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Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6 Atish D

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Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6 Atish D Published OnlineFirst November 29, 2016; DOI: 10.1158/0008-5472.CAN-16-0455 Cancer Tumor and Stem Cell Biology Research Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6 Atish D. Choudhury1,2,3, Anna C. Schinzel1,3, Maura B. Cotter1, Rosina T. Lis1,4, Katherine Labella1, Ying Jie Lock1, Francesca Izzo1,3, Isil Guney1,2, Michaela Bowden1, Yvonne Y. Li1, Jinal Patel3, Emily Hartman3, Steven A. Carr3, Monica Schenone3, Jacob D. Jaffe3, Philip W. Kantoff1,2, Peter S. Hammerman1,2,3, and William C. Hahn1,2,3 Abstract In prostate cancer, the development of castration resistance castration in a mouse xenograft model system. Tumors in is pivotal in progression to aggressive disease. However, which castration resistance was conferred by NEK6 were understanding of the pathways involved remains incomplete. predominantly squamous in histology with no evidence of In this study, we performed a high-throughput genetic screen AR signaling. Gene expression profiling suggested that NEK6 to identify kinases that enable tumor formation by androgen- overexpression stimulated cytoskeletal, differentiation, and dependent prostate epithelial (LHSR-AR) cells under andro- immune signaling pathways and maintained gene expression gen-deprived conditions. In addition to the identification of patterns normally decreased by castration. Phosphoproteome known mediators of castration resistance, which served to profiling revealed the transcription factor FOXJ2 as a novel validate the screen, we identified a mitotic-related serine/ NEK6 substrate, with FOXJ2 phosphorylation associated with threonine kinase, NEK6, as a mediator of androgen-indepen- increased expression of newly identified NEK6 transcriptional dent tumor growth. NEK6 was overexpressed in a subset targets. Overall, our studies establish NEK6 signaling as a of human prostate cancers. Silencing NEK6 in castration- central mechanism mediating castration-resistant prostate cancer. resistant cancer cells was sufficient to restore sensitivity to Cancer Res; 77(3); 1–13. Ó2016 AACR. Introduction the SRC inhibitor dasatinib (6). Cabozantinib, a small-molecule inhibitor of MET and VEGFR2, exhibited clinical activity in Prostate cancer is the second most common cause of cancer metastatic CRPC (7) but a phase III trial failed to demonstrate death in men, and the majority of these deaths occur in patients improvement in overall survival (8). with metastatic castration-resistant prostate cancer (CRPC). Stud- Tumor samples from patients with CRPC show increased levels ies of metastatic tissue from these patients reveal that tumors can of tyrosine phosphorylation as compared with treatment-na€ve become resistant either through persistent activation of the andro- prostate cancer (9), and the analysis of serine/threonine and gen receptor (AR) pathway (1) or by progression to a state tyrosine phosphorylation events in metastatic CRPC demonstrat- described as androgen pathway–independent prostate cancer ed a number of kinases that are differentially expressed or acti- (APIPC; ref. 2) with or without evidence for neuroendocrine vated in prostate cancer metastases (10). However, there is limited transdifferentiation. evidence for activating kinase point mutations in CRPC (11), Constitutive activation of kinases such as ERBB2, MAPK, PI3K/ suggesting that kinase pathways are activated by other (structural Akt, and Src has been implicated in mediating resistance to genetic, epigenetic, microenvironmental) mechanisms. Kinase hormonal therapies through both AR-dependent and AR-inde- signaling has previously been implicated in treatment resistance pendent mechanisms (3). However, inhibitors of many of these in other cancer types through reactivation of the targeted signaling kinases have failed to demonstrate significant clinical benefitin pathway, activation of parallel signaling pathways and through unselected patient populations, such as in trials of the ERBB2 inducing transdifferentiation phenotypes (12); kinases conferring inhibitor lapatinib (4), the MTOR inhibitor everolimus (5), and resistance are obvious druggable targets for potential therapeutic intervention. Because kinase signaling pathways whose activation 1Dana-Farber Cancer Institute, Boston, Massachusetts. 2Harvard Medical School, could lead to castration resistance in patients with prostate cancer Boston, Massachusetts. 3Broad Institute of Harvard and MIT, Cambridge, Mas- have not been comprehensively catalogued, we have performed sachusetts. 