Molecular and Cellular Pathobiology ITGBL1 Is a Runx2 Transcriptional Target and Promotes Breast Cancer Bone Metastasis by Activating the TGFb Signaling Pathway

Bone metastasis affects more than 70% of advanced breast cancer patients, but the molecular mechanisms of this process remain unclear. Here, we present clinical and experimental evidence to clarify the role of the integrin b-like 1 (ITGBL1) as a key contributor to bone metastasis of breast cancer. In an in vivo model system and in vitro experiments, ITGBL1 expression promoted formation of osteomimetic breast cancers, facilitating recruitment, residence, and growth of cancer cells in bone microenvironment along with osteoclast maturation there to form osteolytic lesions. Mechanistic investigations identified the TGFb signaling pathway as a downstream effector of ITGBL1 and the transcription factor Runx2 as an upstream activator of ITGBL1 expression. In support of these findings, we also found that ITGBL1 was an essential mediator of Runx2induced bone metastasis of breast cancer. Overall, our results illuminate how bone metastasis occurs in breast cancer, and they provide functional evidence for new candidate biomarkers and therapeutic targets to identify risk, to prevent, and to treat this dismal feature of advanced breast cancer. Cancer Res; 75(16); 3302–13. 2015 AACR. Introduction Breast cancer cells preferentially metastasize to the bone, leading to osteolytic lesions. To form visible metastatic bone lesions, breast cancer cells undergo processes, including recruitment to the bone, survival, and clonal expansion in the bone microenvironment, as well as osteoclast activation to resorb the bone matrix (1). Increasing evidence has demonstrated that the ectopic expression of bone-remodeling genes or gene signatures in primary breast cancer cells increases the risk of bone metastasis (2); however, the underlying molecular mechanisms remain largely unknown. Integrin beta-like 1 (ITGBL1), which was first cloned and characterized from an osteoblast cDNA library (3), encodes a ten integrin EGF-like repeat domain-containing protein (TIED). The ITGBL1 protein is highly homologous to the N-terminal EGF-like stalk fragment of integrin b (4), but contains neither a transmembrane domain nor an RGD (Arg–Gly–Asp)-binding domain, suggesting that ITGBL1 performs functions distinct from those of integrin. In our previous gene-expression profiling dataset of breast cancer tissues, ITGBL1 was coexpressed with genes encoding proteins involved in bone remodeling and bone metastasis (5). Garcia and colleagues (6) found that ITGBL1 is overexpressed in bone metastatic subclone cells compared with their parental cells and is included in the "osteoblast-like gene expression signature" and "bone metastatic gene signature." These evidences implied that ITGBL1 might contribute to the development of the osteoblast-like (osteomimetic) phenotype and bone metastasis of breast cancer cells. Currently, information regarding the role and molecular mechanism of ITGBL1 expressed by breast cancer cells in bone metastasis remains limited. Runx2 is a critical transcription factor for osteogenic lineage commitment andbone formation. It switches on the expressionof several bone matrix–remodeling genes by binding to the osteoblast-specific cis-acting element 2 (OSE2; refs. 7, 8). Runx2 and its target genes are highly expressed in breast cancer tissues and play pivotal roles in breast cancer bonemetastasis (9–12).On the basis of the evidence that ITGBL1 is coexpressed with RUNX2 in breast cancer cells (6) and the fact that there are OSE2 motifs in the ITGBL1 promoter, we hypothesized that ITGBL1 might be a transcriptional target of Runx2 and mediate Runx2-driven bone metastasis. In this study, we present both in vivo and in vitro evidence to clarify the roles and the underlying molecular mechanisms of ITGBL1 in breast cancer bone metastasis. Moreover, we identify ITGBL1 as a transcriptional target of Runx2, mediating the Runx2-driven bone metastatic potential of breast cancer cells. Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin, China. Key Laboratory of Breast Cancer Prevention andTreatment of theMinistryof Education,TianjinMedical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin, China. Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin, China. Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin, China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). CorrespondingAuthor:Yu-Mei Feng, Tianjin Medical University Cancer Institute and Hospital, Huan-Hu-Xi Road, He-Xi District, Tianjin 300060, China. Phone: 86-22-2334-0123, ext. 6002; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-15-024