YB-1 Evokes Susceptibility to Cancer Through Cytokinesis Failure, Mitotic Dysfunction and HER2 Amplification

Oncogene (2011) 30, 3649–3660 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction and HER2 amplification AH Davies1, I Barrett2, MR Pambid1,KHu1, AL Stratford1, S Freeman3, IM Berquin4, S Pelech5,6, P Hieter2, C Maxwell7 and SE Dunn1 1Laboratory of Oncogenomic Research, Departments of Pediatrics and Experimental Medicine, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada; 2Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada; 3Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; 4Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 5The Brain Research Centre, Division of Neurology, University of British Columbia, Vancouver, British Columbia, Canada; 6Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada and 7Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada Y-box binding protein-1 (YB-1) expression in the Introduction mammary gland promotes breast carcinoma that demon- strates a high degree of genomic instability. In the present Cancer arises from the progressive evolution of a cell study, we developed a model of pre-malignancy to from normalacy through intermediate pre-malignant characterize the role of this gene during breast cancer states to finally become invasive and metastasize initiation and early progression. Antibody microarray (Hanahan and Weinberg, 2000). Cells position them- technology was used to ascertain global changes in signal selves for this progression to malignancy by accumulat- transduction following the conditional expression of YB-1 ing genetic alterations that result in the activation of in human mammary epithelial cells (HMEC). Cell cycle- oncogenes and inactivation of tumor suppressors. associated proteins were frequently altered with the most Studies of human mammary epithelial cells (HMEC) dramatic being LIM kinase 1/2 (LIMK1/2). Conse- are beginning to provide key insight into these early quently, the misexpression of LIMK1/2 was associated genetic events to ascertain their role in fueling breast with cytokinesis failure that acted as a precursor to carcinogenesis (Romanov et al., 2001; Tlsty et al., 2004). centrosome amplification. Detailed investigation revealed Y-box binding protein-1 (YB-1) is a transcription/ that YB-1 localized to the centrosome in a phosphory- translation factor that is overexpressed in a plethora of lation-dependent manner, where it complexed with peri- cancers, including human breast carcinoma (40%) (Wu centrin and c-tubulin. This was found to be essential in et al., 2006; Habibi et al., 2008). A pro-tumorigenic role maintaining the structural integrity and microtubule for YB-1 is supported by its ability to directly bind Y-box nucleation capacity of the organelle. Prolonged exposure promoter elements of a variety of genes, notably epidermal to YB-1 led to rampant acceleration toward tumorigen- growth factor receptor (EGFR), ErbB2 (HER2), cyclin A esis, with the majority of cells acquiring numerical and and cyclin B1 (Jurchott et al., 2003; Wu et al., 2006; structural chromosomal abnormalities. Slippage through Stratford et al., 2007). Moreover, a function for YB-1 in the G1/S checkpoint due to overexpression of cyclin E regulating cell cycle progression is beginning to emerge promoted continued proliferation of these genomically through its ability to alter the expression of genes involved compromised cells. As malignancy further progressed, we at the G1/S boundary (Basaki et al.,2010;Yuet al., 2010). identified a subset of cells harboring HER2 amplification. The transcriptional activity of YB-1 is dependent upon Our results recognize YB-1 as a cancer susceptibility phosphorylation at its Ser-102 residue mediated by Akt/ gene, with the capacity to prime cells for tumorigenesis. PKB, and even more potently by p90 ribosomal S6 kinase Oncogene (2011) 30, 3649–3660; doi:10.1038/onc.2011.82; (RSK) (Sutherland et al., 2005; Stratford et al., 2008). published online 21 March 2011 To establish the importance of YB-1 in malignant transformation, transgenic mice were developed where expres- Keywords: YB-1; premalignancy; centrosome; cell cycle; sion was targeted to the lactating mammary gland HER2 amplification; breast cancer (Bergmann et al., 2005). The resulting mouse mammary tumors formed with 100% penetrance, and close examina- tion revealed substantial centrosome amplification and chromosomal instability (Bergmann et al., 2005). Given these findings, concurrent with the high prevalence of Correspondence: Dr SE Dunn, Departments of Pediatrics, University YB-1 in breast cancer, we hypothesized that it has an of British Columbia, 