Keywords: colorectal, cancer, carcinogenesis, proteasome, ubiquitin, β-catenin, APC

The proteasome is a multi-protein complex that regulates the stability of hundreds of cellular proteins and thus, it is implicated in virtually all cellular functions. Most of the times, to be recognized and processed by the proteasome, a protein has to be linked to a chain of ubiquitin molecules. Cell proliferation, apoptosis, angiogenesis and motility, processes with particular importance for carcinogenesis are regulated by the ubiquitin-proteasome system (UPS). In colorectal epithelium, UPS plays a role in the regulation of the Wnt/ β-catenin /APC/ TCF4 signaling which regulates proliferation of colorectal epithelial cells in the bottom of the crypts and the inhibition of this proliferation as cells move towards colon villi tips. In most colorectal cancers APC (Adenomatous Polyposis Coli) disabling mutations interfere with the ability of the proteasome to degrade β-catenin leading to uninhibited cell proliferation. Other key molecules in colorectal carcinogenesis such as p53, Smad4 and components of the k-ras pathways are also regulated by the UPS. In this review I discuss the role of UPS in colorectal carcinogenesis and colorectal cancer prognosis and aspects of its inhibition for therapeutic purposes.

Introduction

The proteasome is a multiprotein cellular complex which modulates or degrades the majority of cellular proteins. It has been discovered by Hershko, Ciechanover and Rose who have been awarded the Chemistry Nobel prize in 2004 for this discovery.

As a regulator of a variety of protein substrates, the proteasome plays a role in virtually every cellular function including processes of paramount importance for carcinogenesis such as proliferation, apoptosis, angiogenesis and motility /metastasis [START_REF] Wolf | The proteasome: a proteolytic nanomachine of cell regulation and waste disposal[END_REF]. Due to its involvement in these processes, proteasome function is a requirement for the survival of normal and neoplastic cells and indeed during apoptosis proteasome is inhibited [START_REF] Sun | Caspase activation inhibits proteasome function during apoptosis[END_REF][START_REF] Adrain | Caspase-dependent inactivation of proteasome function during programmed cell death in drosophila and man[END_REF]. In order to be recognized by the proteasome and be processed, a protein substrate needs to be linked to a chain of the 76 amino-acid protein ubiquitin [START_REF] Pickart | Back to the future with ubiquitin[END_REF]. Ubiquitin linkage is completed in a series of three reactions carried out by different enzymes called ubiquitin activating enzymes or E1, ubiquitinconjugating enzymes or E2 and ubiquitin ligases or E3, respectively. The proteasome has three enzymatic activities, chymotryptic, tryptic and glutamyl-peptidyl activity.

The importance of each activity varies depending on the substrate [START_REF] Kisselev | Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate[END_REF]. Protein ubiquitination is a reversible process and there exist de-ubiquitinizing enzymes that catalyze proteins de-ubiquitination preventing degradation [START_REF] Amerik | Mechanism and function of deubiquitinating enzymes[END_REF]. The proteasome itself possesses such activity which in this case serves the function of recycling ubiquitin for further use and facilitating protein substrate processing [START_REF] Pickart | Back to the future with ubiquitin[END_REF].

Colorectal carcinoma is one of the most common cancers and its prevalence is greater in developed countries compared with countries in development for reasons related to diet. Despite the introduction of several new drugs for the treatment of colorectal cancer over the last decade that have prolonged survival of advanced stage patients, the disease, when metastatic, remains always lethal and new treatments are urgently needed. Several lines of investigation are actively pursued worldwide in this disease and one of them researching the role of the ubiquitin-proteasome system in colorectal cancer will be the subject of this review.

UPS in the regulation of proliferation and differentiation of colonic epithelium

The Wnt signaling pathway is a signal transduction pathway with particular importance in colorectal development and in the regulation of proliferation and continuous renewal of the adult colorectal epithelium [START_REF] Clevers | Wnt breakers in colon cancer[END_REF][START_REF] Ilyas | Wnt signalling and the mechanistic basis of tumour development[END_REF]. Ligation of Wnt to the

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M A N U S C R I P T membrane receptor Frizzled inhibits the assembly of the destruction complex that would lead to proteasomal degradation of β-catenin, allowing the transcription factor to enter the nucleus and begin, in co-operation with the transcription factor TCF4 (Tcell Factor 4), transcription of target genes that lead to proliferation and differentiation of the cell. Among β-catenin/TCF4 targets are cyclin D, c-myc, akt kinase and transcription factor AP-1 (Activating Protein-1) subunits c-jun and fra-1 [START_REF] Shtutman | The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway[END_REF][START_REF] Wong | β-catenin-A linchpin in colorectal carcinogenesis?[END_REF][START_REF] Dihlmann | Regulation of AKT1 expression by beta-catenin/Tcf/Lef signaling in colorectal cancer cells[END_REF][START_REF] Mann | Target genes of βcatenin/ T cell-factor/ lymphoid-enhancer-factor signalling in human colorectal carcinomas[END_REF]. Increased β-catenin activity is observed in the bottom of colonic crypts where epithelial stem cells proliferate and move towards colonic villi giving rise to differentiated colonic epithelium. Cells in the villi tips display lower β-catenin activity while they express higher levels of APC (Adenomatous Polyposis Coli), a key component of β-catenin destruction complex [START_REF] Fodde | APC, signal transduction and genetic instability in colorectal cancer[END_REF]. β-catenin-dependent transcription plays a role in development in many human tissues but the particular importance of Wnt signaling in colorectal epithelium and carcinogenesis may be derived from the fact that another Wnt regulated transcription factor, Hath1 (Human Atonal Homolog 1) has a restricted expression in human lower gastro-intestinal tract [START_REF] Tsuchiya | Reciprocal targeting of Hath1 and beta-catenin by Wnt glycogen synthase kinase 3beta in human colon cancer[END_REF]. Hath1 is a basic helix-loop-helix transcription factor that promotes differentiation of colorectal epithelial cells and mucin production by gastro-intestinal goblet cells [START_REF] Leow | Hath1, downregulated in colon adenocarcinomas, inhibits proliferation and tumorigenesis of colon cancer cells[END_REF][START_REF] Park | HATH1 expression in mucinous cancers of the colorectum and related lesions[END_REF]. It is regulated by proteasomal degradation in a reverse to β-catenin way [START_REF] Aragaki | Proteasomal degradation of Atoh1 by aberrant Wnt signalling maintains the undifferentiated state of colon cancer[END_REF]. Wnt signaling pushes the balance of GSK3β (a kinase that participates in β-catenin phosphorylation in order to become ubiquitinated subsequently) activity away from βcatenin and towards phosphorylation of Hath1 which is then ubiquitinated and degraded in the proteasome. Hath1 has been found to be down-regulated in colorectal cell lines and colonic neoplastic tissues in contrast to normal adult human colorectum where it is abundantly expressed [START_REF] Tsuchiya | Reciprocal targeting of Hath1 and beta-catenin by Wnt glycogen synthase kinase 3beta in human colon cancer[END_REF]. Thus, the UPS plays a critical role in the availability of both transcription factors β-catenin and Hath1 that direct two different transcription programs and regulate proliferation and differentiation in colorectal epithelium.

