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Plk4 Promotes Cancer Invasion and Metastasis Published OnlineFirst November 21, 2016; DOI: 10.1158/0008-5472.CAN-16-2060 Cancer Molecular and Cellular Pathobiology Research Plk4 Promotes Cancer Invasion and Metastasis through Arp2/3 Complex Regulation of the Actin Cytoskeleton Karineh Kazazian1,2,3, Christopher Go1,4, Hannah Wu5, Olga Brashavitskaya1,6, Roland Xu1, James W. Dennis1,4,6, Anne-Claude Gingras1,4, and Carol J. Swallow1,2,3,6 Abstract The polo family serine threonine kinase Plk4 has been breast cancer cells. In an unbiased BioID screen for Plk4 proposed as a therapeutic target in advanced cancers based interactors, we identified members of the Arp2/3 complex on increased expression in primary human cancers, facilita- and confirmed a physical and functional interaction between tion of tumor growth in murine xenograft models, and Plk4 and Arp2 in mediating Plk4-driven cancer cell move- centrosomal amplification induced by its overexpression. ment. This interaction is mediated through the Plk4 Polo-box However, both the causal link between these phenomena 1-Polo-box 2 domain and results in phosphorylation of and the feasibility of selective Plk4 inhibition remain unclear. Arp2 at the T237/T238 activation site, which is required for Here we characterize Plk4-dependent cancer cell migration Plk4-driven cell movement. Our results validate Plk4 as a ther- and invasion as well as local invasion and metastasis of apeutic target in cancer patients and reveal a new role for Plk4 in cancer xenografts. Plk4 depletion suppressed cancer invasion regulating Arp2/3-mediated actin cytoskeletal rearrangement. and induced an epithelial phenotype in poorly differentiated Cancer Res; 77(2); 1–14. Ó2016 AACR. Introduction spindles and mitotic irregularities, aneuploidy, and ultimately hepatocellular carcinoma, whereas loss-of-heterozygosity at the Polo like kinase 4 (Plk4) is a serine threonine kinase that Plk4 locus is seen in 50% of human hepatocellular carcinomas localizes to the centriole throughout the cell cycle, and is essential (12, 13). In sum, these results highlight the critical importance for centriole duplication (1–4). Increased expression of Plk4 is of tight regulation of Plk4 expression and activity, and the need described in a variety of common human epithelial malignancies, to better understand its function(s). and is associated with inferior survival in several large cohorts of Mechanistically, the oncogenic properties of overexpressed breast cancer patients (5–7). In particular, high levels of Plk4 Plk4 have been attributed to the centriole amplification associated mRNA are found in the triple-negative human breast cancers that with its upregulation (9, 14, 15). Human cancers characteristically are resistant to conventional systemic therapy, stimulating interest harbor supernumerary centrosomes (16–19), although the path- in Plk4 as a therapeutic target (8). Upregulation of Plk4 expression ogenic role of this phenomenon has long been debated (20, 21). independently induces aneuploidy, loss of cell polarity, and However, controlling centriole duplication may not be the only hyperplasia in some nontransformed cell lines and proliferative þ À function of Plk4 relevant to tumorigenicity. In Plk4 / MEF lines tissues (9, 10). In the context of p53 dysfunction, increased Plk4 that spontaneously acquire tumorigenicity over serial passaging, expression contributes to aneuploidy and tumorigenesis (10, 11). we reported a seemingly paradoxical association with changes in Yet, Plk4 also works as a haploinsufficient tumor suppressor in þ/À transcripts predictive of impaired cellular motility (22). More- some contexts: Plk4 mice are predisposed to form multipolar þ À over, the Plk4 / MEFs displayed impaired migration, and siPlk4 þ þ suppressed spreading and invasion in Plk4 / MEFs (22). Here we demonstrate that invasive and metastatic progression of 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, human breast cancer line MDA-MB-231 in murine xenografts is Ontario, Canada. 2Department of Surgery, University of Toronto, Toronto, dependent on Plk4 expression. An unbiased BioID screen for Plk4 3 Ontario, Canada. Institute of Medical Science, University of Toronto, interactors identified the actin-regulating complex Arp2/3 com- Toronto, Ontario, Canada. 4Department of Molecular Genetics, University of 5 ponent Arp2 as a potential mediator of Plk4-induced motility. Toronto, Toronto, Ontario, Canada. Department of Pathology, University of – Toronto, Toronto, Ontario, Canada. 