Tbx2 Is Overexpressed and Plays an Important Role in Maintaining Proliferation and Suppression of Senescence in Melanomas Keith W. Vance, 1 Suzanne Carreira, 1 Gerald Brosch, 2 and Colin R. Goding 1 1 Signaling and Development Laboratory, Marie Curie Research Institute, Surrey, United Kingdom and 2 Department of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria Abstract The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and over- expressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly impor- tant question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senes- cence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21 Cip1 (CDKN1A ) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to dis- placement of histone deacetylase 1, up-regulation of p21 Cip1 expression, and the induction of replicative senescence in CDKN2A -null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas. (Cancer Res 2005; 65(6): 2260-8) Introduction The fact that oncogenic Ras can induce senescence in primary fibroblasts (1) has led to the notion that senescence represents a powerful defense against cancer (2, 3). Indeed, recent evidence indicates that senescence in human cells in response to oncogenic stress exemplified by activated Ras leads to the formation of senescence-associated heterochromatin foci (SAHF) in an Rb-dependent fashion (4). The CDKN2A locus encoding both the INK4A and ARF genes can act to promote senescence, and both genes have been established as important tumor suppressors in human cancer (5). Mutation or deletion of either, or frequently both, the INK4a and ARF genes is associated with melanoma (6–8), a highly dangerous form of skin cancer with an alarming increase in incidence. The key role of CDKN2A in the melanocyte lineage is underscored by the observation that in contrast to fibroblasts (9), a single copy of a deletion at this locus disrupts senescence in mouse melanocytes (10), and targeted expression of activated Ras to melanocytes in transgenic mice generates melanomas only if the INK4a gene is absent (11). Moreover, the INK4a gene is necessary for the normal senescence of primary human melanocytes that occurs in the absence of elevated p21 Cip1 (CDKN1A , hereafter termed p21) or p53 levels (12); human melanocytes deficient for INK4a have an extended life span but do eventually senesce under conditions where p21 expression is induced. Thus, the key role of the CDKN2A locus and the INK4a gene in particular in melanocyte senescence is likely to explain its loss or mutation in melanoma. The T-box gene family (for reviews, see refs. 13–16) illustrates an important example of proteins that are involved both in controlling fundamental developmental decisions and are also misregulated in cancer. In addition to their role in development, increasing evidence suggests that Tbx2 and the highly related factor Tbx3 play a role in cancer progression. The TBX2 gene maps to 17q23, a region that is frequently mutated in ovarian carcinomas (17, 18) and TBX2 is also amplified in pancreatic cancer cell lines (19) and is preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors (20). Furthermore, Tbx3 is also overexpressed in breast cancer lines (21) and can cooperate with Ras and Myc to transform cells and disrupt pathways required for apoptosis (22). Significantly, in defined experimental systems using CDKN2A wild-type primary fibroblasts or striatal cells overexpression of either Tbx2 and Tbx3 can inhibit senescence, most likely through their ability to repress expression of the ARF and p21 promoters (23–27). However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the require- ment for Tbx2/3-mediated repression of ARF in suppressing senescence. In this study, we show that activation of a dnTbx2 molecule induces senescence in CDKN2A -null B16 melanoma cells. Senescence is accompanied by an increase in the levels of the Tbx2 target gene p21 and displacement of histone deacetylase 1 (HDAC1) from the p21 promoter. In human melanoma cells, dnTbx2 induces the formation of SAHFs and severely reduced proliferation. These results identify a molecular mechanism for transcriptional repression by Tbx2 and suggest that continued Tbx2 activity is required to prevent CDKN2A -independent senescence in transformed cells. Requests for reprints: Colin R. Goding, Signaling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom. Phone: 44-1883-722306; Fax: 44-1883-714375; E-mail: c.goding@mcri.ac.uk. I2005 American Association for Cancer Research. Cancer Res 2005; 65: (6). March 15, 2005 2260 www.aacrjournals.org Research Article Research. on April 24, 2020. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from