Functional Impairment of Melanoma-associated p16 Mutants in Melanoma Cells despite Retention of Cyclin-dependent Kinase 4 Binding

Purpose: Melanoma-associated germ-line mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16 have been identified in >100 melanoma-prone families. To predict the melanoma risk for carriers of specific mutations, it is useful to test the function of the mutant proteins in biochemical assays; however, it is unclear how well these results correlate with their cellular effects. We examined the relationship between loss of CDK binding by mutant proteins and various measures of cellular growth in melanoma cells. Experimental Design: The cellular activities of four melanoma-associated p16 mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4. Results: The cell cycle-inhibitory activity of all of the mutants was profoundly reduced, and partially retained capacity for CDK4 binding in functional assays did not correlate with significant preservation of cell cycle-regulatory function. Conclusion: Testing of p16 interactions with CDKs in protein-binding assays is an unreliable predictor of mutant p16 function in cells. In addition to exhibiting reduced stability, these mutant proteins may also be defective in interaction with cellular targets other than CDKs. INTRODUCTION The CDKN2A gene on chromosome band 9p21 encodes the CDK inhibitor p16. Somatic alterations affecting this gene occur in 50% of human tumors (1, 2). Inherited CDKN2A mutations are associated with susceptibility to melanoma and, in some cases, pancreatic carcinoma (3, 4). To date, at least 48 germ-line p16 mutations have been identified in 100 melanoma-prone families world-wide. Although these mutations may segregate closely with the disease in melanomaprone families, demonstration of functional defects in the p16 protein remains clinically important in distinguishing disease-associated mutations from population polymorphisms. p16 was initially identified as a binding partner of CDK4 (5). Although several functions of p16 have been described in recent years, including the repression of the transcription factor NF B (6), it is widely assumed that the main role of p16 is to inhibit CDKs. In particular, p16 binds to and inhibits the activity of CDK4 and CDK6. These kinases regulate the progression of eukaryotic cells through the G1 phase of the cell cycle. CDK4 and CDK6 initiate the phosphorylation of pRb, which permits cells to enter DNA replication in S phase. CDK4 may also be involved in cell cycle progression through the G2 cell cycle phase and into mitosis (M phase; Ref. 7). To investigate various p16 mutations for their ability to bind and inhibit CDK4 and CDK6, a number of CDK-binding and kinase assays have been applied. These assays used p16 and CDKs fused to GST (8), polyhistidines (9), or proteins translated in vitro (9–11). Alternatively, in the yeast two-hybrid system, the proteins were fused to regulatory yeast peptides (12–14). These tests confirmed that most cancer-associated p16 mutations are functionally impaired. However, different assays revealed variable degrees of functional loss for identical p16 mutations. For example, the Gly101Trp mutation is strongly associated with melanoma in 20 families world-wide, but its cyclin D1/CDK4 inhibitory activity in different functional assays ranged from 5 to 73% of the wild-type protein (8, 9, 12, 14). Functional deficiency of p16 mutant proteins has also been shown after the gene or protein was introduced into a number of nonmelanoma and melanoma cell lines (15, 16). However, these studies were also limited because the cell cycle-inhibitory effect of functional p16 selects against cells expressing the transgene and thereby affects longterm studies. This growth-inhibitory effect can be overcome by use of an inducible expression system, and Stone et al. (17) Received 4/16/01; revised 7/2/01; accepted 7/6/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Therese Becker was supported by scholarships of Sydney University (Overseas Postgraduate Research Scholarship, University Postgraduate Scholarship) and the Millennium Foundation, Westmead Hospital. The project was supported by the NSW Cancer Council, the National Health and Medical Research Council of Australia, and the Melanoma and Skin Cancer Research Institute, University of Sydney. 2 To whom requests for reprints should be addressed, Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, NSW 2145, Australia. Phone: 61 2 9845 9058; Fax: 61 2 9845 9102; E-mail: [email protected]. 3 The abbreviations used are: CDK, cyclin-dependent kinase; NF B, nuclear factor B; pRb, retinoblastoma protein; GST, glutathione Stransferase; IPTG, isopropyl-1-thio-D-galactopyranoside; SA, senescence-associated; CTD, COOH-terminal domain. 