The 26S Proteasome Complex: An Attractive Target for Cancer Therapy

Abstract

The 26S proteasome complex engages in an ATP-dependent proteolytic degradation of a variety of oncoproteins, transcription factors, cell cycle specific cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other key regulatory cellular proteins. Thus, the proteasome regulates either directly or indirectly many important cellular processes. Altered regulation of these cellular events is linked to the development of cancer. Therefore, the proteasome has become an attractive target for the treatment of numerous cancers. Several proteasome inhibitors that target the proteolytic active sites of the 26S proteasome complex have been developed and tested for anti-tumor activities. These proteasome inhibitors have displayed impressive anti-tumor functions by inducing apoptosis in different tumor types. Further, the proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory responses, and DNA repair response. A number of proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Many other proteasome inhibitors with different efficiencies are being developed and tested for anti-tumor activities. Several proteasome inhibitors currently in clinical trials have shown significantly improved anti-tumor activities when combined with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90) inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the 26S proteasome complex in carcinogenesis and different proteasome inhibitors with their potential therapeutic applications in treatment of numerous cancers.

Introduction

The 26S proteasome complex is a non-lysosomal proteolytic machine in eukaryotes (1, 2). It consists of a 20S core particle (CP) and a 19S regulatory particle (RP). The 20S CP confers the proteolytic activities of the proteasome, whereas the 19S RP shows an ATP-dependence and specificity for ubiquitin protein conjugates. The 20S CP resembles a cylinder composed of four rings (two α and two β rings) (1, 3). These rings are flush with each other, giving the 20S CP a seven-fold symmetry. Each α ring is composed of seven different α components (α1-α7). Similarly, seven different β components (β1–β7) form a β ring. Three of the seven β-components in the β ring are catalytically active, and are named by their substrate specificities: chymotrypsin-like (β5), trypsin-like (β2), and post-acidic or caspase-like (β1). The chymotrypsin-like activity cleaves proteins after hydrophobic residues, while the trypsin and caspase-like activities cleave after basic and acidic residues, respectively (4, 5). The substrate protein is translocated into the catalytic chamber of the 20S CP with the help of the 19S RP. The substrate protein is targeted to the 26S proteasome via its polyubiquitylation (Figure 1). The ubiquitin chains are added to the protein substrate by three enzymes: ubiquitin-activating E1, ubiquitin-conjugating E2, and ubiquitin-ligase E3 (Figure 1). E1 ubiquitin activating enzyme binds with an ubiquitin molecule, passes the ubiquitin to E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase enzyme enables the linking of C-terminal glycine residues of ubiquitin to lysine (K) residue on the substrate protein (Figure 1). Polyubiquitylation occurs through the linkage on one of the seven K residues of ubiquitin. The 19S RP recognizes the K48-linked polyubiquitylated-substrate protein, unfolds it, and finally feeds it into the catalytic chamber of the 20S CP for proteolysis in an ATP-dependent manner (6, 7, 8). Further, the 19S RP cleaves off the ubiquitin from the substrate protein, and recycles it for future use. While K48-mediated polyubiquitylated-form of the substrate protein is recognized and degraded by the proteasome, K63-linked polyubiquitylation is not targeted for degradation by the 26S proteasome complex, but plays a crucial role in cellular signaling.

Figure 1.

The schematic diagram showing ubiquitylation of substrate protein and its subsequent degradation by the 26S proteasome complex. A ubiquitin activating enzyme (E1) first forms a thio-ester bond with ubiquitin (a highly conserved protein with 76 amino acids) in an ATP-dependent manner. Ubiquitin then binds to a ubiquitin conjugating enzyme (E2). Subsequently, the carboxy-terminus of ubiquitin forms an isopeptide bond with a K residue on the substrate protein in the presence of a ubiquitin ligase enzyme (E3). Multiple ubiquitylation cycles result in polyubiquitylation of the substrate protein. The 26S proteasome complex recognizes, unfolds and degrades polyubiquitylated-substrate protein into small peptides. Ub, ubiquitin.

The proteasome complex is found in both the cytoplasm and nucleus of eukaryotic cell where they regulate the ebb and flow of proteins involved in progression through the cell cycle, inflammatory mechanisms, antigen presentation, signal transduction, apoptosis, and other key regulatory cellular processes. Through these processes, the 26S proteasome complex plays a crucial role in maintaining normal cellular functions. The proteasome exists in two isoforms: the constitutive proteasome or the 26S proteasome, and the immunoproteasome (9). While the constitutive proteasome is found in most cells, the immunoproteasome is tissue-specific and abundant in immune-related cells. The immunoproteasome is formed in response to cytokine signaling. The immunoproteasome differs from its more common counterpart in that it contains a variation of the normal β-components. The β1, β2 and β5 components of the constitutive proteasome are replaced by β1i (LMP2), β2i (MECL1 or LMP10) and β5i (LMP7) in the immunoproteasome. The immunoproteasome also has an 11S regulatory structure or PA28 instead of the 19S RP of the 26S proteasome. Stimulation from the γ-interferon (IFN-γ) can instigate the switch of constitutive β-components to the immuno β-components. Similarly, tumor necrosis factor-alpha (TNF-α) has also been shown to induce the expression of immuno β-components and 11S regulatory cap to form the immunoproteasome. Such modifications help the immunoproteasome to generate antigenic peptides in a major histocompatibity complex (MHC) class I-mediated immune response (10).

The 26S proteasome complex in different cellular events and carcinogenesis

The 26S proteasome regulates many cellular functions, the most prominent of which include the advancement through mitosis, growth, chemotaxis, antigen presentation, angiogenesis, apoptosis, and the expression of several genes which in turn regulate other processes. These mechanisms influenced by the 26S proteasome are some of the processes altered or deregulated in cancers. The most prominent substrates and related molecules of the 26S proteasome involved in cellular processes and carcinogenesis are discussed below.