Common lesions in colorectal pathogenesis and regulations by the UPS Many molecular lesions that lead to colorectal carcinogenesis have been identified

and at least two different pathways from normal colonic epithelium to colorectal carcinoma have been proposed. Mutations in APC is one of the primary events setting the transition from normal epithelium to a pathologic lesion termed Aberrant Crypt Focus (ACF), in a first pathway of colorectal carcinogenesis. ACFs are the first

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pathologic lesions that can be microscopically seen and are characterized by crypts with larger size than the normal ones, an increased pericryptic space and a thicker layer of epithelium [START_REF] Pretlow | Mutant KRAS in aberrant crypt foci (ACF): Initiation of colorectal cancer?[END_REF][START_REF] Mclellan | Aberrant crypts : potential preneoplastic lesions in the murine colon[END_REF]. ACFs transition to adenomas happens when an activating mutation to the proto-oncogene K-ras is added. For adenoma to carcinoma transformation, neutralization of the tumor suppressors p53 and Smad4 (also known as DPC4-Deleted in Pancreatic Cancer 4) is taking place usually by mutation of the one allele and loss of heterozygosity of the remaining normal allele. This sequence of molecular events is exemplified by the hereditary syndrome Familial Adenomatous Polyposis (FAP) and is also the sequence leading to carcinogenesis in 85-90% of sporadic human colorectal cancers. APC is the gene mutated in the germ line in families with FAP leading to the formation of dozens or even hundreds of polyps throughout the colon of the affected family members. In the majority of the remaining 10 to 15% of sporadic colorectal cancers a different sequence of molecular events is the culprit. This is a sequence that is also taking place in a hereditary syndrome, Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and is due to mutations in one of the genes involved in mismatch repair (MMR), the process that detects and repairs a misaligned base in the double helix of DNA. MMR genes MSH2, MLH1, MSH6 and PMS2 are most frequently mutated, triggering microsatellite instability consisting of multiple DNA defects that are most prominent in areas with long repeats of the same nucleotide (known as microsatellites). These areas are more prone to sequence mistakes, which, due to repair proteins defects, are not corrected. As these mutations accumulate the regulatory mechanisms of the cells are paralyzed leading to malignancy.

Many of the molecular lesions leading to colorectal carcinogenesis are directly related to the Ubiquitin-Proteasome System. APC is part of the complex that executes the phosphorylation of β-catenin in order for this transcription factor to become ubiquitinated and be recognized by the proteasome for degradation (Fig. 1A). Thus, mutations of APC in FAP or the majority of sporadic colorectal cancers allows stabilization of β-catenin which can enter the nucleus and execute its transcription function, transcribing genes leading to cell proliferation and inhibition of apoptosis.

Activating mutations of k-ras in colorectal cancer leads to the activation of several transduction pathways that start from this oncogene. Raf kinase activated by k-ras triggers the MAPK cascade which leads to activation of transcription factors such as AP1 [START_REF] Fang | The MAPK signalling pathways and colorectal cancer[END_REF]. Components of this transcription factor such as c-jun and c-fos as well as kinases of the cascade such as ERK3 are proteasome substrates (Fig. 1B). PI3K

kinase is also a target of activation by k-ras and its activation leads to activation of the akt kinase. Akt is a serine / threonine kinase with multiple targets such as mdm2 (a E3 ligase for p53), Bad (an anti-apoptotic member of the bcl-2 family), caspase 9, mTOR, IKK, and GSK3β kinases and transcription factor FKHR [START_REF] Vivanco | The phosphatidylinositol 3-kinase-akt pathway in human cancer[END_REF]. Through its effects on these targets, akt inhibits apoptosis and favors proliferation. Both akt and phosphatase PTEN, its inhibitor, are proteasome substrates [START_REF] Adachi | Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells[END_REF][START_REF] Wu | Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells[END_REF].