4Brigham and Women's Hospital, Boston, Massachusetts. an in vivo functional genomic screen to identify novel pathways fi Note: Supplementary data for this article are available at Cancer Research that may be involved and identi ed NEK6 as a kinase that Online (http://cancerres.aacrjournals.org/). mediates androgen-independent tumor growth. Corresponding Author: William C. Hahn, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Dana 1538, Boston, MA Materials and Methods 02215. Phone: 617-632-2641; Fax: 617-632-4005; E-mail: Cell lines and antibodies [email protected] LH, LHSR-AR, and LHMK-AR cells (13) were cultured in pros- doi: 10.1158/0008-5472.CAN-16-0455 tate epithelial basal medium (PrEBM) with prostate epithelial Ó2016 American Association for Cancer Research. growth medium (PrEGM) SingleQuot Kit Supplements & Growth www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 29, 2016; DOI: 10.1158/0008-5472.CAN-16-0455 Choudhury et al. Factors (Lonza). LNCaP, C4-2, CL-1, and DU145 were cultured neck; pellets were removed at the time of castration. For castration, in phenol red–free RPMI (Gibco) supplemented with 10% FBS, testes were ligated and removed through a lower abdominal 1 mmol/L sodium pyruvate, and 10 mmol/L HEPES. LAPC4 incision. Doxycycline diet at 625 ppm (Test Diet) was fed to mice cells were cultured in IMDM supplemented with 10% FBS and for target dose of 0.6 to 2 mg/kg. 1 nmol/L R1881. VCaP and PC-3 cells were cultured in DMEM: F12 (1:1) with L-glutamine (Hyclone) þ 10% FBS. RWPE-1 cells Tumor processing and gene expression analysis were cultured in keratinocyte-SFM þ EGF and bovine pituitary Tumors were harvested fresh and fragments were immediately extract (Gibco). fixed in 10% neutral-buffered formalin solution (Sigma) for Antibodies used for immunoblotting were: NEK6 (AbCam paraffin embedding or flash frozen in liquid nitrogen and stored ab76071 for Fig. 1C; Santa Cruz sc-134833 for Supplementary in À80C. Protein lysates were generated by mechanical disrup- Fig. S1A; AbCam ab133494 otherwise), AR (Santa Cruz, sc-7305), tion in RIPA buffer (Cell Signaling) and passaged through a Hsp90 (Cell Signaling 4875), b-actin (Santa Cruz sc-47778 HRP), QiaShredder column; RNA was isolated using the Qiagen RNeasy V5 (Invitrogen P/N 46-0708), FOXJ2 (LifeSpan LS-C31294), Purification Kit with passage through a QiaShredder column AKT1 (Cell Signaling 9272), RAF1 (BD Biosciences 610151), and on-column DNase digestion per manufacturer's protocol. p-T412-RPS6KB1 (Cell Signaling 9234), p-S727-STAT3 (GeneTex RNA-Seq, comparative marker selection using GENE-E software, 61050), cleaved PARP (Cell Signaling 9541), p53 (Cell Signaling and gene set enrichment analysis (GSEA) pre-ranked were per- 9282), and p21 (Santa Cruz sc-397). formed as described in the Supplementary Methods. Kinase library and androgen independence screen Immunohistochemistry LHSR-AR cells were infected with each virus from the previously Prostate cancer microarray slides immunostained for NEK6 described human kinase library (14) in 96-well format; cells from were scanned and scored by automated spectral imaging analysis 9 to 10 of these wells were pooled together to generate 61 total per the Supplementary Methods. Paraffin-embedded subcutane- pools. A total of 2 Â 106 cells from each pool in 200 ml 1:1 PBS: ous tumor sections were immunostained using anti-AR (Dako # Matrigel (Corning 354234) were implanted into each of 3 sub- M3562, dilution 1:200, 1-hour incubation, citrate and microwave cutaneous sites of a female BALB/C nude mouse (Charles River). retrieval, EnVision detection. BioGenex platform); anti–b-catenin DNA was isolated from tumors using the Qiagen Blood Spin Mini [Santa Cruz sc-1496, dilution 1:100, T3 H1(30), Leica platform], Kit. For tumor formation in male mice, mice were implanted with anti-KRT13 (GeneTex EPR3671), anti-NEK6 (as for TMAs but at testosterone pellet (Innovative Research NA-151) into the lateral 1:100 dilution). A B P = 0.034* 1,200 160 140 1,000 P = 0.001* a 120 ) ) Figure 1. 3 3 800 P = 0.005* 100 NEK6 confers androgen-independent tumor formation in a xenograft model 600 80 of androgen-dependent prostate cancer. A, Effects of NEK6 expression 60 on the rate of tumor formation at 60 400 Volume (mm Volume (mm 40 days in the indicated LHSR-AR cells in 200 female and castrated mice. Of note, if 20 the tumor denoted with "a" is excluded, the difference in rate of 0 0 tumor formation between parental LHSR-AR LHSR-AR +NEK6 +NEK6 LHMK-AR +NEK6 LHSR-AR cells and cells expressing female castrated female castrated female female NEK6 in castrated mice remains # Tumors 0/6 0/6 6/6 5/6 # Tumors 2/15 10/15 statistically significant (P ¼ 0.009). B, Effects of expressing NEK6 on the rate of tumor formation at 60 days for C LHSR-AR +NEK6 parental LHMK-AR cells and cells expressing NEK6 in female mice. 100 +dox –dox +dox –dox C, Left, Inducible expression of NEK6
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