br Experimental Procedures br Author Contributions
Acknowledgments We thank Drs. Shinichi Aizawa and Frank Costantini for providing mice, and Yuuki Honda, Mayumi Yamamoto, Nanako Goto, Chiaki Kaieda, Masato Tanaka, and Kanako Motomura for excellent technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Society for the Promotion of Science (grant numbers 23112002, 25713014, and 16H01850), Health Labor Sciences Research Grants in Japan, and the Core Research for Evolutional Science and Technology Program of the Japan Agency for Medical Research and Development (AMED-CREST).
Introduction Breast cancer subtypes with particular molecular signatures, e.g., HER2+ and basal/triple-negative subtypes, have a worse prognosis with increased rates of recurrence and metastasis, likely due to an expansion of cancer stem d-xylose (CSCs), alternatively referred to as tumor-initiating cells (TICs) (Blick et al., 2010; Park et al., 2010; Ricardo et al., 2011). Breast CSCs are characterized by the markers CD44+/hi/CD24−/low (Al-Hajj et al., 2003; Blick et al., 2010; Ricardo et al., 2011) and by expression of genes that promote epithelial-mesenchymal transition (EMT) (Blick et al., 2010; Mani et al., 2008), which is critical for cancer progression and metastasis (Choi et al., 2013; Sarrio et al., 2008; Sheridan et al., 2006; Thiery, 2002; Tsai and Yang, 2013). Aggressive cancers of other tissues of origin such as thyroid, colorectum, pancreas, and skin also demonstrate expansion of the CD44+/hi CSC population (Dou et al., 2007; Erfani et al., 2016; Jing et al., 2015; Liu and Brown, 2010; Parmiani, 2016). In contrast to the majority of cells in a tumor, CSCs/TICs have the ability to form tumor xenografts (Al-Hajj et al., 2003; Iqbal et al., 2013). Moreover, CSCs are relatively chemoresistant and become enriched after chemotherapy, leading to the theory that CSCs drive cancer recurrence and metastasis (Alamgeer et al., 2014; Iqbal et al., 2013; Lawson et al., 2015; Lee et al., 2011). Improvements in cancer therapy to achieve durable cancer remission or cure will require novel therapies that are cytotoxic to CSCs (Das et al., 2008). There is growing interest in the role of sumoylation in regulating pathways critical to oncogenesis, cancer growth, and progression (Bettermann et al., 2012). Sumoylation is a process resulting in the reversible binding of a small ubiquitin-like modifier (SUMO) to a lysine residue in the target protein (Geiss-Friedlander and Melchior, 2007). Sumoylation is mediated through a cascade involving an activating enzyme (i.e., SAE1/2), E2-conjugating enzyme (i.e., UBC9), and E3 ligase (i.e., PIAS family) (Bettermann et al., 2012; Hay, 2005). Experimental methods to inhibit the SUMO pathway have relied on elimination of enzymes in the SUMO pathway or use of compounds that inhibit sumoylation enzymes, such as anacardic acid (Fukuda et al., 2009). Sumoylation has profound effects on gene expression, which likely involves post-translational modification of transcription factors by SUMO conjugation (Gill, 2005). EMT, and its converse, mesenchymal-epithelial transition, are regulated by transcription factors, many of whose activity is in turn regulated by SUMO conjugation (Bogachek et al., 2015a). We recently reported that sumoylation of transcription factor activator protein 2α (TFAP2A) in basal breast cancer alters its transcriptional activity and that SUMO-unconjugated TFAP2A acquires activity that results in a profound alteration of the expression profile away from the CSC/EMT phenotype and toward that of the well-differentiated phenotype, clearing of the CD44+/hi/CD24−/low CSC population, and repressing the TIC potential (Bogachek et al., 2014). Treatment of mice with anacardic acid inhibited the outgrowth of basal breast cancer xenografts, demonstrating the proof of principle that small-molecule SUMO inhibitors might form the basis of CSC-specific therapy (Bogachek et al., 2014, 2015b). Another recent study reported that knockdown of the SUMO enzyme PIAS1 repressed the TIC breast cancer population through epigenetic chromatin alterations resulting in gene silencing of cyclin D2, estrogen receptor, and WNT5A (Liu et al., 2014). Further studies have reported that knockdown of sumoylation enzymes impaired the outgrowth of colorectal cancer xenografts (He et al., 2015), suggesting the broad application of SUMO inhibitors in cancer therapy.