In patients with breast cancer

In patients with breast cancer, ZRANB1 gene amplification was found in a subset of cases, and ZRANB1 protein levels correlated with EZH2 protein levels and poor survival (both TNBC and non-TNBC). Moreover, unlike normal mammary epithelial cells, all human breast cancer cell lines examined (both TNBC and non-TNBC) showed downregulation of EZH2 protein upon ZRANB1 knockdown, suggesting that ZRANB1 is a general regulator of EZH2 in breast cancer cells. On the other hand, however, ZRANB1 may have higher functional importance and better therapeutic potential in TNBC than in non-TNBC, given that knockdown of ZRANB1 led to severe growth defect in all 11 TNBC cell lines, whereas ER+ or HER2+ breast cancer cell lines showed variable responses (either responsive or resistant) to ZRANB1 depletion. This suggests that some of the non-TNBC Doxorubicin are not dependent on ZRANB1 and EZH2 for their proliferation. In the present study, a small-molecule ZRANB1 inhibitor, NSC112200, destabilized EZH2 through ubiquitination and the proteasome, and it inhibited cell viability through, at least in part, ZRANB1. In contrast, it had no effect on EZH2 protein level and little effect on viability in normal mammary epithelial cells with no detectable ZRANB1 expression. It should be noted that NSC112200 is not water soluble (dissolved in DMSO or corn oil) and that it inhibits ZRANB1’s DUB activity, destabilizes EZH2, and kills TNBC cells at micromolar, but not nanomolar, concentrations. Moreover, some of the NSC112200-treated C57BL/6 mice exhibited acute responses (low body temperature, less active) in preliminary animal testing (data not shown), indicating some toxicity. Thus, NSC112200 can serve as a tool compound but may not behave in a drug-like manner in vivo. Nevertheless, here we provide a proof of principle that the EZH2 deubiquitinase identified in this study is amenable to inhibition by small molecules. This represents a starting point to target ZRANB1. Considering that NSC112200 was from in silico screening, further development of a clinical ZRANB1 inhibitor may involve lead compound identification via high-throughput chemical screening and the use of the structure-activity relationship (SAR) analysis to optimize the activity, selectivity, pharmacokinetics, safety profile, and physical properties of lead compounds, which will enable full evaluation in animal models.
Experimental Procedures Further details and an outline of resources used in this work can be found in the Supplemental Experimental Procedures.
Acknowledgments We thank Xiang Zhang for providing luciferase-expressing LM2 cells, Dihua Yu for providing SKBR3 cells, Jae-Il Park for providing EZH2 constructs, Qi Cao for thoughtful suggestions, and Hyemin Lee for assistance with flow cytometric analysis. We also thank MD Anderson’s shRNA and ORFeome Core, Small Animal Imaging Facility, Characterized Cell Line Core Facility, and Pharmaceutical Chemistry Facility for technical assistance. L.M. is supported by NIH grants R01CA166051 and R01CA181029, a Cancer Prevention and Research Institute of Texas (CPRIT) grant RP150319, and a Stand Up To Cancer Innovative Research Grant (403235). Y.H. is supported by a CPRIT grant RR140053, an Innovation Award from the American Heart Association (16IRG27250155), a John S. Dunn Foundation Collaborative Research Award, and the Texas A&M University Start-up Funds. X.Y. was supported by NIH grants GM086937 and GM100777. M.J.Y. was supported in part by NIH R01CA164346 and R01CA200703 and CPRIT RP140402. Z.X. is supported by a Rosalie B. Hite Graduate Fellowship. P.Z. is a scholar in the National 1000 Young Talents Program of China.
Introduction Lymph nodes of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) show diffuse architectural effacement by a proliferation of small neoplastic lymphocytes with variably prominent scattered paler proliferation centers (PCs). The PCs are composed of a mixture of small lymphocytes, prolymphocytes and paraimmunoblasts [1, 2, 3].