Lung cancer osteopontin isoforms exhibit angiogenic functional heterogeneity

Abstract

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.

Figure 1

OPN mRNA expression in human lung cancer and immortalized bronchial epithelial cells. Primers amplify each of the three isoforms which are then separated by electrophoresis on 2% agarose gel. OPNa produces the 305 bp dominant band seen at the top. OPNb amplifies a 263 bp fragment seen below the dominant band in A549, H157, Calu-3, H460, and H1299. OPNc amplifies a 224 bp fragment that migrates below the OPNb and is not seen in these cell lines. Amplification of pcmv2 plasmid specific to each isoform serves as positive controls and amplification of PPIA serve as control for equal loading. OPN expression correlates with OPN secretion as detected by ELISA after 72 hours in serum-free media. (BrEpC: immortalized bronchial epithelial cells, adeno: adenocarcinoma, SCLC: small cell NSCLC, NSCLC: non-small cell NSCLC, PPIA:peptidylprolyl isomerase A).

Figure 2

RT-PCR demonstrating forced expression of individual OPN isoforms in four NSCLC cell lines. Pooled populations of transfected cells were used in RT-PCR and angiogenesis assay. H153 and H358 have low native OPN expression. A549 and H60 have high native OPN expression.

Figure 3

Representative sections showing tubule formation and mean tubule lengths for BCE’s plated on Matrigel™ and cultured in conditioned media collected from NSCLC cell lines transfected with the individual OPN isoforms and pCMV empty vector controls. H153 and H358 have low native OPN expression, and A549 and H460 have high native OPN expression. OPNa overexpression in all cell lines resulted in significantly more tubule formation compared to controls. OPNb overexpression also significantly increased tubule length in all cell lines compared to controls, but to a lesser extent then OPNa. OPNc overexpression resulted in a significant decrease in tubule formation compared to controls in all four NSCLC cell lines.

Figure 4a

Bar graft depicting the effect of the overexpression of individual OPN isoforms compared to pCMV empty vector controls on mean BCE tubule length (μ) in four NSCLC cell lines. Asterisk denotes a significant difference between OPN isoform overexpression in cell lines and pCMV controls. Mean tubule length ± standard deviation and p-value are listed below bar graft. H153 and H358 wild type cells had significantly shorter BCE tubule lengths than A549 and H460, which have high native OPN expression. Tubule formation was significantly increased in all cell lines with forced overexpression of OPNa and OPNb compared to controls. Conversely, overexpression of OPNc resulted in significantly fewer tubules formed compared to controls in all cell lines.

Figure 4b

Bar graft depicting associated changes in VEGF secretion (pg/ml) with overexpression of individual OPN isoforms compared to pCMV empty vector controls on in four NSCLC cell lines. Asterisk denotes a significant difference between OPN isoform and pCMV controls. Mean VEGF concentrations ± standard deviation and p-value are listed below bar graft. H153 and H358 have low native OPN expression and A549 and H460 have high native OPN expression. Significant increases in VEGF secretion were associated with OPNa overexpression compared to controls. Conversely, OPNc overexpression was associated with in significant decreases in VEGF secretion compared to controls in all cell lines. OPNb overexpression was associated with no significant change in VEGF secretion in any of the four cell lines.

Figure 4c

Bar graft depicting the effect of the overexpression of individual OPN isoforms compared to pCMV empty vector controls on OPN secretion (pg/ml) in four NSCLC cell lines. Asterisk denotes a significant difference between OPN isoform overexpression in cell lines and pCMV controls. Mean OPN concentrations ± standard deviation and p-value are listed below bar graft. In H153, H358 and H460 overexpression of OPNa and OPNb had no significant impact on OPN protein concentrations in conditioned media. In A549 overexpression of OPNa and OPNb caused significant step-up in OPN protein secretion. Overexpression of OPNc caused a significant increase in OPN secretion in all cell lines.OPN protein is measured by ELISA which is not isoform specific.

Figure 5

Gene array analysis demonstrated OPNa overexpression was associated with a two-fold increase in relative expression of VEGF while OPNc overexpression was associated with a 40% decrease in VEGF expression compared to controls. RT-PCR validation of gene array with regard to divergent VEGF expression with overexpression of the OPN isoforms is demonstrated here. Overexpression of OPNa in all four cell lines resulted in elevated VEGF mRNA expression, while OPNc overexpression decreased VEGF expression in all cell lines compared to wild type cells. Elevated VEGF mRNA expression was appreciated in control cell lines with high native OPN (A549 and H460) compared to cell lines with low native OPN expression (H153 and H358).