An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo.
OBJECTIVE
To elucidate the clinical significance of osteopontin and the effect of conditional down-regulation of osteopontin in esophageal squamous cell carcinoma (ESCC), we investigated osteopontin expression in tumors and tested an inducible osteopontin-short-hairpin RNA (shRNA) expression vector in an ESCC cell line.


METHODS
Osteopontin mRNA expression was extracted from gene expression profiles of 23 tumors determined by cDNA microarray and was analyzed. Paraffin sections of 144 tumors were immunohistochemically investigated. Osteopontin protein expression in 34 cell lines was examined by Western blot. A doxycycline-inducible osteopontin-shRNA vector was stably transfected into HSA/c cells to assess the role of osteopontin in cell motility, invasion in vitro, tumor formation, and lymph node metastasis in nude mice.


RESULTS
cDNA microarray revealed that high osteopontin mRNA expression was associated with poor survival of ESCC patients (P = 0.029). In immunohistochemistry, osteopontin protein expression was associated with poor prognosis (P < 0.001), distant lymph node metastasis (P = 0.0004), tumor staging (P = 0.027), and histologic grade (P = 0.024). Multivariate analysis showed that osteopontin overexpression was the strongest independent prognostic factor among nine clinicopathologic variables (P < 0.001). Among cell lines tested, 30 had overexpressed osteopontin protein compared with a normal esophageal epithelial cell line. An inducible shRNA vector against osteopontin successfully down-regulated osteopontin expression by 71% to 88% and repressed cell motility by 69% to 97%, cell invasion by 59% to 71%, tumor formation by 56% to 92%, and lymph node metastasis by 50% to 67% in HSA/c cells.


CONCLUSIONS
Our findings suggest that osteopontin overexpression may play an important role in progression of ESCC and osteopontin could be a potential target of ESCC therapy.
Ito
Tetsuo
T
Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Hashimoto
Yosuke
Y
Tanaka
Eiji
E
Kan
Takatsugu
T
Tsunoda
Shigeru
S
Sato
Fumiaki
F
Higashiyama
Motoshige
M
Okumura
Tomoyuki
T
Shimada
Yutaka
Y
D000284:Administration, Oral; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004318:Doxycycline / Q000008:administration & dosage / Q000494:pharmacology / Q000627:therapeutic use; D004938:Esophageal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008207:Lymphatic Metastasis / Q000235:genetics / Q000517:prevention & control; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D053495:Osteopontin; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012795:Sialoglycoproteins / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D004318:Doxycycline