Nuclear Factor-KappaB (NF-κB)

NF-κB was originally discovered as a regulator for the expression of the kappa light-chain gene in murine B-lymphocytes (11). Later on, NF-κB has been found in nearly all animal cell types. NFκB is a transcription factor, and is involved in the activation of the genes encoding for cytokines, chemokines, growth factors, cell-adhesion molecules, and surface receptors (12–15) (Figure 2). Through transcriptional regulation of a number of genes, NFκB controls various immune and inflammatory responses. Further, it suppresses apoptosis, and induces angiogenesis, cell proliferation and migration (Figure 2), and thereby plays a crucial role in tumorigenesis (16–18). NFκB is a heterodimer of p50 and p65. The 26S proteasome is involved in generating p50 from the precursor protein p105. p50 then binds to p65 and becomes the active dimer or NFκB. In the cytoplasm, IκB binds to NFκB and inhibits the translocation of NFκB to the nucleus for gene activation (Figure 2). External stimuli (e.g. ionizing and ultraviolet irradiation, pathogens, stress, free radicals, and cytokines) induce phosphorylation of IκB (Figure 2). This phosphorylation triggers polyubiquitylation of IκB for degradation by the 26S proteasome complex (Figure 2). The proteasomal degradation of IκB promotes the translocation of NFκB to the nucleus to switch on the transcription of its target genes (Figure 2). Thus, the 26S proteasome complex plays a pivotal role in regulating the function of NFκB and associated key intra- and inter-cellular events. Therefore, misregulation of NFκB function would lead to various types of cancers. Incidentally, several cancers like breast cancer, myeloma, prostate cancer, and leukemias show constitutive activity of NF-κB (14, 19–24). This confers chemoresistance and increased aggression in phenotypes through the continued expression of factors associated with anti-apoptosis, angiogenesis, cell growth/proliferation, and metastasis (14, 24–26). In addition to its involvement in cancer, NF-κB is also linked to inflammatory and autoimmune diseases, septic shock, viral infection, improper immune development, processes of synaptic plasticity and memory as well as neurodegenerative and heart diseases (27–29).

Figure 2.

The schematic diagram showing the regulation NF-kB functions by the 26S proteasome complex.

Apoptosis

Cancer is characterized by an uncontrolled growth and spread of abnormal cells. Thus, the induction of apoptosis would promote the killing of abnormal cancer cells. However, cancer cells often have a disregulation of apoptotic signaling pathways, leading to the suppression of apoptosis. Such an aberrant regulation of apoptosis provides a survival advantage to the cancer cells and therefore resistance to chemotherapy. Intriguingly, the key factors involved in controlling the apoptosis are regulated by the 26S proteasome complex (9, 30). For example, the levels of the pro-apoptotic factors such as p53, Bax, and NOXA are increased following inhibition of the proteolytic function of the 26S proteasome. Further, the inhibition of the proteasome activity has been shown to downregulate the anti-apoptotic factors such as Bcl-2 and IAP (inhibitor of apoptosis) proteins. Therefore, the inhibition of the proteolytic function in cancer cells would promote the apoptosis by upregulating the functions of the pro-apototic factors and suppressing the anti-apoptotic factors, thereby killing the cancer cells. Indeed, inhibition of the proteolytic function of the 26S proteasome has been shown to enhance the apoptosis in a number of cancer cells (9) (Figure 3).

Figure 3.

The schematic diagram showing the effects of the proteasome inhibition on different pathways, contributing to cancer prevention.

As mentioned above, p53 is a pro-apoptotic factor that plays a critical role in apoptosis. Normally an unstable protein, its enhanced levels result in the initiation of the apoptosis cascade. Down-regulation of 26S proteasomal degradation causes the pro-apoptotic accumulation of p53, and hence induction of apoptosis (Figure 3). Activation of an E3 ubiquitin ligase, MDM2, ubiquitylates p53 and subsequently leads to its proteasomal degradation (31). This downregulation of p53 activity leads to tumor progression and drug resistance. Further, in high-risk HPV (human papilloma virus)-related cancer cases, the E6 oncoprotein and E6-AP (E6 associated protein, an E3 ubiquitin ligase) bind to p53, and ubiquitylate it for subsequent 26S proteasomal degradation (32, 33). Thus, the 26S proteasome complex plays a key role in regulating the level of p53, and through it apoptosis and cancer progression. Therefore, the inhibition of the proteolytic function of the 26S proteasome in cancer cells would augment the level of p53, and would eventually impair the cancer progression by inducing apoptosis (Figure 3). Unsurprisingly, the inhibition of the proteasome activity has been shown to induce p53-dependent apoptosis in renal cancer cell lines (34), colon cancer (35), melanoma, and multiple myeloma (36). Proteasome inhibition has also been demonstrated to activate the downstream target genes of p53 such as Bax, p21, PUMA (p53 upregulated modulator of apoptosis) and Fas ligand (37).

Cell cycle

Regulation of cell cycle progression is a pivotal step in controlling carcinogenesis. In general, rapid cell cycle progression leads to cell proliferation and uncontrolled growth, ultimately developing transformed cancerous cells. Cyclins and cyclin dependent kinases (CDKs) tightly control the progression of cell cycle. However, the functions of several cyclins, CDKs, and their interplay are regulated by the proteolytic activity of the 26S proteasome complex, conferring proteasomal regulation of cell cycle progression in a number of ways. For example, CDK inhibition by tumor suppressor p27 downregulates cyclins D and E, and subsequently negatively controls the cell cycle progression through G1/S phase (38) (Figure 3). The 26S proteasome complex is involved in the degradation of p27, and thus promotes cell cycle progression (Figure 3). The E3 ubiquitin ligase, Skp-2 (S-phase kinase protein 2) targets p27 for ubiquitylation for its 26S proteasomal degradation. Due to this, low levels of p27 would lead to rapid cell cycle progression and hence oncogenesis. In fact, low levels of p27 are observed in various malignancies such as lymphoma, breast, lung, colon, prostate, ovarian, and brain cancers (39). Further, high levels of Skp-2 contributing to enhanced proteasomal degradation of p27 have been demonstrated in several cancers including non-small cell lung carcinoma (40). Similarly, the 26S proteasome is also involved in the regulation of CDK-inhibiting protein p21 levels, hence controlling cell cycle progression (41–43) (Figure 3).

Like cyclins D and E, cyclins A and B are also regulated by the 26S proteasome. The anaphase-promoting complex/cyclosome (APC/C) serves as an E3 ubiquitin ligase, and ubiquitylates both cyclins A and B, marking them for degradation by the 26S proteasome complex (44–46). The 26S proteasomal degradation of cyclins A and B guarantees that the cell completes mitosis and can enter the next cell cycle. In fact, cyclin B is rapidly degraded by the proteasome as the cell exits mitosis (47–49). Clearly, an alteration of the proteasomal degradation of cyclins A and B, or their enhanced level would be correlated with a number of cancers. Indeed, cyclin B has been found to be overexpressed in numerous cancer cell lines (50).

