Molecular and Cellular Pathobiology Tropomodulin 1 Expression Driven by NF-kB Enhances Breast Cancer Growth

Triple-negative breast cancers (TNBC),which include the basallike and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-kB activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-kB target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-kB–inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-kB and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of b-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-kB activation and MMP13 induction, which accounts in part for the NF-kB–dependent malignant phenotype of TNBC. Cancer Res; 75(1); 62–72. 2014 AACR. Introduction Gene-expression analyses have defined five breast cancer subtypes (luminal-like, ERBB2-enriched, basal-like, claudinlow, and normal breast-like), each of which is thought to be derived from a distinct differentiation stage of mammary epithelial cells, thereby displaying unique prognostic features (1–3). The luminal-like subtype is characterized as either an estrogen receptor–positive (ERþ) or a progesterone receptor– positive (PRþ) phenotype, whereas the basal-like and claudinlow subtypes constitute the majority of triple-negative cancers (TNBCs; ER , PR , and ERBB2 ), show a higher malignancy than other subtypes, and exhibit a poor prognosis against various methods of therapy. Triple-negative breast cancers (TNBC) were identified on the basis of low/absent expression of luminal differentiation markers, and enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Therefore, although luminal-like cells appear more differentiated and form tight cell–cell junctions, TNBCs appear less differentiated and have a more mesenchymal-like appearance. TNBCs are much more frequently highly invasive (4) and show higher proliferative activity as reflected in high Ki-67 expression when compared with luminal-like subtypes (5). Therefore, it is necessary to find molecular signatures and signaling pathways that contribute to the malignancy of TNBCs. The NF-kB family of transcriptional factors plays a critical role in inflammation, immunoregulation, and cell differentiation (6, 7). This family consists of five members, including p50, p52, RELA (p65), RELB, and c-REL, which form homomeric or heteromeric dimers to activate transcription of the target genes. NF-kB is made transcriptionally inactive by being sequestered in the cytoplasm when it forms complexes with the IkB family, including IkBa, IkBb, IkBe, and the p105 and p100 precursors of p50 and p52, respectively. Nuclear translocation ofNF-kB can be driven by two distinct signaling pathways. In the canonical pathway, a large number of stimuli, including various cytokines and bacterial and viral products, induce IkB kinase (IKK) b-catalyzed phosphorylation and proteasomal degradation of IkBa, followed by nuclear translocation of mainly p50-RELA heterodimers (8). The noncanonical pathway is activated by receptors that are crucial in the formation of lymphoid organs and lymphocyte development, such as the lymphotoxin b receptor, the receptor activator of NF-kB (RANK), and CD40. This pathway induces the IKKacatalyzed phosphorylation of the C-terminal half of p100 that sequesters RelB in the cytoplasm, which leads to polyubiquitination-dependent processing of p100 to p52 and the nuclear translocation of the p52-RELB heterodimers (9). Accumulating evidence indicates that aberrant NF-kB activation leads to tumorigenesis and cancer malignancy through the expression of genes involved in survival, metastasis, and angiogenesis (6, 10). We have previously reported that TNBCs undergo constitutive and strong activation of NF-kB, whose activation is transient in normal cells upon various physiologic stimuli (11, 12). Furthermore, the adenovirus-mediated expression of a nondegradable IkBa super-repressor (IkBaSR), in which Ser-32 and Ser-36 (the residues phosphorylated by IKKb) were substituted with alanine, blocked NF-kB activation, and thereby inhibited the growth of Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan. Division of CancerCell Research, InstituteofMedical Science,Universityof Tokyo, Tokyo, Japan. Department of Life Science and Medical Bio-science, Waseda University,Tokyo, Japan. Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan. Graduate School of Medicine, Kochi University, Kochi, Japan. Corresponding Author: Jun-ichiro Inoue, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 813-5449-5275; Fax: 813-5449-5421; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-3455 2014 American Association for Cancer Research. Cancer Research Cancer Res; 75(1) January 1, 2015 62 on July 18, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 14, 2014; DOI: 10.1158/0008-5472.CAN-13-3455