3282 Vol. 7, 3282–3288, October 2001 Clinical Cancer Research Research. on September 22, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from reported a detailed study of the effects of induced wild-type p16 expression in melanoma cells. Consequently, we chose to thoroughly assess the function of melanoma-associated mutant p16 proteins by stably introducing inducible constructs of the wild-type or mutant CDKN2A genes into melanoma cells lacking endogenous p16. The functional loss of all tested melanoma-associated p16 mutations was much greater than CDK4 binding studies suggested. We postulate that these mutants are defective in interaction with cellular targets additional to CDKs. MATERIALS AND METHODS CDKN2A cDNA and Mutagenesis. The originally cloned CDKN2A cDNA (5) was extended to full length (18) by PCR. Overlapping PCR, modified from Aiyar and Leis (19), was used to engineer four p16 mutations. The Arg24Pro (G071C) and Ala36Pro (G106C) variants were originally identified in our laboratory (20, 21), and the Arg24Pro mutation has now been reported in nine melanoma-prone families world-wide (11, 13, 20–25). The Met53Ile (G159C) variant was also originally detected in an Australian melanoma kindred (26), and melanoma association has now been reported in 12 families world-wide (11, 13, 21, 22, 24, 26–28). The Val126Asp (T377A) variant was one of the first CDKN2A mutations identified; its function has been examined with different CDK binding and kinase assays and after introduction into cell lines. It has been associated with melanoma in at least three families worldwide (24, 29, 30). Protein Expression in Bacteria. CDKN2A cDNA was cloned into the pGEX-5X-I vector (Amersham Pharmacia), and the insert and adjacent vector sequences were completely sequenced. Apart from the inserted point mutations, all CDKN2A constructs had identical sequences. GST-fusion p16 (GSTp16) was expressed in Escherichia coli (JM109), purified using glutathione-Sepharose beads (as described by the supplier) and stored in glycerol lysis buffer [25% glycerol, 100 mM NaCl, 1% Triton, 1 mM DTT, 1 mM EDTA, 50 mM Tris (pH 8)]. CDK4 cDNA was obtained by reverse transcription-PCR from total RNA derived from human lymphoblastoid cells. The CDK4 cDNA insert was cloned into the pQE31 vector (Qiagen) and completely sequenced. CDK4 fused to six histidine residues (His-CDK4) was expressed in E. coli (BL21). p16 CDK4 Binding Assays. Equal amounts of wild-type GST-p16, mutant GST-p16, or GST alone were immobilized on glutathione-Sepharose beads, and excess proteins were washed away. Bacterial lysate, containing excess levels of expressed His-CDK4, was added to the protein-coated Sepharose beads. The final binding buffer concentrations were as follows: 50 mM Tris (pH 8), 8.3% glycerol, 0.33% Triton, 33.3 mM NaCl, 0.33 mM DTT, and 0.33 mM EDTA. Incubation was performed for 1.5 h at room temperature or at 37°C with gentle agitation. The Sepharose beads were then washed three times with binding buffer. SDS-PAGE loading buffer was added, and samples were boiled for 5 min. Proteins were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. CDK4 was detected by Western analysis with the antibody, clone DCS-35 (Neomarkers). Cell Culture and Transfection. The human WMM1175 melanoma cell line was originally isolated from a s.c. metastatic tumor of an individual with a family history of melanoma. The WMM1175 cell line is homozygously deleted for the CDKN2A region on chromosome 9p21 (31), but expresses wild-type CDK4 and pRb (32). Cells were grown in DMEM (Trace) with 10% FCS (Trace). An IPTG-inducible mammalian expression system (Lac-switch system; Stratagene) was used to obtain melanoma cell clones carrying the stably integrated CDKN2A gene under IPTG-inducible expression control. A WMM1175 clone transfected with the lac repressor vector, p3 SS, and expressing high levels of the lac repressor protein was selected for further transfection (33). Full-length CDKN2A cDNA (wild type or mutant) was cloned into the expression plasmid, pOPRSVICAT, and transfected using the calcium phosphate precipitation method. Transfected cells were selected with hygromycin (250 g/ml) and geneticin (500 g/ml; Life Technologies, Inc.). Expression of the transgene in selected clones was induced by 4 mM IPTG. The growth rate was monitored over 7 days, cell cycle distribution was studied after 24, 48, 72, and 96 h of p16 induction (Becton Dickinson FACScan), and colonyforming ability was tested 14 days after induction. SA-Galactosidase Staining. Clones induced to express wild-type p16 protein with 4 mM IPTG were screened for expression of SA-galactosidase, as described (34). Senescent human fibroblasts were used as controls for SA-galactosidase staining.