Smad4, a signal transducer of the TGFβ pathway is frequently involved in the pathogenesis of colorectal cancer and is a proteasome substrate [START_REF] Xu | Mutations in the tumor suppressors Smad2 and Smad4 inactivate transforming growth factor β signalling by targeting Smads to the ubiquitinproteasome pathway[END_REF](Fig. 1C). The same holds true for another tumor suppressor mutated in colorectal cancer, p53. p53's related protein p73 and p53's co-factor ASPP/53BP2 are also proteasome substrates [START_REF] Coutts | The p53 response: Emerging levels of co-factor complexity[END_REF].

HIF-1α (Hypoxia Inducible Factor 1α) is a transcription factor involved in angiogenesis and cell survival under hypoxic conditions and is regulated by the proteasome. In normoxia UPS degrades HIF-1α (Fig. 2). The E3 ligase promoting its ubiquitination for proteasomal degradation, VHL (Von Hippel-Lindau) is genetically mutated in the eponymous syndrome and frequently somatically mutated in renal carcinoma. About 10% of colorectal carcinomas bear VHL mutations and in some of these cases a parallel hyper-expression of HIF-1α was detected by immunohistochemistry [START_REF] Kuwai | Mutation of the von Hippel-Lindau (VHL) gene in human colorectal carcinoma : association with cytoplasmic accumulation of hypoxia-inducible factor (HIF)-1α[END_REF].

As evidenced from this discussion pathways involved in colorectal cancer are regulated by the proteasome in multiple levels and with multiple ways. It is this multiplicity of influences, though, that makes the final result of proteasome manipulation difficult to predict. For example, proteasome inhibition will activate βcatenin by preventing its degradation, a pro-carcinogenic effect, while it would have opposing effects in the k-ras/PI3K/akt pathway due to both akt and negative regulator PTEN being a proteasome substrate. Nevertheless the final result of multiple cellular dysfunctions from proteasome deregulation or inhibition would be expected to be homeostatic loss and cellular death which would be predicted to become more prominent in cells already unstable such as the neoplastic cells.

Effects of proteasome inhibitors on colorectal cancer in vitro and in vivo

Given the importance of the proteasome on cell regulation including the regulation of several neoplastic processes, many studies have sought to investigate the effect of proteasome inhibitors on colorectal cancer.

Proteasome inhibitors have been shown to inhibit proliferation and induce apoptosis in a variety of cell types. Hematologic cancer derived cells are among the most sensitive to proteasome inhibition [START_REF] Drexler | Activation of the cell death program by inhibition of proteasome function[END_REF]. Both multiple myeloma and lymphoma cells display inhibition of their proliferation after exposure to proteasome inhibitors and indeed bortezomib is already in clinical use in multiple myeloma patients and in advanced development in lymphoma. Proteasome inhibition also leads to inhibition of proliferation in a variety of solid tumors such as breast, prostate, head and neck and colorectal in vitro and in vivo [START_REF] Teicher | The proteasome inhibitor PS-341 in cancer therapy[END_REF][START_REF] Adams | Proteasome inhibitors: a novel class of potent and effective antitumor agents[END_REF][START_REF] Almond | The proteasome: a novel target for cancer chemotherapy[END_REF][START_REF] Sunwoo | Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-kappaB, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma[END_REF][START_REF] Voutsadakis | Combined treatment of colorectal cancer cell lines with aspirin and bortezomib[END_REF]. The inhibitory effects of proteasome inhibitors on cell proliferation have been attributed to diverse mechanisms. Inhibition of antiapoptotic transcription factor NF-κB is a prominent mechanism proposed to explain the inhibitory effects of proteasome inhibitors. NF-κB is positively regulated by the UPS because its inhibitor IκB is a proteasome substrate and can be ubiquitinated and degradated by an active UPS system. In addition, NF-κB family members p100 and p105 are cleaved to their active counterparts p52 and p50 respectively by the proteasome [START_REF] Chen | Ubiquitin signalling in the NF-κB pathway[END_REF]. The same E3 ubiquitin ligase, β-TrCP, functions in the ubiquitination of IκB for complete proteasomal degradation and in the ubiquitination of p100 and p105 for partial proteasomal cleavage and activation. The final result of both processes is NF-κB transcription activation. This activation is of particular relevance in colorectal cancer where, due to β-catenin activation, β-TrCP is upregulated, being a β-catenin transcriptional target [START_REF] Spigelman | Wnt/ β-catenin signalling induces the expression and activity of βTrCP ubiquitin ligase receptor[END_REF]. Indeed NF-κB up-regulation is observed during colorectal carcinogenesis and can be at least in part due to proteasomal activity [START_REF] Lind | Nuclear factor-kappa B is upregulated in colorectal cancer[END_REF][START_REF] Yu | Increased expression of RelA/ Nuclear factor-κB protein correlates with colorectal tumorigenesis[END_REF].

Another transcription factor, p53 that is important for apoptosis and cell cycle arrest under stress conditions, is regulated by the UPS and may mediate inhibitory effects of proteasome inhibitors. p53 has been termed the guardian of the genome for its paramount importance in inducing cell cycle arrest when DNA is damaged in order to be repaired or apoptosis when the damage is irreversible [START_REF] Yu | The transcriptional targets of p53 in apoptosis control[END_REF]. p53 is regulated by ubiquitination with the mediation of several E3 ligases, the most prominent and first described being mdm2 (also referred as hdm2 in humans). Other E3 ligases of p53 include COP1 [START_REF] Dornan | The ubiquitin ligase COP is a critical negative regulator of p53[END_REF], Pirh2 [START_REF] Leng | Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation[END_REF], ARF-BP1/Mule [START_REF] Chen | ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor[END_REF] and hUREB (human Upstream Regulatory Element Binding protein 1). hUREB is a HECT (Homolog to E6BP Carboxy-Terminal) domain ubiquitin ligase expressed in colorectal carcinoma cells and its expression is inversely correlated with the expression of p53 [START_REF] Yoon | Over-expression of human UREB1 in colorectal cancer: HECT domain of human UREB1 inhibits the activity of tumor suppressor p53 protein[END_REF]. Forced expression of the HECT domain of hUREB1 in these cells destabilizes p53.

