TPX2 in melanoma: honing on drivers of amplification on chromosome 20q11.2

1687 We previously performed BAC based array-comparative genomic hybridization (aCGH) on 83 metastatic melanoma lesions (27 melanoma cell lines, 34 frozen and 22 formalin-fixed, paraffin embedded clinical melanoma samples). In this analysis, we found recurrent and characteristic losses of chromosome 5q, 6q, 9,10q, 11q, 8q and gains of chromosomes 1q, 6p, 7, 8p, 17p and 20q. Moreover, in this sample set, we were able to prove with statistical significance (p BRAF mutation is associated with amplifications of chromosomes 7 and 20. Copy number gain of the long arm of chromosome 20 also has been shown in other genome-wide analyses of melanoma corroborating our hypothesis that genes important for melanoma progression reside on chromosome 20q. Multiplex ligation dependent probe amplification (MLPA) is useful technique for confirming DNA copy number changes previously observed in aCGH. Thus, to define more precisely the amplified genes on 20q, MLPA was performed in 19 melanoma cell lines and 27 frozen metastatic melanomas. The genes that MLPA specifically assesses on 20q11.2 include BCL2L1 , TPX2 , SRC , MYBL2 , ZNF217 , CYP24 , STK6 , BCAS1 and GNAS among others. The amplifications seen on MLPA were quantified using Coffalyser (MRC-Holland) software. Fine mapping of chromosome 20q using MLPA demonstrated copy number increases for several genes. However, one gene, TPX2 , was found to be more frequently amplified than any other. TPX2 amplifications were found in 13/19 (68%) melanoma cell lines and 12/27 (44%) of melanoma samples. Additionally, using expression profiling data generated in-house and the Oncomine Database, we found TPX2 was significantly over-expressed (p=2.45E-10) in most cell lines and melanoma samples. TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromatin and the targeting of Aurora-A kinase to the spindle apparatus. TPX2 binding not only prevents the inactivation of AurA but also increases the kinase activity of the activated AurA. These results suggest a role for TPX2 as a potential candidate oncogene of importance in melanoma and a possible molecular therapeutic target. Additional studies are underway to demonstrate the functionality of TPX2 in primary melanoma cell lines.