Further, the 26S proteasome complex has been shown to regulate cell cycle progression via an oncogenic transcription factor, Forkhead Box M1 (FoxM1). FoxM1 induces the expression of the genes that are involved in cell cycle progression (Figure 3). It is expressed at a low level in normal cells. However, its overexpression can lead to a rapid cell cycle progression. In fact, FoxM1 has been shown to be overexpressed in numerous cancers such as non-small cell lung carcinoma (51) breast cancer (52), colorectal cancer (53), glioblastomas (54), pancreatic carcinomas (55) and squamous cell carcinomas (56). Additionally, the inhibition of the proteolytic activity of the 26S proteasome has been demonstrated to suppress the expression of FoxM1 and its transcriptional activity in cancer cell lines (57, 58) (Figure 3). Thus, the proteasome plays an important role in regulation of oncogenesis via controlling the expression and activity of FoxM1.

Endoplasmic reticulum (ER) stress

Following the translation of mRNA at the ER, proteins are folded into their functional forms. When proteins are not folded properly, they are directed to 26S proteasomal degradation. If misfolded or unfolded proteins are not degraded by the proteasome, they form aggregates and lead to the ER stress. The ER stress triggers unfolded protein response (UPR) to reduce the accumulation of unfolded proteins and restore the ER function. When protein aggregation or ER stress persists, especially in cancerous cells with high rates of protein synthesis, the UPR signaling switches from the pro-survival to pro-apoptotic. Consequently, the 26S proteasome complex also plays an important role in regulating the ER stress and cell survival. Therefore, inhibition of the proteasomal function in cancer cells would promote apoptosis and have an anti-tumor function (Figure 3). In fact, the inhibition of the proteolytic activity of the 26S proteasome has been shown to induce pro-apoptotic ER stress in multiple myeloma (59), pancreatic (60), head and neck cancer (61), and non-small cell lung carcinoma (62).

Androgen receptor (AR)

AR is a ligand-dependent transcription factor, and belongs to the family of nuclear receptors. It plays an important role during differentiation and growth of the prostate and accompanying urogenital structures (24, 63). In presence of its ligand androgen, it binds to the promoters of a set of genes and regulates their expression (24, 63). With the ability to upregulate or downregulate under certain conditions, AR influences the expression of many genes. Some include: keratinocyte growth factor, probasin, prostate specific antigen (PSA), p21, Kallikrein, ornithine decarboxylase, and the AR gene itself. When the strict regulation of AR slips, it causes tumorigenesis, especially prostate cancer. Androgen plays a crucial role in the development of prostate cancer by activating AR. Prostate cancer is the second leading cause of cancer death among American males. Due to this, great importance is placed on androgen and its part in the development of prostate cancer. When AR is inactivated in AR-dependent prostate cancer, those cells can no longer replicate DNA or enter S phase, causing cell death (24) (Figure 3). Interaction between calmodulin (CaM) and AR can cause CaM-dependent protein kinases to phosphorylate AR, thus manipulating its molecular stability and nuclear localization via the 26S proteasome (24). Proteasome inhibitor MG-132 arrests AR interaction with co-regulators ARA70 or TIF2, as well as preventing nuclear translocation of AR, repressing AR transactivation (24, 63). Further, the inhibition of the proteolytic activity of the 26S proteasome by bortezomib reduces the basal level of AR, and subsequently induces the apoptosis of androgen-dependent human prostate cancer LNCaP cells (24). However, some prostate cancers grow in the absence of androgen, named androgen-independent AR-positive prostate cancer. Interestingly, decreasing active AR levels in these cells still inhibits growth (24). Together, these studies have implicated the 26S proteasome complex in regulating AR expression and function, and hence carcinogenesis. Thus, proteasome inhibition has anti-tumor effects via modulating the activity of AR. Likewise, proteasome inhibition has also been shown to regulate GR (glucocorticoid receptor) and ER (estrogen receptor) target genes in MCF-7 breast cancer cells (64). Further, Alarid and colleagues (65) have recently provided a new link of proteasomal function in estrogen signaling in breast cancers, by demonstrating the repression of ER gene expression in response to proteasome inhibition.

Chemokines

Chemokines are a family of chemotactic cytokines, and cause cells to move along a chemical gradient. This mechanism is very important in terms of angiogenesis, cell migration, and metastasis, all playing enormous roles in cancer (66–69). The role of chemokines in metastasis is the outcome of modified presentation of G-protein coupled receptors. When the production of the chemokine receptor CXCR4 is upregulated in cancer cells (e.g., metastatic breast cancer, ovarian cancer and malignant melanoma cells), the cell migration is increased towards tissues that constitutively express the cognate chemokines ligand CXCL12, like liver, bone marrow, lymph nodes, and lung. To the contrary, the chemokine CXCL14/BRAK exhibits tumor suppressing activity but its expression is often modified in several cancers including prostate as well as squamous cell carcinomas of the head, neck, and cervix (69–71). The ubiquitin-proteasome system is responsible for degrading chemokine CXCL14 in cancer and other immortalized cells, but the proteasome does not degrade CXCL14 in normal epithelial cells (69). Treatment with proteasome inhibitor lactacystin resulted in the expression of CXCL14 in cancer cell lines that had previously shown impaired CXCL14 expression. Also, when LNCaP prostate cancer cells were treated with proteasome inhibitor MG-132, polyubiquitylated CXCL14 was found. These results demonstrate that cancer cells prevent their own CXCL14 expression as well as degradation by the proteasome. When overexpressed, CXCL14 inhibits angiogenesis and hence tumor growth (69, 72) (Figure 3). Further, normal and lower levels of CXCL14 would increase the chances of dendritic cells penetrating the tumor, leading to detection of the tumor by the immune system (69, 71–73). Thus, CXCL14 has anti-tumor activities via impairing angiogenesis and enhancing the detection of tumor by the immune system. The absence of CXCL14 in head and neck and prostate cancers favors cancer progression. Therefore, the inhibition of the proteolytic function of the proteasome in the cancer cells would increase the level of CXCL14, and hence would produce anti-tumor effects (Figure 3).

