitope-tagged p16INK4A mutation constructs in U2-OS (A) and NM-39 (B) cells. Each p16INK4A -FLAG tagged construct was transiently introduced into p16-null human osteosarcoma (U2-OS) and melanoma (NM-39) cells. Cells were then immunostained with a monoclonal mouse ␣-FLAG M2 antibody and then exposed to a fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody (green). Nuclei were visualized by Hoechst staining (blue). The subcellular distribution of the p1623Asp showed areas of intense fluorescence which were classified as cytoplasmic aggregates, for large areas localised in the cytoplasm, or as nuclear speckles for small discrete areas localised in the nucleus. On the left, values of protein distribution are reported below cell schemes. LM, light microscopy.
4. Discussion Key to an effective cancer prevention strategy is the early identification of high risk individuals [22]; thus, important decisions on the clinical management of cancer susceptibility syndromes are made based on whether a subject carries a pathogenic mutation or not. In addition to disease causing genetic alterations (i.e. deletions and truncations), mutational screening often finds missense substitutions, which are provisionally named unclassified variants. As recognized earlier [23], these variants are a potentially serious problem [24], and specific recommendations by the IARC Working Group on unclassified genetic variants in high risk cancer susceptibility genes are reported in the November special issue of Human Mutation. Besides specific mutations affecting the p16INK4A and/or p14ARF products of the CDKN2A locus, which are firmly established as causes of increased individual risk of melanoma, over 110 germline variants have been identified [25] and additional novel missense mutations continue to be discovered. Pathogenicity of a variant is defined by both, direct evidence, like its segregation with disease, and its absence in control DNA, or by indirect proof, as severe amino acid change, occurrence in a highly conserved, relevant protein domain, functional assays, and in silico tools [26]. As for the direct criteria, the CDKN2A p.Gly23Asp (p.G23D) missense mutation was not present in 186 healthy donor samples, while it was detected in only one of the two tested melanoma patients of kindred 4 (Fig. 1) with 3 melanoma cases, as well as in a melanoma-free relative, affected by B-NHL. Interestingly, also in a French family, the variant did not segregate with disease [17], but no functional assays were reported to assess its putative role. Lack of segregation with disease, albeit rare, could occur in high risk cancer families, and phenocopies might be present in melanoma families, likely due to the relevant role played by low penetrance genes (i.e. MC1R), acting together with environmental factors (i.e. sun exposure). On the other hand, the presence of melanoma-free carriers could be ascribed to incomplete gene pene-
trance; previous studies have, indeed, demonstrated that CDKN2A mutations might have extremely variable penetrance depending on the various geographical areas (i.e. UV irradiation) in which carriers live [27], as it was also assumed that every single mutation might have its own penetrance [18]. Moreover, we can not exclude that the occurrence of the B-NHL in the melanoma-free carrier could be related to the presence of the p.Gly23Asp mutation, as a result of its variable expressivity; obviously, lack of any correlation between p1623Asp and B-NHL is also a possibility. In the p.Gly23Asp variant, the mutated amino acid is located in the evolutionary conserved domain of the first ankyrin repeat, that binds specifically to and blocks the function of CDK4/6, thus likely disrupting the ankyrin repeat unit and the p16 binding site. As shown in Fig. 2, and in line with a recent report [19] three different approaches gave consistent results, disclosing either a direct (Fig. 2A and B) or indirect (Fig. 2C) p1623Asp impairment for CDK4 binding. Structural changes could be responsible also of the findings reported in Fig. 3; notably, the immunostaining assay showed a p1623Asp mislocalization pattern, reminiscent of protein aggregation. As previously observed by Rizos et al. [12], it is unlikely that mislocalization is simply the result of high protein concentration, since overexpressed wild type or innocent p16 variants did not display altered subcellular distribution. Together with cytoplasmic aggregates in the p1623Asp U2-OS cells (Fig. 3A), we detected, not surprisingly, nuclear speckles in p1623Asp NM-39 cells (Fig. 3B), since p16wt is diffusely present in both nucleus and cytoplasm of neuroblastoma [28] and melanoma [15] cell lines. These various patterns of p16 localization, far from being understood, rather suggest that cell type-dependent properties may account for some of the inconsistent functional data on p16INK4A mutants in the literature. Finally, as shown in Fig. 4, there is a relevant difference between p16wt and p1623Asp behaviour in proliferation assays. Using all three approaches (Fig. 4A–C) the p.G23D variant showed a clear
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Fig. 4. Loss of p1623Asp proliferation inhibition in U2-OS cells. (A) Flow cytometry analysis of U2-OS cells transfected with p16wt and p1623Asp and control (EGFP) constructs. Percent of cells in each cell cycle phase are indicated. (B) G418-resistant U2-OS cells expressing p1623Asp , p16wt or EGFP, monitored for a period of 10 days at indicated time points. (C) Crystal violet staining of colonies after 2 weeks under G418 selection of U2-OS cells transfected with p1623Asp , p16wt or lacZ. The number of colonies (average of two replicates ± standard deviation) is indicated at the bottom.
