Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth Huy Q
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Published OnlineFirst November 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0224 Cancer Molecular and Cellular Pathobiology Research Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth Huy Q. Ta1, Melissa L. Ivey1, Henry F. Frierson Jr2,3, Mark R. Conaway3,4, Jaroslaw Dziegielewski3,5, James M. Larner3,5, and Daniel Gioeli1,3 Abstract Prostate cancer is the second leading cause of cancer death in that CHK2 affects prostate cancer proliferation, partly, through American men, and curing metastatic disease remains a significant the AR. Remarkably, we further show that CHK2 is a novel AR- challenge. Nearly all patients with disseminated prostate cancer repressed gene, suggestive of a negative feedback loop between initially respond to androgen deprivation therapy (ADT), but CHK2 and AR. In addition, we provide evidence that CHK2 virtually all patients will relapse and develop incurable castration- physically associates with the AR and that cell-cycle inhibition resistant prostate cancer (CRPC). A high-throughput RNAi screen increased this association. Finally, IHC analysis of CHK2 in to identify signaling pathways regulating prostate cancer cell prostate cancer patient samples demonstrated a decrease in growth led to our discovery that checkpoint kinase 2 (CHK2) CHK2 expression in high-grade tumors. In conclusion, we pro- knockdown dramatically increased prostate cancer growth and pose that CHK2 is a negative regulator of androgen sensitivity and hypersensitized cells to low androgen levels. Mechanistic inves- prostate cancer growth, and that CHK2 signaling is lost during tigations revealed that the effects of CHK2 were dependent on the prostate cancer progression to castration resistance. Thus, perturb- downstream signaling proteins CDC25C and CDK1. Moreover, ing CHK2 signaling may offer a new therapeutic approach CHK2 depletion increased androgen receptor (AR) transcriptional for sensitizing CRPC to ADT and radiation. Cancer Res; 75(23); 1– activity on androgen-regulated genes, substantiating the finding 13. Ó2015 AACR. Introduction growth will lead to new therapies or the improvement of current treatments for the management of CRPC. Prostate cancer is the most common noncutaneous malignancy Current research has demonstrated that androgen receptor in American men. With approximately 30,000 deaths yearly, (AR) signaling continues to play a crucial role in CRPC progres- prostate cancer is the second leading cause of cancer mortality, sion. AR overexpression is sufficient to drive androgen-dependent following lung carcinoma. The standard treatment for advanced prostate cancer cells to castration resistance and facilitate prolif- prostate cancer is radiation and androgen deprivation therapy eration in low androgen levels (1). In addition to its cognate (ADT). Despite the high response rate to ADT, the outgrowth of steroid hormone, AR can be regulated by interactions with a castration-resistant cells will invariably occur with progression to constellation of coregulatory and signaling molecules (2). Several a lethal disease and a relative 5-year survival rate of 28% (www. mechanisms, including extensive networks between androgen cancer.org). Even with the recent advances in ADT that have led to and peptide growth factor signaling pathways, multiple genetic improvements in overall survival, castration-resistant prostate mutations, and the genetic plasticity of cancer, contribute to the cancer (CRPC) is still incurable. Therefore, insights into the inherent and acquired resistance to ADT (3). Previous studies have mechanisms by which androgens stimulate prostate cancer shown that signal transduction pathways can stimulate AR acti- vation. These studies illustrate that modulating signaling path- ways in prostate cancer models regulates castration-resistant 1Departments of Microbiology, Immunology, and Cancer Biology, Uni- growth, AR transcription, AR protein stability, AR DNA binding, 2 versity of Virginia, Charlottesville,Virginia. Department of Pathology, and AR phosphorylation (4–6). Together, these reports suggest University of Virginia Health System, Charlottesville, Virginia. 