The cyclin dependent kinases (CDKs) inhibitor p21 is a transcriptional target of p53 and is induced after treatment of colorectal cancer cells with the proteasome inhibitors LLnL and lactacyctin [START_REF] Chen | Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors[END_REF]. Furthermore, p21 is a target of proteasome degradation and thus proteasome inhibition directly stabilizes this cell cycle inhibiting protein [START_REF] Maki | Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation[END_REF]. CDK inhibitor p27 KIP1 is also a proteasome substrate and mutations in residues that prevent its ability to be ubiquitinated for proteasomal degradation decrease progression of intestinal polyps to carcinomas in mice [START_REF] Timmerbeul | Testing the importance of p27 degradation by the SCF skp2 pathway in murine models of lung and colon cancer[END_REF]. E3 ligase SCF Skp2 mediates p27 degradation (Fig. 3). p27 levels do not play a role in tumor initiation but rather in tumor progression, given the fact that polyp formation is not affected by inhibition of p27 degradation in these mice. Moreover the reverse is true and p27 reduction is associated with colorectal tumor progression in APC mutant mice [START_REF] Philipp-Staheli | Pathway-specific tumor suppression: reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice[END_REF].

The underlying molecular lesions that lead to tumor initiation may influence p27 ability to inhibit tumor progression because in contrast to what happens in APC mutant mice, in Smad3 mutant mice, p27 levels are not reduced with tumor progression [START_REF] Philipp-Staheli | Pathway-specific tumor suppression: reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice[END_REF].

Several members of the bcl-2 regulators of apoptosis are proteasome substrates (Fig. 3). Bik/NBK, a BH3-only pro-apoptotic member of the family is induced after treatment of several colon cancer cell lines with the proteasome inhibitors bortezomib and MG132 [START_REF] Zhu | Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors[END_REF][START_REF] Zhu | Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation[END_REF]. This treatment-induced Bik/NBK accumulation led to cell apoptosis which was reduced when cells were pre-treated with Bik/NBK siRNA [START_REF] Zhu | Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors[END_REF].

Pro-apoptotic bcl-2 family members Bax and Bak are also up-regulated by treatment of HCT116 colorectal cancer cells with bortezomib [START_REF] Yu | Differential apoptotic response to the proteasome inhibitor bortezomib (Velcade, PS-341) in Bax-deficient and p21deficient colon cancer cells[END_REF] while anti-apoptotic members bcl-2 and bcl-xl are not affected [START_REF] Zhu | Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors[END_REF][START_REF] Yu | Differential apoptotic response to the proteasome inhibitor bortezomib (Velcade, PS-341) in Bax-deficient and p21deficient colon cancer cells[END_REF]. It is worth mentioning that the same effect of pro-apoptotic bcl-2 members stabilization after proteasome inhibition has been observed in other cell types [START_REF] Nikrad | The proteasome inhibitor bortezomib sensitizes cells to killing by death receptor ligand TRAIL via BH3-only proteins Bik and Bim[END_REF][START_REF] Li | Bax degradation by the ubiquitin/ proteasome-dependent pathway: Involvement in tumor survival and progression[END_REF][START_REF] Chen | Induction of G1 arrest and apoptosis in human Jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27 Kip1 , p21 Cip1/WAF1 , and Bax proteins[END_REF] and may be a universal anti-neoplastic mechanism of proteasome inhibition.

Proteasome inhibitors in combination with other agents in colorectal carcinoma

Proteasome inhibitors have been tested in colorectal cancer in combination with chemotherapeutic and other agents in order to augment their tumor inhibiting effects through reversal of drug resistance or additive cytotoxity with different mechanisms.

Topoisomerase II is an enzyme involved in DNA replication and repair and is inhibited by chemotherapeutic agents such as etoposide and the anthracyclines. Diverse pharmacologic, dietary and other agents have been described to have antineoplastic effects in colon that may be mediated through proteasome inhibition.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of Cox inhibiting agents used clinically as analgesic and anti-inflammatory drugs. They also possess antineoplastic properties which are to a significant extent Cox inhibition-independent [START_REF] Yin | Cyclooxygenaseindependent effects of aspirin on HT-29 human colon cancer cells, revealed by oligonucleotide microarrays[END_REF]. In colorectal cancer Cox inhibitors have chemo-preventive actions established from epidemiologic studies [START_REF] Sandler | A randomized trial of aspirin to prevent colorectal adenoma in patients with previous colorectal cancer[END_REF][START_REF] Baron | A randomized trial of aspirin to prevent colorectal adenomas[END_REF][START_REF] Imperiale | Aspirin and the prevention of colorectal cancer[END_REF] in addition to their tumor suppressive and antineoplastic properties [START_REF] Brown | COX-2: A molecular target for colorectal cancer prevention[END_REF][START_REF] Gupta | Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2[END_REF]. Anti-neoplastic actions of NSAIDs in colorectal cancer are evident in both Cox expressing and non-expressing cells and diverse mechanisms have been proposed to explain this Cox-independent anti-cancer activity such as direct IKK-β inhibition [START_REF] Yin | The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta[END_REF], spermidine/spermine acyl-transferase (SSAT) induction [START_REF] Babbar | Cyclooxygenaseindependent induction of apoptosis by sulindac sulfone is mediated by polyamines in colon cancer[END_REF][START_REF] Ignatenko | Suppression of polyamine catabolism by activated K-ras in human colon cancer cells[END_REF], Peroxisome Proliferator Activated Receptor γ (PPARγ) activation [START_REF] Lehmann | Peroxisome Proliferator-activated receptors α and γ are activated by indomethacin and other non-steroidal anti-inflammatory drugs[END_REF] and reduction of β-catenin transcriptional activity [START_REF] Dihlmann | Reduction of β-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated β-catenin[END_REF]. Proteasome inhibition is an additional mechanism that has been found to play a role in aspirin induced apoptosis [START_REF] Dikshit | Aspirin induces apoptosis through the inhibition of proteasome function[END_REF]. Proteasome inhibition resulted in stabilization of proteasome substrates such as Bax, IκB-α, p53 and p27. This inhibition by aspirin was dose dependent and the most significant effects were seen with doses at or above 10 mM which are the levels where Cox-independent proliferation inhibition is generally observed and which are not easily attainable in the clinical setting, at least for prolonged times. Other previously described Cox-independent effects of NSAIDs in colorectal cancer may be proteasome inhibition-dependent. For example the aforementioned reduction of βcatenin transcriptional activity is accompanied by an accumulation of phosphorylated

