Expression of Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma.
BACKGROUND
The side population (SP) of cancer cells is reportedly enriched with cancer stem cells (CSCs), however, the functional role and clinical relevance of CSC marker molecules upregulated in the SP of head and neck squamous carcinoma (HNSCC) cells are yet to be elucidated. Patients with clinical stage I/II (T1-2N0M0) tongue squamous cell carcinoma (TSCC) typically undergo partial glossectomy; however, development of delayed neck metastasis (DNM) tends to reduce their survival. In the present study, we aimed to determine the CSC markers in the SP of HNSCC cells along with their functions in cellular behaviors, and to clarify the association of these markers with DNM.


METHODS
Flow cytometry was applied to isolate SP from main population (MP) in HNSCC cells. The expression of the CSC markers was examined by semi-quantitative RT-PCR and immunocytochemistry. In vitro proliferation, migration, and invasion assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Oct3/4 and Nanog were evaluated using surgical specimens from 50 patients with stage I/II TSCC.


RESULTS
SPs were isolated in all three cell lines examined. Expression levels of Oct3/4 and Nanog were higher in SP cells than MP cells. Additionally, cell migration and invasion abilities were higher in SP cells than MP cells, whereas there was no difference in proliferation. Univariate analysis showed that expression of Oct3/4 and Nanog, vascular and muscular invasion, and mode of invasion were significantly correlated with DNM. Multivariate logistic regression revealed that Oct3/4 expression (risk ratio = 14.78, p = 0.002) and vascular invasion (risk ratio = 12.93, p = 0.017) were independently predictive of DNM. Regarding the diagnostic performance, Oct3/4 showed the highest accuracy, sensitivity, and NPV of 82.0 %, 61.5 %, and 86.8 %, respectively, while vascular invasion showed the highest specificity and PPV of 94.6 % and 71.4 %, respectively.


CONCLUSIONS
These results suggest that Oct3/4 and Nanog represent probable CSC markers in HNSCC, which contribute to the development of DNM in part by enhancing cell motility and invasiveness. Moreover, along with vascular invasion, expression of Oct3/4 can be considered a potential predictor for selecting patients at high risk of developing DNM.
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. habunoboru@gmail.com.|Department of Otorhinolaryngology, Tachikawa Hospital, Tokyo, Japan. habunoboru@gmail.com.
Noboru
N
Habu
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. yorihisa@ja2.so-net.ne.jp.
Yorihisa
Y
Imanishi
Department of Pathology, Keio University School of Medicine, Tokyo, Japan. kameyama@a5.keio.jp.
Kaori
K
Kameyama
Department of Pathology, Keio University School of Medicine, Tokyo, Japan. shimoda@a2.keio.jp.
Masayuki
M
Shimoda
Department of Otorhinolaryngology, National Tokyo Medical Center, Tokyo, Japan. toku@sb4.so-net.ne.jp.
Yutaka
Y
Tokumaru
Department of Otorhinolaryngology, Saiseikai Utsunomiya Hospital, Utsunomiya, Japan. kojisakamoto355@yahoo.co.jp.
Koji
K
Sakamoto
Department of Otorhinolaryngology, Saiseikai Yokohamashi Nanbu Hospital, Yokohama, Japan. ryoichi421@nifty.com.
Ryoichi
R
Fujii
Department of Otorhinolaryngology, Yokohama Municipal Citizen's Hospital, Yokohama, Japan. shigetomi@mva.biglobe.ne.jp.
Seiji
S
Shigetomi
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. rynonic@jt9.so-net.ne.jp.
Kuninori
K
Otsuka
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. y-ichiro-orlhns@hotmail.co.jp.
Yoichiro
Y
Sato
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. ywatanabefhu@gmail.com.
Yoshihiro
Y
Watanabe
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. ozakkyy123@gmail.com.
Hiroyuki
H
Ozawa
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. tomitamt@gmail.com.
Toshiki
T
Tomita
National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan. fujiimasato@kankakuki.go.jp.
Masato
M
Fujii
Department of Otorhinolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, 160-8582, Shinjuku, Tokyo, Japan. ogawak@a5.keio.jp.
Kaoru
K
Ogawa
D015415:Biomarkers; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D008297:Male; D000071317:Nanog Homeobox Protein; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D012307:Risk Factors; D058985:Side-Population Cells / Q000378:metabolism; D000077195:Squamous Cell Carcinoma of Head and Neck; D014062:Tongue Neoplasms / Q000235:genetics* / Q000473:pathology*
D015415:Biomarkers; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger