Published OnlineFirst July 27, 2017; DOI: 10.1158/1078-0432.CCR-17-0913 Biology of Human Tumors Clinical Cancer Research NumbÀ/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling Yanjing Guo1, Kai Zhang2, Chaping Cheng2, Zhongzhong Ji2, Xue Wang2, Minglei Wang1, Mingliang Chu1, Dean G. Tang3, Helen He Zhu1, and Wei-Qiang Gao1,2 Abstract Purpose: To elucidate the role and molecular mechanism of Results: We show here that Numb was downregulated and Numb in prostate cancer and the functional contribution of negatively correlated with prostate cancer advancement. Func- À Numb /low prostate cancer cells in castration resistance. tionally, Numb played an inhibitory role in xenograft prostate Experimental Design: The expression of Numb was assessed tumor growth and castration-resistant prostate cancer develop- using multiple Oncomine datasets and prostate cancer tissues ment by suppressing Notch and Hedgehog signaling. Using from both humans and mice. The biological effects of the a Numb promoter–based lentiviral reporter system, we were able À overexpression and knockdown of Numb in human prostate to distinguish Numb /low prostate cancer cells from Numbhigh À cancer cell lines were investigated in vitro and in vivo.In cells. Numb /low prostate cancer cells were smaller and quiescent, addition, we developed a reliable approach to distinguish preferentially expressed Notch and Hedgehog downstream and between prostate cancer cell populations with a high or low stem-cell–associated genes, and associated with a greater resis- endogenous expression of Numb protein using a Numb pro- tance to ADT. The inhibition of the Notch and Hedgehog signal- À moter–based lentiviral reporter system. The difference between ing pathways significantly increased apoptosis in Numb /low À Numb /low and Numbhigh prostate cancer cells in the response cells in response to ADT. À to androgen-deprivation therapy (ADT) was then tested. Conclusions: Numb /low enriches a castration-resistant The likely downstream factors of Numb were analyzed using prostate cancer cell subpopulation that is associated with luciferase reporter assays, immunoblotting, and quantitative unregulated Notch and Hedgehog signaling. Clin Cancer Res; real-time PCR. 1–13. Ó2017 AACR. Introduction standing of the molecular etiology of castration resistance remains limited, leading to limited intervention approaches The most recent statistics suggest that aging and a Western- for the treatment of this deadly disease. In addition, prostate ized lifestyle are associated with an increasing trend of prostate cancer cells display great intratumor heterogeneity. Their cancer incidence and mortality rates worldwide (1, 2). Andro- response to androgen deprivation therapy is also radically gen deprivation therapy in conjunction with surgery or radia- different from each other (4–6). The targeted eradication of tion is the mainstream treatment strategy for advanced prostate cell subpopulations with greater castration resistance may serve cancers (3). However, the emergence of castration-resistant as an optimal strategy for the treatment of CRPC. The identi- prostate cancer (CRPC) remains the predominant cause of fication of those cell subpopulations and the understanding death in prostate cancer patients. Unfortunately, our under- of their molecular characteristics will be a first step achieving this goal. fi Drosophila 1State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Numb was originally identi ed and investigated in . Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao The Numb protein has been found to be distributed asym- Tong University, Shanghai, China. 2School of Biomedical Engineering and Med-X metrically during divisions of sensory organ precursors (SOP) Research Institute, Shanghai Jiao Tong University, Shanghai, China. 3Depart- in Drosophila embryos, resulting in different cell fates of ment of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Carlton daughter cells (7, 8). Upon the first division of SOP, Numb and Elm Streets, Buffalo, New York. is selectively distributed into the anterior daughter cell, which Note: Supplementary data for this article are available at Clinical Cancer differentiates into neurons or glia, whereas the posterior Research Online (http://clincancerres.aacrjournals.org/). daughter cell without inheritance of Numb differentiates into Corresponding Authors: Wei-Qiang Gao, Shanghai Jiao Tong University, 160 outer support cells (9–11). The critical importance of Numb Pujian Road, Shanghai, 200127, China. Phone: 86-158-2112-6637; Fax: 86-21- during progenitor cell differentiation and cell fate determina- 6838-3916; E-mail: [email