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β-catenin in cells [START_REF] Dihlmann | Reduction of β-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated β-catenin[END_REF], the form that would normally be processed for ubiquitination and proteasome degradation.

Butyrate is a short chain fatty acid that is among the fatty acids produced in the intestine by the bacterial metabolism of dietary fiber. Fiber protects against colorectal neoplasia and this effect may be mediated by butyrate which induces cancer cell apoptosis [START_REF] Avivi-Green | Apoptosis cascade proteins are regulated in vivo by high intracolonic butyrate concentration: correlation with colon cancer inhibition[END_REF][START_REF] Hague | Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer[END_REF][START_REF] Augenlicht | Short chain fatty acids and colon cancer[END_REF]. Butyrate has been shown to inhibit proteasome activity and inhibit NF-κB transcription activity through IκB-α stabilization [START_REF] Yin | Butyrate suppression of colonocyte NF-κB activation and cellular proteasome activity[END_REF]. Probiotics, nonpathogenic bacteria of the intestine also inhibit the proteasome [START_REF] Petrof | Probiotics inhibit nuclear factor-κB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition[END_REF]. Such a probiotic combination called VSL#3 and composed of Streptococcus thermophilus as well as lactobacillus and bifidobacteria species inhibits proteasome activity of mouse colonic cells in vitro, stabilizes IκB-α and as a result inhibits NF-κB and decreases the production of MCP-1 (Monocyte Chemoattractant Protein 1), a transcription target of NF-κB and mediator of inflammation. In this manner probiotics have beneficial effects in conditions such as inflammatory bowel disease, infective and antibioticassociated diarrhea [START_REF] Gionchetti | Probiotics in infective diarrhoea and inflammatory bowel diseases[END_REF] and pouchitis [START_REF] Gionchetti | Oral bacteriotherapy as maintainance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial[END_REF]. By suppressing inflammation in a generally pro-inflammatory environment and through production of butyrate and other short chain fatty acids or possibly additional means of inhibiting the proteasome, probiotics help maintaining the good health of colon and prevent neoplasia which is promoted by inflammation.

UPS components and substrates as prognostic indicators in colorectal cancer

Given the important role of UPS in many processes related to colorectal carcinogenesis, it comes as no surprise that several components of the system as well as protein substrates are differentially expressed in individual colorectal cancer cases with differing aggressiveness and provide prognostic information.

β-TrCP (beta-Transducin repeat-Containing Protein) is an E3 ligase that is involved in the proteasomal degradation of β-catenin and in the partial cleavage of the NF-κB family precursor proteins p105 and p100 to yield their active counterparts p50 and p52 respectively [START_REF] Chen | Ubiquitin signalling in the NF-κB pathway[END_REF][START_REF] Fong | Genetic evidence for the essential role of β-Transducin repeat-containing protein in the inducible processing of NF-κB2/p100[END_REF][START_REF] Lang | βTrCP-mediated proteolysis of NF-κB1 p105 requires phosphorylation of p105 serines 927 and 932[END_REF]. Moreover β-TrCP is a target gene of β-catenin/TCF4 transcription and, as a result, is up-regulated in colorectal cancer where β-catenin is activated due to APC mutations and disabling of the proteasomal degradation machinery. Indeed a study has shown that increased β-TrCP levels were associated with β-catenin activation, nuclear NF-κB activity and decreased apoptosis in colorectal cancer cells [START_REF] Ougolkov | Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin, and NF-κB in colorectal cancer[END_REF]. In addition tumors from patients with metastatic stage disease displayed higher levels of β-TrCP mRNA compared to tumors from patients without metastases, witnessing for a role of β-TrCP-involved pathways in the aggressiveness of colorectal cancer [START_REF] Ougolkov | Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin, and NF-κB in colorectal cancer[END_REF].