and coherent behaviour always overlapping with the p16-negative (or mutant) control. Ancillary to direct and functional approaches are in silico based methods that exploit different tools and algorithms [24]. Using some of these in silico tools (i.e. SNAP, Polyphen, SIFT, BLOSUM62), concordant results on p1623Asp behaviour were obtained, ranging from “non-neutral” to “deleterious” (data not shown). Assessing pathogenicity of Unclassified Genetic Variants (UVs) is a major scientific issue emerging worldwide. A panel of experts has recently suggested to approach the problem of UVs classification with as many assays as possible, provided that they explore different functional aspects, and that the evidences obtained are independent from each other [29–31]; UV classification should thus take advantage of all possible means of assessment, due to the clinical implications involved when considering a variant pathogenic rather than neutral. In conclusion, using a multi-evidence approach of independent assays, according to the new IARC recommendations [24], p1623Asp is here defined as a “loss of function” mutation [26], which most likely predisposes carriers to melanoma development. The mutant protein, indeed, is not able to bind CDK4 and to prevent pRB hyperphosphorylation. Since, at the very end, the p16wt physiological role results in the inhibition of cell cycle progression (Fig. 4), it is our opinion that a specific attention should be given to assays that analyze this property in detail, even though for any functional assay validation studies and definition of “gold standards” are very much needed [32].
Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Ministero del Lavoro, della Salute, e delle Politiche Sociali: Programma Integrato Oncologia N.5; Alleanza Contro il Cancro (N.ACC2/R6.9); Lega Italiana per la Lotta contro i Tumori (LILT), Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio di Padova e Rovigo. MCS and MC were supported by a fellowship from FIRC (Fondazione Italiana per la Ricerca sul Cancro). References [1] M. Serrano, G.J. Hannon, D. Beach, A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4, Nature 366 (1993) 704–707. [2] Y. Zhang, Y. Xiong, W.G. Yarbrough, ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways, Cell 92 (1998) 725–734. [3] A. Kamb, D. Shattuck-Eidens, R. Eeles, Q. Liu, N.A. Gruis, W. Ding, C. Hussey, T. Tran, Y. Miki, J. Weaver-Feldhaus, M. McClure, J.F. Aitken, D.E. Anderson, W. Bergman, R. Frants, D.E. Goldgar, A. Green, R. MacLennan, N.G. Martin, L.J. Meyer, P. Youl, J.J. Zone, M.H. Skolnick, L.A. Cannon-Albright, Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus, Nat. Genet. 8 (1994) 23–26. [4] C.J. Hussussian, J.P. Struewing, A.M. Goldstein, P.A. Higgins, D.S. Ally, M.D. Sheahan, W.H. Clark Jr., M.A. Tucker, N.C. Dracopoli, Germline p16 mutations in familial melanoma, Nat. Genet. 8 (1994) 15–21.
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Functional impairment of p16INK4A due to CDKN2A p.Gly23Asp missense mutation Maria Chiara Scaini a,∗ , Elisabetta Rossi a , Paula Lobao Antunes de Siqueira Torres g , Daniela Zullato b , Monia Callegaro a , Cinzia Casella a , Monica Quaggio a , Simona Agata b , Sandro Malacrida f , Vanna Chiarion-Sileni c , Antonella Vecchiato d , Mauro Alaibac e , Marco Montagna b , Graham J. Mann g , Chiara Menin b , Emma D’Andrea a,b a
Section of Oncology, Department of Oncology and Surgical Sciences, University of Padova, via Gattamelata, 64, I-35128 Padova, Italy Molecular Diagnostic Immunology and Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy c Medical Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy d Surgical Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy e Unit of Dermatology, University of Padova, Italy f Department of Biology, University of Padova, Italy g Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute and Melanoma Institute Australia, Westmead, NSW, Australia b
a r t i c l e
i n f o
Article history: Received 6 March 2009 Received in revised form 11 August 2009 Accepted 12 August 2009 Available online 25 August 2009 Keywords: Familial melanoma CDKN2A locus Functional assays p16INK4A unclassified variant
a b s t r a c t The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16INK4A and p14ARF , involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16INK4A properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16wt , p1623Asp , or the p1632Pro pathogenic variant. We found that p1623Asp was less efficient than p16wt in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p1623Asp in familial melanoma. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The CDKN2A locus maps to chromosome 9p21 and codes, by alternative splicing of different first exons (1␣ and 1), for two oncosuppressors: p16INK4A involved in cell cycle regulation as a cyclin dependent kinase (CDK4 and CDK6) inhibitor, and p14ARF that acts in p53 stabilization by binding and sequestering MDM2 [1,2]. CDKN2A is the most common, high penetrance, susceptibility gene identified to date in melanoma families [3,4]. The CDKN2A mutation detection rate in Italian high risk melanoma families is about 20–40%, but it might vary from 5% to 60% according to population characteristics and family selection criteria. A high number
∗ Corresponding author. Tel.: +39 0498215855; fax: +39 0498072854. E-mail address: [email protected] (M.C. Scaini). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.08.007
