Lung cancer osteopontin isoforms exhibit angiogenic functional heterogeneity

Objective

Osteopontin is a multifunctional phosphoprotein with an important but poorly understood role in non–small cell lung cancer pathogenesis. We hypothesize that osteopontin isoforms (OPNa, OPNb, and OPNc) have divergent roles in non–small-cell lung cancer angiogenesis and divergent impact on vascular endothelial growth factor secretion.

Methods

We examined mRNA expression using reverse transcriptase-polymerase chain reaction primers for 3 osteopontin isoforms in non–small-cell lung cancer and immortalized bronchial epithelial cell lines, and correlated expression with osteopontin secretion into media detected by enzyme-linked immunosorbent assay. Angiogenic properties conferred by osteopontin isoforms were evaluated by transfecting cDNA plasmids specific to each isoform and controls into non–small-cell lung cancer cell lines, H153 and H358 (low endogenous osteopontin) and A549 and H460 (high endogenous osteopontin), analyzing conditioned media on a bovine capillary endothelial platform, and measuring vascular endothelial growth factor levels by enzyme-linked immunosorbent assay.

Results

OPNa mRNA expression correlated with osteopontin secretion in cell lines (r = 0.912, P = .0006). OPNa overexpression significantly increased tubule length compared with controls, OPNb had a similar, but less pronounced effect, and OPNc significantly decreased tubule length compared with controls in each cell line. OPNa overexpression was associated with significant increases in vascular endothelial growth factor secretion, whereas OPNb had no effect and OPNc overexpression was associated with significant decreases in vascular endothelial growth factor compared with controls in each cell line.

Conclusion

We demonstrated divergent effects of osteopontin isoforms on non–small-cell lung cancer angiogenesis and vascular endothelial growth factor secretion. OPNa overexpression was associated with increased bovine capillary endothelial tubule length and vascular endothelial growth factor secretion, whereas OPNc was associated with decreases in both. These findings may lead to therapeutic strategies for selective isoform inhibition in non–small cell lung cancer.

CTSNet classification: 9, 10, 10.1, 29

Abbreviations and Acronyms: BCE, bovine capillary endothelial, bp, base pair, DMEM, Dulbecco's Modified Eagle Medium, ELISA, enzyme-linked immunosorbent assay, NSCLC, non–small-cell lung cancer, OPN, osteopontin, RT-PCR, reverse transcriptase-polymerase chain reaction, VEGF, vascular endothelial growth factor