Cell-surface receptors

Cell-surface receptors, growth factors and their signaling pathways play important roles in carcinogenesis. Cell-surface receptors have been demonstrated to be ubiquitylated and degraded by the 26S proteasome complex. Proteasomal degradation of the cell-surface receptors is very relevant in cancer chemotherapy. For example, tyrosine kinase receptors are degraded by the 26S proteasome, and such degradation provides anti-tumor activity to herbimycin A which inhibits multiple tyrosine protein kinases (74). Similarly, protein kinase C (PKC) inhibitor downregulates PKC via 26S proteasomal degradation (75, 76). Other cell-surface receptors such as T-cell antigen receptor (TCR) and platelet-derived growth factor (PDGF) are also degraded by the 26S proteasome. These receptors are ubiquitylated in reponse to ligand binding for proteasomal degradation (77, 78). Thus, the 26S proteasome complex plays an important role in regulating the stabilities of the cell-surface receptors, and hence their signaling pathways.

AP-1

The proto-oncogene products c-JUN and c-FOS interact to form the transcription factor, AP-1 (activator protein 1). These two proto-oncoproteins form AP-1 as a heterodimer of c-JUN and c-FOS or a homodimer of c-JUN, and are known to be degraded by the 26S proteasome complex (79–84). The delta region, a 27 amino acid long segment of c-JUN, enables ubiquitylation and proteasomal degradation of the protein. This segment is missing in v-JUN, the transforming retroviral counterpart of c-JUN. As a result, v-JUN is upregulated, and such an increased stability is very likely to contribute to its oncogenicity (79). Therefore, an impaired proteasomal degradation of c-JUN can lead to oncogenesis. Moreover, the proteasomal degradation of c-JUN is essential to maintain normal function of AP-1 factors. The AP-1 factors play important roles in handling oxidative stresses (85, 86). AP-1 upregulation has been correlated with drug resistance in several cancer cell lines (87–89).

DNA repair

The DNA damage and regulation of its repair mechanisms are strongly correlated with carcinogenesis. The proteasome complex plays a crucial role in DNA repair (90). The inhibition of the proteolytic function would impair DNA repair (Figure 3), and trigger apoptosis. Therefore, DNA damaging agents have been used to kill cancer cells. Further, the combination of DNA damaging agents with proteasome inhibitor would have synergistic effects in killing cancer cells. Indeed, proteasome inhibitors have shown more effective anti-tumor activities when combined with the DNA damaging agents such as radiation and camptothecin (CPT) (9, 90).

MHC-restricted class I antigens

As mentioned above, MHC-restricted class I antigens are vitally processed by the immunoproteasome and presented on the cell surface for recognization by cytotoxic T lymphocytes. Proteins LMP2, LMP7, and LMP10 are interchanged with the three components of the 20S catalytic core to form the immunoproteasome in the instance of IFN-γ induction (91–95). Substitution of LMP2, LMP7, and LMP10 yields more types of peptides expressed on the cell surface (96, 97). Thus, the low levels of LMP2, LMP7, and LMP10 can lead to a decrease in MHC class I-restricted peptide presentation, and cause an escape from immune surveillance, leading to cancer. Indeed, very low levels of LMP2, LMP7, and lower antigen presentation are found in 3 small-cell lung carcinoma lines (98). Further, the mouse T-cell lymphoma cell line SP-3 has been shown to display an underexpression of LMP2 and an impairment of antigen presentation (99). Oncogenic viruses have been found to down-regulate LMP2 and LMP7 upon viral transformation of the cell (100). However, the expression of LMP2 and LMP7 has been shown to be increased with an enhancement of antigen presentation in these cancer cell lines by expressing IFN-γ following transfection (99, 101). By expressing fewer peptides, cancerous cells may avoid detection by the immune system. Thus, the immunoproteasome plays an important role in antigen presentation, and its malfunction would lead to the escape from immune surveillance (Figure 3), and hence cancer. In fact, a low level of immunoproteasome activity is present in certain cancer cells (98–100). A down-regulation of LMP2 and LMP7 has also been observed in hepatocellular carcinoma (102). Likewise, a decreased level of 26S proteasomal activity has also been observed in lung cancer stem-like cells in vitro (103). Further, an altered level of the proteasome component MB1 (β5) is found in ovarian cancer (104).

As discussed above, the 26S proteasome is involved in nearly every kind of pathway cancer uses for survival and growth. By understanding these pathways and their relationship with the proteasome, it becomes clear that the manipulation of the 26S proteasome in turn would determine the fate of cancer cells. Undoubtedly, the proteasomal inhibition has been proven to be an attractive anti-cancer tool as discussed below.

Proteasome inhibition to treat cancer

When proteasome inhibitors prevent the proteasome from activating NF-κB, factors of angiogenesis, survival, and growth are down-regulated while apoptosis is up-regulated in multiple tumor cell lines (16, 105–115) (Figure 3). This effect is also noticed in chemotherapy-resistant cells, additionally due to disruption of proteasomal regulation of caspases and Bcl-2. Further, proteasome inhibition enhances the levels of p21 and p27 (116, 117) (Figure 3). Such enhancement inhibits CDKs and consequently arrests cell cycle, halting the growth of cancer cells (Figure 3). The inhibition of the proteolytic function of the 26S proteasome has also been shown to impair the development of new blood vessels from endothelial cells or angiogenesis (Figure 3) that is a vital factor for tumor growth and metastasis (115, 118). Disruption of angiogenesis by proteasome inhibition also occurs by decreasing microvessel density and the expression of vascular endothelial growth factor (VEGF) (115, 118, 119) (Figure 3). Thus, the proteasome inhibition impairs angiogenesis as well as disturbs cellular homeostasis, hence leading to an anti-tumor activity. Overall, these studies demonstrated that the inhibition of the proteolytic function of the 26S proteasome induces apoptosis and cell cycle arrest, and represses angiogenesis as well as metastasis (Figure 3). In fact, apoptosis and other anti-tumor effects from proteasome inhibition have been observed in various cancer cell lines and xenograft models including lymphoma, leukemia, melanoma, pancreatic, prostate, head and neck, breast, and lung cancers (36, 119–126). Further, the cancer cells are more sensitive to the cytotoxic effects of the proteasome inhibition as compared to the normal cells (127, 128). Also, cessation of all proteasomal function is not required to achieve anti-tumor effects (14, 129). Together, these studies have implicated the proteasome inhibition as an attractive way of treating cancer cells (Figure 3). Therefore, a large number of studies are focused on a variety of proteasome inhibitors for effectively treating cancer.