of melanoma patients within a family, earlier age at onset, and the presence of multiple primary melanomas (MPM) are all factors that strongly influence mutation detection rates regardless of the technical approach employed. CDKN2A mutations can affect both p16INK4A and p14ARF functions because the two proteins share exon 2 and part of exon 3, even though they are read in different frames. To date, p14ARF -only mutations are limited to a few melanoma families. A GenoMEL study on 466 families with at least three melanoma patients reported a frequency of p14ARF mutations of 1–2%, while 38% of the families had mutations which involved p16INK4A ; overall, 70% were missense or nonsense mutations, 23% were insertions or deletions, 5% were splicing, and 2% were regulatory mutations [5]. The GenoMEL study group used computational algorithms, such as the Grantham scale or BLOSUM62 matrix, to evaluate the severity of the missense mutations. In general, functionally impaired variants had larger chemical changes, suggesting
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that the biochemical nature of the amino acid change may be a useful predictor of functional loss [5,6]. Evolutionarily conserved amino acid may serve also as an indicator of functionally critical mutations that may be cancer-related. Nonetheless, the most important criterion for the pathogenic role of a CDKN2A variant remains the evaluation of its effect on cell cycle regulation. Structurally, p16INK4A is comprised of four ankyrin-type motifs: these domains are composed of two antiparallel helices and a loop, creating a cleft that binds to and blocks the function of CDK4 and CDK6. When associated with D-type cyclins, CDK4/6 promote cell cycle progression through the G1 phase by contributing to the phosphorylation and functional inactivation of the retinoblastoma gene product, pRb (reviewed in Refs. [7,8]). Different assays have been applied to investigate the clinical relevance of CDKN2A variants. Most of these tests, as the CDK4/6 binding and kinase activity inhibition assays, have been performed by in vitro translation of mutant proteins [9], as well as by yeast [10,11] or mammalian [12] two-hybrid binding assays. Alternatively, cell cycle-inhibition or colony-forming efficiency has been evaluated by ectopic expression of the wild-type and variant p16 into various cell lines [13–15]. We identified the CDKN2A p.Gly23Asp (c.68G>A) variant in a melanoma-prone family with three melanoma cases; only one of the two tested melanoma patients was a p.Gly23Asp carrier, likewise a melanoma-free first degree relative, affected by B-cell Non-Hodgkin Lymphoma (B-NHL). This variant has previously been reported as a somatic mutation in pancreatic carcinoma [16] and it was also detected in a French [17] and in an Italian melanomaprone family [18]. While cosegregation of p.Gly23Asp with disease was present in the Italian family, only one out of three melanoma cases carried the variant in the French family, thus mining its pathogenic relevance. The p.Gly23Asp mutation affects one of the amino acids belonging to the ankyrin consensus sequence and, as recently shown by Kannengiesser et al. [19], reduces the p16 binding to CDK4, thus likely affecting its cell cycle inhibitory activity. Other amino acid changes have been reported in codon 23 [5,20,21] and some evidence indicates that they might be involved in melanoma predisposition. Therefore, the functional effect of this variant deserves further investigation, and, above all, a reliable and clear cut functional assessment of this variant should explore all p16 properties, namely its ability to inhibit pRb phosphorylation, to evenly localize in the cell, and to cause cell cycle arrest. Here we report that p1623Asp is functionally impaired in all of its abilities, as assessed by different experimental approaches. 2. Materials and methods 2.1. Sample collection and CDKN2A mutational analysis The fourth pedigree, identified while recruiting melanoma-prone families from North Eastern Italy, was named “kindred 4”, and included in the screening for CDKN2A (GenBank AF527803.1; MIM# 600160) constitutive mutations. Written informed consent was obtained from the subjects prior to participation. DNA was isolated from blood and/or from lymphoblastoid cell line (LCL) using standard phenol–chloroform extraction procedure. The entire coding sequence of CDKN2A (exons 1␣, 1, 2, and 3), plus the 5 and 3 UTR were analyzed. All regions were amplified by PCR from genomic DNA using primers complementary to the flanking intron sequences and overlapping their 5 and 3 UTR; primer sequences were derived from “primer 3”, http://biotools.umassmed.edu/bioapps/primer3 www.cgi, and they are available upon request. Amplified products were sequenced using Big Dye terminator v1.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and analyzed on the ABI Prism 3130 sequencer (Applied Biosystems). As control population, we collected 186 blood samples from geographically matched healthy donors. After DNA extraction (NucleoSpin Blood Kit, Macherey-Nagel, GmbH & Co., Duren, Germany), DHPLC analysis (Denaturing High Performance Liquid Chromatography) of the amplified exon 1␣ was performed using p.Gly23Asp and wild-type samples, as controls. Data analysis was based on visual inspection of the chromatograms and comparison with normal and mutant controls included in each run.
