Overexpression of protein O-fucosyltransferase 1 (POFUT1)
in human oral cancer: correlation with tumor progression
Tomoyoshi Koyama1, Satoshi Yokota1, Katsunori Ogawara2, Yukinao Kouzu2, Tomomi Shida1, Ryota
Kimura1, Takao Baba1, Morihiro Higo2, Atsushi Kasamatsu2, Yosuke Endo-Sakamoto2, Masashi Shiiba3,
Katsuhiro Uzawa1,2*, and Hideki Tanzawa1,2
1
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan
Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan
3
Department of Clinical Oncology, Graduate School of Medicine, Chiba University, Japan
2
*Address correspondence to: Katsuhiro Uzawa, Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana,
Chuo-ku, 260-8670, Chiba, Japan
E-mail: uzawak@faculty.chiba-u.jp ; telephone: +81-43-226-2300; fax: +81-43-226-2151
Abstract. Purpose：Protein O-fucosyltransferase 1 (POFUT1) is closely related to glycosylation, one of the post-translational
modifications, for target proteins. However, detail mechanism of POFUT1 in carcinogenesis is still unknown. The purpose of this
study was to examine POFUT1 expression levels in oral squamous cells carcinoma (OSCC) cell lines and human clinical OSCC
samples. In addition to in vitro data that POFUT1 mRNA and protein levels were significantly up-regulated in OSCC cell lines
compared with human normal oral keratinocytes, we found up-regulation of POFUT1 in clinical OSCC samples. Furthermore,
POFUT1-positive OSCC cases were correlated with advanced tumor size compared with POFUT1-negative OSCC cases. Consistent
with clinical behaviors, POFUT1 knockdown cells showed lower cellular proliferation rate, indicating that POFUT1 would
contribute an important role for tumor progression. Taken together, POFUT1 may be a potential diagnostic marker and a therapeutic
target for OSCCs.
Key words: oral squamous cell carcinoma, protein O-fucosyltransferase 1, tumor progression
on these data, we proposed that POFUT1 might be a key
regulator of tumor progression in OSCCs.
1. Introduction
A number of etiologic factors have been implicated in the
development of oral squamous cells carcinomas (OSCCs), such
as the use of tobacco, alcohol, or the presence of incompatible
prosthetic materials [1, 2, 3]. However, some patients develop
OSCC without risk factors, suggesting that host susceptibility
may play a role. Molecular alterations in a number of
oncogenes and tumor suppressor genes associated with the
development of OSCC could be important clues for addressing
these issues [2, 4]. Comprehensive and exhaustive expression
studies of numerous genes, including functionally unknown
genes, are essential for understanding the complexity and
polymorphisms of OSCC [5-8].
Protein O-fucosyltransferase 1 (POFUT1), located on
chromosome 20q11.21, produces an O-fucose modification on
EGF-like repeats of a number of cellular surface and secreted
proteins [9]. Recent studies have reported that POFUT1
affected anterior-posterior somite patterning in mammalian
embryos [10] and regulated T-, myeloid-, and B-lineage
differentiation in mammals [11]. Several groups have reported
the relationship between POFUT1 expression and malignancies,
such as acute myeloid leukemia, myeloid dysplastic syndrome,
glioblastoma, and colon cancer [12-15].
In the current study, further experiments showed clinical
relationship between POFUT1 levels and OSCC status. Based
2. Materials and methods
2.1. OSCC cell lines and tissue samples
Five cell lines, derived from human OSCCs, were
purchased from the Human Science Research Resources Bank
(Osaka, Japan). Primary cultured human normal oral
keratinocytes (HNOKs) were obtained from three healthy
donors [16-25]. All cells were grown in Dulbecco’s modified
Eagle medium/F-12 HAM (Sigma, St. Louis, MO,USA)
supplemented with 10% fetal bovine serum (Sigma) and 50
units/ml penicillin and streptomycin (Sigma)[26-28].
Many tissue samples from unrelated Japanese patients with
primary OSCC who were treated at the Chiba University
Hospital were obtained during surgical resection. The resected
tissues were divided into two parts, one of which was frozen
immediately and stored at -80˚C until RNA isolation, and the
second of which was fixed in 10% buffered formaldehyde
solution for pathologic diagnosis and immunohistochemistry
(IHC). Histopathologic analysis of the tissues was performed
according to the World Health Organization criteria by the
Department of Pathology, Chiba University Hospital.
