Platelet-derived growth factor receptor alpha (PDGFRα) targeting and relevant biomarkers in ovarian carcinoma

Abstract

Objective: Platelet-derived growth factor receptor alpha (PDGFRα) is believed to be associated with cell survival. We examined (i) whether PDGFRα blockade enhances the antitumor activity of taxanes in ovarian carcinoma and (ii) potential biomarkers of response to anti-PDGFRα therapy.

Methods: PDGFRα expression in 176 ovarian carcinomas was evaluated with tissue microarray and correlated to survival outcome. Human-specific monoclonal antibody to PDGFRα (IMC-3G3) was used for in vitro and in vivo experiments with or without docetaxel. Gene microarrays and reverse-phase protein arrays with pathway analyses were performed to identify potential predictive biomarkers.

Results: When compared to low or no PDGFRα expression, increased PDGFRα expression was associated with significantly poorer overall survival of patients with ovarian cancer (P=0.014). Although treatment with IMC-3G3 alone did not affect cell viability or increase apoptosis, concurrent use of IMC-3G3 with docetaxel significantly enhanced sensitization to docetaxel and apoptosis. In an orthotopic mouse model, IMC-3G3 monotherapy had no significant antitumor effects in SKOV3-ip1 (low PDGFRα expression), but showed significant antitumor effects in HeyA8-MDR (high PDGFRα expression). Concurrent use of IMC-3G3 with docetaxel, compared with use of docetaxel alone, significantly reduced tumor weight in all tested cell lines. In protein ontology, the EGFR and AKT pathways were downregulated by IMC-3G3 therapy. MAPK and CCNB1 were downregulated only in the HeyA8-MDR model.

Conclusion: These data identify IMC-3G3 as an attractive therapeutic strategy and identify potential predictive markers for further development.

Keywords: EGFR; IMC-3G3; MAPK; Ovarian cancer; Platelet-derived growth factor receptor alpha.

Fig. 1

PDGFRα expression and clinical outcomes of ovarian carcinoma. A) Proportion of PDGFRα-expressing tumors in serous ovarian carcinoma. B) Proportion of expressed protein PDGFRα and FIGO stage. C) Proportion of protein PDGFRα-expressing tumors in ovarian carcinoma and disease recurrence. D) Overall survival and PDGFRα protein expression. Red, protein PDGFRα-expressing tumors; blue, protein PDGFRα non-expressing tumors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

PDGFRα blockade and docetaxel sensitization in ovarian carcinoma. A) PCR analysis for 9 ovarian carcinoma cell lines. B) Western blot of 10 ovarian carcinoma cell lines. Densitometry was used for quantification of PDGFRα expression. Bars represent the ratio of PDGFRα to 2774, the cell line that showed the least PDGFRα expression among tested cell lines. C) Neutralization of phosphorylation of PDGFRα with IMC-3G3 (100 μg/mL) demonstrated in SKOV3-ip1 cells. D) Neutralization of PDGFRα phosphorylation with IMC-3G3 (40 mg/kg/mouse, i.p.) in HeyA8 tumor-bearing mice (n = 3 per group). Mice were killed at 12 and 48 h after the second injection of IMC-3G3. E) Cell viability assay for IMC-3G3 monotherapy in 5 ovarian carcinoma cell lines. Treatment was given for 72 h. No change in cell viability was seen in any of the cell lines tested. F) Cell viability assay for the combination treatment with docetaxel and IMC-3G3 in the ES2 cell line. Treatment was given for 72 h. Sensitization to docetaxel was shown to be dose-dependent with IMC-3G3. G–K) Cell viability assay results for 5 different cell lines treated with docetaxel alone (solid line) and docetaxel with IMC-3G3 (100 μg/mL, dashed line). All cell lines except 2774 showed statistically significant sensitization to docetaxel with IMC-3G3 co-treatment. L) Correlation between the extent of PDGFRα protein expression and the extent of docetaxel sensitization; the correlation was statistically significant (r = 0.94, P < 0.01). M–N) Apoptosis assay for docetaxel and IMC-3G3 is shown at the 72-hour time point in SKOV3-ip1 (M) and HeyA8 (N). The proportion of total apoptosis was determined by flow cytometry. Although there was no change in the proportion of apoptosis in IMC-3G3 monotherapy, apoptosis in docetaxel used with IMC-3G3, compared with apoptosis in docetaxel used alone, increased significantly (* p < 0.05, ** p < 0.01). The dot and error bar represent the mean with SE in (E) and (G) through (K). The bar represents the mean with SE in (F), (M), and (N).

Fig. 3

Antitumor effects of PDGFRα blockade in ovarian carcinoma. A) In vivo therapy experiments for 4 different ovarian carcinoma cell lines. Treatment was given for 5–6 weeks (weekly docetaxel i.p. and IMC-3G3 three times a week i.p.). A short-term (2-week) experiment was conducted in the ES2 cell line. ^†Significant antitumor effect with IMC-3G3 monotherapy was seen in HeyA8, HeyA8-MDR, and ES2. No antitumor effect was seen with IMC-3G3 monotherapy in SKOV3-ip1 tumor-bearing mice. *Significant sensitization of docetaxel antitumor effect was seen in all 4 tested cell lines with co-administration of IMC-3G3 and docetaxel. The bar represents mean with SE. B) Salvage therapy experiment with drug-resistant ovarian carcinoma cell line (HeyA8-MDR-Luc). Treatment was initiated 17 days after cell injection. Borderline significance was seen in the IMC-3G3 monotherapy group as well as in the docetaxel with IMC-3G3 treatment group when compared with the control group (P = 0.07 and .054, respectively). Error bars represent SE. C) 3D tumor vascular imaging is shown in HeyA8-MDR tumor. Docetaxel and IMC-3G3 combination therapy was given for treatment tumor.

Fig. 4

Reverse-phase protein array for PDGFRα blockade in ovarian carcinoma. Heatmap of reverse-phase protein array (RPPA) results in HeyA8-MDR (PDGFRα-sensitive cell line) treated with PDGF-AA and IMC-3G3 at an early time point (6 h) and late time point (24 h).

Fig. 5

PDGFRα blockade and protein interactions in ovarian carcinoma. A–B) Network ontology of proteins (A) increasing the expression with PDGF-AA treatment and neutralized with IMC-3G3 pretreatment, and (B) not responding to IMC-3G3 in the HeyA8-MDR cell line. C–D) Network ontology of proteins (C) increasing the expression with PDGF-AA treatment and neutralized with IMC-3G3 pretreatment, and (D) not responding to IMC-3G3 in the SKOV3-ip1 cell line. Proteins with parentheses (p) represent phosphoproteins.

Fig. 6

PDGFRα targeting and relevant biomarkers in ovarian carcinoma. A–B) Western blot analysis for 12 antibodies is shown for HeyA8-MDR (A) and SKOV3-ip1 (B). Experiment time point was at 24 h. IgG Ab, human immunoglobulin (100 μg/mL). C–D) Relative expression of protein in Western blot analysis compared with the control group of each targeted biomarker. ImageJ was used for densitometry measurement and standardized with the control group. Protein activity was evaluated for the phosphoprotein/total protein ratio in PDGFRα, AKT, MAPK, and FAK. C) Proteins that showed a common response to IMC-3G3 therapy included PDGFRα, EGFR, and AKT. D) Protein that showed different responses between HeyA8-MDR and SKOV3-ip1 in IMC-3G3 therapy included MAPK, FAK, and CCNB1.