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2.2. Cell culture Three different cell lines were used in this study. The NM-39, a human melanoma cell line, and the U2-OS, a human osteosarcoma cell line, both with no detectable p16INK4A or p14ARF ; and the SaOS 2, a p53 and pRb-null human osteosarcoma cell line. The cells were maintained at 37 ◦ C and 5% CO2 atmosphere in Dulbecco’s modified Eagle’s Medium (SAFC Biosciences, MO, USA), and/or Mc Coy’s 5A Medium Modified (Sigma–Aldrich, MO, USA) for U2-OS, supplemented with 10% foetal bovine serum (FBS, GIBCO-Invitrogen Corporation) and 1.5 mM Ultraglutamine (Cambrex Bio-Science, Wokingham, UK; CAS registry number: 56859) was used.
2.3. Construction of expression vectors The cDNA of the appropriate lymphoblastoid cell line was amplified using primers F (5 -CACCGCGGGGAGCAGCATGGAG-3 ), and R (5 CCTGTAGGACCTTCGGTGAC-3 ) generating a 553 bp product encompassing the region from exon 1␣ ATG codon to exon 3 TGA codon. PCR products were suitable for cloning in the pcDNATM 3.1 D/V5-His-TOPO® expression vector (Invitrogen, CA, USA). For immunostaining, kinase activity assay and cell cycle tests, the p16-null human osteosarcoma cell line, U2-OS, was transfected with the appropriate plasmid DNA (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP, pcDNA3.1-lacZ) and then maintained in medium containing 300 g/ml G418 (G418, 50 mg/ml active Geneticin® ; GIBCO-Invitrogen Corporation; CAS registry number: 108321422), unless otherwise indicated. Alternatively, the constructs harbouring the desired nucleotide change (p.Gly23Asp and p.Leu32Pro) were engineered by polymerase chain reactionmediated mutagenesis (QuikChange® II XL Site-Directed Mutagenesis Kits, Stratagene, CA, USA, following manufacturer’s instructions). All mutants were ligated to the N terminus of the FLAG epitope encoded by the pFLAG-CMV-5b vector (Sigma–Aldrich) and fully sequenced. The constructs were also cloned in frame with the GAL4 nuclear localization signal in the pM vector (Clontech, CA, USA): after a PCR reaction using primers p16 ATG fwd (5 -GAATTCCCGCCACCATGGAGCCGGCGGCGGGGA-3 ) and p16 TGA rev (5 -GGATCCCGATCGGGGATGTC-3 ), plasmids were digested with the suitable restriction enzymes BamHI and EcoRI (New England Biolabs, MA, USA) and ligated to the destination plasmid.
2.4. CDK4 binding and kinase activity assay Two different approaches were used to determine impairment of p1623Asp ability for CDK4 binding. For the mammalian two-hybrid assay, p16wt and its variant constructs (p1632Pro and p1623Asp ) were each cloned in frame with the GAL4 nuclear localization signal plus the GAL4 binding domain in the pM vector (Clontech). The CDK4 cDNA was cloned in frame with the SV40 nuclear localization sequence plus the GAL4 activation domain in the pVP16 vector. SaOS 2 cells were then seeded in six-well plates (1 × 105 cells/well) and after 24 h co-transfected with either p16 wild-type or its variants constructs, CDK4 vector, a transfection efficiency control vector, -Gal, and the vector coding for the reporter gene, pG5luc (1.5 g pMBD DNA, 1.5 g CDK4-pVP16, 300 ng pG5luc and 200 ng -Gal, 1 l Lipofectamine 2000). Forty hours after transfection the interaction of p16INK4A and CDK4 was quantified with a luciferase assay using a microplate scintillation and luminescence counter (Top Count NXT, PerkinElmer Life Sciences, MA, USA). For immunoprecipitation assay, U2-OS transfectants were obtained with 1 l Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, added with 2.5 g of the appropriate plasmid DNA (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP) and then maintained in medium containing 300 g/ml G418 (50 mg/ml active Geneticin® ; GIBCO-Invitrogen Corporation) added 24 h after transfection. Seventy-two hours after transfection, U2-OS cells were lysed in NP-40 buffer (PBS, 1% NP-40, CAS registry number: 9036195, 2 mM EDTA, CAS registry number: 62339), plus proteases inhibitors (Complete Mini, Roche Life Sciences, Basel, Switzerland), scraped and centrifuged. The supernatant was mixed with 2 g of anti-p16 antibody (C-20: sc468; Santa Cruz Biotechnology, CA, USA) and the immunocomplexes absorbed to 100 l of a 3:1 slurry of protein G-Sepharose (Amersham Biosciences UK Ltd, Little Chalfont, Buckinghamshire, UK). The beads were re-suspended in sodium dodecyl sulphate (SDS, CAS registry number: 151213) sample buffer, boiled, electrophoresed on 12% SDS-Polyacrylamide (CAS registry number: 9003058) gel and subjected to Western Blotting analysis with antibodies anti-CDK4 (H-22:sc-601, Santa Cruz Biotechnology) to detect the presence of the co-immunoprecipitated CDK4, and anti-p16. For the pRb phosphorylation analysis, U2-OS cells transfected with the proper construct (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP) using the same conditions described for immunoprecipitation, were collected in SDS sample buffer supplemented with proteases (Roche Life Sciences) and phosphatases inhibitors (200 M Na3 VO4, CAS registry number: 13721-39-6; 50 mM NaF, CAS registry number: 7681-49-4) 72 h after transfection. Western Blot analysis (6% SDS-PAGE) was performed with monoclonal antibodies against total Rb (␣ pRb; 4H1, Cell Signaling Technology, MA, USA) and tubulin (␣ tubulin; Clone B-5-1-2; Sigma–Aldrich, MO, USA), and with a polyclonal antibody against hyperphosphorylated pRb (␣ ppRb; Ser807 and Ser811: 9308; Cell Signaling Technology).
