Biologic impact of proteasome inhibition in MM cells - from the aspects of preclinical studies

Abstract

The ubiquitin-proteasome pathway (UPP) is a major protein degradation system that maintains homeostasis of intracellular proteins, involved in DNA repair, cell cycle regulation, cell proliferation, and drug resistance. Since numerous proteins are processed by proteasomes, their inhibition triggers dramatic disruption of protein homeostasis. Consequently, accumulation of polyubiquitinated proteins triggers different types of cellular stress responses, followed by growth arrest and cytotoxicity. Importantly, multiple myeloma cells are considered to have lower threshold against these stresses than other cell types, which makes MM cells sensitive to proteasome inhibitors.

1. Introduction

Lysosomal (autophagy) and proteasomal protein degradation pathways are two major pathways maintaining intracellular protein catabolism and homeostasis; however, which proteins are specifically processed by proteasomes and/or lysosomes is still not totally understood, despite recent advanced proteomics technologies ^1,2. The 26S proteasome is an ATP-dependent, multifunctional proteolytic complex that differs in many respects from typical proteolytic enzymes. It consists of a proteolytic core, the 20S proteasome, sandwiched between two 19S regulatory complexes. The 19S proteasome regulatory complexes control the access of substrates to the proteolytic core. The 20S proteasome is a multicatalytic protease and forms a hollow cylinder comprised of four stacked rings. Each outer ring is composed of 7 different α-subunits and each inner ring is composed of 7 distinct β-subunits. Moreover, each β-ring contains caspase-like, trypsin-like, and chymotrypsin-like proteolytical active sites. The 20S proteasome degrades oligonucleotide and protein substrates by endoproteolytic cleavage. Immunoproteasomes are alternative β forms (β1i, β2i, and β5i) expressed in subsets of hematopoietic cells in response to pro-inflammatory stimuli (ie, interferon-γ) and have an important role for generating peptide antigens for MHC class I presentation. Recent studies have shown that inhibitors of immunoproteasome also blocks MM cell growth in vitro and in vivo ^3,4.

Different classes of proteasome inhibitors have been developed according to reversible or irreversible inhibition of chymotrypsin-like, trypsin-like, and/or caspase-like activities. They all induce inhibition of 20S proteasome activity by blockade of the 20S β-subunits. Therefore these proteasome inhibitors, regardless of class, have similar biologic impact in preclinical in vitro and in vivo studies against MM cells. Recent studies have both defined the mechanisms of protein degradation by proteasome and provided the framework for therapeutic applications in MM. Proteasome inhibitors can also target other cellular components in the bone marrow microenvironment. In this chapter, the authors describe biologic impact of proteasome inhibition specifically in MM cells.

2. Biologic impact of proteasome inhibition in MM cells

Proteasomes degrade numerous proteins involved in MM cell proliferation, survival, and drug resistance; therefore, the biologic impact of proteasome inhibition is broad and has highly complex. Selected targets are discussed in this section.

(1) Induction of cell cycle arrest and apoptosis

As described above, the UPP is a major proteolytic system regulating a broad spectrum of proteins mediating cell cycle. These proteins include cyclin dependent kinase inhibitors (p21^Cip1 and p27^Kip1), cyclin D, cyclin E, cdc25, Wee1 and p53 ^5–7. Upregulation of these proteins by proteasome inhibition results in cell cycle arrest.

A hallmark of proteasome inhibitory effect in MM cells is induction of apoptosis. Indeed many proteasome inhibitors, including bortezomib, trigger extrinsic and intrinsic apoptotic pathways with caspase-9 and caspase-8 activation, respectively. Although the molecular mechanisms whereby proteasome inhibitors induce extrinsic apoptotic pathway have not yet been fully delineated, proteasome inhibitors, similar to CD95 receptor (Fas/APO-1) and tumor necrosis factor receptor 1, trigger c-Jun NH2-terminal kinase (JNK) and caspase-8 activation. Conversely, JNK inhibitor partially blocks proteasome inhibitor-induced apoptosis ^8,9.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) playing an important role in cell stress-induced apoptosis. For example, ASK1 activates JNK and p38MAPK in response to different types of stress, including endoplasmic reticulum (ER) stress. Indeed, previous studies have shown that bortezomib triggers ER stress ¹⁰, which can induce ASK1 followed by JNK activation. These results suggest that ASK1-JNK axis plays a crucial role in extrinsic apoptotic pathway. Most recently, Laussmann et al. demonstrated that proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8 in non-small cell lung cancer cells ¹¹, which further suggests another potential mechanism whereby proteasome inhibitors may trigger the extrinsic apoptotic pathway in MM cells.

