Mechanistic Insights into the Role of IGFBP-2 in Glioblastoma

Abstract

Insulin like Growth Factor Binding Proteins (IGFBPs) 1 to 6 have important physiological functions of regulating half life and bioavailability of Insulin like Growth Factors (IGFs). Consequently, these have been known to play important roles in embryonic development, postnatal growth and disease conditions like cancer. However, the physiological roles of IGFBPs are diverse and not restricted only to the IGF regulation. These molecules are found to be tumor suppressors or promoters depending on the physiological contexts. IGFBP-2 has been established as a tumor promoter and found to be unregulated in several cancers including breast, ovarian, prostate cancer and glioblastoma (GBM). Various in vitro and in vivo studies have convincingly demonstrated the role of IGFBP-2 in inducing tumor cell proliferation, migration, invasion and chemoresistance. Increased plasma and tissue levels of IGFBP-2 have been associated with poor clinical outcome with respect to patients’ response to the therapy, relapse and overall survival. Various studies so far have demonstrated the role of IGFBP-2 in promoting glioma cell proliferation, migration, invasion, chemoresistance and determining stamens of GICs (Glioma Initiating Cells). However, the exact mechanisms underlying these functions remain unknown. Apart from being a diagnostic and prognostic indicator, IGFBP-2 has also been proposed as a therapeutic target. Therefore it is essential to understand mechanistic insights into pro-tumorigenic functions of IGFBP-2. Apart from the conventional function of regulating IGFs, IGFBP-2 has been shown to have several IGF independent functions. In a previous study, we reported IGFBP-2 as an upstream regulator of β-catenin signaling pathway in breast cancer. Interestingly, this study linked the association of higher expression of IGFBP-2 and β-catenin with the lymph node metastasis status of breast cancer. β-catenin signaling has been considered as one of the most important pro-tumorigenic pathways in several cancers including glioblastoma. Considering the importance of IGFBP-2 and β-catenin signaling pathways in glioblastoma, it becomes important to evaluate regulation of β-catenin activity by IGFBP-2 in glioma and address its clinical relevance. With this aim, the objectives of this study are,  To study mechanism of IGFBP-2 mediated regulation of β-catenin signaling in glioma cells and prognostic significance of IGFBP-2 and β-catenin expression in GBM tissues.  Isolation of human single chain variable fragment (scFv) against IGFBP-2 and its characterization as an inhibitor for IGFBP-2 pro-tumorigenic functions. Towards this, we established stable IGFBP-2 knockdown U251 cell line and IGFBP-2 over expressing LN229 and U87 cell lines. IGFBP-2 modulation in these glioma cell lines did not alter the rate of proliferation but there was a significant effect on cellular migration and invasion. In case of U251 cell line, there was a significant decrease in the intracellular levels of β-catenin while in IGFBP-2 over expressing cell lines there was a marked increase in intracellular β-catenin suggesting that IGFBP-2 is involved in the regulation of β-catenin in these cells. It was observed that this regulation of β-catenin was not because of its transcriptional regulation or regulation of canonical Wnt ligands Wnt1, Wnt2 and Wnt3a. To further delineate the pathway and understand the mechanism behind regulation of β-catenin, upstream regulators of β-catenin were analyzed. GSK3β is an important negative regulator of β-catenin which primes it for ubiquitination and proteasomal degradation. Phosphorylation of GSK3β at Ser9 position renders this enzyme inactive. In our study, it was observed that there was a significant downregulation of p-GSK3β in U251 cells with IGFBP-2 knockdown and upregulation in IGFBP-2 over expressing cell lines. Overexpression of IGFBP-2 in LN229 and U87 cell lines resulted in considerable decrease in the GSK3β mediated phosphorylation of β-catenin. This study unequivocally established that regulation of β-catenin by IGFBP-2 is via inactivation of GSK3β. Furthermore, regulation of GSK3β was found to be due to action of FAK following binding of IGFBP-2 to integrins. The expression pattern of IGFBP-2 and β-catenin protein in the tumor tissues of 112 GBM patients was studied and its correlation with patient survival was analysed. In this analysis it was observed that co-expression of IGFBP-2 and β-catenin is a strong predictor of patient prognosis. These results further implied the importance of understanding IGFBP-2 and β-catenin association in GBM pathology. One of the interesting observations in our study is that, not only full length IGFBP-2 protein but also C-terminal domain of IGFBP-2 was sufficient to regulate β-catenin and other IGFBP-2 mediated functions. This strongly asserts the importance of C-terminal region of IGFBP-2 as a tumor promoter. Towards an attempt to develop an inhibitor for IGFBP-2 actions, we screened a human single chain variable fragment (scFv) library using phage display technique. From this screening, one scFv (B7J) was identified which was a binder of full length IGFBP-2 as well as C-terminal domain of IGFBP-2. This scFv showed inhibition of IGFBP-2-cell surface interaction and also efficiently inhibited IGFBP-2-induced signaling pathways like ERK, FAK and GSK3β/β-catenin. B7J treatment also neutralized regulation of IGFBP-2 transcriptional targets like MMP2 and CD24. Gelatin zymography indicated the ability of B7J to decrease matrix metalloprotease activity in the conditioned medium of glioma cells. These effects ultimately reflected on the IGFBP-2-induced cellular migratory and invasive behaviour which was largely abrogated by B7J scFv treatment. Considering the therapeutic importance of scFvs because of their small size, better tumor penetration and tumor retention capacity than full length antibody molecules, such kind of strategy could be of great importance in the management of GBM. Altogether, this study provides a mechanistic insight of IGFBP-2 mediated actions involving integrin/FAK/GSK3β/β-catenin pathways and the possible role of this crosstalk in the aggressiveness of glioblastoma. This study also provides a proof of principle that an inhibitor like anti IGFBP-2 scFv could be of importance for controlling invasive glioblastoma.