Six1 Overexpression in Mammary Cells Induces Genomic Instability and Is Sufficient for Malignant Transformation Ricardo D. Coletta, 1 Kimberly L. Christensen, 5 Douglas S. Micalizzi, 5 Paul Jedlicka, 2 Marileila Varella-Garcia, 3 and Heide L. Ford 1,4,5 Departments of 1 Obstretrics and Gynecology, 2 Pathology, 3 Medical Oncology, 4 Biochemistry and Molecular Genetics, and 5 Program in Molecular Biology, University of Colorado Health Sciences Center, Aurora, Colorado Abstract Homeoproteins are transcription factors that act as master regulators of development and are frequently dysregulated in cancers. During embryogenesis, the Six1 homeoprotein is essential for the expansion of precursor cell populations that give rise to muscle and kidney, among other organs. Six1 overexpression is observed in numerous cancers, resulting in increased proliferation, survival, and metastasis. Here, we investigate whether Six1 can play a causal role in mammary tumor initiation . We show that Six1 overexpression in MCF12Amammaryepithelialcellspromotesmultipleproper- ties associated with malignant transformation, including increased proliferation, genomic instability, and anchorage- independent growth. We further show that this transforma- tion is dependent on up-regulation of its transcriptional target, cyclin A1, which is normally expressed in the embryonic mammary gland but dramatically reduced in the adult gland. Six1-transformed MCF12A cells are tumorigenic in nude mice, forming aggressive tumors that are locally invasive and exhibit peritumoral lymphovascular invasion. In human breast carcinomas, expression of Six1 and cyclin A1 mRNA correlate strongly with each other (P < 0.0001), and expression of Six1 and cyclin A1 each correlate with Ki67, a marker of proliferation (P < 0.0001 and P = 0.014, respectively). Together, our data indicate that Six1 over- expression is sufficient for malignant transformation of immortalized, nontumorigenic mammary epithelial cells, and suggest that the mechanism of this transformation involves inappropriate reexpression of cyclin A1 in the adult mammary gland. [Cancer Res 2008;68(7):2204–13] Introduction The homeobox gene superfamily encodes for transcription factors that play important roles in development (1). As ‘‘master regulators’’ of development, homeoproteins control diverse cellular processes by regulating the expression of many downstream target genes. Thus, it is typical for an individual homeoprotein to confer pleiotropic effects on cell behavior. These effects include alterationsinproliferation,survival,migration,andinvasionamong others (2). Because these processes are also all important in human cancers, misexpression of homeobox genes is likely to play a causal role in tumor onset and/or progression. However, although aberrant expression of numerous homeobox genes has been observed in various solid tumor and hematologic malignancies, it is still unclear if such misexpression is generally a cause or consequence of tumorigenesis (3, 4). In support of a causal role for homeoproteins in the initiation of cancer, overexpression of several homeoproteins has been shown to result in oncogenic transfor- mation of human cells (5–7). Most recently, Oct-4 was shown to play a critical role in the genesis of germ cell tumors, in a dose- dependent manner (8). This elegant study provided further insight into how homeobox genes can be oncogenic, yet distinct from ‘‘classic oncogenes’’ in several ways that make them attractive therapeutic targets: they are tissue (and often developmentally) restricted, they can be associated with early stages of carcinogen- esis, and they are sensitive to dosage (9). In this paper, we provide evidence that the Six1 homeoprotein plays a causal role in mammary tumor initiation, leading to highly aggressive mammary tumors in vivo . The Six1 homeoprotein is essential for the development of numerous organs through its effects on cell proliferation, survival, and cellular migration (10–13). Six1 plays a role in the expansion of precursor cell populations during organogenesis, after which its expression is lost in most adult differentiated tissues (14). Aberrant overexpression of Six1 is observed in numerous human cancers, where it leads to increased proliferation, survival, and metastasis (15–17). The pro-proliferative role of Six1 has been shown in breast and ovarian carcinoma, as well as in rhabdomyosarcoma (15–17). In breast cancer cells, Six1 promotes entrance into and progression through S phase and attenuates the DNA damage–induced G 2 cell cycle checkpoint (14, 15). Together, these properties of Six1 may contribute to its role in tumorigenesis. Several of the known transcriptional targets of Six1 play a role in cell growth and proliferation, including cyclin A1, cyclin D1, and c-myc (10, 15, 18). We have previously shown that the embryonic and tissue-restricted cyclin A1 mRNA is regulated by Six1 in the normal mammary gland (15). Both genes are highly expressed in the embryonic mammary gland but are dramatically decreased in the adult-differentiated mammary gland, suggesting a role for Six1 and cyclin A1 in early mammary gland development (15). Interestingly, cyclin A1 is up-regulated by Six1 in breast cancer cell linesandiscriticalfortheSix1-mediatedincreaseinproliferationin these cells (15). Aside from controlling proliferation, cyclin A1 influencesnumerousothercellularprocessesincludingcellsurvival, DNA repair, and angiogenesis (19–21). Its role in these processes suggests that cyclin A1 misexpression could contribute to several tumorigenic phenotypes that are found in a spectrum of different tumors. Indeed, cyclin A1 is highly expressed in testicular, ovarian, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). R.D. Coletta and K.L. Christensen contributed equally to this work. PresentaddressforR.D.Coletta:DepartmentofOralDiagnosis,SchoolofDentistry, State University of Campinas, Piracicaba, Sa ˜o Paulo, Brazil. Requestsforreprints: Heide L. Ford, University of Colorado at Denver and Health Sciences Center at Fitzsimons, Department of Obstetrics and Gynecology, Mailstop 8309, PO box 6511, Aurora, Colorado 80045. Phone: 303-724-3509; Fax: 303-724-3512; E-mail: heide.ford@uchsc.edu. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-3141 Cancer Res 2008; 68: (7). April 1, 2008 2204 www.aacrjournals.org Research Article Research. on October 6, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from