There is a wide variety of natural and synthetic proteasome inhibitors. These inhibitors are clustered into five groups: peptide aldehydes, peptide vinyl sulphones, peptide boronates, peptide epoxyketones, and β-lactones (lactacystin and its derivatives). Small-molecule proteasome inhibitors mimic the peptide substrates of the active sites in the 20S catalytic core subunit of the 26S proteasome complex. Lactacystin is a microbial metabolite isolated from Streptomyces, and is the first compound found to have an inhibiting effect on the proteasome. Lactacystin effectively and irreversibly inhibits the β5-component of the proteasome by selectively modifying N-terminal threonine residues, and also reversibly binds to the β1- and β2-components. MG-132 (Z-Leu-Leu-Leu-aldehyde) and PSI (Z-Ile-Glu-(OtBu)-Ala-Leu-aldehyde) are two of the first proteasome inhibitors synthesized. These are peptide aldehydes that reversibly bind to the β2- and β5 components by forming covalent hemiacetal adducts. At high concentrations, they also inhibit calpains and cathepsins proteases. However, these compounds exhibit low specificity and high metabolic instability, limiting to use as research reagents. The peptide vinyl sulfone proteasome inhibitor has a vinyl sulfone group which is less reactive than the aldehyde group of the peptide aldehyde proteasome inhibitor. The vinyl sulfone group irreversibly binds to the active site. One most potent peptide vinyl sulfone proteasome inhibitor is AdaAhx3-LLL-vs. This inhibitor binds to the active sites of both the constitutive and immunoproteasome with almost equal efficiencies (130, 131). Peptide boronates, epoxomicin (peptide epoxyketone), and lactacystin have shown higher specificity to the proteasome, and therefore show the most promise for drug development. Two of the peptide boronates, MG-262 and bortezomib, form more stable tetrahedral intermediates with N-terminal threonine residues of the 20S CP, lending them a greater efficacy.

In addition to the synthetic and natural proteasome inhibitors, a variety of proteasome inhibiting compounds can also be found in foods. Some of these inhibitors are: apigenin, epigallocatechin-gallate (EGCG), and ajoene. Apigenin is a polyphenolic flavone found in a broad range of fruits and vegetables (132, 133). It has demonstrated chemopreventive properties in several cancer models such as lung, skin, cervical, prostate, and leukemia by scavenging the free radicals, anti-inflammation, and proteasome inhibition (134–141). It comes as no surprise that frequent ingestion of apigenin and other polyphenolic compounds correlates with a lowered cancer risk and even a suppression of tumor growth (132, 133). Apigenin achieves its proteasomal inhibiting effect by interrupting the chymotrypsin-like activity of the β5-component of the proteasome (133). In a study using MDA-MB-231 breast cancer cells, Chen et al. (133) found that at the highest concentration tested, apigenin reduced proliferation and viability of cancer cells by 50% after 24 hours. Western blot analysis confirmed that apigenin caused 40% proteasome inhibition, a buildup of ubiquitylated-Bax and IκBα, as well as increased caspase-3, caspase-7, and cleaved PARP (poly ADP-ribose polymerase) levels, indicating apoptosis (133, 136, 141). In addition to a significant proteasome inhibition, apigenin also seems to induce the expression of death receptor 5 and an apoptosis-inducing TNF-associated ligand in leukemia, prostate, and colon cancer cells without having a toxic effect on normal cells (133, 142). This cancer-targeted toxicity is echoed in animal models in vitro and in vivo (133). Like apigenin, EGCG present in green, but not black, tea has been demonstrated to inhibit the proteolytic function of the 26S proteasome. It is a polyphenolic compound, and has an anti-tumor activity (143, 144). Further, ECGC has been shown to attenuate the release of pro-inflammatory cytokines, thereby terminating inflammation (143, 144). Likewise, ajoene is an organo sulfur compound present in garlic (130). It has been shown to inhibit the trypsin-like activity of the 20S CP of the proteasome complex (145). It induces apoptosis as well as cell cycle arrest of tumor cells by inhibiting G2/M phase. Consequently, ajoene has cytotoxic effects in tumor cells (146).

Like the proteasome inhibitors found in foods, naturally occurring gallium has also shown anti-neoplastic activity in clinical trials in bladder cancer, lymphomas, and a variety of other malignancies (147–152). Gallium III complex demonstrates anti-tumor activity via the inhibition of the proteasomal activity of the 26S proteasome (152). Further, gallium disturbs iron homeostasis by competing with Fe³⁺ for uptake into cells, the mediator for which is the transferrin receptor system that is overexpressed in cancerous cells (152, 153–155). Chen et al (152) have demonstrated that a certain gallium complex tested inhibited 81% of proteasomal activity in C4–2B prostate cancer cells. This complex also induces apoptosis, as evidenced by PARP cleavage, TUNEL positivity, nuclei condensation, and activation of caspase-3/caspase-7 (152).

Bortezomib: a proteasome inhibitor in the clinic to treat cancer

As mentioned above, the proteasome complex plays crucial roles in many important biological events, and its malfunction is strongly correlated to carcinogenesis. Thus, the proteasome inhibitors have shown a broad spectrum of anti-proliferative and pro-apoptotic activities against haematological and solid tumors. However, many of these proteasome inhibitors have low potency, specificity or stability (156–161). Therefore, new proteasome inhibitors with greater potency and selectivity were developed. Thirteen boron-containing proteasome inhibitors were synthesized, and subsequently screened for anti-cancer activity using a panel of 60 human tumor cell lines of National Cancer Institute, USA (162). One compound showed extremely high potency against a wide range of cancer cell lines. This compound is known as bortezomib, velcade, or PS-341 (originally synthesized as MG-341 at a company called Myogenics, and marketed as velcade by Millennium Pharmaceuticals, Inc., Cambridge, MA, USA) (Table 1). Bortezomib is a water-soluble dipeptide boronic acid which contains pyrazinoic acid, phenylalanine and Leucine with boric acid instead of a carboxylic acid.

Table 1.

26S Proteasome inhibitors with theirs tagets and clinical status.