Proteasome inhibitors also activate the intrinsic apoptotic pathway. Previous studies have shown that mitochondria-mediated dysregulation of intracellular Ca2+ is one of the mechanisms for activation of caspases in MM cell lines ¹². Noxa is a BH-3 only member of the Bcl-2 family and its expression id regulated by p53. Noxa, in a BH3 motif-dependent, translocates to mitochondria and inhibits Bcl-2 family member proteins, resulting in the activation of caspase-9 and apoptosis. We have previously shown that bortezomib triggers apoptosis in MM cells independent of p53 status ^8,13. Consistent with these studies, Qin et al. demonstrated that proteasome inhibitors trigger p53-independent Noxa expression and apoptosis ¹⁴. Importantly, proteasome inhibitors not only directly activate caspases, but also downregulate anti-apoptotic proteins, which in turn further accelerate caspase activation by proteasome inhibitors. X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein (IAP) family and is the best characterized and most potent direct endogenous caspase inhibitor. Therefore XIAP is considered to be a key regulator of the apoptotic threshold in cancer cells. Specifically, XIAP binds to caspase-3, -7 and -9 and blocks their activities. Previous studies have shown that bortezomib downregulates (caspase-dependent cleavage) XIAP and induces significant cytotoxicity in MM cells ¹⁵. Myeloid cell leukemia-1 (Mcl-1) is other known well characterized anti-apoptotic protein in MM cells and Gomez-Bougie et al. reported that bortezomib-induced MM cell apoptosis is associated with Mcl-1 cleavage, regardless of Mcl-1L (long form) accumulation ¹⁶.

(2) Induction of autophagy

Autophagy is another major protein catabolic system via lysosomal machinery, and either inhibition or increased autophagy can cause cytotoxicity. Previous studies have shown that proteasome inhibitors activate autophagy as a protective/compensatory response, to maintain homeostasis of protein catabolism. Specifically, polyubiquitinated proteins are degraded via both proteasomes and aggresomes, in which protein aggregates are processed by autophagy. Upon inhibition of proteasome activity, aggresomal protein degradation is activated. In the aggresomal protein degradation pathway, histone deacetylase 6 (HDAC6) is an essential element; therefore inhibiting both the proteasome and HDAC6 triggers accumulation of ubiquitinated proteins, followed by cell stress and cytotoxicity in MM cells ^17,18.

(3) Induction of endoplasmic reticulum stress

Proteasome inhibitors trigger accumulation of misfolded and unfolded proteins, which induces ER stress followed by the unfolded protein response (UPR). The UPR is the major protective and compensatory mechanism triggered during ER stress and three ER stress sensor proteins (IRE1α, PERK and ATF6) have been identified on ER membrane. Importantly, bortezomib indirectly (via ER stress) activates IRE1α to generate spliced (active) form of a transcription factor X-box binding protein-1 (XBP-1) ¹⁹. Recent studies also demonstrated that proteasome inhibitor upregulates BiP (Grp78) and C/EBP homologous protein (CHOP), associated with apoptosis ²⁰. Moreover, bortezomib-triggered apoptosis is dependent on caspase-2 activation, followed by breakdown of mitochondrial transmembrane potential and release of cytochrome-C ²¹.