Clinicopathologic staging was determined by the TNM
classification of the International Union against Cancer.
1
2.2. mRNA expression analysis
Total RNA was isolated from cells using Trizol Reage
nt (Invitrogen, Carlsbad, CA,USA) and was reverse-transcri
bed by Ready-to-Go You-Prime first-strand beads (GE Hea
lthcare, Little Chalfont, UK) and Oligo (dT) primer (Invitr
ogen). Real-time quantitative reverse transcriptase-polymeras
e chain reaction (qRT-PCR) was performed to evaluate the
expression level of POFUT1 mRNA in the OSCC cell lin
es and HNOKs. The expression levels were determined usi
ng primers and probes that were designed using the Unive
rsal Probe Library (Roche Diagnostics GmbH, Mannheim,
Germany) following the manufacturer’s instructions. The pr
imer sequences for POFUT1 were forward 5’-CTGATGAC
CCGATGGTAAGC-3’; reverse 5’-AAGCCTCCTTTCACCA
ACCT-3’; and universal probe #78. All qRT-PCR analyses
were performed using a LightCycler® 480 PCR system (
Roche Diagnostics GmbH). The transcript amount for POF
UT1 was estimated from the respective standard curves an
d normalized to the glyceraldehyde-3-phosphate dehydrogen
ase (GAPDH) (forward, 5’-CATCTCTGCCCCCTCTGCTGA
-3’; reverse, 5’-GGATGACCTTGCCCACAGCCT-3’; and u
niversal probe #60) transcript amount determined in corres
ponding samples.
xylene, and mounted. As a negative control, triplicate sections
were immunostained without exposure to primary antibodies,
which confirmed the staining specificity. To quantify the state
of POFUT1 protein expression in those components, we used
the IHC score system described previously [16-25,29]. The
stained cells were counted in at least five random fields at 400 x
magnification in each section. We counted 100 cells per one
field of vision. The staining intensity (0, negative; 1, weak; 2,
moderate; 3, intense) and the number of positive cells in the
field of vision were multiplied to calculate the IHC score. The
formula for obtaining the IHC score was: IHC score = 1 x (the
number of weak stained cells in the field) + 2 x (number of
moderately stained cells in the field) + 3 x (number of intensely
stained cells in the field). Cases with a POFUT1 IHC score
exceeding 71.5 (the maximal score within +3 standard
deviations (SD) of the mean of normal tissues) were defined as
POFUT1-positive. Two independent pathologists, neither of
whom had knowledge of the patients’ clinical status, made
these judgments.
2.5. shRNA experiments
OSCC cell lines, cell line-1 and cell line-4, in which
POFUT1 protein expression was higher than in the other cell
lines, were stably transfected with POFUT1 shRNA
(shPOFUT1) or control shRNA (mock) (Santa Cruz
Biotechnology) using Lipofectamine LTX and Plus Reagents
(Invitrogen). The stable transfectants were isolated by the
culture medium containing 1 mg/ml Puromycin (Invitrogen).
Two to 3 weeks after transfection, viable colonies were picked
up and transferred to new dishes. shPOFUT1 and mock cells
were used for further functional experiments. To investigate the
effect of POFUT1 knockdown on cellular proliferation,
migration, and invasiveness, we performed cell growth assay,
wound healing assay, and boyden chamber assay, respectively.
These experiments were carried out according to our previous
reports [25, 30, 31].
2.3. Protein expression analysis
The cells were washed twice with cold tris-buffered saline
(TBS) and centrifuged briefly. The cell pellets were incubated
at 4˚C for 30 min in a lysis buffer (7 M urea, 2 M thiourea, 4%
w/v CHAPS, and 10 mM Tris pH 7.4). Protein extracts were
electrophoresed on 4-12% Bis-Tris gel, transferred to
nitrocellulose membranes (Invitrogen), and blocked for 1 hour
at room temperature in Blocking One (Nacalai Tesque, Tokyo,
Japan). The membranes were washed three times with 0.1%
Tween 20 in TBS and incubated with rabbit anti-human
POFUT1 polyclonal antibody (OriGene Technologies,
Rockville, MD, USA) overnight at 4°C. The membranes were
washed again and incubated with an anti-rabbit IgG (H+L)
horseradish peroxidase conjugate (Promega, Madison, Wis,
USA) as a secondary antibody for 1 hour at room temperature.