26S Proteasome inhibitor	Target in 26S proteasome	Clinical status
Bortezomib	Chymotrypsin-like activity	Approved for MM and MCL.
Carfilzomib	Chymotrypsin-like activity	Phase III in MM. Phase I in AML, ALL, CLL. Phase Ib/II in solid tumors.
ONX0912	Chymotrypsin-like activity	Phase I in solid tumors
NPI-0052	Chymotrypsin-like, Trypsin-like, Caspase-like activities	Phase I in MM, solid tumors, refractory lymphoma, and non-small cell lung carcinoma.
CEP-18770	Chymotrypsin-like activity	Phase I/II in MM.
MLN9708	Chymotrypsin-like activity	Phase I/II in MM. Phase I in lymphoma and non-haematological malignancies.

MM, multiple myeloma; MCL, mantle cell lymphoma; AML, acute myeloid leukaemia; ALL, acute lymphoblastic leukaemia; and CLL, chronic lymphocytic leukaemia.

Bortezomib is a stable proteasome inhibitor that binds covalently and reversibly with the β5 component of the 20S catalytic core subunit of the proteasome forming tetrahedral intermediates on the N-terminal threonine residues (156). Further, it does not have any known activity against other cellular proteases (163). Due to these qualities, bortezomib entered into clinical phase I trials (19, 20, 164–166). In phase I clinical trials, bortezomib demonstrated an effective proteasome inhibition with fair tolerance levels, and thus moved into phase II clinical trials. Bortezomib had success in this clinical phase with refractory multiple myeloma patients (167), spurring its rapid approval by the Food and Drug Administration (FDA) and European Medicines Agency (EMEA). Interestingly, in the phase III clinical trials in refractory multiple myeloma, the survival rate of the patients treated with bortezomib exceeded that of the patients treated with dexamethasone (168). Also, this proteasome inhibitor is consistently able to surmount the factors that normally cause treatment resistance including γ-radiation and the chemotherapeutic agent CPT-11 by preventing the activation of NF-κB (18, 169). Though accumulation of p53 can initiate apoptosis, bortezomib kills tumor cells independently of p53 levels, even drug resistant multiple myeloma cell lines with mutant p53 (16, 170).

Bortezomib specifically shows a high efficacy in multiple myeloma, non-small lung cancer, mantle cell lymphomas, and pancreatic cancer (125, 164, 167, 171, 172). Though not all tumor cell types react similarly to bortezomib, its substantial activity in a variety of cancer cell lines and tumor types in clinical trials propelled it into FDA approval. The FDA first approved bortezomib in 2003 for the third-line treatment of multiple myeloma (173). Later on, it was approved for the first-line treatment in 2008. It has also been approved in treating mantle cell lymphoma (a fast-growing cancer that begins in the cells of the immune system) in 2006 (174). As the first FDA approved proteasome inhibitor, bortezomib exhibits an around 1000-fold improvement over its aldehyde predecessors and more specificity to the β5 component of the 20S catalytic core of the proteasome. Although bortezomib has shown a significant anti-tumor activity, it is also used to overcome chemoresistance (175–177). Due to this, bortezomib has been successfully combined with several other agents such as doxorubicin, thalidomide, melphalan, and dexamethasone. Thus, there is a great hope in developing better combinatorial therapy without increasing toxicity in treating numerous cancer patients. Currently, there are a large number of clinical trials going on for combinatorial therapy involving bortezomib against haematological malignancies and solid tumors.

Combinatorial therapies have shown great potential for cancer treatment. The combination of bortezomib with other drugs such as Hsp90 inhibitor, HDAC inhibitor, Akt inhibitor, and lenalidomide have more clinical benefits as compared to bortezomib alone (115, 178). Bortezomib with DNA damaging agent works well with relapsed and/or refractory cancer patients (110, 115, 179, 180). Hsp90 inhibitor has been shown to overcome bortezomib resistance in mantle cell lymphoma (108, 178). Lenalidomide has been combined with steroids, proteasome inhibitors, mTOR (target of rapamycin) inhibitors, humanized monoclonal antibodies, and Akt inhibitors. Lenalidomide with bortezomib or Akt inhibitor has shown very impressive responses in the cancer patients (115). Overall, the combinatorial therapies have shown a very promising results, and the most successful combination is likely to be approved soon to treat cancer patients.

Although bortezomib has been approved to treat multiple myeloma and mantle cell lymphoma patients, it also sensitizes pancreatic cancer cells to ER stress-mediated apoptosis (30, 181–183). Further, an induction of ER stress is a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. The combination of bortezomib with HDAC inhibitor, SAHA (Suberoylanilide hydroxamic acid), entered clinical trials in 2007. Additionally, the combination of bortezomib with HDAC6 inhibitor (more specific) may have better clinical benefits in treating pancreatic cancer or other solid malignancies (30, 182).

Though bortezomib kills cancer cells, the cellular mechanisms for clinical efficacy of bortezomib are not clearly known. However, several mechanisms-of-action of bortezomib have been implicated in killing cancer cells, which include disruption of cell adhesion- and cytokine-dependent survival pathways (e.g., NF-kB signaling pathway), inhibition of angiogenesis, activation of a misfolded protein stress response (or ER stress), upregulation of pro-apoptotic or downregulation of anti-apoptotic genes. DNA microarray analysis revealed upregulation of genes involved in hypoxia, ER stress/UPR, oxidative stress, apoptosis, and amino acid starvation following proteasomal inhibition (184–194). Thus, bortezomib seems to kill cancer cells by hypoxic response deregulation in tumor cells, mTOR inhibition, and ER stress-induced apoptosis. Further, bortezomib has been shown to upregulate AP-1 activity and activating transcription factor (ATF) families (192, 193, 195–199). ATF4 contributes to apoptosis, thus implicating ATFs in bortezomib-induced apoptosis. Like other cancer therapies, some factors contribute resistance to bortezomib treatment (115). An increased expression of HSPs reduces the efficacy of bortezomib. For example, HSP27 directly correlates with bortezomib resistance (200), and HSP90 inhibition overcomes bortezomib resistance in mantle cell lymphoma (115, 178). Bortezomib has also been shown to promote IFN-α and TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis in human bladder cancer cells (201). Therefore, bortezomib manifests its anti-tumor activity via multiple mechanisms.

Bortezomib is metabolized primarily by cytochrome P450 3A4 (202, 203). Though intensely effective in treating many types of cancer, bortezomib is not without its side effects. Bortezomib has a dose limiting toxicity and pain associated with intravenous administration. Patients treated with bortezomib have experienced peripheral neuropathy, pyrexia, adverse gastrointestinal events, myelosuppression, orthostatic hypotension, asthenia, thrombocytopenia, cardiac and pulmonary disorders, and pain (128, 167, 168, 204, 205). Bortezomib is also associated with a high rate of shingles (206). Further, it has not shown promising results in treating solid tumors (9). These facts have demanded the need to develop a new generation of proteasome inhibitors. In this direction, several proteasome inhibitors have been developed, and are currently under clinical trials as presented below.

