Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0051 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Single Cell Transcriptomics Analysis Identifies Nuclear Protein 1 as a Regulator of Docetaxel Resistance in Prostate Cancer Cells Patricia M. Schnepp1#, Greg Shelley1#, Jinlu Dai1, Nicole Wakim2, Hui Jiang2, Atsushi Mizokami3 and Evan T. Keller1,4, * 1 Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan, 48109, USA 2 Department of Biostatics, University of Michigan, Ann Arbor, Michigan 48109, USA 3 Department of Urology, Kanazawa University, Kanazawa, Japan 4 Biointerfaces Institute, University of Michigan Medical School, Ann Arbor, Michigan, 48109, USA # Co-First Authors Running Title: NUPR1 promotes Docetaxel resistance Keywords: single cell sequencing, prostate cancer, NUPR1 Financial support: This work was supported by the National Cancer Institute of the National Institutes of Health under Award Numbers P01CA093900 to E.T.K and P30CA046592 by use of the Cancer Center Shared Resource: Single Cell Analysis Core. P.M.S. was supported by NCATS grant UL1TR002240. N.W. was supported by NIH NHGRI Genome Science Training Grant T32HG00040. H.J. was partly supported by NIH NCI grant P30CA046592. Corresponding author contact info: Evan T. Keller NCRC B14 RM 116 2800 Plymouth Road Ann Arbor, MI 48105 Tel: 734-615-0280 Email: [email protected] Conflict of interest: The authors have no conflicts of interest to declare. Word Counts: 4910 Total number of figures and tables: 5 1 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0051 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract The majority of prostate cancer (PCa) patients treated with docetaxel develop resistance to it. In order to better understand the mechanism behind the acquisition of resistance, we conducted single cell RNA sequencing (scRNA-seq) of docetaxel sensitive and resistant variants of DU145 and PC3 PCa cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both PCa cell lines. A subset of these genes gave a gene expression profile in the resistant-transcriptome-like sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified potential 321 regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the PCa cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of PCa drug resistance, which provides the rationale to explore NUPR1 and its target genes to for reversal of docetaxel resistance. Implications Using single cell sequencing of PCa, we show that NUPR1 plays a role in docetaxel resistance. 2 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0051 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer- related death in men (1). Advanced disease is typically treated with androgen deprivation therapy (ADT); however, patients develop resistance to ADT in what is termed castration-resistant PCa (CRPC) (2). In 2004, docetaxel was approved for the treatment of CRPC (3,4). However, 50% of patients do not respond to docetaxel therapy and the majority of those that do respond relapse within three years of initiating therapy (3). Thus, understanding mechanisms through which docetaxel resistance develops is critical to improving therapy of CRPC. Docetaxel has been shown to have two main mechanisms of anti-tumor activity; namely, microtubule depolymerization and inhibition of Bcl-2 expression (5,6). However, multiple mechanisms of resistance have previously been identified. Structural changes to β-tubulin block docetaxel from affecting microtubules (7,8). PCa cells can also up-regulate Bcl-2 in order to overcome docetaxel-induced apoptosis (9). However, these previous studies of docetaxel resistance were performed on bulk populations of PCa cells. It is unknown if the resistant cell population develops from a subset of resistant cells already present in the parental population or develop de novo subsequent to therapy. In order to study the process of acquisition of resistant characteristics, different methods of investigation are necessary. Advances in next generation sequencing have allowed for the investigation at the single cell level. Single cell RNA sequencing (scRNA-seq) allows researchers to investigate the variability and complex gene expression across all the individual cells instead of a more homogeneous expression profile from traditional bulk RNA sequencing of tissues. In PCa, CTCs have been shown to have heterogeneous androgen resistance gene profiles within individual patients (10). This single cell heterogeneity may play a role in the clinical and therapeutic heterogeneous response (11). Additionally, with scRNA-seq it is possible to trace the origin of cancer cells to the cell type of origin (12). Similarly, scRNA-seq of docetaxel sensitive versus resistant cells may uncover mechanisms leading toward development of docetaxel resistance in PCa. 3 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0051 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. In this study, we conducted scRNA-seq of docetaxel sensitive and resistant variants of the DU145 and PC3 PCa lines. We analyzed the heterogeneity within and between the cell lines. We identified similar gene expression changes and functional gene pathways across both cell lines that are important to the acquisition of resistance. We also identified potential regulators of the resistant gene expression profile shared between both cell lines and identified a specific mediator of chemoresistance. Our findings uncover the heterogeneity in PCa as well as identifying signaling pathways important for the acquisition of docetaxel resistance. Materials and Methods Cell lines and Reagents Parental (sensitive) DU145 and PC3 PCa cells were obtained from American Type Culture Collection (Manassas, VA). Docetaxel resistant variants of the DU145 and PC3 PCa cells have been previously described (13). Cell identification is confirmed every 6 months using short tandem repeat analysis. Cells are tested every three months for Mycoplasma. Cells were used within 3 passages from the time of thawing. Cells were cultured in RPMI 1640 (Invitrogen Co., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Inc.). Resistant DU145 and PC3 cells were maintained with the addition of docetaxel in DMSO to a final concentration of10 nM while sensitive DU145 and PC3 cells were maintained with the addition of DMSO to a final level of 0.1% Gene Expression Analysis For one week, cells were transferred to docetaxel free media. Cells were trypsinized in 0.05% Trypsin EDTA for 5-10 minutes at 37°C and washed with media. The cell suspension was loaded into in the Fluidigm C1TM machine and processed into single cell cDNA libraries according to manufacturer protocol (PN 101-4981). Briefly, full length mRNA-seq libraries were generated from single cells captured using the Fluidigm C1TM Single Cell mRNA Seq IFC, 10-17µm (PN 100-5760) and Fluidigm C1TM Single-Cell Reagent Kit for mRNA Seq (PN 100-6201). Each chip was visually inspected to identify which wells contained cells. Wells containing one cell were included in library preparation ad sequencing. The 4 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0051 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. capture rate was between 78% - 96% across all chips used in this study. Full length cDNA was converted into sequence ready libraries using SMART-seq v4 Ultra Low Input RNA kit for Fluidigm C1TM System (Takara Bio, Cat 635025) and SeqAmpTM DNA Polymerase (Takara Bio, Cat 638504). Library preparation was completed using Nextera XT DNA library prep kit (Illumina, Cat. FC-131-1096) and Nextera XT DNA Library Prep Index Kit (Illumina, Cat FC-131-1002). Samples following PCR reactions as called for in each kit’s manufacturer’s protocol was purified using Agencourt AMPure XP (Beckman