Tumor and Stem Cell Biology Hbo1 Is a Cyclin E/CDK2 Substrate That Enriches Breast Cancer Stem-like Cells

Expression of cyclin E proteolytic cleavage products, low-molecular weight cyclin E (LMW-E), is associated with poor clinical outcome in patients with breast cancer and it enhances tumorigenecity in mouse models. Here we report that LMW-E expression in human mammary epithelial cells induces an epithelial-tomesenchymal transition phenotype, increases the CD44/CD24 population, enhances mammosphere formation, and upregulates aldehyde dehydrogenase expression and activity. We also report that breast tumors expressing LMW-E have a higher proportion of CD44/CD24 tumor cells as compared with tumors expressing only full-length cyclin E. In order to explore how LMW-E enriches cancer stem cells in breast tumors, we conducted a protein microarray analysis that identified the histone acetyltransferase (HAT) Hbo1 as a novel cyclin E/CDK2 substrate. The LMW-E/CDK2 complex phosphorylated Hbo1 at T88 without affecting its HAT activity. When coexpressed with LMW-E/CDK2, wild-type Hbo1 promoted enrichment of cancer stem-like cells (CSC), whereas the T88 Hbo1 mutant reversed the CSC phenotype. Finally, doxorubicin and salinomycin (a CSC-selective cytotoxic agent) synergized to kill cells expressing LMW-E, but not fulllength cyclin E. Collectively, our results suggest that the heightened oncogenecity of LMW-E relates to its ability to promote CSC properties, supporting the design of therapeutic strategies to target this unique function. Cancer Res; 73(17); 5556–68. 2013 AACR. Introduction Before the G1–S phase boundary, cyclin E associates with cyclin-dependent kinase 2 (CDK2) to further phosphorylate and inactivate the pRb–E2F complex and promotes the Sphase entry (1–3). Cyclin E overexpression by genomic and transcriptional amplification has been reported in multiple cancers, particularly in breast cancer (4–8). In addition, posttranslational proteolytic cleavage of cyclin E mediated by a serine protease generates two low-molecular weight cyclin E (LMW-E) isoforms in cancer cells (9, 10). Compared with full-length cyclin E (EL), the LMW-E isoforms are hyperactive by forming tighter interaction with CDK2 and are more resistant to p21 and p27 inhibition, resulting in higher rate of proliferation (11, 12, 13). Transgenic mice expressing LMW-E isoforms are themselves malignant as approximately 27% of LMW-E transgenic mice develop mammary adenocarcinomas compared with 10% of transgenic mice with EL expression (14). Furthermore, approximately 25% of the LMW-E tumors develop metastasis compared with 8% in the EL tumors (14). We recently showed preferential accumulation of LMW-E/CDK2 complexes in the cytoplasm (15). The altered subcellular localization of LMW-E renders it less susceptible to Fbw7-directed proteasomal degradation. This finding implicates many possibilities that can explain the particular oncogenic characteristics of LMW-E that are distinct from full-length cyclin E, and that these effects could be independent from cell-cycle regulation. Compared with the EL isoform, overexpression of LMW-E induces aberrant acinar morphogensis and mammary tumorigenesis in a CDK2-dependent manner (15, 16). In addition, examination of 395 breast cancer patient tissues identified LMW-E as an independent and powerful prognostic and predictive marker of patient outcome (17). Finally, we recently reported that the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival (16). Here, we provide evidence that the tumorigenicity of LMW-E is mediated through induction of the epithelial-to-mesenchymal transition (EMT) that leads to enrichment of the cancer stem-like cells (CSC) population via the phosphorylation of Hbo1 [HAT binding to ORC1 (origin recognition complex 1)] Authors' Affiliations: Departments of Experimental Radiation Oncology, Radiation Oncology, and Surgical Oncology, The University of TexasMD AndersonCancer Center; and The University of Texas Graduate School of Biomedical Sciences, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). CorrespondingAuthors:KhandanKeyomarsi, TheUniversity of TexasMD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 0066, Houston, TX 77030. Phone: 713-792-4845; Fax: 713-794-5369; E-mail: [email protected]; and Kelly K. Hunt, [email protected] doi: 10.1158/0008-5472.CAN-13-0013 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(17) September 1, 2013 5556 on April 20, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst August 16, 2013; DOI: 10.1158/0008-5472.CAN-13-0013