Evidence of galectin-1 involvement in glioma chemoresistance

Abstract

Glioblastomas (GBMs) are resistant to apoptosis but less so to autophagy; a fact that may at least partly explain the therapeutic benefits of the pro-autophagic drug temozolomide in the treatment of GBM patients. Galectin-1 (Gal1) whose expression is stimulated by hypoxia is a potent modulator of GBM cell migration and a pro-angiogenic molecule. Hypoxia is also known to confer cancer cells with resistance to chemotherapy and radiotherapy and to modulate the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. The present study investigates whether decreasing Gal1 expression (by means of a siRNA approach) in human Hs683 GBM cells increases their sensitivity to pro-autophagic or pro-apoptotic drugs. The data reveal that temozolomide, the standard treatment for glioma patients, increases Gal1 expression in Hs683 cells both in vitro and in vivo. However, reducing Gal1 expression in these cells by siRNA increases the anti-tumor effects of various chemotherapeutic agents, in particular temozolomide both in vitro and in vivo. This decrease in Gal1 expression in Hs683 cells does not induce apoptotic or autophagic features, but is found to modulate p53 transcriptional activity and decrease p53-targeted gene expression including DDIT3/GADD153/CHOP, DUSP5 ATF3 and GADD45A. The decrease in Gal1 expression also impairs the expression levels of seven other genes implicated in chemoresistance: ORP150, HERP, GRP78/Bip, TRA1, BNIP3L, GADD45B and CYR61, some of which are located in the ER and whose expression is also known to be modified by hypoxia. This novel facet of Gal1 involvement in glioblastoma biology may be amenable to therapeutic manipulation.

Introduction

Malignant gliomas, especially glioblastomas (GBMs), are characterized by the diffuse invasion of distant brain tissue by a myriad of single migrating cells with reduced levels of apoptosis (type I programmed cell death (PCD)) and consequent resistance to the cytotoxic insults of pro-apoptotic drugs (Lefranc et al., 2005). In contrast, GBM cells are less resistant to autophagy-related cell death (type II PCD) than to apoptosis (Lefranc et al., 2005, Lefranc et al., 2006). Current recommendations are therefore that patients with glioblastomas should undergo maximum surgical resection followed by concurrent radiation and chemotherapy with the pro-autophagic drug temozolomide (Kanzawa et al., 2004, Lefranc et al., 2006, Stupp et al., 2005, Mirimanoff et al., 2006).

Galectin-1 (Gal1) which is a lectin with specificity for β-galactosides (Liu and Rabinovich, 2005, Camby et al., 2006), markedly influences glioma cell migration both in vitro and in vivo (Camby et al., 2001, Camby et al., 2002, Camby et al., 2005). High-grade glioma patients whose gliomas markedly express Gal1, survive for a significantly shorter period than individuals whose glioma expresses less Gal1 (Camby et al., 2002). Decreasing Gal1 expression in human orthotopic GBM xenografts significantly increases the survival of GBM tumor-bearing mice (Camby et al., 2002). Gal1 expression is increased under hypoxic conditions (Le et al., 2005, Case et al., 2007), which seems to induce pseudopalisades formation in GBMs (Rong et al., 2006). Hypoxia also confers cellular resistance to conventional chemotherapy and accelerates malignant progression (Ozawa et al., 2001, Shannon et al., 2003, Le et al., 2005).

Gal1 is negatively regulated by p53 (Puchades et al., 2007) and Gal1 reciprocally has been shown to modify p53 biological functions (Camby et al., 2005). p53 triggers apoptosis in response to cellular stress including chemotherapy (Liu et al., 2007, Strano et al., 2007), while loss of p53 functionality leads to chemoresistance (Strano et al., 2007). The reciprocal control exerted by Gal1 or p53 on the other, could be implicated in GBM chemoresistance, as could also be the case with respect to the partnership between Ras and Gal1. Ras is implicated in gliomagenesis (Kapoor and O'Rourke, 2003, Uhrbom et al., 2005) and it modulates glioma aggressiveness (Goldberg and Kloog, 2006). Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane micro-domains and various intra-cellular compartments (Ashery et al., 2006). Such interactions are dictated by individual elements in the carboxy-terminal domain of the Ras proteins, among which, one is recognized by Gal1, galectin-3 (Gal3) and cGMP phosphodiesterase delta (Ashery et al., 2006). Gal1 thereby promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3K) and Ral guanine nucleotide exchange factor (RalGEF), while Gal3 promotes K-Ras signaling to both Raf and PI3K (Ashery et al., 2006). When K-Ras-GTP interacts with Gal3 it gains a conformation that promotes activation of Raf, PI3K and a third signal that attenuates ERK activation (Elad-Sfadia et al., 2004). Gal1 could therefore be involved in these Ras-related pathways implicated in GBM resistance to apoptosis (Lefranc et al., 2005, Blum et al., 2006).

We investigated in the present study whether decreasing the expression of Gal1 in human Hs683 GBM cells could increase their sensitivity to the pro-autophagic effects of temozolomide or to the pro-apoptotic effects of various cytotoxics including lomustine (CCNU), carmustine (BCNU), procarbazine (PCB) and vincristine (VCR), which are drugs used to treat GBM patients (Lefranc et al., 2005, Lefranc et al., 2006). An anti-Gal1 siRNA approach was employed to decrease the expression of Gal1 in human Hs683 GBM cells. Full genome-wide microarray analysis (with proteomic validation by western blotting and immunofluorescence analyses) was further used to identify chemoresistance-related genes (with a special emphasis on p53-targeted ones) whose expression was modified in Gal1-deficient compared to native Hs683 GBM cells.