Another member of an E3 ubiquitin ligase complex that has been associated with prognosis in colorectal carcinoma is skp2 (S phase kinase protein 2). Skp2 together with a regulator protein Cks1 (Cyclin-dependent kinase subunit 1) are effecting the ubiquitination of Cdk inhibitor p27 KIP1 . Overexpression of the two proteins has been observed in patients with poor prognosis colorectal carcinoma [START_REF] Shapira | The prognostic impact of the ubiquitin ligase subunits Skp2 and Cks1 in colorectal carcinoma[END_REF]. Skp2 and Cks1 are strongly correlated with overall survival of patients with colorectal cancer independently of grade and stage. A very interesting finding in this study was that patients with advanced stage colorectal carcinoma but a high Skp2 expression had better overall survival than patients with early (stage I and II) disease but low Skp2 expression in immunohistochemistry [START_REF] Shapira | The prognostic impact of the ubiquitin ligase subunits Skp2 and Cks1 in colorectal carcinoma[END_REF]. This result illustrates eloquently the paramount importance of the molecular making of a tumor and specifically of UPS beyond gross pathologic extent in colorectal carcinogenesis. Whether p27 is the only ubiquitination target of Skp2 and Cks1 that defines the importance of the two proteins in the prognosis of colorectal cancer is not known. Skp2 has been implicated in the ubiquitination and degradation of CDK inhibitor p21 which may also play a role in colorectal cancer pathogenesis [START_REF] Bloom | To be or not to be… ubiquitinated?[END_REF][START_REF] Yu | Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21 CIP1/WAF1 and cyclin D proteins[END_REF]. Nevertheless, the role of p27 is well established and several studies describe the effect of its expression in the aggressiveness of colorectal carcinoma [START_REF] Loda | Increased proteasome-dependent degradation of the cyclindependent kinase inhibitor p27 in aggressive colorectal carcinomas[END_REF][START_REF] Thomas | Downregulation of p27 is associated with development of colorectal adenocarcinoma metastases[END_REF]. Patients with all stages of colorectal cancer whose tumors expressed p27 by immunohistochemistry had a median survival of over ten years while those patients with tumors not expressing p27 had a median survival of less than six years [START_REF] Loda | Increased proteasome-dependent degradation of the cyclindependent kinase inhibitor p27 in aggressive colorectal carcinomas[END_REF]. Specifically for patients with stage II disease, the median survival was greater than 219 months in patients with tumors having more than 50% p27 positive cells compared with a median survival of 140 months in patients with tumors with less than 50% p27 positive cells and 69 months in patients with no p27 expression at all [START_REF] Loda | Increased proteasome-dependent degradation of the cyclindependent kinase inhibitor p27 in aggressive colorectal carcinomas[END_REF]. p27 expression in these patients with colorectal cancer was modulated by differential proteasomal degradation and not by different degrees of mRNA production. Extracts from tumors expressing different p27 levels were able to degrade recombinant purified p27 in different degrees when incubated ex vivo, mimicking the in vivo situation. In a series of 86 patients with early stage colorectal cancer undergoing surgery for the primary tumor with a curative intent, from the 48 patient with more than 20% p27 staining in their tumor by

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immunohistochemistry, 47 remained free from recurrence whilst from 38 patients with less than 20% p27 positive cells 21 patients had tumor recurrence [START_REF] Galizia | Determination of molecular marker expression can predict clinical outcome in colon carcinomas[END_REF]. In another study of 89 stage Duke A to C colorectal cancer patients, using a 50% positivity for p27 as the cut-off point, patients with high expression displayed a statistically significant better cancer-specific mortality [START_REF] Palmqvist | Prognostic significance of p27 Kip1 expression in colorectal cancer : A clinico-pathological characterization[END_REF]. In patients with metachronous metastases from colorectal cancer, p27 was found to be down-regulated in the metastatic tumor cells compared with the primary tumors, a phenomenon not seen in metastatic colorectal disease diagnosed at the same time (synchronous) with the primary tumors, where p27 expression was low in both primary and metastatic sites [START_REF] Thomas | Downregulation of p27 is associated with development of colorectal adenocarcinoma metastases[END_REF]. p27 down-regulation in metachronous colorectal metastases was due to increased protein degradation given that mRNA expression by in situ hybridization remained unchanged, speaking for the role of UPS in this protein degradation. The level of p27 during the different phases of the cell cycle in normal tissues is not regulated in the transcription level, as its mRNA levels remain stable through-out the cell cycle, but in the protein stability and degradation level which is effectuated by phosphorylation, ubiquitination and proteasomal degradation [START_REF] Montagnoli | Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation[END_REF][START_REF] Hershko | Prognostic role of p27 Kip1 deregulation in colorectal cancer[END_REF]. It is this regulation that fails in colorectal cancer due to skp2 and cks1 over-expression (and possibly additional mechanisms) leading to decreased p27 stability and increasing tumor aggressiveness.

p97 protein also known with the alternative names VCP (Valosin Containing Protein) and cdc48 is an ATPase of the AAA family (ATPase Associated with diverse cellular Activities) [START_REF] Hanson | AAA+ proteins: have engine, will work[END_REF] and constitutes an integral part of the proteasome forming an homohexameric ring [START_REF] Briggs | p97 and ubiquitin: A complex story[END_REF]. p97 is able, through interactions with diverse partner proteins, to recognize poly-ubiquitinated substrates for protein degradation and plays important roles in endoplasmic reticulum-associated degradation (ERAD), mitotic spindle disassembly and post-mitotic membrane fusion [START_REF] Briggs | p97 and ubiquitin: A complex story[END_REF][START_REF] Meyer | A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways[END_REF][START_REF] Kondo | p47 is a cofactor for p97-mediated membrane fusion[END_REF][START_REF] Cao | The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis[END_REF][START_REF] Buchberger | Cdc48 (p97) and its cofactors[END_REF]. In colorectal carcinoma patients whose tumors showed high p97 expression were found to have statistically significantly higher probability of distant metastases development and lower disease free and overall survival compared with patients whose tumors showed low p97 expression by immunohistochemistry [START_REF] Yamamoto | Expression of valosin-containing protein in colorectal carcinomas as a predictor for disease recurrence and prognosis[END_REF]. In this series of 129 patients, p97 expression was classified as high or low with the staining of normal colonic mucosa as the reference point. p97 has been shown to be involved in the activation of transcription factor NF-κB by taking part in the recognition of ubiquitinated I-κBα for

