YB-1 Expression and Phosphorylation Regulate ... Oncogenes and Tumor Suppressors YB-1 Expression and

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  • Oncogenes and Tumor Suppressors

    YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT Corinna Kosnopfel1, Tobias Sinnberg1, Birgit Sauer1, Christian Busch1,2, Heike Niessner1, Anja Schmitt3, Stephan Forchhammer1, Cornelia Grimmel4, Peter R. Mertens5, Stephan Hailfinger3, Sandra E. Dunn6, Claus Garbe1, and Birgit Schittek1

    Abstract

    Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gainmigratory and invasive properties promoting theirmetastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a pow- erful inducer of EMT in breast carcinoma cells. Interestingly, EMT- like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo. Intriguingly, this effect seems to be mainly mediated by cyto- plasmic YB-1 that does not exhibit phosphorylation at serine-102

    (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture sys- tems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.

    Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149–60. �2018 AACR.

    Introduction Malignantmelanoma is an aggressive neoplasm accounting for

    the majority of skin cancer–related deaths despite its comparably low incidence (1). While localized melanomas can be effectively treated by surgical resection and are associated with five-year survival rates of 98%, the outlook in case of metastatic disease is rather bleak with a current five-year survival rate of 17% (1). Unfortunately, malignant melanomas exhibit a very high pro- pensity to metastasize, which is already prevalent at stages of low primary tumor thickness (2) and hypothesized to be a conse- quence of its melanocytic origin (3). During embryonic develop-

    ment, highly migratory neural crest cells delaminate from the neural tube andmigrate to the skin, hair follicles, or other specific locations within the developing embryo, where they eventually give rise to melanocytes (4). In line with originating from exceed- ingly motile cells, melanocytes are supposed to retain latent migratory capabilities, which could easily be reactivated in the process of melanoma progression (3).

    The epithelial-to-mesenchymal transition (EMT) is a highly conserved program during embryonic morphogenesis and is associated with epithelial cells acquiring mesenchymal character- istics including an enhanced physiologic migratory capability. In addition to its crucial role for mesoderm formation as well as neural crest development and function, EMT is also frequently exploited by epithelial cancer cells during the invasive phase of metastasis (5).

    The Y-box binding protein 1 (YB-1, YBX1) is a multifunctional member of the cold-shock domain (CSD) protein family, which can act both as a transcription factor in the nucleus and as a translational regulator in the cytoplasm (6). The subcellular localization of YB-1 is governed by regulatory sequences in the C-terminal region of the protein including two noncanonical nuclear localization signals (NLS) as well as a cytoplasmic reten- tion site (CRS; refs. 7, 8). Owing to a dominant effect of the CRS under normal cellular conditions, YB-1 is predominantly found in the cytoplasm (9). Upon environmental stresses and in response to promitogenic stimuli, however, YB-1 translocates to the nucle- us (9–11). The serine/threonine kinase AKT and the p90 ribo- somal S6 kinase (RSK) are capable of phosphorylating YB-1 at a

    1Division of Dermatooncology, Department of Dermatology, University of T€ubingen, T€ubingen, Germany. 2Dermateam, Winterthur, Switzerland. 3Inter- faculty Institute of Biochemistry, University of T€ubingen, T€ubingen, Germany. 4FACS Core Facility, Department of Dermatology, University of T€ubingen, T€ubingen, Germany. 5Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University, Magdeburg, Germany. 6Phoenix Molecular Designs, Vancouver, British Columbia, Canada.

    Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

    Corresponding Author: Birgit Schittek, Division of Dermatooncology, Depart- ment of Dermatology, University of T€ubingen, Liebermeisterstr. 25, T€ubingen D-72076, Germany. Phone: 4970-7129-80832; Fax: 4970-7129-5187; E-mail: birgit.schittek@uni-tuebingen.de

    doi: 10.1158/1541-7786.MCR-17-0528

    �2018 American Association for Cancer Research.

