A gene signature predictive for outcome in advanced ovarian cancer identifies a survival factor: microfibril-associated glycoprotein 2

Abstract

Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. Here, we identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors. Independent evaluation confirmed the association of a prognostic gene microfibril-associated glycoprotein 2 (MAGP2) with poor prognosis, whereas in vitro mechanistic analyses demonstrated its ability to prolong tumor cell survival and stimulate endothelial cell motility and survival via the alpha(V)beta(3) integrin receptor. Increased MAGP2 expression correlated with microvessel density suggesting a proangiogenic role in vivo. Thus, MAGP2 may serve as a survival-associated target.

Figure 1. Identification and Validation of a Prognostic Gene Expression Signature Correlating with Survival in 53 Microdissected Late-Stage High-Grade Papillary Serous Ovarian Tumors

(A) Hierarchical clustering of 53 advanced stage, high-grade serous adenocarcinomas using expression values for genes possessing a Cox score >10 (gene expression: red, upregulated; blue, downregulated; survival: blue, short survival; red, long survival). (B) Genes presented in this table possessed a large Cox score (>10). Only the probe set with the highest Cox score is presented for MAGP2. (C) Kaplan Meier analysis of the predictor demonstrated a significant difference in survival time (p = 0.0029). (D) Kaplan Meier survival analysis of 49 patients using qRT-PCR validation data obtained for the top 11 survival-signature genes confirmed the two groups retained significantly different survival endpoints (p = 0.0107).

Figure 2. Assessment of Putative Signaling Events Contributing to Patient Survival

(A) Pathway analysis of select differentially regulated genes identified in the 53 microdissected serous tumors, as compared to 10 normal OSE brushings (red, upregulated; blue, downregulated; gray, no change; red halo, survival-associated gene). (B) Heat map demonstrating association between survival signature genes identified in the pathway and overall patient survival. Increased expression of one or more genes greatly reduced patient survival. (C) qRT-PCR validation of select genes identified in the pathway analysis for all 53 tumor samples and 10 normal OSE. Error bar represents mean fold change ± SD.

Figure 3. The MAGP2 Locus Is Amplified in Serous Ovarian Tumors and Its Expression Products Are Significantly Correlated with Overall Survival and Chemoresponse

(A) Oligonucleotide microarray CGH for 42 of the serous tumors established the median MAGP2 copy number (2.5) on chromosome 12p. MAGP2 is highlighted in red. (B) qPCR correlated with the CGH microarray data for 29 of the serous tumors (r = 0.61, p = 0.004). (C) Kaplan Meier survival analysis of MAGP2 mRNA expression using the 53 patients with stage III/IV high-grade serous ovarian cancer demonstrated a significant difference in patient outcome (p = 0.001). (D) Low-level MAGP2 protein staining was observed in normal surface epithelia (A), epithelial and stromal components of benign ovarian cysts (C), and in some high-grade serous tumors (B). Strong MAGP2 staining was observed in a small proportion of high-grade serous ovarian tumor tissues (D). Arrowheads indicate the epithelial layer of the normal ovary and benign ovarian cyst (S, stroma; T, tumor cells). (E) Kaplan Meier survival analysis of MAGP2 protein expression using 53 patients with stage III/IV high-grade serous ovarian cancer. A statistically significant difference was observed between the outcome groups (p = 0.05). (F) Independent TMA validation of MAGP2 expression in 64 serous ovarian cancers. A, high-level staining; B, moderate staining; C, low-level staining. (G) Box plot analysis of MAGP2 protein expression levels among chemotherapy responders and non-responders. A significant correlation was found between poor response and increased MAGP2 protein levels (p = 0.018).

Figure 4. MAGP2 Is Expressed in Ovarian Cancer Cells and Enhances Adhesion and Survival In Vitro

(A) Western blot analysis of MAGP2 protein isolated from 2 normal HOSE cultures and 12 ovarian cancer cell lines. (B) Flow cytometry analysis of α_Vβ₃ cell surface receptor levels in select high and low MAGP2-expressing cell lines using monoclonal antibodies against α_Vβ₃ or IgG₁ isotype control. (C) Silver staining for total protein obtained from yeast clone 10 supernatant identifies recMAGP2 as the principal secreted product following purification. (D) Increased adhesion of A224 ovarian cells in the presence of purified recMAGP2 (p < 0.02), with reduced adhesion following anti-α_Vβ₃ integrin antibody treatment (p < 0.01). (E) α_Vβ₃ integrin receptor-negative UCI107 cells displayed no significant increase in adhesion in the presence of recMAGP2. (F) OVCA429 cell viability was evaluated with increasing concentrations of recMAGP2. Increased cell survival was seen at higher concentrations of recMAGP2 reaching a maximum at 200 ng/ml (p = 0.004). For (E) and (F), data are the mean ± SD of three independent experiments. (G) Survival signaling in OVCA429 cells in response to treatment with recMAGP2. Differentially regulated genes were identified (red, upregulated; blue, downregulated; gray, no change).