protected]; and Helen He Zhu, tion in hematopoiesis, neurogenesis, cardiac morphogenesis, [email protected] and myogenesis in vertebrates was demonstrated subsequently doi: 10.1158/1078-0432.CCR-17-0913 (12–15). Recently, a growing body of evidence has suggested Ó2017 American Association for Cancer Research. that Numb may act as a tumor suppressor in various tumor www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 27, 2017; DOI: 10.1158/1078-0432.CCR-17-0913 Guo et al. Plasmids Translational Relevance The Penco Numb-DsRed-DR retroviral reporter was gener- Castration-resistant prostate cancer (CRPC) remains one of ously donated by Dr. Dean G. Tang (25). The doxycycline the most deadly and incurable cancer types worldwide. Tumor (dox)-inducible lentiviral plasmid Notch-ICD was a gift from cells in samples from CRPC patients display tremendous Rudolf Jaenisch (Addgene plasmid # 61540; ref. 26). We used heterogeneity. Identification of a prostate cancer cell subpop- shRNAtoknockdowntheexpressionofNumbandPTCH1. ulation with greater castration resistance is a key to the devel- The shRNAs were cloned into lentiviral GV298 vector with opment of targeted anti-CRPC treatment strategies. Our pres- IRES-Cherry and puromycin-selective markers (Shanghai ent study reveals that a prostate cancer cell subpopulation GENECHEM Co., Ltd). Detailed information regarding the with low expression of Numb, which is an evolutionarily sequences of shNumb and shPTCH1 is provided in Supple- conserved cell fate determinant, displays greater resistance to mentary Table S4. The fluorescent Numb promoter lentiviral androgen deprivation. Numb is downregulated in prostate reporter was generated by replacing the pCMV gene in the cancer and is negatively associated with the progression of PLVX-AcGFP1-N1 vector (Clontech, Catalog Nos.632154) with prostate cancer. Our in vitro and in vivo studies demonstrate the Numb-2K promoter sequence (from-2913 to þ84). The that Numb plays a suppressive role in prostate cancer and truncated Numb promoter (1K-4K) sequences were amplified castration resistance by inhibiting Notch and Hedgehog sig- andclonedintoapGL3basicluciferasevector(Promega naling. Inhibitors that targeting Notch and Hedgehog signal- #E1751) using standard recombinant DNA methods. The À ing can effectively deplete Numb /low CRPC cells. Collectively, RBP-Jk, GLI and TCF/LEF1 luciferase reporter lentiviruses were these findings shed new light on the development of effective purchased from Shanghai Genomeditech Co., Ltd. Detailed anti-CRPC treatment strategies. information regarding the plasmid components and response elements of the RBPJk, GLI and TCF/LEF1 luciferase reporter is provided in Supplementary Tables S5 and S6. types, including hepatocellular carcinoma, malignant pleural Luciferase reporter assay Numb mesothelioma, and breast cancer (16–18). The downregula- promoter (1K-4K) luciferase reporters were co-trans- tion of Numb is associated with a poor prognosis in hepato- fected into cells with an internal control plasmid expressing fi fl cellular carcinoma (19), salivary gland carcinoma (20), and Renilla luciferase respectively. The re y luciferase and Renilla esophageal squamous cell carcinoma (21). However, the luciferase activities were measured using a luminometer with the expression of Numb is greatly heterogeneous among different Dual-luciferase Reporter Assay System (Promega, E1910). The fi fl fi cellswithinatumormass(22–24). The molecular mechanism re y luciferase activity was quanti ed and normalized to the underlying the downregulation of Numb and the functional Renilla activity. difference between Numb highly expressed and underex- In vivo pressed cancer cells is not well elucidated. xenograft assay m In this study, we aim to elucidate the role of Numb in CRPC The cells were harvested and suspended in 50 L serum-free m development and decipher the transcriptional regulation of the medium and mixed with 50 L Matrigel (BD Biosciences). Then, fl Numb gene in prostate cancer. On the basis a promoter analysis of the mixture was injected subcutaneously into the anks of the Numb gene, we designed a fluorescent protein reporter lenti- 4-week-old BALB/c nude mice (SLAC). The tumor growth was viral system to distinguish between prostate cancer cells with a monitored and recorded weekly after the inoculation. The À high or low Numb expression. We find that Numb /low enriches a volume of the tumor was calculated using the following formula: Â Â 2 fi small subpopulation of smaller and quiescent cells that prefer- 0.5 tumor length tumor width . The mice were sacri ced entially express Notch and Hedgehog downstream and stem-cell–