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proteasomal degradation [START_REF] Dai | Involvement of Valosin-containing Protein, an ATPase co-purified with IκBα and 26S proteasome, in ubiquitin-proteasome-mediated degradation of IκBα[END_REF][START_REF] Asai | VCP (p97) regulates NF-κB signalling pathway, which is important for metastasis of osteosarcoma cell line[END_REF], an event that may explain the association of its hyper-expression with colorectal cancer prognosis.

p21 WAF1 is another CDK inhibitor and proteasome substrate and is degraded by the proteasome through both ubiquitin-dependent and independent pathways [START_REF] Li | Ubiquitin-and ATP-independent proteolytic turnover of p21 by the REGγproteasome pathway[END_REF][START_REF] Jin | MDMX promotes proteasomal turnover of p21 at G1 and early S phases independently of, but in cooperation with, MDM2[END_REF][START_REF] Bloom | Proteasomemediated degradation of p21 via N-terminal ubiquitination[END_REF][START_REF] Touitou | A degradation signal located in the C-terminus of p21 WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome[END_REF].

In colorectal cancer, several studies investigated p21 expression correlation with prognosis. In a series of 192 colorectal cancer patients of all stages, absent or low (less than 10% of cells) p21 expression was statistically significantly associated with poorer disease free and overall survival than high expressors in univariate analysis, although this prognostic significance was lost in multivariate analysis [START_REF] Viale | p21 WAF1/CIP1 expression in colorectal carcinoma correlates with advanced disease stage and p53 mutations[END_REF]. In another series of 126 patients that used 50% as the cut-off point between p21 high and low expression cases, a statistically significant poorer survival of low p21 expressors was maintained even in multivariate analysis [START_REF] Holland | Subcellular localisation of cyclin D1 protein in colorectal tumours is associated with p21 WAF1/CIP1 expression and correlates with patient survival[END_REF]. Yet in another study that included only rectal stage I to III cancer patients, low p21 expression was associated with higher recurrence rate and disease-free survival [START_REF] Schwandner | p21, p27, cyclin D 1 , and p53 in rectal cancer: immunohistology with prognostic significance?[END_REF]. Nevertheless, other investigators have not found p21 expression to be correlated with clinical outcomes in colorectal cancer [START_REF] Mckay | Analysis of key cell-cycle checkpoint proteins in colorectal tumours[END_REF][START_REF] Watanabe | Molecular predictors of survival after adjuvant chemotherapy for colon cancer[END_REF]. This may be related to a more complex regulation of p21 than p27 and the fact that p21 is regulated not only by proteasomal degradation but also by transcription control. In fact it is a transcriptional target of p53 (a proteasome substrate itself and bearing inactivating mutations in many advanced colorectal cancer cases) and indeed p21 mRNA has been found to be decreased in colorectal cancer [START_REF] Matsushita | Reduced messenger RNA expression level of p21CIP1 in human colorectal carcinoma tissues and its association with p53 gene mutation[END_REF].

Clinical studies of UPS inhibition in colorectal cancer

Following encouraging in vitro and in vivo xenograft data of proteasome inhibition in colorectal cancer [START_REF] Teicher | The proteasome inhibitor PS-341 in cancer therapy[END_REF], a phase II study of the proteasome inhibitor bortezomib, the only currently clinically available proteasome inhibiting drug, was undertaken in patients with metastatic colorectal cancer [START_REF] Mackay | A phase II trial with pharmacodynamic endpoints of the proteasome inhibitor bortezomib in patients with metastatic colorectal cancer[END_REF]. Nineteen patients who had received one to three lines of chemotherapy were treated in this study with an intravenous dose proteasome inhibition [START_REF] Aghajanian | A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies[END_REF] while most clinical responses in other solid tumors were observed with proteasome inhibition above 70% [START_REF] Papandreou | Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer[END_REF]. This level of inhibition was obtained with doses of 1.6 mg/m 2 twice weekly which was the dose recommended for phase II studies in a previous phase I studies [START_REF] Papandreou | Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer[END_REF]. Nevertheless in another phase I dose escalation study of two different schedules (twice weekly two out of three weeks or four out of six weeks) of bortezomib in solid tumors, which included 16 colorectal cancer patients, two patients with stable disease had received doses of 0.5 mg/m 2 and 0.9 mg/m 2 , doses that resulted in a mean proteasome inhibition of 36% and 52% at 1

hour respectively [START_REF] Dy | A phase I and pharmacologic trial of two schedules of the proteasome inhibitor, PS-341 (Bortezomib, Velcade), in patients with advanced cancer[END_REF]. All other patients treated in this study had progressive disease. This study also established that the twice weekly schedule two out of three weeks was better tolerated than the twice weekly four out of six weeks schedule and allowed better delivery of intended doses at the scheduled time.