Figure 5. RecMAGP2 Stimulates Adhesion, Survival, Motility, and Invasion of HUVECs In Vitro

(A) HUVECs cultured on recMAGP2 showed increased adhesion (p < 0.0002) when compared to anti-α_Vβ₃ integrin antibody preincubation. (B) Enhanced survival of HUVECs treated with recMAGP2 (p < 0.05). (C) Dose-dependent HUVEC motility in response to recMAGP2 protein was significant at all concentrations evaluated (p ≤ 0.01). (D) Anti-α_Vβ₃ integrin antibody attenuated re-cMAGP2-stimulated HUVEC motility through α_Vβ₃ receptor inhibition (p < 0.002), whereas anti-α₅ and anti-β₁ integrin antibody treatments did not. (E) Successful processing and secretion of wild-type supMAGP2 and RGD motif mutant proteins by 293T cells (1, signal sequence; 2, RGD motif; 3, N-linked carbohydrate consensus sequence; the underlined letter refers to the mutated RGD motif amino acid). (F) supMAGP2 stimulated HUVEC motility (p = 0.002), whereas the three mutant constructs had no significant effect. (G) Proper processing and secretion of wild-type OVsupMAGP2 protein by UCI107 ovarian cancer cells. (H) Ovarian cancer cell-derived OVsupMAGP2 induced HUVEC motility (p < 0.001). (I) Increased invasion of recMAGP2-treated HU-VECs into matrigel, as compared to untreated cells (p < 0.05). For (A)–(D), (F), (H), and (I), the data represents the mean ± SD of three independent experiments.

Figure 6. Identification of Putative Signaling Events Mediating the Effects of MAGP2 on HUVECs

(A) Hierarchical clustering of recMAGP2-treated (n = 3) and untreated (n = 3) HUVECs following supervised class comparison. The top 50 differentially regulated probe sets are displayed (red, upregulated; blue, downregulated). (B) Signaling events stimulated in HUVECs treated with recMAGP2 as compared to untreated cells. Differentially regulated genes were identified (red, upregulated; blue, downregulated; gray, no change). (C) recMAGP2 treatment of HUVECs increased the proportion of phosphorylated FAK, as compared to untreated cells. Error bar represents the area/second mean ± SD of three independent experiments. (D) Time course changes in [Ca²⁺] levels in a single recMAGP2 induced in a single HUVEC. (E) Mean normalized time course changes in [Ca²⁺] induced by recMAGP2 in HUVECs. (F) Mean normalized time course of changes in [Ca²⁺] induced by recMAGP2 in HUVECs pre-treated with GRGDSP competitor peptide.

Figure 7. MAGP2 Expression Correlates with Tumor Size and CD34⁺ Microvessel Density in Serous Ovarian Cancer Tumors and Tissue Specimens

(A) Western blot demonstrated knockdown of MAGP2 in five clones isolated from stably transfected SKOV3 cells, as well as three empty vector clones. (B) MAGP2 knockdown resulted in significantly decreased tumor size in mice. The median weight of tumors from SKOV3 cells is 0.18 g; knockdown of MAGP2 results in a significant decrease in tumor weight (0.02 g; p < 0.01). (C) MAGP2 knockdown resulted in decreased MAGP2 expression and CD34⁺ microvessel density in tumors developed in mice. Scale bars represent 50 μm. (D) A section of a high-grade serous adenocarcinoma showing strong MAGP2 expression in tumor cells (T) but not in the stromal component (S) or the endothelial cells (arrowheads). The scale bar represents 100 μm. (E) Immunolocalization of CD34⁺ microvessels in a human serous ovarian adenocarcinoma tumor section. (F) MAGP2 expression in a late-stage high-grade human serous ovarian adenocarcinoma tumor section. (G) Correlation between CD34⁺ and MAGP2 expression.