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2.5. Immunostaining To perform subcellular distribution assays, U2-OS or NM-39 cells were seeded on coverslips in six-well plates (1 × 105 cells/well) and, after 24 h, transfected with 1 g of each p16 constructs, cloned in frame with the FLAG epitope, and 1 l Lipofectamine 2000 (Invitrogen). After 48 h the cells were fixed in 4% formaldehyde (CAS registry number: 50000) in phosphate-buffered saline (PBS) for 15 min and then permeabilised with 0.2% Triton-X (CAS registry number: 9002931) in PBS for 10 min at room temperature. The cells were immunostained for 50 min with rabbit IgG anti-FLAG antibodies (Sigma–Aldrich) diluted 1:300 in PBS/FCS, followed by another 50 min exposure to goat IgG anti-rabbit, conjugated with Alexa-488 (Invitrogen), secondary antibodies, diluted 1:1000 in PBS/FCS. Nuclei were visualized by Hoechst 33258 staining (2 g/ml, CAS registry number: 23491454). The coverslips were mounted in glycerol (CAS registry number: 56815):PBS (9:1) containing 0.2 N n-propyl-gallate (CAS registry number: 121799) to reduce bleaching. The slides were observed under a fluorescence microscope Olympus BX51 (Shinjuku Monolith, Tokyo, Japan), equipped with a RT-SE18 digital camera. Subcellular distribution was determined from a total of 600 fluorescent cells, in at least three independent transfection experiments. 2.6. Cell cycle inhibition assay U2-OS cells trasfected with p16 (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt ) and control (pcDNA3.1-EGFP) constructs were trypsinized 72 h after transfection, pelleted, re-suspended in 1 ml of GM solution (1.1 g/l glucose (CAS registry number: 50997), 8 g/l NaCl (CAS registry number: 7647145), 0.4 g/l KCl (CAS registry number: 744740-7), 0.2 g/l Na2 HPO4 .2H2 O (CAS registry number: 7558794), 0.15 g/l KH2 PO4 (CAS registry number: 7778770), 0.5 mM EDTA) and fixed in 3 ml of cold ethanol (CAS registry number: 64175). By using 600 g/ml of G418, almost all cells (>98%) were fluorescent. Before the flow cytometry analysis (FACScalibur, BD, NJ, USA; “Modfit” software), the pellet was treated with 5 U/ml of RNase A (Roche Diagnostics, Basel, Switzerland) and incubated for 2 h with 0.1 g/ml Propidium Iodide (CAS registry number: 25535164). 2.7. Cell proliferation assays G418-resistant U2-OS cells, transfected as in the previous experiment, were maintained under G418 selection (300 g/ml) and monitored for a period of 10 days. Using trypan blue dye (CAS registry number: 72571), manual cell counts (average of two replicates) were performed at different time points. Alternatively, U2-OS cells
expressing p1623Asp , p16wt , or control (lacZ) were analysed 2 weeks after transfection; 15000 cells were plated in duplicate and grown under 300 g/ml G418 selection. They were then washed twice with PBS and stained with 1.5% crystal violet solution (CAS registry number: 548629). Total colony number (±standard deviation) was determined from two replicates.