Finally, the membranes were detected using SuperSignal West
Pico Chemiluminescent substrate (Thermo, Rockford, Ill, USA)
and immunoblotting was visualized by exposing the membranes
to ATTO Light-Capture II (ATTO, Tokyo, Japan). The signal
intensities were quantitated using the CS Analyzer, version 3.0
software (ATTO).
3. Results and Discussion
3.1. Evaluation of POFUT1 expression in OSCC cell lines
Our previous microarray data indicated that POFUT1 was
one of the most markedly up-regulated genes in OSCC cell
lines [29]. To assess POFUT1 protein expression as well as
mRNA, we performed qRT-PCR and immunoblotting analyses
using five OSCC-derived cell lines (cell line-1, -2, -3, -4, and
-5)). POFUT1 mRNA was significantly (P<0.05) up-regulated
in all OSCC cell lines compared to the HNOKs. Representative
results of the immunoblotting analysis showed that a significant
increase in POFUT1 protein, 44 kDa, was observed in all
OSCC cell lines compared to the HNOKs. These results
suggested that POFUT1 would be a critical tumor marker in
OSCC cell lines.
2.4. Immunohistochemistry (IHC)
IHC of 4-µm sections of paraffin-embedded specimens was
performed using rabbit anti-human POFUT1 polyclonal
antibody. Upon incubation with the primary antibody, the
specimens were washed three times in TBS and treated with
Envision reagent (DAKO, Carpinteria, CA,USA) followed by
color development in 3,3’-diaminobenzidine tetrahydrochloride
(DAB, DAKO). The slides then were counterstained lightly
with hematoxylin, dehydrated with ethanol, cleaned with
3.2. Evaluation of POFUT1 expression in primary OSCCs
Similar to the data from the OSCC cell lines, qRT-PCR
analysis showed that POFUT1 mRNA expression was
2
down-regulated in more than 80% of the primary OSCCs
compared to the matched normal oral tissues. We then
analyzed the POFUT1 protein expression in primary OSCCs
using the IHC scoring system. Representative IHC data for
POFUT1 protein showed that strong POFUT1
immunoreactivity was detected in the cytoplasm in OSCCs,
whereas the normal tissues showed almost negative
immunostaining.
The correlations between the tumor progression of the
patients with OSCC and the status of the POFUT1 protein
expression using the IHC scoring system were examined.
Among the clinical classifications, POFUT1-positive cases
exhibited more tumor progression compared with
POFUT1-negative cases (P<0.05). The POFUT1 IHC scores
of T3/T4 were much higher than those of T1/T2. These
results suggested a strong association between POFUT1
level and progression of OSCC .
3.3. Functional analysis of POFUT1 knockdown cells
Since POFUT1 expression was up-regulated in the
OSCC cell lines, we assumed that POFUT1 might play an
important role in OSCCs. To assess the POFUT1 functions
in OSCCs, shRNA experiment was carried out in two OSCC
cell lines. Expressions of POFUT1 mRNA and protein in
shPOFUT1 cells were significantly (P<0.05) lower than in
mock cells. We also performed cellular proliferation,
migration, and invasiveness assays to evaluate the biologic
effects of shPOFUT1 cells. Cellular proliferation assay
indicated that cell growth of shPOFUT1 decreased
compared with mock cells, whereas we did not find any
difference between POFUT1 knockdown and control cells
by migration and invasiveness assays. These results strongly
supported our present data that POFUT1-negative OSCC
cases related to small size of tumors.
Fig. 1. Hypothesis of POHUT1 in OSCC cells.
In cells expressing the POFUT1, the O-fucose is extended by
POFUT1 enzymatic activity, thereby altering the ability of
specific ligands to activate Notch. The Notch receptor is
activated by binding to a ligand presented by a neighboring cell.