    Molecular Cancer Research

    www.aacrjournals.org 1149

    on July 10, 2020. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

    Published OnlineFirst May 9, 2018; DOI: 10.1158/1541-7786.MCR-17-0528

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  • serine residue (S102) within the nucleic acid–binding cold-shock domain (10, 12, 13). Intriguingly, this phosphorylation eventwas reported to impair the association of YB-1 with mRNAs in the cytoplasm and to simultaneously induce its nuclear translocation and activity as a transcription factor in breast and ovarian cancer, thuspromoting tumor cell proliferation and chemoresistance (10, 12–15). Apart from its nuclear function, also increased expression of cytoplasmic unphosphorylated YB-1 proved to mediate a potent protumorigenic effect by stimulating the translation of mRNAs encoding EMT-regulating transcription factors in a breast cancer model (16). Indeed, both nuclear and cytoplasmic YB-1 have been repeatedly associated with poor prognosis and disease recurrence in a variety of human malignancies (6).

    On the basis of earlier work providing first evidence for increased YB-1 expression during melanoma progression and for a functional role of the cold-shock domain protein in the biology of metastatic melanoma cells (17–19), we now more specifically addressed the impact of YB-1 protein levels and its S102 phosphorylation status on the aggressive phenotype of malignant melanoma reflected by patient survival as well as melanoma cell tumorigenicity, migratory activity, and invasive capacity.

    Materials and Methods Culture of human cells

    The human metastatic melanoma cell lines MeWo and A375 were purchased from ATCC (20) and authenticated via Short Tandem Repeat (STR) profiling (Leibniz Institute DSMZ); 1205LU was kindly provided by the laboratory of M. Herlyn (The Wistar Institute, Philadelphia, PA; ref. 21), which routinely per- forms STR profiling using the AmpFlSTR Identifiler PCR Ampli- ficationKit (Life Technologies). The cultivation ofmelanoma cells was conducted as described previously (22). All melanoma cell lines were used no longer than 2 months upon thawing of the frozen stock. Mycoplasma testing was regularly performed using the Venor GeM Classic Mycoplasma Detection Kit (Minerva Biolabs).

    YBX1 geneknockout (CRISPRYBX1)was carriedout byCRISPR/ Cas9–mediated genome engineering as described previously (18). 1205LU CRISPR YBX1 #1 and #2 (from two separate approaches using different sgRNA sequences targeting YBX1) represent pooled populations of cells with effective knockout and unaffected cells, while theA375 andMeWoCRISPR YBX1 cells constitute respective single-cell clones with effective YBX1 knockout.

    Lentiviral gene transfer was used for the generation of mela- noma cells with constitutive overexpression of HA-tagged wild- type YB-1 (HA-YB-1; cloned in pWPI; ref. 17), of cells with inducible overexpression of 3XFLAG-tagged wild-type YB-1 (YB-1WT) or S102 phospho-site mutant YB-1 (YB-1S102D, YB-1S102A; ref. 23; subcloned into pLVX-Tight-Puro and cotrans- duced with pLVX-Tet-On Advanced, both Clontech) as well as of melanomacellswith inducible expressionof YB-1–specific shRNA (shYB-1, TRIPZ, clone V2THS_232997) or nonsilencing shRNA (NonSil, TRIPZ, #RHS4743; both Dharmacon/GE Healthcare). Transgene expression was induced by 2 mg/mL doxycycline (AppliChem) in the culture medium.

    Lentiviral gene transfer Production of lentiviral particles and transduction of melano-

    ma cells were conducted as described previously (17).

    In vivo tumor growth assay To assess melanoma growth in vivo, 5 � 105 1205LU cells with

    stable HA-YB-1 overexpression or transduced with the empty vector were subcutaneously injected into NOD/SCID mice. Tumor size was monitored up to 56 days after injection by measurement of tumor length and width using a caliper. The tumor volume (V ¼ 0.4 � length � width2) was calculated as a correlate for tumor growth.

    For the in vivo tumor growth assay assessing the effect of YB-1 knockdown, 1� 106 1205LU shYB-1 cells in 100 ml PBSwith 10% Matrigel were subcutaneously injected into SCID hairless outbred (SHO)mice. Themicewere randomized into two groups (