3. Results 3.1. Identification of p.Gly23Asp mutation While screening for patients with familial predisposition for malignant melanoma, we identified a family (kindred 4, Fig. 1) with a history of melanoma as well as other cancers. Mutational analysis of CDKN2A exon 1␣ of the proband (indicated by arrow in Fig. 1) was uninformative, while analysis of the second melanoma patient (indicated by asterisk), and of her father with B-NHL disclosed the presence of the germline missense mutation p.Gly23Asp in both of them. This variant was not found in 186 healthy controls, thus excluding the possibility of a common benign polymorphism. The nucleotide change (c.68G>A) located in p16-specific exon 1␣, belongs to an evolutionary conserved domain located in the first ankyrin repeat that specifically binds to CDK4/6, thus inhibiting kinase function. To investigate if this variant impairs the activity of the p16 protein, different functional assays were performed. 3.2. Loss of CDK4 binding and pRb hyperphosphorylation CDK4 is the main known target of p16INK4A , and its kinase activity is inhibited by mutual binding. Therefore, p1623Asp variant was analyzed for loss of its binding ability with a subsequent accumulation of the hyperphosphorylated pRb form (ppRb). The in vivo binding activity of this variant was assessed using a mammalian two-hybrid assay. Each p16 construct (the pathogenic p1632Pro ,
Fig. 1. The high risk melanoma kindred 4. Mutational analysis of the proband (indicated by arrow) was uninformative, while analysis of the second melanoma patient (indicated by asterisk), and her father with B-NHL, disclosed the presence of the germ-line missense mutation p.Gly23Asp in both of them. Age at pedigree compilation, age of melanoma onset and type of tumor are shown under each symbol. CDKN2A mutation status, when determined, is also indicated.
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phosphorylated (ppRb) and total pRb (Fig. 2C). The specific ␣ ppRb antibody detects endogenous levels of pRb only when phosphorylated at serine 807/811, i.e. when cells progress from G1 to S phase. As expected, the expression of wild-type p16 caused a reduction in the amount of the hyperphosphorylated form of pRb (lane 3). Furthermore, the bands corresponding to pRb and ppRb are visible only in p16wt cells but not in p1623Asp cells (lane 2), where only the ppRb band is present, thus suggesting that p1623Asp is much less efficient than p16wt in blocking cell cycle progression. 3.3. Different subcellular distribution of p1623Asp Each of the p16 construct (p1623Asp and p16wt ) was cloned in frame with the FLAG epitope in the pFLAG-CMV-5b vector. The subcellular distribution of each construct was evaluated in transiently transfected U2-OS osteosarcoma cells (Fig. 3A) and NM-39 melanoma cells (Fig. 3B). As expected, p16wt localized evenly throughout the cell, in both cell lines; on the contrary, p1623Asp presented a different subcellular distribution when compared to the wild-type pattern (Fig. 3A and B). Cytoplasmic aggregates (large areas of intense fluorescence) could be detected in cells transfected with the variant, possibly indicating an incorrect folding of the mutant protein during post-translational processing in the ER and Golgi apparatus. Besides the cytoplasmic aggregates observed in the U2-OS cells (Fig. 3A), nuclear speckles (small discrete areas of intense fluorescence) could also be detected in NM-39 cells, transfected with the p1623Asp variant (Fig. 3B), thus suggesting that even though pleomorphic, aggregates of this variant are readily detectable in different cell lines. 3.4. Loss of G1 cell cycle arrest in p1623Asp cells
Fig. 2. Binding of p16 to CDK4 and inhibition of pRb phosphorylation. (A) Twohybrid system assay in p53 and pRb-null SaOS 2 cells. Binding of variant proteins was determined as a percentage relative to the binding activity of the wild-type (p16wt ) protein. At least four independent transfection experiments were performed in duplicate for each construct. (B) Western Blotting detection of CDK4 in U2-OS cell lysates immunoprecipitated with p16. Cells expressing p1623Asp , p16wt or a control EGFP plasmid (negative control), were lysed and immunoprecipitated with a polyclonal antibody anti-p16. Western Blotting was performed using a polyclonal antibody anti-CDK4. Hela cells lysates, expressing endogenous p16wt , were used as positive control. (C) Analysis of pRb phosphorylation inhibition by p16 constructs and control vector in U2-OS cells. Samples were immunoblotted with antibody against total Rb (␣ pRb), hyperphosphorylated pRb (␣ ppRb) and tubulin (␣ tubulin) as shown on the left; the positions of hyper- and hypophosphorylated forms of the protein are indicated by arrows on the right.
