Deregulation of the Insulin-Like Growth Factor Type 1 Receptor (IGF-1R) in Transformed Follicular Lymphomas: Implications for Novel Therapy.

Abstract

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin lymphoma in the western hemisphere. The vast majority of cases are incurable and transformation to diffuse large B-cell lymphoma (DLBCL) is an important cause of death. The molecular and biologic mechanisms underlying FL transformation are largely uncharacterized. In this study, we utilized a global quantitative proteomics approach for the identification of differentially expressed proteins associated with follicular lymphoma transformation. Five matched pairs of clonally identical cases of follicular lymphoma and their transformed counterparts (DLBCL) arising in the same individual were utilized. Quantitative analysis of differentially expressed proteins was performed by isotope-coded affinity tagging (ICAT™) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Equivalent quantities of total cell lysates obtained from the FLs and the DLBCLs were ICAT™ labeled, and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin, and analyzed by automated reverse phase nanospray LC-MS/MS. Our proteomic studies revealed upregulation of the IGF-1R (3–5 fold) in the transformed lymphomas. Western blot analysis using an antibody to the a-subunit of IGF-1R revealed overexpression in the transformed lymphoma samples as compared to their preceding FL counterparts (discovery set). Similarly, IGF-1R upregulation was demonstrated in an additional independent set of 6/7 DLBCL samples as compared to their preceding FL counterparts. Immunohistochemical studies were performed on formalin-fixed paraffin-embedded tissue sections of 15 matched pairs of FL and their transformed DLBCL counterparts. The neoplastic cells of FL demonstrated negligible levels of IGF-1R whereas in 5/15 cases, the neoplastic cells of DLBCL demonstrated strong cytoplasmic and membranous expression of IGF-1R. We carried out studies to determine the functional role of IGF-1R in the survival of lymphoma cells in vitro. Blocking antibodies to IGF-1R caused a significant reduction of cell viability in all three transformed FL cell lines (SUDHL-4, OCI-LY1, Karpas 4224) as determined by MTT assays. In contrast, antibodies against EGFR, EphA and Frizzled 8 protein did not affect the cell viability of any of the transformed FL cell lines, indicating specificity. Furthermore, knockdown of IGF-1R expression in SUDHL-4 cells by RNA interference resulted in significant reduction in cell viability whereas the control “scramble” siRNA or EGFR siRNA did not have an effect. Cell cycle analysis of the IGF-1R siRNA transfected cells indicated an increase in cells undergoing apoptosis relative to control cells. We utilized a synthetic tyrphostin compound (AG1024) which selectively inhibits the IGF-1R tyrosine kinase activity to determine the effects of pharmacologic inhibition of IGF-1R on the viability of transformed FL cells. Inhibition of IGF-1R resulted in inhibition of cell viability with IC50 of 22mM. This study, for the first time, reveals the role of deregulated expression of IGF-1R in transformed FL and provides a rational basis for the use of IGF-1R blocking agents in the therapy of these neoplasms.