Inhibition of Autophagy Promotes Bortezomib-Mediated Cell Death in Myeloma Cells

Abstract

The 26S proteasome is a key regulator of proteins controlling many important cellular functions, including cell cycle progression, differentiation, gene transcription and apoptosis. Proteasome inhibition is a new therapeutic strategy that has shown promise in the treatment of B cell malignancies, primarily multiple myeloma. We and others have demonstrated that proteasome inhibitors induce endoplasmic reticulum (ER) stress and activate an unfolded protein response (UPR) in transformed cells. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to calcium-dependent apoptotic cell death. Co-treatment of myeloma cells with the mitochondrial uniporter inhibitor ruthenium red (RuR) abrogated bortezomib mediated cell death, indicating that the cytotoxic effects of proteasomal inhibition requires dysregulation of intracellular Ca2+. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy or “self-eating”. Macroautophagy (hereafter referred to as autophagy) is induced by various cellular stresses including nutrient deprivation, metabolic insufficiency, interruption of growth factor signaling, elevated ROS, accumulation of intracellular Ca2+, and the UPR. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis is not well understood. In this study, we investigate the role of the Ca2+ dependent serine protease, calpain, as a mediator of the conversion from autophagic cell survival to accelerated cell death in the ER stress response. We demonstrate that the proteasome inhibitor, bortezomib, initiates autophagy in myeloma cells, and protection from bortezomib-mediated cell death by mitochondrial Ca2+ inhibitors is associated with a promotion and stabilization of the autophagosome. This response can be reversed, and indeed, accelerated, leading to enhanced cell death, by blockade of calpain activity. Inhibition of calpain activity with the tri-peptide zLLY-FMK (Calpain Inhibitor IV, (CiIV) or the non-peptide inhibitor, PD150606, demonstrated a significant increase in the cytotoxic activity of bortezomib. Similarly, elimination of the small catalytic subunit, CAPNS1, using siRNA, enhanced bortezomib-mediated cell death, and prevented autophagosome-lysosomal progression. Furthermore, inhibition of calpain by clinically approved HIV protease inhibitors including Nelfinavir, Ritonavir, Saquinavir, and Indinavir sulfate, significantly increased the cytotoxic activity of bortezomib in vitro. We suggest that disregulation of Ca2+ by bortezomib-mediated ER stress activates the autophagic survival response. Inhibition of mitochondrial Ca2+ uptake by the uniporter inhibitor RuR promotes autophagy, and confers resistance to bortezomib. Conversely, inhibition of the Ca2+-dependent serine protease, calpain, prevents autophagolysosome maturation, and subverts the survival response to cell death. These data are likely to have important clinical implications for the treatment of refractory myeloma and other B cell malignancies.

Avenues to molecular imaging of dying cells: Focus on cancer

“…This question is raised because most inhibitors of autophagy accelerate (and not retard) cell death. [30][31][32][33][34] For this reason, autophagic cell death has now been defined as cell death inhibited by inactivation of autophagy genes or by autophagy inhibitors, such as 3MA, rather than cell death judged by simple morphological classification. 35 This definition is based on studies which have elucidated molecular mechanisms of autophagic cell death.…”

“…Even today, there is much controversy on the question whether in vivo autophagy is a type of cell death or fulfills a pro‐survival function, for example, by limiting cell constituents during nutrient starvation. This question is raised because most inhibitors of autophagy accelerate (and not retard) cell death . For this reason, autophagic cell death has now been defined as cell death inhibited by inactivation of autophagy genes or by autophagy inhibitors, such as 3MA, rather than cell death judged by simple morphological classification .…”