p16wt and p1623Asp ) was cloned in frame with the GAL4 nuclear localization sequence, and transiently co-transfected with a CDK4 expression plasmid, into SaOS 2 cells. As shown in Fig. 2A, the ability of p1623Asp to bind CDK4 is null, and comparable to that of p1632Pro . In a different approach, U2-OS cells expressing p1623Asp , p16wt or EGFP, as control plasmid, were lysed and immunoprecipitated with a polyclonal antibody anti-p16: the presence of co-precipitating CDK4 was then assessed by western blotting using a polyclonal antibody anti-CDK4. Contrary to p16wt , p1623Asp was unable to bind CDK4, as shown in Fig. 2B. These results were consistent with those obtained by the mammalian two-hybrid binding assay, and showed once more no affinity of p1623Asp for CDK4. A further proof of the p1623Asp loss of CDK4 binding activity was obtained evaluating its effect on pRb phosphorylation. Total protein samples from U2-OS cells transfected with pcDNA3.1p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP were fractionated in a 6% SDS-PAGE and immunoblotted with antibodies against hyper-
To further investigate the loss of p1623Asp proliferationinhibitory functions, we evaluated the cell cycle distribution in three experimental U2-OS cell samples (pcDNA3.1-p1623Asp , pcDNA3.1-p16wt , pcDNA3.1-EGFP), using flow cytometry (Fig. 4A). Cell selection (98% of fluorescent cells) was obtained in 72 h using twice the standard G418 concentration (600 g/ml). The assay, in six replicates (as reported in Fig. 4A), confirmed the same conclusions reached previously, showing a higher percentage of G1 cells in p16wt (69.4%) than in p1623Asp and EGFP (∼50%) cultures; consequently, the percentage of S-phase cells was higher in p1623Asp and EGFP (∼35%) transfected cells than in control p16wt (19%) cultures. 3.5. Loss of cell proliferation arrest in p1623Asp cells G418-resistant U2-OS cells expressing p1623Asp , p16wt or EGFP plasmid were also monitored for a period of 10 days; their proliferation curves are reported in Fig. 4B, as an example of three independent experiments. Clearly, cells expressing p16wt expanded more slowly, due to the expression of the functional oncosuppressor. On the other hand, p1623Asp cells grew better than their wild-type counterpart p16wt cells; additionally, their growth curve precisely overlapped with that of the cells transfected with a control plasmid (EGFP), that has no effect on cell cycle regulation. Finally, we tested whether the higher proliferation rate of p1623Asp cells, when compared with p16wt cells, was also reflected by the ability of the cells to form colonies when seeded at low density. After 2 weeks under G418 selection, colony formation ability was much lower in p16wt cells than in p1623Asp cells (Fig. 4C). In fact, the number of p1623Asp colonies was similar (>200 colonies/plate) to the control clones (pcDNA3.1-lacZ) and much higher than that of p16wt cells (∼50 colonies/plate), thus confirming, once more, that the expression of p1623Asp was unable to arrest cell proliferation.
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Fig. 3. Cellular distribution of FLAG epitope-tagged p16INK4A mutation constructs in U2-OS (A) and NM-39 (B) cells. Each p16INK4A -FLAG tagged construct was transiently introduced into p16-null human osteosarcoma (U2-OS) and melanoma (NM-39) cells. Cells were then immunostained with a monoclonal mouse ␣-FLAG M2 antibody and then exposed to a fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody (green). Nuclei were visualized by Hoechst staining (blue). The subcellular distribution of the p1623Asp showed areas of intense fluorescence which were classified as cytoplasmic aggregates, for large areas localised in the cytoplasm, or as nuclear speckles for small discrete areas localised in the nucleus. On the left, values of protein distribution are reported below cell schemes. LM, light microscopy.
4. Discussion Key to an effective cancer prevention strategy is the early identification of high risk individuals [22]; thus, important decisions on the clinical management of cancer susceptibility syndromes are made based on whether a subject carries a pathogenic mutation or not. In addition to disease causing genetic alterations (i.e. deletions and truncations), mutational screening often finds missense substitutions, which are provisionally named unclassified variants. As recognized earlier [23], these variants are a potentially serious problem [24], and specific recommendations by the IARC Working Group on unclassified genetic variants in high risk cancer susceptibility genes are reported in the November special issue of Human Mutation. Besides specific mutations affecting the p16INK4A and/or p14ARF products of the CDKN2A locus, which are firmly established as causes of increased individual risk of melanoma, over 110 germline variants have been identified [25] and additional novel missense mutations continue to be discovered. Pathogenicity of a variant is defined by both, direct evidence, like its segregation with disease, and its absence in control DNA, or by indirect proof, as severe amino acid change, occurrence in a highly conserved, relevant protein domain, functional assays, and in silico tools [26]. As for the direct criteria, the CDKN2A p.Gly23Asp (p.G23D) missense mutation was not present in 186 healthy donor samples, while it was detected in only one of the two tested melanoma patients of kindred 4 (Fig. 1) with 3 melanoma cases, as well as in a melanoma-free relative, affected by B-NHL. Interestingly, also in a French family, the variant did not segregate with disease [17], but no functional assays were reported to assess its putative role. Lack of segregation with disease, albeit rare, could occur in high risk cancer families, and phenocopies might be present in melanoma families, likely due to the relevant role played by low penetrance genes (i.e. MC1R), acting together with environmental factors (i.e. sun exposure). On the other hand, the presence of melanoma-free carriers could be ascribed to incomplete gene pene-