3Cancer Center Member, University of Virginia, Charlottesville, Virginia. that the ability of signaling cascades to influence AR function may 4Department of Public Health Sciences, University of Virginia, Char- have a significant role in CRPC progression, and that CRPC may 5 lottesville,Virginia. Department of Radiation Oncology, University of not be effectively treated by ligand-directed therapy alone. Virginia, Charlottesville, Virginia. Comparative genomic hybridization studies of cancer have Note: Supplementary data for this article are available at Cancer Research shown that loss of genetic material is much more common than Online (http://cancerres.aacrjournals.org/). gains or amplifications, suggesting that tumor suppressor genes Corresponding Author: Daniel Gioeli, University of Virginia, Jordan Hall Room 2- play a crucial role in tumor development (7). Individuals with 16, 1300 Jefferson Park Avenue, PO Box 800734, Charlottesville, VA 22908. defects in DNA damage response (DDR) signaling lose the natural Phone: 434-982-4243; Fax: 434-982-0689; E-mail: [email protected] protection against tumorigenesis and are more susceptible to doi: 10.1158/0008-5472.CAN-15-0224 cell transformation and cancer. Checkpoint kinase 2 (CHK2), a Ó2015 American Association for Cancer Research. serine/threonine kinase and candidate tumor suppressor gene, is www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst November 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0224 Ta et al. essential in the cell-cycle checkpoint activated in response to RPMI-1640 (Invitrogen) with 10% heat-inactivated serum. LHS radiation-induced DNA double-strand breaks (DSB; refs. 8, 9). cells (a gift from Dr. William Hahn, Department of Medical Activated CHK2 phosphorylates multiple downstream effectors Oncology, Dana-Farber Cancer Institute and Harvard Medical involved in regulating cell-cycle arrest, apoptosis, DNA repair, School, Boston, MA) were grown in ProstaLife Epithelial Cell mitotic spindle assembly, and chromosomal stability (9, 10). Rare Medium (Lifeline). For growth and RNA experiments, phenol-red CHK2 germline mutations have been identified in several familial free RPMI-1640 media with 5% charcoal-stripped serum (CSS; cancers and rare somatic mutations have been reported in various Gemini) was used. Commercial DNA fingerprinting kits (DDC carcinomas, including those arising in the prostate, breast, and Medical) verified cell lines. The following STR markers were colon (11–13). These mutations affect the stability or kinase activity tested: CSF1PO, TPOX, TH01, Amelogenin, vWA, D16S539, of CHK2, suggesting that they may be involved in the pathogenesis D7S820, D13S317, and D5S818. Allelic score data revealed a of these neoplasms. LOH studies suggest that CHK2 mutations may pattern related to the scores reported by the ATCC, and consistent contribute to tumor development through haploinsufficiency (14). with their presumptive identity. Although loss of the q arm on chromosome 22 where CHK2 is Antibodies: CHK2, CDC25C, CDK1, ERK1/2, and HA (Cell located has been observed in breast, colorectal, and ovarian cancers, Signaling Technology); AR21 (in-house). Western blotting per- mutation of the remaining allele was not associated with this loss formed as previously described (21, 22). (15). However, the function of the remaining normal allele can be CHK2 primers were based on the human CHEK2 sequence disrupted (16). CHK2 splice variants lacking kinase activity or obtained from Genbank (BC004207.2; Supplementary Fig. S1A). mislocalized to the cytoplasm have been detected in breast cancer PCR was performed using iProof High-Fidelity DNA Polymerase (17). Reduced expression or total loss of CHK2 protein that was (Bio-Rad) and MGC Human CHEK2 Sequence-Verified cDNA as associated with mutation or promoter methylation has been found the template (Thermo Scientific). Amino-terminal triple HA-tagged in many cancers including those of the breast, urinary bladder, wild-type (wt) CHK2 was ligated into AgeI and EcoRI sites of the colon, ovary, and lung (15, 18, 19). Reports of other tumors lacking lentiviral expression vector pLJM1 (Addgene plasmid 19319) using CHK2 expression without any detectable mutation or promoter T4 DNA ligase (Promega), transformed into Stbl3 bacteria (Life methylation suggest that additional epigenetic, posttranscriptional, Technologies), and clones were sequenced for verification. or posttranslational mechanisms can decrease CHK2 protein levels TRC lentiviral human shRNAs targeting CHK2, CDC25C, and (20). Thus, these studies provide support that CHK2 behaves as a CDK1, and pLKO vector control (Thermo Scientific). tumor suppressor gene. In this study, we demonstrated that, in addition to dramatically CyQuant growth assays fl increasing prostate cancer proliferation, CHK2 depletion hyper- Assay was performed as previously described (22). Brie y, fi sensitized prostate cancer cells to castrate androgen levels. Con- shRNA or pLKO control virus was added to bronectin-coated m versely, CHK2 overexpression decreased cell growth. The CHK2- (1 g/mL) 96-well plates. Cells were plated in RPMI-1640 plus 5% m mediated effects on growth required the downstream signaling CSS with vehicle, 0 to 0.05 nmol/L R1881, and/or 10 mol/L fi proteins CDC25C and CDK1. We also found