6Laboratory Medicine and Pathobiology, Further analysis revealed Polo-box 1-Polo-box 2 dependent University of Toronto, Toronto, Ontario, Canada. binding of Plk4 to Arp2, phosphorylation of Arp2 at T237/ Note: Supplementary data for this article are available at Cancer Research T238 by Plk4, and dependence of Plk4-driven cell migration on Online (http://cancerres.aacrjournals.org/). Arp2. Corresponding Author: Carol J. Swallow, Lunenfeld-Tanenbaum Research Insti- tute, 1225-600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Phone: Materials and Methods 416-586-1558; Fax: 416-586-8392; E-mail: [email protected] Cell culture, transfection doi: 10.1158/0008-5472.CAN-16-2060 Cells were grown at 37C in DMEM (HeLa, MDA-MB-231, Ó2016 American Association for Cancer Research. HEK293T), RPMI1640 (MDA-MB-435), or McCoy 5A medium www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 21, 2016; DOI: 10.1158/0008-5472.CAN-16-2060 Kazazian et al. (U2OS) supplemented with 10% FBS. MDA-MB-231 HTB-26 and Scientific). Immunofluorescence images were collected using HeLa cell lines were obtained from ATCC in 2013 and 2009, the Olympia Deconvolution fluorescence microscope and soft- respectively. HEK293T and MDA-MB-435 cell lines were a kind WoRx software (Applied Precision). Images were collected gift from the Tony Pawson laboratory (obtained in 2010; Lunen- using a 100 or 60  1.4 NA oil objective (Olympus). feld Tanenbaum Research Institute, Mount Sinai Hospital, Tor- onto, Canada), and U2OS cells were a kind gift from the Laurence Spreading assays Pelletier laboratory (obtained in 2013; Lunenfeld Tanenbaum HeLa cells were seeded onto 6-well plates (150,000 cells/well) Research Institute, Mount Sinai Hospital, Toronto, Canada). with a glass coverslip or 96-well plates (2,500 cells/well). At These cell lines were not further authenticated in our laboratory. 3 hours, cells were fixed and immunostained, and 96-well plates Transient transfection was performed using Lipofectamine2000 were scanned and images acquired on the INCell Analyzer 6000, or LipofectamineRNAiMAX (Invitrogen) according to manufac- equipped with Nikon Plan Fluor 10Â/NA 0.45 objective and turer's instructions; a pool of four siRNAs and the individual 2,048  2,048 sCMOS camera. Automated cell size measurements constructs were utilized (Dharmacon; Supplementary Table S1). were performed using a custom image analysis routine (Colum- bus2.3, PerkinElmer). Hoechst-stained nuclei were detected, fol- Stable cell lines lowed by segmentation of cell borders based on F-actin signal, Stable cell lines were generated as Flp-In HeLa or U2OS T-REx and determination of cell area. cell pools or clones. The jetPRIME DNA transfection reagent (Polyplus) was used to transfect cells grown in 6-well plates with Xenograft studies in mice 300 ng DNA and 3 mg pOG44 overnight, as per manufacturer's All protocols were approved by the Toronto Center for Phe- 6 instructions. Transfected cells were selected using 200 mg/mL nogenomics (TCP) Animal Care Committee. A total of 1.0  10 hygromycin (Multicell, 450141XL). HeLa, MDA-MB-231, or MDA-MB-231 RFP or Plk4 shRNA cells were injected subcutane- MDA-MB-435 cells expressing two Plk4 (SHCLNG-NM_014264; ously in the right flank of 5-week-old NOD SCID/J1303 mice Sigma), luciferase or RFP (Sigma) shRNAs were generated through (Jackson Laboratory). Tumor growth was monitored by palpa- lentiviral infection. Lentiviruses were produced as described (23), tion, size was measured with calipers, and volume calculated and used to infect cells for 24 hours, followed by puromycin assuming an ellipsoid shape. Mice were sacrificed at 4- to 6-week (1 mg/mL: HeLa and MDA-MB-231, 5 mg/mL: MDA-MB-435) or 7- to 10-week postinjection. Tumors were harvested and split mediated drug selection. for fixation in 10% buffered formalin phosphate (SF100-20; Thermo Fisher Scientific), or frozen in liquid nitrogen for RNA RNA extraction, real-time RT-PCR extraction. The right lung lobes, left liver lobe, and intraabdom- fi RNA was isolated using the RNeasy Mini Kit (Qiagen), inal metastases were harvested and xed for histology. treated with RNase-free DNase (Invitrogen), and reverse tran- scribed with SuperScript II Reverse Transcriptase (18064-014; Histology and IHC fi m Invitrogen) using Random Primers (48190-011; Invitrogen) Fixed tissue was paraf n-embedded, sectioned (4 m), depar- fi according to manufacturer's instructions. Real-time RT-PCR af nized in xylene, and rehydrated in graded ethanol. For tumors, m was performed using SYBR Green PCR Master Mix (Applied serial coronal sectioning was performed at four levels, 200 m Biosystems) on an ABI 7900HT apparatus. All quantifications apart. Samples were stained with hematoxylin and eosin (H&E) or were normalized to control endogenous GAPDH or RPII the indicated antibodies (Supplementary Methods) according to (Primers; Supplementary Table S2). Data generated by PCR standard protocols using the Veristain Gemini Automated Slide fi software (SDS2.2.2; Applied Biosystems) were analyzed using Stainer (Thermo Scienti c) at the TCP Pathology Core, and ÀDDC examined with a Leica DMR upright microscope by a Royal the 2 t method (24). College–certified pathologist in a blinded manner. fi Cell protrusion quanti cation Quantification of lung micrometastasis and Ki67 HeLa cells transfected with siRNA for 48 hours or with FLAG- Images were taken at Â20 magnification by light microscopy, Plk4-wild-type (WT) or FLAG were serum-starved for 8 hours, and the number and size of lung micrometastases determined  6 m fi then 1 10 cells were plated on 1- m pore size lters (PET using ImageJ.
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