An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo.

Abstract

Purpose: 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. Experimental Design: 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.

Significance of nerve growth factor overexpression and its autocrine loop in oesophageal squamous cell carcinoma

“…et al, 1999; Ito et al, 2006). Thus far, it has been revealed that HSA/c produced and secreted quite high amounts of NGF ( Figure 3B-E).…”

Genomewide mRNA profiling of esophageal squamous cell carcinoma for identification of cancer biomarkers

“…Based on staining intensity, we have classified 118 ESCC cases into four subgroups: 3.38% (4/118) tumors with negative expression, 4.23% (5/118,+) tumors with weak expression, 22.03% (26/118,++) tumors with moderate expression, and 70.33% (83/118,+++) tumors with strong expression. Earlier studies done on ESCC using immunohistochemical labeling reported overexpression of OPN in 15% (22/144,+++) 34 or 48% (84/175) of ESCC cases. 59 In concordance with earlier studies, we could identify overexpression of OPN in 97% of the cases in our study which proves OPN could be used as a biomarker in ESCC.…”

Osteopontin, E-cadherin, and β-catenin expression as prognostic biomarkers in patients with radically resected gastric cancer

“…When the opinions of the two evaluators were different, consensus was reached by discussion. The degree of cytoplasmatic immunoreactivity for osteopontin was evaluated according to the modified method proposed by Ito et al [15]. Staining was evaluated in ten fields per section at 9200 magnification of two slides from each specimen to classify samples into three groups according to the osteopontin expression intensity: 0/1?…”