Proteasome inhibitors in clinical trials to treat cancer

There are several promising proteosome inhibitors that are currently in clinical trials. These are: Carfilzomib (PR-171), ONX0912 (PR-047), Marizomib (NPI-0052), CEP-18770, and MLN9708 (Table 1). Several immunoproteasome inhibitors (Table 2) have also been developed, which have shown impressive results in the pre-clinical studies. These inhibitors are described below.

Table 2.

Immunoproteasome inhibitors with theirs tagets and pre-clinical results.

Immunoproteasome inhibitor	Target in immunoproteasome	Pre-clinical results
PR-957	Chymotrypsin-like activity	Inhibits inflammatory response.
PR-924	Chymotrypsin-like activity	Inhibits tunor growth in animal models without significant toxicities. Inhibits growth of primary cell lines and primary tumor cells. Anti-tumor activity against MM.
IPSI-001	Caspase-like activity	Inhibits haematological malignancies in in vitro models. Inhibits proliferation in myeloma patient samples. Overcomes other drug resistance.

Carfilzomib

Carfilzomib (also known as PR-171) is an epoxomicin-based proteasome inhibitor with improved pharmaceutical properties. Proteolix Inc. (California, USA) has developed carfilzomib as a second generation proteasome inhibitor to treat multiple myeloma patients (129). Carfilzomib irreversibly binds to the catalytic site of the proteasome, and inhibits the chymotrypsin-like activity. Unlike bortezomib, carfilzomib has shown minimal cross-reactivity with the other catalytic sites of the 20S CP. Further, carfilzomib shows minimal reactivity with other protease classes. Thus, carfilzomib has a better selectivity than bortezomib for chymotrypsin-like activity of the 26S proteasome in in vitro and in vivo studies (129, 207, 208). Carfilzomib has also shown better tolerability and dosing flexibility in xenograft models (129, 208). Pre-clinical studies indicate that carfilzomib is active against models of solid tumors, lymphomas, and myeloma (129, 208–210). Carfilzomib inhibits cell proliferation, and induces apoptosis which is associated with activation of JNK (c-Jun N-terminal protein kinase), depolarization of mitochondrial membrane, release of cytochrome C, and activation of both intrinsic and extrinsic caspase pathways in patient-derived multiple myeloma cells as well as neoplastic cells from patients with other hematologic malignancies (129, 209, 210). The phase I clinical trials of carfilzomib demonstrated that multiple myeloma patients who have relapsed or progressed following a number of therapies (including bortezomib and stem cell transplant) can also achieve durable anti-tumor responses with carfilzomib. Carfilzomib is well tolerated in patients at doses that suppress chymotrypsin-like proteasome activity by >80% in whole blood. The phase II clinical trials of carfilzomib provided promising results in patients with relapsed or refractory multiple myeloma. Currently, clinical phase III trials are ongoing for carfilzomib in multiple myeloma (9, 211, 212). Further, carfilzomib is now under clinical phase I trials for acute myeloid leukemia (AML), acute lymphoblastic leukaemia (ALL), and chronic lymphocytic leukaemia (CLL) (9, 129). It is also in phase 1b/II trials in solid tumors (118, 213). Like bortezomib, carfilzomib may work better in combination with other therapies. In fact, it has been shown to function better in leukemia and lymphoma in combination with HDAC inhibitors in vitro (214, 215). Carfilzomib has also been shown to interact synergistically with HDAC inhibitors in mantle cell lymphoma cells (211). Furthermore, carfilzomib acts synergistically with dexamethasone, and has shown an increased level of anti-multiple myeloma activity as compared to bortezomib (129).

ONX0912

Both bortezomib and carfilzomib are administered intravenously. However, an oral proteasome inhibitor could be easily administered in the multi-drug treatment regimens. Proteolix, Inc. has developed an oral analogue, ONX0912 (also known as PR-047) that has N-cap with significant pre-clinical anti-tumor activities (216). This agent shows an improved therapeutic window over carfilzomib in experimental animal models. It has been demonstrated to reduce tumor progression and prolong survival in animal models of multiple myeloma, non-Hodgkin’s lymphoma and colorectal cancer (216–218). Further, it has been shown to enhance the anti-tumor activity in combination with HDAC inhibitor, lenolidomide and bortezomib (216–218). This proteasome inhibitor is currently under clinical trials. The clinical phase I trials of this compound are also ongoing in advanced solid tumors (9).

NPI-0052

NPI-0052 (also known as salinosporamide A or marizomib) is an irreversible second generation proteasome inhibitor, and orally bioactive (219). It has been developed by Nereus Pharmaceuticals, Inc. (San Diego, CA, USA) (220). It is a non-peptide, β-lactone compound that is related to lactacystin. It has been derived from the marine bacterium Salinospora tropica (221), and possesses anti-tumor activity through caspase-8 activation (222, 223). It stimulates apoptosis predominantly via caspase-8-mediated pathway (222, 223). Thus, NPI-0052 induces apoptosis via mechanisms that are unique from those evoked by bortezomib (108, 222, 223). NPI-0052 also differs from bortezomib or carfilzomib in terms of its inhibitory effects on the three major enzymatic activities of the 20S CP. It binds irreversibly to all catalytic sites for proteolysis of the 26S proteasome (223). At the maximum tolerated dose without apparent toxicity, NPI-0052 shows as high as 90% proteasome inhibition as compared to 70% inhibition by bortezomib (169, 219). The proteasome inhibition by NPI-0052 increases progressively over 24 hours, and remained essentially unchanged for 72 hours. On the other hand, the proteasome inhibition by bortezomib reaches the maximum level of inhibition at 1.5 hours, and then significantly decreases over the next 24 hours (219). Therefore, NPI-0052 appears to be a more effective compound in treating cancer patients. The cellular response to NPI-0052 occurs much earlier than bortezomib. Further, it has shown effectiveness in multiple myeloma cell lines that are resistant to bortezomib (223). It has also been demonstrated to be significantly effective in pre-clinical studies in Waldenstrom’s macroglobulinemia, acute leukaemia, CLL, prostate, pancreatic and colon cancers (219, 222, 224–228). However, NPI-0052 may be less specific since its analog lactacystin binds to several proteasome subunits as well as inhibits other cellular proteases. Although NPI-0052 blocks a wider range of proteasome activities, it appears to be less toxic to normal cells (223, 229). In mice implanted with human myeloma tumor cells, NPI-0052 was well tolerated and showed prolonged survival as well as significantly reduced the rate of cancer recurrences. Further, the cancer cells were killed more effectively by the combination of NPI-0052 with bortezomib and HDAC inhibitors, MS-275 and valproic acid (VPA) without additional toxicity to normal cells (222). The clinical phase I trials of NPI-0052 are ongoing in advanced solid tumors, refractory lymphoma and non-small cell lung carcinoma (9).