γ-Secretase complex then cleaves the Notch transmembrane
domain to release the Notch intracellular domain (NICD).
NICD then enters the nucleus where it associates with the
DNA-binding protein to regulate several genes, including
oncogenes [32-34]. Therefore, POFUT1 is likely to be not only
a molecular marker for tumor growth but also an efficacious
treatment strategy for preventing progression in OSCCs.
Competing interests
The authors declare that they have no competing interests.
3.4. Hypothesis of the POHUT1 role in OSCC
Our hyposesis of the POFUT1 role in OSCC is showed
in Fig. 1. In cells expressing the POFUT1, the O-fucose is
extended by POFUT1 enzymatic activity, thereby altering
the ability of specific ligands to activate Notch. The Notch
receptor is activated by binding to a ligand presented by a
neighboring cell. γ-Secretase complex then cleaves the
Notch transmembrane domain to release the Notch
intracellular domain (NICD). NICD then enters the nucleus
where it associates with the DNA-binding protein to regulate
several genes, including oncogenes [32-34]. Therefore,
POFUT1 is likely to be not only a molecular marker for
tumor growth but also an efficacious treatment strategy for
preventing progression in OSCCs.
Acknowledgement
We thank Lynda C. Charters for editing the manuscript.
References
[1]
[2]
[3]
[4]
[5]
[6]
3
Macfarlane GJ, Zheng T, Marshall JR, et al. Alcohol,
tobacco, diet and the risk of oral cancer: a pooled analysis
of three case-control studies. Eur J Cancer B Oral Oncol
1995;31B: 181-7.
Fearon ER, Vogelstein B. A genetic model for colorectal
tumorigenesis. Cell 1990;61:759–67.
Mashberg A, Boffetta P, Winkelman R, et al. Tobacco
smoking, alcohol drinking, and cancer of the oral cavity
and oropharynx among U.S. veterans. Cancer
1993;72:1369–75.
Marshall CJ. Tumor suppressor genes. Cell
1991;64:313–26.
Severino P, Alvares AM, Michaluart P Jr, et al. Global
gene expression profiling of oral cavity cancers suggests
molecular heterogeneity within anatomic subsites. BMC
Res Notes 2008;1: 113-21.
Scully C, Field JK ,Tanzawa H. Genetic aberrations in
oral or head and neck squamous cell carcinoma
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
(SCCHN): 1. Carcinogen metabolism, DNA repair and
cell cycle control. Oral Oncol 2000;36: 256-63.
Scully C, Field JK , Tanzawa H. Genetic aberrations in
oral or head and neck squamous cell carcinoma 3:
clinico-pathological applications. Oral Oncol 2000;36:
404-13.
Sticht C, Freier K, Knöpfle K, et al. Activation of MAP
kinase signaling through ERK5 but not ERK1 expression
is associated with lymph node metastases in oral
squamous cell carcinoma (OSCC) . Neoplasia 2008;10:
462-70.
Wang Y, Lee GF, Kelley RF, et al. Identification of a
GDP-L-fucose: polypeptide fucosyltransferase and
enzymatic addition of O-linked fucose to EGF domains.
Glycobiology 1996;6: 837-42.
Schuster-Gossler K, Harris B, Johnson KR, et al. Notch
signalling in the paraxial mesoderm is most sensitive to
reduced POFUT1 levels during early mouse development.
BMC Dev Biol 2009;9:6.
Stanley P, Guidos CJ. Regulation of Notch signaling
during T- and B-cell development by O-fucose glycans.
Gurvich N, Perna F, Farina A, et al. L3MBTL1 polycomb
protein, a candidate tumor suppressor in del(20q12)
myeloid disorders, is essential for genome stability. Proc
Natl Acad Sci USA 2010;107: 22552-7.
Mackinnon RN, Selan C, Wall M, et al. The paradox of
20q11.21 amplification in a subset of cases of myeloid
malignancy with chromosome 20 deletion. Genes
Chromosomes Cancer 2010; 49: 998-1013.
Kroes RA, Dawson G, Moskal JR. Focused microarray
analysis of glyco-gene expression in human
glioblastomas. J Neurochem 2007;103 Suppl 1: 14-24.