(4) Inhibition of NF-kB

Since IκBα is a substrate of the proteasome, the initial rationale to use proteasome inhibitors in MM was blockade of NF-κB activity; and importantly, NF-κB is one of the major transcription factors contributing to MM pathogenesis. Previous studies have revealed that NF-κB activity in MM is mediated via the canonical and the non-canonical, pathways ^22–24. These NF-κB pathways are characterized by protein complexes consisting of dimers of different combinations of Rel family proteins of RelA (p65), RelB, c-Rel, p50 (NFκB1), and p52 (NFκB2). Typically, p50/RelA and p52/RelB heterodimers mediate the canonical and non-canonical pathways, respectively. Importantly, both pathways can be further activated in the context of the bone marrow microenvironment ²⁵. IκBα is an IκB family protein which can block the canonical pathway by inhibiting p50/RelA nuclear translocation ²⁶ and is a substrate of proteasome. Therefore, proteasome inhibitors block IκBα degradation, thereby resulting in inhibition of canonical NF-κB activity. Moreover, conversion of p50 (NFκB2) from precursor protein p100 is also proteasome dependent; therefore, proteasome inhibitors also can block the non-canonical pathway ²⁵. Interestingly, previous studies have demonstrated that bortezomib can activate the canonical NF-κB pathway by downregulation of IκBα ^27,28. Taken together, these results suggest that bortezomib inhibits non-canonical, but can activate the canonical, pathway. Therefore bortezomib-induced cell growth inhibition may not be solely triggered by NF-κB inhibition, depending on the basal level of NF-κB activity maintained by canonical versus non-canonical pathway. In addition, bone marrow stromal cells (BMSCs) promote proliferation, survival and conventional drug resistance in MM cells via both MM-BMSC adhesion (cell adhesion-mediated drug resistance) and secretion of soluble factors (ie, IL-6). Inhibition of NF-κB activity inhibits both MM cell adhesion to BMSCs via downregulation of adhesion molecules (ie, ICAM-1) and secretion of these growth/anti-apoptotic factors in both cell types ^29,30.

(5) Downregulation of growth factor receptors

Interleukin-6 (IL-6) is one of the major cytokines mediating MM cell growth, survival, and drug resistance via downstream extracellular signal-regulated kinases (ERK), Janus kinase (JAK) 2/signal transducers and activators of transcription 3 (STAT3), and phosphatidylinositol-3 kinase/Akt signaling pathways in the BM milieu ³¹. Importantly, bortezomib induces caspase-dependent downregulation of gp130 (CD130), the β-subunit of IL-6 receptor, thereby abrogating IL-6 mediated signaling pathways ³².

(6) Inhibition of DNA repair

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a subunit of DNA-PK and has an important role in repair of DNA double stranded breaks. Cells lacking DNA-PK or in which DNA-PK is inhibited lack proper non-homologous end-joining (NHEJ). Ataxia telangiectasia mutated kinase (ATM) is also a serine/threonine kinase regulating cell cycle checkpoints and DNA repair. Like DNA-PK, DNA double-strand breaks also activate ATM. Both DNA-PKcs and ATM can phosphorylate p53 at serine 15, which inhibits the MDM2 binding to p53 and promotes both nuclear accumulation and activation of p53 in response to DNA damage. Importantly, both DNA-PKcs and ATM are cleaved and inactivated by bortezomib in MM cells ⁸, providing further rationale for combined treatment with proteasome inhibitors and DNA damaging agents.

3. Future directions

The proteasome degrades numerous proteins; however, which proteins are specifically substrates of the proteasome and/or lysosome is not yet totally understood. Recent advances in proteomics studies, including stable isotope labeling with amino acids in cell culture (SILAC) and ubiquitin proteomics, together with increased availability and understanding of proteasome inhibitors, histone deacetylase 6 inhibitors, and/or lysosome inhibitors, will allow for identification of proteins processed in different mechanisms. These studies will both define molecular mechanisms regulating sensitivity or resistance to proteasome inhibitors and allow for novel treatment strategies targeting protein homeostasis in MM and other cancers.

Figure 1. Mechanism of action of proteasome inhibitor-induced MM cell growth inhibition.

Proteasome inhibitors upregulate p53 and induce JNK activation, followed by activation of caspases, which further triggers DNA damage (double-strand break) followed by activation of p53. Activated caspase-3 also cleaves DNA-PKcs and ATM/ATR, as well as gp130, resulting in impaired DNA repair and response to IL-6, respectively. Proteasome inhibitors trigger ER stress and induce activation of IRE1α followed by XBP1 splicing, thereby increasing its transcriptional activity. Proteasome inhibitors block inducible canonical NF-κB activity by cytokines/chemokines or cell adhesion. However, it can directly downregulate IκBα and canonical NF-κB activation, as well as non-canonical NF-κB activity by inhibiting proteasome-dependent p100 conversion to p52.