trance; previous studies have, indeed, demonstrated that CDKN2A mutations might have extremely variable penetrance depending on the various geographical areas (i.e. UV irradiation) in which carriers live [27], as it was also assumed that every single mutation might have its own penetrance [18]. Moreover, we can not exclude that the occurrence of the B-NHL in the melanoma-free carrier could be related to the presence of the p.Gly23Asp mutation, as a result of its variable expressivity; obviously, lack of any correlation between p1623Asp and B-NHL is also a possibility. In the p.Gly23Asp variant, the mutated amino acid is located in the evolutionary conserved domain of the first ankyrin repeat, that binds specifically to and blocks the function of CDK4/6, thus likely disrupting the ankyrin repeat unit and the p16 binding site. As shown in Fig. 2, and in line with a recent report [19] three different approaches gave consistent results, disclosing either a direct (Fig. 2A and B) or indirect (Fig. 2C) p1623Asp impairment for CDK4 binding. Structural changes could be responsible also of the findings reported in Fig. 3; notably, the immunostaining assay showed a p1623Asp mislocalization pattern, reminiscent of protein aggregation. As previously observed by Rizos et al. [12], it is unlikely that mislocalization is simply the result of high protein concentration, since overexpressed wild type or innocent p16 variants did not display altered subcellular distribution. Together with cytoplasmic aggregates in the p1623Asp U2-OS cells (Fig. 3A), we detected, not surprisingly, nuclear speckles in p1623Asp NM-39 cells (Fig. 3B), since p16wt is diffusely present in both nucleus and cytoplasm of neuroblastoma [28] and melanoma [15] cell lines. These various patterns of p16 localization, far from being understood, rather suggest that cell type-dependent properties may account for some of the inconsistent functional data on p16INK4A mutants in the literature. Finally, as shown in Fig. 4, there is a relevant difference between p16wt and p1623Asp behaviour in proliferation assays. Using all three approaches (Fig. 4A–C) the p.G23D variant showed a clear
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Fig. 4. Loss of p1623Asp proliferation inhibition in U2-OS cells. (A) Flow cytometry analysis of U2-OS cells transfected with p16wt and p1623Asp and control (EGFP) constructs. Percent of cells in each cell cycle phase are indicated. (B) G418-resistant U2-OS cells expressing p1623Asp , p16wt or EGFP, monitored for a period of 10 days at indicated time points. (C) Crystal violet staining of colonies after 2 weeks under G418 selection of U2-OS cells transfected with p1623Asp , p16wt or lacZ. The number of colonies (average of two replicates ± standard deviation) is indicated at the bottom.
and coherent behaviour always overlapping with the p16-negative (or mutant) control. Ancillary to direct and functional approaches are in silico based methods that exploit different tools and algorithms [24]. Using some of these in silico tools (i.e. SNAP, Polyphen, SIFT, BLOSUM62), concordant results on p1623Asp behaviour were obtained, ranging from “non-neutral” to “deleterious” (data not shown). Assessing pathogenicity of Unclassified Genetic Variants (UVs) is a major scientific issue emerging worldwide. A panel of experts has recently suggested to approach the problem of UVs classification with as many assays as possible, provided that they explore different functional aspects, and that the evidences obtained are independent from each other [29–31]; UV classification should thus take advantage of all possible means of assessment, due to the clinical implications involved when considering a variant pathogenic rather than neutral. In conclusion, using a multi-evidence approach of independent assays, according to the new IARC recommendations [24], p1623Asp is here defined as a “loss of function” mutation [26], which most likely predisposes carriers to melanoma development. The mutant protein, indeed, is not able to bind CDK4 and to prevent pRB hyperphosphorylation. Since, at the very end, the p16wt physiological role results in the inhibition of cell cycle progression (Fig. 4), it is our opinion that a specific attention should be given to assays that analyze this property in detail, even though for any functional assay validation studies and definition of “gold standards” are very much needed [32].
Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Ministero del Lavoro, della Salute, e delle Politiche Sociali: Programma Integrato Oncologia N.5; Alleanza Contro il Cancro (N.ACC2/R6.9); Lega Italiana per la Lotta contro i Tumori (LILT), Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio di Padova e Rovigo. MCS and MC were supported by a fellowship from FIRC (Fondazione Italiana per la Ricerca sul Cancro). References [1] M. Serrano, G.J. Hannon, D. Beach, A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4, Nature 366 (1993) 704–707. [2] Y. Zhang, Y. Xiong, W.G. Yarbrough, ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways, Cell 92 (1998) 725–734. [3] A. Kamb, D. Shattuck-Eidens, R. Eeles, Q. Liu, N.A. Gruis, W. Ding, C. Hussey, T. Tran, Y. Miki, J. Weaver-Feldhaus, M. McClure, J.F. Aitken, D.E. Anderson, W. Bergman, R. Frants, D.E. Goldgar, A. Green, R. MacLennan, N.G. Martin, L.J. Meyer, P. Youl, J.J. Zone, M.H. Skolnick, L.A. Cannon-Albright, Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus, Nat. Genet. 8 (1994) 23–26. [4] C.J. Hussussian, J.P. Struewing, A.M. Goldstein, P.A. Higgins, D.S. Ally, M.D. Sheahan, W.H. Clark Jr., M.A. Tucker, N.C. Dracopoli, Germline p16 mutations in familial melanoma, Nat. Genet. 8 (1994) 15–21.
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