Loo LW, Tiirikainen M, Cheng I, et al. Integrated
analysis of genome-wide copy number alterations and
gene expression in microsatellite stable, CpG island
methylator phenotype-negative colon cancer. Genes
Chromosomes Cancer 2013;52: 450-66.
Koike H, Uzawa K, Grzesik WJ, et al. GLUT1 is highly
expressed in cementoblasts but not in osteoblasts.
Connect Tissue Res 2005;46: 117-24.
Kasamatsu A, Uzawa K, Nakashima D,
et al.
Galectin-9 as a regulator of cellular adhesion in human
oral squamous cell carcinoma cell lines. Int J Mol Med
2005;16: 269-73.
Endo Y, Uzawa K, Mochida Y, et al. Sarcoendoplasmic
reticulum Ca(2+) ATPase type 2 downregulated in human
oral squamous cell carcinoma. Int J Cancer 2004;110:
225-31.
Sakuma K, Kasamatsu A, Yamatoji M, et al . Expression
status of Zic family member 2 as a prognostic marker for
oral squamous cell carcinoma. J Cancer Res Clin Oncol
2010;136: 553-9.
Yamatoji M, Kasamatsu A, Kouzu Y, et al.
Dermatopontin: a potential predictor for metastasis of
human oral cancer. Int J Cancer 2012;130: 2903-11.
Yamatoji M, Kasamatsu A, Yamano Y, et al. State of
homeobox A10 expression as a putative prognostic
marker for oral squamous cell carcinoma. Oncol Rep
2010;23: 61-7.
Kato Y, Uzawa K, Yamamoto N, et al. Overexpression
of Septin1: possible contribution to the development of
oral cancer. Int J Oncol 2007;31: 1021-8.
[23] Kouzu Y, Uzawa K, Koike H, et al. Overexpression of
stathmin in oral squamous-cell carcinoma: correlation
with tumour progression and poor prognosis. Br J Cancer
2006;94: 717-23.
[24] Iyoda M, Kasamatsu A, Ishigami T, et al. Epithelial cell
transforming sequence 2 in human oral cancer. PLoS One
2010;5: e14082.
[25] Onda T, Uzawa K, Nakashima D, et al.
Lin-7C/VELI3/MALS-3: an essential component in
metastasis of human squamous cell carcinoma. Cancer
Res 2007;67: 9643-8.
[26] Saito Y, Kasamatsu A, Yamamoto A, et al. ALY as a
potential contributor to metastasis in human oral
squamous cell carcinoma. J Cancer Res Clin Oncol
2013;139: 585-94.
[27] Koike K, Kasamatsu A, Iyoda M, et al. High prevalence
of epigenetic inactivation of the human four and a half
LIM domains 1 gene in human oral cancer. Int J Oncol
2013;42: 141-50.
[28] Usukura K, Kasamatsu A, Okamoto A, et al. Tripeptidyl
peptidase II in human oral squamous cell carcinoma. J
Cancer Res Clin Oncol 2013;139:123-30.
[29] Yamano Y, Uzawa K, Shinozuka K, et al.
Hyaluronan-mediated motility: a target in oral squamous
cell carcinoma. Int J Oncol 2008;32: 1001-9.
[30] Imam JS, Buddavarapu K, Lee-Chang JS, et al.
MicroRNA-185 suppresses tumor growth and progression
by targeting the Six1 oncogene in human cancers.
Oncogene 2010;29: 4971-9.
[31] Ogoshi K, Kasamatsu A, Iyoda M, et al. Dickkopf-1 in
human oral cancer. Int J Oncol 2011;39: 329-36.
[32] Raphael K, Ma.Xenia G. llagan: The canonical Notch
Signaling Pathway: Unfolding the Activation Mechanism.
Cell 2009;137: 216-33.
[33] Mark S, Uehara K, Changhui G, et al. Role of Pofut1 and
O-Fucose in Mammalian Notch Signaling. J. Biol. Chem
2008;283:13638-51.
[34] Shi S, Stanley P. Protein O-fucosyltransferase 1 is an
essential component of Notch signaling pathways. PNAS
2003;100:5234-9.
4
5