950 West 28th Avenue, Room 3083, Vancouver, essential role in breast tumorigenesis. British Columbia, Canada V5Z 4H4. Genomic instability, in the form of alterations to E-mail: [email protected] Received 10 October 2010; revised 4 February 2011; accepted 14 chromosome number and structure, is a characteristic February 2011; published online 21 March 2011 feature of almost all types of cancer (Nigg, 2002; YB-1 is a cancer susceptibility gene AH Davies et al 3650 Fukasawa, 2007; Holland and Cleveland, 2009; Nigg Table 1 Cell cycle-associated proteins putatively regulated by YB-1 and Raff, 2009). However, whether this represents Protein Antibody Localization % CFCa a cause or consequence of tumorigenesis remains mysterious. To address this contentious issue and begin LIMK1/2 Y507 þ T508/Y504 þ T505 Centrosome 365 to establish a paradigm for malignant transformation, ZAP70 Y315 þ Y319 Centrosome 230 it has become imperative to study cancer during the RSK1/2 S380/S386 Kinetochore 269 CDK1/2 Y15 Centrosome 161 earliest pre-malignant stages, which, to date, have Cdc34 Pan-specific Mitotic spindle 148 remained wholly uncharacterized. A large body of Cofilin Pan-specific Centrosome 135 evidence has recently been compiled indicating p53 Pan-specific Cytoplasm/nucleus 118 that amplification of centrosomes has the potential to CDK9 Pan-specific Nucleus 116 PP2A Pan-specific Centrosome 102 cause mitotic defects that lead to chromosomal insta- ZAP70 Y292 Centrosome 102 bility (Nigg, 2006; Basto et al., 2008; Ganem et al., CDK6 Pan-specific Centrosome 98 2009). According to this model, centrosome abnormal- PKA Pan-specific Centrosome À67 ities would need to emerge early during neoplastic progression. Ultimately, through Darwinian selection, a a%CFC refers to the percentage change from control (uninduced karyotype adept at enhancing tumor progression would HTRY cells). materialize and expand (Fujiwara et al., 2005; Shi and King, 2005). Of all sporadic breast cancer, 20–30% including HER2, strongly supported the fidelity of the exhibit amplification of HER2, prompting us to address screen. We prioritized subsequent analysis on the active if this is a common feature of pre-malignancy that arises LIM kinases (pLIMK1/2T508/T505), as they exhibited the through targeted genomic instability preceding clonal most profound increase in level (365%) following YB-1 outgrowth (Slamon et al., 1987). induction. This correlation was validated both by In this study, we examined the role of YB-1 during immunoblotting (Figure 1a) and immunofluorescence pre-malignancy to uncover the molecular events that staining (Figure 1b). In a reciprocal experiment, define the earliest transitions in breast cancer initi- silencing YB-1 with small interfering RNA (siRNA) in ation and progression. A comprehensive understanding MDA-MB-231 and SUM149 breast cancer cell lines of these processes will usher the development of novel repressed the phosphorylation of LIMK1/2 at Thr-508/ therapeutics that target the process, rather than the Thr-505, with minimal effect on total LIMK1 expression consequences, of tumorigenesis. (Figure 1c). To confirm these results, we utilized a complimentary pharmacological approach for suppression of YB-1 activity using a signal transduction inhibitor to RSK. Our prior work indicated that inhibition Results of RSK directly impaired YB-1 phosphorylation and activity (Stratford et al., 2008). Treating MDA-MB-231 YB-1 alters the expression and activity of cell cells with the RSK inhibitor BI-D1870 (a gift cycle-associated proteins from Ching-Shih Chen, The Ohio State University, To address the potential contribution of YB-1 in the Columbus, OH, USA) yielded complete suppression of initiation of tumorigenesis, we engineered non-malignant pLIMK1/2T508/T505, highlighting that pYB-1S102 was H16N2 HMECs that conditionally expressed the gene necessary to promote LIMK1/2 activation (Figure 1d). under control of a tetracycline-inducible promoter These data were mirrored using siRNA against RSK1 (designated HTRY cells). HMECs containing an induci- and RSK2 (Supplementary Figure S2). Previous reports ble LacZ construct served as a matched control implicated LIMK1/2 as centrosomal proteins (Sumi (designated HTRZ cells). The ectopic expression level et al., 2006; Chakrabarti et al., 2007) and, accordingly, of YB-1 achieved in this model closely recapitulated that we wanted to examine the localization in our HTRY cell observed in established cancer cell lines (Supplementary model. At 96-h post-YB-1 induction, we observed Figure S1A). Further characterization revealed that both punctate pLIMK1/2T508/T505

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