Proteotoxic crisis, the ubiquitin-proteasome system, and cancer therapy

Raymond J Deshaies 0 0 Division of Biology and Biological Engineering and Howard Hughes Medical Institute, California Institute of Technology , Box 114-96, Pasadena, CA 91107 , USA Genomic alterations may make cancer cells more dependent than normal cells on mechanisms of proteostasis, including protein folding and degradation. This proposition is the basis for the clinical use of proteasome inhibitors to treat multiple myeloma and mantle cell lymphoma. However, proteasome inhibitors have not proved effective in treating other cancers, and this has called into question the general applicability of this approach. Here, I consider possible explanations for this apparently limited applicability, and discuss whether inhibiting other broadly acting components of the ubiquitin-proteasome system - including ubiquitin-activating enzyme and the AAA-ATPase p97/VCP - might be more generally effective in cancer therapy. - homeostasis (proteostasis) [2], including the UPS (Figure 1). Genome sequencing has revealed that cancer genomes are typically littered with dozens to hundreds of point mutations in protein coding sequences [3]. Many of these mutated proteins are likely to present significant folding challenges, with increased degradation of the mutant protein via the UPS being one possible outcome. In addition, cancer cell genomes often contain large duplications, deletions, inversions, and translocations as well as altered copy numbers of entire chromosomes (aneuploidy). It has been estimated that over 90% of human solid tumors contain cells with more than two copies of one or more chromosomes [4]. These excess chromosomes continue to be expressed, and therefore protein synthesis in aneuploid cancer cells is often imbalanced, with proteins encoded by extra chromosomes being produced in excess over proteins encoded by chromosomes that are present in two copies [5,6]. This is particularly a problem for proteins that assemble to form stoichiometric complexes like the ribosome. In such cases, the excess proteins almost certainly cannot attain stable conformations, and hence are degraded by the UPS [7,8]. In theory, this creates in cancer cells a heightened dependence on protein quality-control (PQC) mechanisms, including protein chaperones, the UPS, and autophagy [9-12]. In agreement with this, approximately one-third of single chromosomal aneuploidies in yeast cells render them hypersensitive to proteasome inhibitors [13], and some yeast cells that adapted to aneuploidy were found to contain mutations that derepress the UPS [6]. These data suggest that agents that inhibit PQC pathways should be more toxic to cancer cells than normal cells, and might be used to treat a broad variety of cancers. In the remainder of this review, I will refer to this idea as the proteotoxic crisis approach to cancer therapy. Here, I will focus on targeting PQC pathways of the UPS as a means to induce proteotoxic crisis in cancer cells. Other reviews have focused specifically on targeting chaperones or autophagy as a means to treat cancer [11,12]. Figure 1. Proteotoxic crisis in cancer cells. (A) In normal cells, the natural load of degradation substrates on the left is in balance with the capacity of the cellular ubiquitin-proteasome system (UPS), signified by the proteasome on the right. (B) In cancer cells, the load is increased due to expression of mutant proteins and/or expression of excess proteins due to aneuploidy. This results in an imbalance where the degradation load exceeds the capacity of the UPS. Bortezomib validates the proteotoxic crisis hypothesis but raises questions about its generality The proteasome inhibitor bortezomib provided the first direct evidence that it is possible to inhibit the UPS in a manner that is lethal to at least some cancer cells while mostly sparing normal cells [14]. Before discussing bortezomib in detail, a primer on the structure and mechanism of the 26S proteasome is in order. The catalytic core of the proteasome is a 20S cylinder, the inside of which contains two copies each of the active sites 1, 2, and 5 (Figure 2) [15]. A second form of the proteasome, referred to as the immunoproteasome, is enriched in cells of the hematopoietic lineage and has a specialized function in immune cells, but an essentially analogous composition in which the 1, 2, and 5 sites are replaced by the closely related 1i, 2i, and 5i sites. The 5/5i sites (also known as the chymotrypsin-like sites) are inhibited by bortezomib with high potency, whereas the 1 (caspase-like) sites have approximately 10-fold lower affinity and the 2 sites are not appreciably targeted under normal conditions [16-18]. Substrates enter the 20S cylinder through its ends, which are capped with structures referred to as 19S regulatory particles (RPs). A 20S cylinder capped at each end with a 19S RP is referred to as the 26S proteasome. Assembly of the 26S proteasome is enabled by pockets at the ends of the 20S cylinder into which are inserted short carboxy-terminal tails that emanate from a heterohexameric ring of Rpt1-6 subunits in the 19S RP. Degradation substrates are tethered to the 26S proteasome via their ubiquitin chain, which binds to one or more of a set of receptor proteins, some of which (for example, Rpn10 and Rpn13) are intrinsic to the 19S RP, while others (for example, hRad23, hPLIC) shuttle on and off. It is thought that substrates are bound to the 26S proteasome in a manner that enables them to be grasped by the Rpt1-6 proteins, which are AAA ATPases that use the energy derived from ATP hydrolysis to unfold substrates, open the normally closed gate at the end of the 20S cylinder to admit substrate, and translocate the substrate through a pore in the center of the Rpt ring and into the internal chamber of the 20S cylinder. As substrate is being translocated through the Rpt ring, the Rpn11 subunit of the 19S RP, which is positioned immediately above the channel through the Rpt ring, scans for ubiquitin chains. Rpn11 is a protease that removes ubiquitin chains as the substrate translocates by, which is thought to prevent the chains from clogging up the entry channel into the proteasome. Inhibition of 20S peptidase activity with bortezomib is highly cytotoxic to the plasma cell cancer multiple myeloma (MM) [20], and bortezomib has been an effective therapy for treating patients with this disease as well as mantle cell lymphoma (MCL) [21-23]. However, despite its considerable success as a therapy for MM and MCL, bortezomib has not been approved for treating other cancers. This is not for lack of effort: over 700 bortezomib trials have or are being run [24], including many in indications other than MM and MCL, in attempts to identify cancers that might respond favorably. This clinical experience is consistent with in vitro data: although brief exposure to proteasome inhibitors is highly cytotoxic to MM cells, it is not more cytotoxic to solid tumor cell lines than it is to non-transfor (...truncated)