CEP-18770

It is a boronic acid-based proteasome inhibitor. Like bortezomib, it is a reversible proteasome inhibitor, and primarily inhibits the chymotrypsin-like activity of the proteasome (118, 230). It is a water soluble and orally bioactive proteasome inhibitor (118, 230). CEP-18770 abrogates the production of VEGF in multiple myeloma cells (118). Such a decreased level of VEGF production inhibits cell migration and vasculogenesis from the endothelial progenitors (118). Further, the role of CEP-18770 in angiogenesis is corroborated by its direct inhibitory effect on endothelial cell proliferation, survival, and capillary tubular morphogenesis (118). CEP-18770 has also been shown to promote apoptosis in human multiple myeloma cell lines (118, 230). It is a potent inhibitor of constitutive and TNF-α-triggered NF-kB activation (118). CEP-18770 has been demonstrated to have a significantly reduced toxicity toward human bone marrow progenitors, bone marrow stromal cells, and normal human intestinal cells as compared to bortezomib (118). Although CEP-18770 has a significant anti-tumor activity, it is more effective in combination with bortezomib and melphalan in animal tumor models (231). The clinical phase I trials of CEP-18770 have been completed for solid tumors and non-Hodgkin’s lymphoma (9). Currently, it is under phase I/II clinical trials for multiple myeloma (9).

MLN9708

It is a small molecule boron-containing peptide inhibitor (Millennium Pharmaceuticals, Inc.). In contrast to bortezomib, MLN9708 is orally bioavailable (232). Like bortezomib, it inhibits the chymotrypsin-like activity of the proteasome. However, the proteasome dissociation half-life of MLN9708 is shorter than bortezomib. Thus, it has improved tissue distribution, pharmacokinetics, pharmacodynamics, and anti-tumor activity in xenograft models (233). It is biologically inactive. However, it is hydrolyzed quickly in plasma to MLN2238 that is biologically active. It has shown strong anti-cancer activity against numerous cancer cell lines (232, 233). It has also been demonstrated to be effective in human prostate xenograft, colon cancer and lymphoma models (233). Very recently, Chauhan et al (232) have demonstrated that MLN9708 has synergistic anti-multiple myeloma activity when combined with bortezomib, HDAC inhibitor, lenalidomide or dexamethasone. This proteasome inhibitor is currently in phase I clinical trials in patients with lymphoma and non-haematological malignancies (9). Further, clinical phase I/II trials of MLN9708 for multiple myeloma are ongoing (9).

Immunoproteasome inhibitors

The immunoproteasome is present in immune cells at a lower level. Thus, the inhibition of the immunoproteasome will provide specificity over constitutive proteasome. Such specificity will attenuate the toxicities associated with constitutive proteasome inhibition. Several immunoproteasome inhibitors such as PR-957, PR-924 and IPSI-001 have been developed. Pre-clinical studies of these inhibitors have shown impressive anti-tumor and anti-inflammatory responses. PR-957 (also known as ONX0914) has recently been developed by Proteolix, Inc (234, 235). Like carfilzomib, it is a peptide epoxyketone proteasome inhibitor. It inhibits chymotrypsin-like activity of the immunoproteasome. PR-957 inhibits the functions of IL-1 (interleukin-1), IL-6 and TNF. Further, it blocks the production of IL-23 by activated monocytes and interferon-γ and IL-2 by T cells. Therefore, PR-957 has immunosuppressive effects (234, 235). Hence, PR-957 may be effective against autoimmune diseases in conjunction with cancer treatment. PR-957 induces an anti-inflammatory response at a low dose as compared to the non-selective inhibitors such as bortezomib and carfilzomib (234, 236, 237). Like PR-957, PR-924 is a peptide epoxyketone proteasome inhibitor, and inhibits chymotrypsin-like activity of the immunoproteasome (238). It impairs the growth of multiple myeloma cell lines and primary tumor cells. It has also been shown to inhibit the tumor growth in animal models without significant toxicities. Unlike PR-957 and PR-924, IPSI-001 is a peptide aldehyde type of inhibitor (239). It inhibits preferentially the β1i component of the immunoproteasome. It has been shown to inhibit the haematological malignancies in in vitro models. It also potently inhibits proliferation in myeloma patient samples (239). Further, IPSI-001 overcomes conventional and novel drug resistance (239). Together, these immunoproteasome inhibitors have great potential to be in the clinic in future with more selectivity and less toxicity.

Concluding remarks

Here, we have discussed the 26S proteasome complex in different key cellular events and carcinogenesis. It is clear from a large number of studies that the 26S proteasome complex regulates a multitude of cellular processes like cell cycle progression, inflammation, antigen presentation, apoptosis, DNA repair, transcription, and indirectly: cell growth, chemotaxis, angiogenesis, and cell adhesion. Many of these mechanisms are altered to the benefit of cancer cells. For this reason, the 26S proteasome complex has become an attractive target for cancer therapy. In fact, the proteasome inhibition has led to an increased apoptosis and other anti-tumor effects such as cell cycle arrest, and inhibition of angiogenesis and metastasis in various cancer cell lines and xenograft models. The proteasome inhibitor bortezomib is in the clinic to treat multiple myeloma and mantle cell lymphoma patients. Several proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Additional proteasome inhibitors with different efficacies are being developed and tested for anti-tumor activities. Several proteasome inhibitors have shown significantly improved anti-tumor activities when combined with other drugs such as HDAC inhibitor, Akt inhibitor, DNA damaging agent, Hsp90 inhibitor, and lenalidomide. In summary, proteasome inhibitors alone or in combination with other therapies have shown very promising results to treat cancer patients in the clinic more effectively.