Published OnlineFirst October 24, 2014; DOI: 10.1158/1535-7163.MCT-14-0447 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Elevated LIM Kinase 1 in Nonmetastatic Prostate Cancer Reflects Its Role in Facilitating Androgen Receptor Nuclear Translocation Katerina Mardilovich1, Mads Gabrielsen1, Lynn McGarry1, Clare Orange2, Rachana Patel1, Emma Shanks1, Joanne Edwards3, and Michael F. Olson1 Abstract Prostate cancer affects a large proportion of the male popula- inhibiting proliferation and increasing apoptosis in androgen- tion, and is primarily driven by androgen receptor (AR) activity. dependent prostate cancer cells more effectively than in androgen- First-line treatment typically consists of reducing AR signaling by independent prostate cancer cells. LIMK inhibition blocked hormone depletion, but resistance inevitably develops over time. ligand-induced AR nuclear translocation, reduced AR protein One way to overcome this issue is to block AR function via stability and transcriptional activity, consistent with its effects on alternative means, preferably by inhibiting protein targets that proliferation and survival acting via inhibition of AR activity. are more active in tumors than in normal tissue. By staining Furthermore, inhibition of LIMK activity increased aTubulin prostate cancer tumor sections, elevated LIM kinase 1 (LIMK1) acetylation and decreased AR interactions with aTubulin, indi- expression and increased phosphorylation of its substrate Cofilin cating that the role of LIMK in regulating microtubule dynamics were found to be associated with poor outcome and reduced contributes to AR function. These results indicate that LIMK survival in patients with nonmetastatic prostate cancer. A LIMK- inhibitors could be beneficial for the treatment of prostate cancer selective small molecule inhibitor (LIMKi) was used to determine both by reducing nuclear AR translocation, leading to reduced whether targeted LIMK inhibition was a potential prostate cancer proliferation and survival, and by inhibiting prostate cancer cell therapy. LIMKi reduced prostate cancer cell motility, as well as dissemination. Mol Cancer Ther; 14(1); 246–58. Ó2014 AACR. Introduction (CRPC) that may quickly progress to metastatic disease with high mortality rates (4). Therefore, a major goal is to identify potential Prostate cancer is the most commonly diagnosed malignancy targets for the development of prostate cancer therapies that target and second leading cause of cancer-related deaths in American AR function in a hormone-independent manner. Such treatments men (1). At the molecular level, prostate cancer development and might not only delay the progression of prostate cancer to CRPC, progression are driven primarily by activity of the androgen but could possibly also be used for the treatment of CRPC, which receptor (AR), a steroid hormone receptor typically localized in maintains and relies upon active AR (4). the cytoplasm in the absence of hormone stimulation (2). In the Targeting the microtubule cytoskeletal network is one approach presence of ligand, ARs translocate to the nucleus to activate the that has been used to achieve the goal of reducing AR signaling. transcription of target genes that control cell-cycle progression, Docetaxel, a microtubule-stabilizing drug commonly used for the cell growth, and survival. As a result, the first line of therapy in treatment of prostate cancer, has been shown to exert its cytotoxic prostate cancer has been to decrease AR activity by hormone effect on prostate cancer cells by inhibiting microtubule-mediated depletion (3). Unfortunately, hormone-ablation therapy often AR nuclear translocation in addition to its direct antimitotic leads to the development of castration-resistant prostate cancer activity (5, 6). Two major issues with docetaxel treatment are that resistance develops over time, and its general antimitotic and microtubule-stabilizing actions result in strong side effects, 1Beatson Institute for Cancer Research, Glasgow, United Kingdom. including alopecia, neutropenia, and anemia. Therefore, an 2Pathology Department, Division of Cancer Sciences and Molecular appealing objective for future prostate cancer drug development Pathology, Western Infirmary, University of Glasgow, Glasgow, United is to identify alternative microtubule regulators, which if inhibited 3 Kingdom. Institute of Cancer Sciences, College of Medical,Veterinary would affect AR function with low nonspecific cytotoxicity. In and Life Sciences, University of Glasgow, Glasgow, United Kingdom. particular, if this target was more active in prostate cancer, its Note: Supplementary data for this article are available at Molecular Cancer inhibition would improve drug