American Association for Cancer Research
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Supplementary Data from Numb−/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling

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posted on 2023-03-31, 20:31 authored by Yanjing Guo, Kai Zhang, Chaping Cheng, Zhongzhong Ji, Xue Wang, Minglei Wang, Mingliang Chu, Dean G. Tang, Helen He Zhu, Wei-Qiang Gao

Supplementary Mehods, Materials, figures and tables Figure S1. Immunofluorescent staining of Numb in PCa tissues. Figure S2. Classical Wnt signaling was not affected by Numb in PCa cells. Figure S3. The mRNA levels of Hes1 and GLI1 negatively correlated with Numb in PCa tissues. Figure S4. Identification of Numb proximal promoter. Figure S5. Notch signaling exerted no significant effect on the transcription of Numb gene. Figure S6. Effects of the treatment of DAPT, cyclopamine, or a combination of DAPT and cyclopamine on the GFP status of Numb-promoter-GFP reporter lentivirus transfected C4-2B cells. Figure S7. Treatment with a combination of DAPT and cyclopamine resulted in a shift of Numb-/low to Numbhigh cells. Figure S8. The percentage of cells with nuclear localized AR protein was significantly lower in Numb-/low PCa cells than Numbhigh cells. Figure S9. Blockage of Notch and Hedgehog signaling by DAPT and cyclopamine significantly enhanced the apoptosis of Numb-/low cells induced by flutamide-mediated androgen deprivation. Supplementary Table 1. Detailed clinical information of human prostate cancer patients in this study Supplementary Table 2. Primer sequences for qRT-PCR Supplementary Table 3. Antibodies used in Apoptosis Assay, Immunoblotting and Immunofluorescence experiments. Supplementary Table 4. shPTCH1 and shNumb sequences. Supplementary Table 5. Information of plasmids components of RBP-Jκ, GLI and TCF/LEF1 luciferase reporter Supplementary Table 6. Information of response elements of RBP-Jκ, GLI and TCF/LEF1 luciferase reporter

Funding

Chinese Ministry of Science and Technology

National Natural Science Foundation of China

Science and Technology Commission of Shanghai Municipality

NSFC

Young Eastern Scholar

Shanghai Rising-Star Program

School of Medicine, Shanghai Jiao Tong University

Ren Ji Hospital

History

ARTICLE ABSTRACT

Purpose: To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb−/low prostate cancer cells in castration resistance.Experimental Design: The expression of Numb was assessed using multiple Oncomine datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated in vitro and in vivo. In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter–based lentiviral reporter system. The difference between Numb−/low and Numbhigh prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR.Results: We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a Numb promoter–based lentiviral reporter system, we were able to distinguish Numb−/low prostate cancer cells from Numbhigh cells. Numb−/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell–associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb−/low cells in response to ADT.Conclusions: Numb−/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. Clin Cancer Res; 23(21); 6744–56. ©2017 AACR.

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