Despite the good rational and the encouraging pre-clinical data, it becomes clear from the above phase I and II studies of bortezomib that in colorectal cancer the maximal tolerated doses do not produce significant antitumor results. This may be related to both pharmacodynamic parameters and to innate tumor resistance to single drugs which is a clinical problem with several anti-neoplastic agents and has led to the development of the concept of combination cancer therapy. Indeed combinations of bortezomib with other agents is the most promising approach for further development of the drug in colorectal cancer as well as in other solid malignancies and has begun to be explored. Based on pre-clinical data of in vitro synergism of pharmaceutical proteasome inhibition with topoisomerase I inhibitors [START_REF] Desai | Ubiquitin/ 26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells[END_REF][START_REF] Cusack | Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: Implications for systemic nuclear factor-κB inhibition[END_REF] (see also section on combination), a phase I dose escalation study of bortezomib with irinotecan has been undertaken in 51 patients with advanced solid tumors [START_REF] Ryan | A phase I study of bortezomib plus irinotecan in patients with advanced solid tumors[END_REF]. Twenty three patients with colorectal cancer were included. Bortezomib dose of 1.3 mg/m 2 on days 1, 4, 8 and 11 plus irinotecan 125 mg/m 2 on days 1 and 8 every three weeks were found to be the maximal tolerated doses for the combination and were recommended for phase II studies. Thirty four patients were evaluable for response. No objective responses were obtained in the entire cohort but 10 of 34 patients (29%) had stable disease for at least 6 weeks. Among these 10 patients five colorectal cancer patients were included (22%, given that these patients were heavily pre-treated (median of 3 prior chemotherapy regimens, range 0 to 7) and some of them had previously received irinotecan.

Three other phase I studies combining bortezomib with gemcitabine, docetaxel or the combination of carboplatin and paclitaxel have been published in full paper [START_REF] Ryan | Phase I clinical trial of bortezomib in combination with gemcitabine in patients with advanced solid tumors[END_REF][START_REF] Messersmith | Phase I trial of bortezomib in combination with docetaxel in patients with advanced solid tumors[END_REF][START_REF] Ma | A phase I and pharmacologic study of sequences of the proteasome inhibitor, bortezomib (PS-341, Velcade TM ), in combination with paclitaxel and carboplatin in patients with advanced malignancies[END_REF] and had included small numbers of colorectal cancer patients (three, one and two patients respectively). None of these patients had obtained any response (Table ), a fact probably expected from the borderline activity of gemcitabine, taxanes and carboplatin in colorectal malignancies. A next step for bortezomib development in colorectal cancer would be to investigate its combination with an established drug in this disease such as oxaliplatin, irinotecan or 5-fluoro-uracil and its analogues in less heavily pre-treated patients, possibly with the addition of a targeted anti-VEGF or anti-EGFR therapy such as bevacizumab, cetuximab or panitumumab which have no significant over-lapping toxicities. Another strategy would be the combination of bortezomib and an established cytotoxic drug with novel targeted therapies which have given encouraging pre-clinical results with bortezomib in colorectal cancer, such as Cox inhibitors [START_REF] Voutsadakis | Combined treatment of colorectal cancer cell lines with aspirin and bortezomib[END_REF][START_REF] Choi | Lactacystin augments the sulindac-induced apoptosis in HT-29 cells[END_REF][START_REF] Minami | Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity[END_REF][START_REF] Voutsadakis | Pathogenesis of colorectal carcinoma and therapeutic implications: The roles of the ubiquitin-proteasome system and Cox-2[END_REF] or deacetylase inhibitors [START_REF] Adachi | Synergistic effect of histone deacetylase inhibitors FK228 and m-Carboxycinnamic acid bis-hydroxamide with proteasome inhibitors PSI and PS-341 against gastrointestinal adenocarcinoma cells[END_REF]. Other proteasome inhibitors, such as NPI-0052 [START_REF] Cusack | NPI-0052 enhances tumoricidal response to conventional cancer therapy in a colon cancer model[END_REF] or compounds targeting different components of UPS are in initial phases of development [START_REF] Wang | Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding[END_REF]. A good targeting strategy could be ubiquitin ligases which would have the advantage of being more specific for the stabilization of proteins served by the specific E3 ligase blocked [START_REF] Pagano | When protein destruction runs amok, malignancy is on the loose[END_REF]. Selection of patients to be entered in UPS inhibitor studies based on evidence of increased UPS activity (e.g. decreased p27 expression) could be a fruitful strategy based on the model of K-ras mutations screening before treatment of colorectal cancer patients with EGFR inhibitors [START_REF] Lièvre | KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab[END_REF]. Succesful development of UPS inhibitors will permit patients with metastatic colorectal carcinoma who have already one of the longest prospects of survival among solid tumor patients (20 to 24 months) to obtain further improvement in prognosis with a hope for the ultimate goal of a cure for metastatic cancer.

A

  Topoisomerase II inhibitors resistance is mediated by a down-regulation of the enzyme by proteasome degradation in stress conditions such as glucose deprivation or hypoxia. This is relevant for the tumor environment in vivo because in many tumors extensive areas are hypoxic and nutrient starved, and thus would be expected to display topoisomerase II down-regulation. Inhibition of the proteasome by lactacystin sensitizes HT29 colorectal cancer cell line to topoisomerase II inhibitors etoposide and doxorubicin in vitro[START_REF] Ogiso | Proteasome inhibition circumvents solid tumor resistance to topoisomerase II-directed drugs[END_REF]. Moreover lactacystin partially reverses resistance of HT29 xenografts to etoposide in Balb/c-nude mice in vivo. Reversal of topoisomerase II resistance by proteasome inhibitors is not restricted to colorectal carcinoma. The cell line of ovarian origin A2780 displayed reversal of etoposide resistance when cotreated with lactacystin in glucose starvation conditions[START_REF] Ogiso | Proteasome inhibition circumvents solid tumor resistance to topoisomerase II-directed drugs[END_REF].A similar phenomenon is seen with topoisomerase I inhibitor camptothecin which induces proteasome-dependent down-regulation of topoisomerase I in a variety of cell lines (among which colorectal cancer cell lines) that results in resistance to the antiproliferative actions of the drug. Treatment with the proteasome inhibitor MG132 reverses this resistance by stabilizing topoisomerase I[START_REF] Desai | Ubiquitin/ 26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells[END_REF]. