HARPΔ111-136 enhances radiation-induced apoptosis of U87MG glioblastoma by induction of the proapoptotic protein CHOP

Abstract

Abstract. We previously demonstrated, using the glioblastoma cell line U87MG as an experimental model, that the adenoviral mediated overexpression of the truncated protein HARPΔ111-136 inhibits the proliferation of these cells in vitro as well as tumor growth and angiogenesis in vivo. This study focused on identifying the underlying mechanisms for the observed antitumoral effect. The present study demonstrated that HARPΔ111-136 induced the ATF4/ATF3/CHOP cascade resulting in a strong expression of the proapoptotic protein CHOP, leading to tumor cell apoptosis as demonstrated by PARP cleavage and FACS analysis. siRNA-mediated CHOP gene silencing abolished Ad-HARPΔ111-136 induced apoptosis. Moreover, Ad-HARPΔ111-136 increased the expression of the death receptor DR5 and enhanced U87MG cells sensitivity in vitro to TRAIL a DR5 ligand with subsequent activation of caspase 8. Infection of U87MG cells with Ad-HARPΔ111-136 also enhanced radiation-induced apoptosis. In vivo, the combination of Ad-HARPΔ111-136 and radiation therapy resulted in a striking inhibition (92%) of the growth of U87MG xenografts, resulting from the potent effect on tumor angiogenesis and tumor cell apoptosis as determined by TUNEL analysis. Taken together, our results indicated that the inhibitor HARPΔ111-136 sensitized U87MG cells to apoptosis. IntroductionHeparin affin regulatory peptide (HARP), also called pleiotrophin, is a secreted growth factor known for its oncogenic (1), mitogenic (2) and angiogenic activities (3,4). HARP has been shown to signal through two high affinity receptors, the receptor-type protein tyrosine phosphatase ß/˙(RPTPß/˙) (5,6) and the anaplastic lymphoma kinase (ALK) (7). HARP together with its receptors are strongly overexpressed in several tumor types particularly glioblastoma (3,(8)(9)(10). Glioblastoma multiforme (GBM) is the most aggressive and frequent adult primary brain tumor (11-13). Resistance to apoptosis is a well known feature of glioblastoma cells leading to a poor response to chemotherapy and radiation therapy (14). Since HARP has been considered as a growth and a survival factor for cancer cells (15,16), blocking the HARP pathway could be of particular interest.HARPΔ111-136 is a truncated form of HARP lacking the last 26 amino-acids that acts as a dominant negative effector by heterodimerization with the wild-type HARP protein (17,18). In a recent study (19) we demonstrated using the human glioblastoma cell line U87MG as an experimental model, that overexpression of this truncated protein inhibited in vitro and in vivo proliferation of this tumor cell line as well as in vivo angiogenesis. A replication-defective adenovirus encoding this truncated protein (Ad-HARPΔ111-136) was constructed and our results showed that direct intra-tumoral administration of Ad-HARPΔ111-136 inhibited growth and angiogenesis of established U87MG xenografts in nude mice. However, the underlying mechanisms by which HARPΔ111-136 inhibits tumor proliferation remain to be explored. Herein we identified, in U87MG cells infected w...