Insulin growth factor-binding protein 2 is a candidate biomarker for PTEN status and PI3K/Akt pathway activation in glioblastoma and prostate cancer

Abstract

PTEN is an important tumor-suppressor gene associated with many cancers. Through expression profiling of glioblastoma tissue samples and prostate cancer xenografts, we identified a molecular signature for loss of the PTEN tumor suppressor in glioblastoma and prostate tumors. The PTEN signature consists of a minimum of nine genes, several of which are involved in various pathways already implicated in tumor formation. Among these signature genes, the most significant was an increase in insulin growth factor-binding protein 2 (IGFBP-2) mRNA. Up-regulation of IGFBP-2 was confirmed at the protein level by Western blot analysis and validated in samples not included in the microarray analysis. The link between IGFBP-2 and PTEN was of particular interest because elevated serum IGFBP-2 levels have been reported in patients with prostate and brain tumors. To further investigate this link, we determined that IGFBP-2 expression is negatively regulated by PTEN and positively regulated by phosphatidylinositol 3-kinase (PI3K) and Akt activation. In addition, Akt-driven transformation is impaired in IGFBP2(-/-) mouse embryo fibroblasts, implicating a functional role for IGFBP-2 in PTEN signaling. Collectively, these studies establish that PTEN and IGFBP-2 expression are inversely correlated in human brain and prostate cancers and implicate serum IGFBP-2 levels as a potential serum biomarker of PTEN status and PI3K Akt pathway activation in cancer patients.

Fig. 1.

Identification of a gene expression classifier of PTEN status. Microarrays were used to measure the expression profiles of 25 prostate cancer xenograft and glioblastoma samples, of which 11 were classified PTEN wild type and 14 were classified PTEN mutant. To relate PTEN status to the gene expression data, we used RF predictors and the Kruskal–Wallis test of differential expression. An RF importance measure was used to rank probe sets according to their prognostic importance for PTEN status. (A) The RF variable importance measure is highly correlated (r = 0.28) with a conventional measure of differential expression (minus log 10 of the Kruskal–Wallis test P value). In the SI Appendix, we also consider alternative variable importance measures and the Student t test. We find that our results are highly robust with respect to the gene screening method. (B) The error rate of the RF predictor as a function of the number of most important probe sets. We find that the 10 most important probe sets lead to an apparent error rate of zero. (C) Classical multidimensional scaling plot for visualizing the dissimilarities between the microarray samples (on the basis of the 10 most important probe sets). Prostate cancer samples are labeled “P,” and glioblastoma samples are labeled “B.” PTEN mutant samples are in orange, and wild type is in cyan (turquoise). The plot in C and the supervised hierarchical clustering plot (D) show that the 10 most important probe sets stratify the samples according to their PTEN status.

Fig. 2.

PTEN-null cancers express high levels of the secreted protein IGFBP-2. (A and B) IGFBP2 and PTEN protein levels are inversely correlated by Western blot analysis in previously characterized prostate cancer xenografts with known PTEN status LAPC4 (1), LAPC9 (2), LuCaP 35 (3), LAPC3 (4), LuCaP23 (5), LAPC12 (6), LuCaP41 (7), and LAPC14 (8) (A) and in glioblastoma tissue samples (B). (C and D) Human IGFBP2 levels were measured by RIA in the media of brain, prostate, and breast cancer cell lines (n = 2) (C) and in the sera of mice bearing human prostate and breast cancer xenografts mice (n = 2) (D). Values were normalized to cell confluency in B and to the weight of the excised xenograft tumor in C.

Fig. 3.

IGFBP2 expression is negatively regulated by PTEN and positively regulated by the PI3K/Akt pathway. (A) The levels of PTEN, IGFBP-2, and β-actin protein were measured in PTEN^+/+ and PTEN^−/− MEFs by Western blot analysis. (B) PTEN mutant LnCaP prostate cancer cells were treated with the PI3K inhibitor LY294002 and then were examined for IGFBP-2 protein expression and Akt activation by Western blot analysis. (C) PTEN wild-type DU145 prostate cancer cells were infected with retrovirus-expressing HA-tagged Myr–Akt and then examined for expression of the Akt transgene, IGFBP-2, and β-actin.

Fig. 4.

IGFBP2 plays a functional role in PTEN/Akt signal transduction. (A) LnCaP cells were treated with the PI3K inhibitor LY294002. IGFBP-2 expression was measured by Western blot analysis, and the percent of cells in S phase was determined as described in Methods. LY, LY294002; V, empty vector; I, IGFBP-2 cDNA. (B) IGFBP2^+/+ (plates 1–4) or IGFBP2^−/− (plates 5–8) MEFs infected with retrovirus expressing HA-epitope tagged Mry–Akt (plates 3, 4, 7, and 8), IGFBP2 (plates 2, 4, 6, and 8), or corresponding control vectors and then plated at limiting dilution on plastic as described in Methods. Colonies were visualized by crystal violet staining and counted after 2 weeks of culture. The results for Myr–Akt and Myc are quantified in B and C, respectively.