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  • br Results br Discussion The data presented herein substanti


    Discussion The data presented herein substantially expand the potential of developing CSC-targeted therapy based on inhibition of the SUMO pathway. We show that inhibition of sumoylation by knockdown of UBC9 and PIAS1 effectively repressed expression of MMP14 and CD44, reduced invasiveness, and substantially inhibited tumorigenesis in a basal breast cancer model. Similar effects were demonstrated with a variety of small molecules that inhibit different steps in the SUMO pathway. Serial propagation of tumor xenografts as secondary xenografts has been used to identify the CSC/TIC population (Patel et al., 2012), and our finding that small tumors that developed in animals treated with SUMO inhibitor could not be serially transplanted as secondary xenografts is further evidence that SUMO inhibitors functionally eliminated the CSC/TIC population. Interestingly, the data also suggest that SUMO inhibition induces lasting effects on the CSC population that are maintained after stopping the drug. Parallel experiments in a colorectal cancer cell line model and primary colon cancer isolates demonstrated that identical SUMO-sensitive pathways of gene regulation and physiologic response of tumor growth were present and functional. Our findings are in agreement with other studies showing that knockdown of the SUMO pathway enzymes UBC9 and SAE2 in colon cancer muscarinic agonist or PIAS1 in basal breast cancer reduced growth and inhibited tumorigenesis of xenografts (He et al., 2015; Liu et al., 2014). Our data support a TFAP2A-dependent transcriptional mechanism that is functional in both basal breast and colorectal carcinomas. Previous findings indicated that AP-2 transcription factors regulate the process of EMT and that many transcription factors which induce EMT are regulated by post-translational sumoylation (Bogachek et al., 2015a). In both basal breast cancer (Bogachek et al., 2014) and colorectal cancer (Figure 7), knockdown of TFAP2A abrogated the effects of SUMO inhibition on repression of CD44. Furthermore, the non-sumoylatable K10R TFAP2A mutant was able to repress expression of CD44 and MMP14, whereas overexpression of wild-type TFAP2A failed to repress expression of these genes. The findings lead to the most likely conclusion that SUMO-unconjugated TFAP2A represses CD44 and MMP14 transcription. However, there are other possible mechanisms to account for the findings. First, it should be noted that SUMO inhibition has the potential to reduce the overall expression of TFAP2A (e.g., Figure 7A). Previous studies have shown that knockdown of TFAP2C increases TFAP2A expression, leading to the conclusion that TFAP2C represses TFAP2A expression. Since SUMO-unconjugated TFAP2A acquires transcriptional activity that mimics TFAP2C, it is likely that SUMO-unconjugated TFAP2A autoregulates its own level of expression. This mechanism would account for the reduction of overall TFAP2A expression found with SUMO inhibition. Additionally there may be other transcription factors, some of which may be SUMO sensitive, that are involved in the physiologic findings related to maintenance of the CSC/TIC population. The current findings indicate that additional efforts are needed to elucidate transcriptional mechanisms that maintain the CSC phenotype. Compelling data exist that CD44 is not merely a marker but that CD44 expression drives the cancer phenotype, inducing a propensity for expanded growth, invasion, and metastasis (Godar et al., 2008; Hiraga et al., 2013). Several approaches have been used to directly target CD44 as a means of inhibiting the CSC population. Anti-CD44 antibodies have been utilized as one means of specifically targeting the CSC population (Arabi et al., 2015; Liu and Jiang, 2006; Molejon et al., 2015). Other approaches to target the CSC population via CD44 have been based on altering transcriptional pathways regulating CD44 expression including Wnt (Yun et al., 2016), FOXP3 (Zhang et al., 2015), SMURF1 (Khammanivong et al., 2014), and Bmi-1 (Yu et al., 2014). Our current findings indicate that SUMO inhibitors can repress expression of CD44 in the CSC population through a TFAP2A-dependent mechanism. In some instances, the effect of repression at the RNA level exceeded the reduction in CD44 protein expression. Although repression of CD44 expression is likely mediated through reduced transcription, activation of cryptic transcriptional initiation and altered RNA splicing may account for differences between the degree of repression noted when comparing RNA and protein reduction. Further work is needed to clarify the mechanism of CD44 repression by TFAP2A, which may repress CD44 transcription directly or could affect CD44 expression through secondary mechanisms.