IGFBP ratio confers resistance to IGF targeting and correlates with increased invasion and poor outcome in breast tumors

Abstract

Purpose: To improve the significance of insulin-like growth factor-binding protein 5 (IGFBP-5) as a prognostic and potentially predictive marker in patients with breast cancer.

Experimental design: Increased IGFBP-5 expression was identified in MCF-7 cells resistant (MCF-7R4) to the IGF-1R/insulin receptor (InsR) inhibitor BMS-536924 and its role examined by targeted knockdown and overexpression in multiple experimental models. Protein expression of IGFBP-5 was measured by immunohistochemistry in a cohort of 76 patients with breast cancer to examine correlative associations with invasive tumor fraction and outcome. The use of a combined IGFBP-5/IGFBP-4 (BPR) expression ratio was applied to predict anti-IGF-1R/InsR response in a panel of breast cancer lines and outcome in multiple breast tumor cohorts.

Results: IGFBP-5 knockdown decreased BMS-536924 resistance in MCF-7R4 cells, whereas IGFBP-5 overexpression in MCF-7 cells conferred resistance. When compared with pathologically normal reduction mammoplasty tissue, IGFBP-5 expression levels were upregulated in both invasive and histologically normal adjacent breast cancer tissue. In both univariate and multivariate modeling, metastasis-free survival, recurrence free survival (RFS), and overall survival (OS) were significantly associated with high IGFBP-5 expression. Prognostic power of IGFBP-5 was further increased with the addition of IGFBP-4 where tumors were ranked based upon IGFBP-5/IGFBP-4 expression ratio (BPR). Multiple breast cancer cohorts confirm that BPR (high vs. low) was a strong predictor of RFS and OS.

Conclusion: IGFBP-5 expression is a marker of poor outcome in patients with breast cancer. An IGFBP-5/IGFBP-4 expression ratio may serve as a surrogate biomarker of IGF pathway activation and predict sensitivity to anti-IGF-1R targeting.

Figure 1

IGFBP-5 confers resistance to IGF-1R/InsR inhibition. (A) Comparison of IGF pathway genes in MCF-7 (white bars) vs MCF-7R4 (black bars) cells (B) Western Blot analysis of IGFBP-5 expression in MCF-7 vs. MCF-7R4 cells (C) Immunohistochemistry staining for the nucleus (blue) and IGFBP-5 (red) in MCF-7 (left column) and MCF-7R4 (right column) cells. Colonies (top row) and single cells (bottom row) are shown at different magnifications. (D) Depiction of IGFBP-5 knockdown as measured by qPCR (left) and resultant growth (right) in MCF-7R4 cells. (E) MCF-7 cells overexpressing empty vector (open circles) or IGFBP-5 (closed circles) were exposed to increasing concentrations of BMS-536924 and assessed for colony outgrowth. (F) Growth assessment of MCF-7 cells in response to BMS-536924 (10uM) treatment and addition of recombinant IGFBP-5. (G) Dose response to IGF-1 and LR3-IGF-1 and resulting growth response of MCF-7/Vector (open) vs. MCF-7/IGFBP-5 (closed). (H) Growth response of MCF-7 vs. MCF-7R4 cells to LR3-IGF-1 (10nM), insulin (10nM), Heregulin (10nM), and EGF (10nM). Error bars represent standard deviation, asterisks denote statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001, and results are representative of at least three independent replicates.

Figure 2

IGFBP-5 expression correlates with increased invasion and poor outcome. (A) Breast cancer tumor sections were stained for IGFBP-5 in a cohort of patients from the Mayo Clinic (n=76), and an immunoscore assigned (0 = no expression to 5 = highest expression). (B) Box and whisker plot comparing tumors with high (≥70%) vs. low (<70%) invasive fractions. Statistical analysis was performed by Mann-Whitney test and p-value included. (C) Univariate Kaplan-Meier analysis of metastasis-free survival (MFS), recurrence-free survival (RFS), and overall survival (OS) in the Mayo cohort stratified by high (≥3, red line) vs. low (≤3, blue line) IGFBP-5 immunoscore. (D) IGFBP transcript expression in breast tissues obtained from standard reduction surgery (white bars), invasive breast carcinoma (red bars), and histologically normal adjacent breast tissue (black bars). (E) IGFBP-5/IGFBP-4 expression directly correlates to anti-IGF1R/InsR activity in a panel of breast cancer cell lines. IC₅₀ is plotted alongside the IGFBP-5/IGFBP-4 expression value for each cell line. Pearson correlation shows there is a significant correlation for both BMS-536924 (r = 0.8225, P = 0.0035) and BMS-754807 (r = 0.9452, P < 0.0001) with a higher expression ratio being associated with a decreased response to both agents. Error bars represent standard deviation, asterisks denote statistical significance; ***, P < 0.01; ****, P < 0.0001.

Figure 3

Forest plot of RFS (open) and OS (filled) by BPR expression in multiple breast tumor cohorts, showing HRs (BPR Low/High) and 95% CIs where a HR >1 implies a higher risk of recurrence and mortality in the BPR High group.

Figure 4

Kaplan Meier analysis of the combined cohorts depicting RFS (A) and OS (B) in the BPR Low (gray line) vs. BPR High (black line) groups.