selectivity for prostate cancer Therapeutics Online (http://mct.aacrjournals.org/). tumors over normal tissue and contribute to a greater therapeutic Current address for Clare Orange: Department of Pathology, Laboratory Med- window. icine Building, Southern General Hospital, Glasgow, UK. Although best known as regulators of actin–myosin cytoskel- Corresponding Author: M.F. Olson, The Beatson Institute for Cancer Research, etal dynamics (7), LIM kinases 1 and 2 (LIMK1 and LIMK2) also Garscube Estate, Switchback Road, Glasgow, Scotland G61 1BD, UK. Phone: contribute to the regulation of the microtubule cytoskeleton (8– 44-141-330-3654; Fax: 44-141-942-6521; E-mail: [email protected] 10). LIM kinases are highly homologous serine/threonine kinases doi: 10.1158/1535-7163.MCT-14-0447 that are activated by RhoA/ROCK, Rac/PAK, and Cdc42/MRCK Ó2014 American Association for Cancer Research. signaling pathways (7). The most well-characterized LIMK 246 Mol Cancer Ther; 14(1) January 2015 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 24, 2014; DOI: 10.1158/1535-7163.MCT-14-0447 LIM Kinases in Prostate Cancer substrates are Cofilin proteins, which are inhibited for their actin- Cell lines and antibodies severing activities when phosphorylated on serine 3 (11). There Cell lines were cultured in RPMI media, supplemented with have been previous reports of elevated LIMK1 expression in 10% FBS and 2 mmol/L L-glutamine (GIBCO). LNCaP-AI cells prostate cancer (12–14), in which it was postulated to have a were cultured in phenol red-free RPMI with 10% charcoal-stripped role in promoting metastasis (15). However, there have been no serum (CSS) and 2 mmol/L L-glutamine (GIBCO). The LNCaP, previous studies that systematically evaluated the expression DU145, PC3, RWPE-1 cell lines were from the ATCC, LNCaP-AI levels of LIMK1, LIMK2, or phosphorylation of their common was made in the laboratory of Hing Leung (Beatson Institute for substrate Cofilin as an indicator of kinase activity in primary Cancer Research, Glasgow, UK) and gifted to us, CWR22 cells were prostate cancer tumor samples accompanied by analysis of pros- from Thomas Pretlow (Case Western Reserve University, Cleve- tate cancer clinical outcomes. land OH). All cell lines were obtained at the beginning of the study We undertook immunohistochemical analysis of a prostate in April 2012 and authenticated using the GenePrint 10 system cancer tissue microarray (TMA) comprised of 164 primary pros- STR multiplex assay (Promega) that amplifies 9 tetranucleotide tate cancer and 23 benign hyperplasia samples from 94 individual repeat loci and Amelogenin gender determining marker. Antibo- patients (16), and identified significant associations of elevated dies: Santa Cruz Biotechnology, AR (N-20 and sc-816), LIMK2 (H- LIMK1 expression and phosphorylation of nuclear Cofilin with 78 and sc-5577); Cell Signaling Technology, p-Cofilin (Ser3, reduced survival of patients with nonmetastatic prostate cancer. 3311), LIMK1 (3842); Abcam, Cofilin (ab54532), LIMK1 Moreover, elevated levels of LIMK1 and cytoplasmic phospho- (ab55414); Sigma, aTubulin (clone DM1A, T9026); Millipore, Cofilin were both associated with significantly higher lympho- MMP-1 (04–1112); Leica, MMP-10 (NCL-MMP10); Novus Bio- vascular invasion. To pharmacologically evaluate whether LIMK logicals, acetylated-a Tubulin (NB600-567). could be a potential prostate cancer drug target, we tested a potent and selective LIMK inhibitor (LIMKi; ref. 17). LIMK inhibition Cytotoxicity assays reduced prostate cancer cell motility, suggesting that blocking Cells were plated in 96-well plates at 2,000 cells per well in LIMK activity could be beneficial as an antimetastatic therapeutic triplicate and treated the next day without changing the media. target in prostate cancer. Interestingly, we observed a cytotoxic Drugs were serial diluted in DMSO, before diluting equal amounts effect of LIMK inhibition that was significantly greater in andro- in media at 2Â of the final concentration. Then 100 mLofdrug- gen-dependent prostate cancer cells than in androgen-indepen- containing media was added to each well that already contained dent cells. Treatment of androgen-dependent prostate cancer cells 100 mL of cells. Cells were treated for 72 hours, fixed with 4% with LIMKi reduced AR nuclear translocation and transcriptional paraformaldehyde, and stained with 250 ng/mL DAPI. Plates were activity by altering microtubule dynamics that facilitate AR inter- imaged on a High Content Imaging Operetta system (PerkinElmer) actions with aTubulin, thus inhibiting cell proliferation. There- and nuclei in each well were quantified using Harmony High fore, in addition to a potential role in promoting metastasis, Content