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
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.
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
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:
�2018 American Association for Cancer Research.
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
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
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
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 (