1Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan
2Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan
3Department of Clinical Oncology, Graduate School of Medicine, Chiba University, Japan *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
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
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 . Recent studies have reported that POFUT1 affected anterior-posterior somite patterning in mammalian embryos  and regulated T-, myeloid-, and B-lineage differentiation in mammals . 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 on these data, we proposed that POFUT1 might be a key regulator of tumor progression in OSCCs.
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.
2.2.mRNA expression analysis
Total RNA was isolated from cells using Trizol Reagent (Invitrogen, Carlsbad, CA,USA) and was reverse-transcribed by Ready-to-Go You-Prime first-strand beads (GE Healthcare, Little Chalfont, UK) and Oligo (dT) primer (Invitrogen). Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to evaluate the expression level of POFUT1 mRNA in the OSCC cell lines and HNOKs. The expression levels were determined using primers and probes that were designed using the Universal Probe Library (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s instructions. The primer sequences for POFUT1 were forward 5’-CTGATGACCCGATGGTAAGC-3’; reverse 5’-AAGCCTCCTTTCACCAACCT-3’; and universal probe #78. All qRT-PCR analyses were performed using a LightCycler® 480 PCR system (Roche Diagnostics GmbH). The transcript amount for POFUT1 was estimated from the respective standard curves and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5’-CATCTCTGCCCCCTCTGCTGA-3’; reverse, 5’-GGATGACCTTGCCCACAGCCT-3’; and universal probe #60) transcript amount determined in corresponding samples.
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).
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 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.
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].
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 . 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.
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 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.
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.
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.
The authors declare that they have no competing interests.
We thank Lynda C. Charters for editing the manuscript.
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