Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1

Abstract

Frequent metastasis to the cervical lymph nodes leads to poor survival of patients with oral squamous cell carcinoma (OSCC). To understand the underlying mechanisms of lymph node metastasis, two sublines were successfully isolated from cervical lymph nodes of nude mice through in vivo selection, and identified as originating from poorly metastatic parental cells. These two sublines specifically metastasized to cervical lymph nodes in 83% of mice, whereas OEC-M1 cells did not metastasize after injection into the oral cavity. After gene expression analysis, we identified insulin-like growth factor binding protein 3 (IGFBP3) as one of the significantly up-regulated genes in the sublines in comparison with their parental cells. Consistently, meta-analysis of the public microarray datasets and IGFBP3 immunohistochemical analysis revealed increased both levels of IGFBP3 mRNA and protein in human OSCC tissues when compared to normal oral or adjacent nontumorous tissues. Interestingly, the up-regulated IGFBP3 mRNA expression was significantly associated with OSCC patients with lymph node metastasis. IGFBP3 knockdown in the sublines impaired and ectopic IGFBP3 expression in the parental cells promoted migration, transendothelial migration and lymph node metastasis of orthotopic transplantation. Additionally, ectopic expression of IGFBP3 with an IGF-binding defect sustained the IGFBP3-enhanced biological functions. Results indicated that IGFBP3 regulates metastasis-related functions of OSCC cells through an IGF-independent mechanism. Furthermore, exogenous IGFBP3 was sufficient to induce cell motility and extracellular signal-regulated kinase (ERK) activation. The silencing of integrin β1 was able to impair exogenous IGFBP3-mediated migration and ERK phosphorylation, suggesting a critical role of integrin β1 in IGFBP3-enchanced functions.

Laminin γ2‐enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3‐dependent uptake by lymphatic endothelial cells

“…LN1‐1 cells, derived from OEC‐M1 cells by in vivo selection have elevated abilities of lymph node metastasis and lymphangiogenesis compared to parental OEC‐M1 cells . LEC migration was enhanced when LECs were co‐cultured with LN1‐1 cells versus OEC‐M1 cells (Fig.…”

“…Orthotopic tumor growth assays were conducted as described previously . The detailed procedures are described in Supporting Information materials and methods.…”

“…To understand the precise molecular mechanisms of lymph node metastasis, two independent OSCC sublines were derived from the parental OSCC cells by in vivo selection . These sublines demonstrated stronger capacities for lymphangiogenesis; however, it remains unknown if OSCC cell‐derived EVs play roles in the interaction between OSCC cells and LECs.…”

“…(G) The relative LAMC2 , LAMA3 , and LAMB3 expressions were determined by dividing the detected signal from LN1‐1 sublines by that from their parental OEC‐M1 cells. The data were obtained by averaging different probes from the dataset GSE62326 . Bar, SE; *p < 0.05; **p < 0.01; ***p < 0.001.…”

Endothelial cell colony forming units derived from malignant breast diseases are resistant to tumor necrosis factor-α-induced apoptosis

“…The procedures used in this analysis have been previously described34. Briefly, 500 ng of the total RNA were reverse transcribed and subjected to second-strand cDNA synthesis, followed by in vitro transcription and complementary RNA labelling with biotin-dUTP.…”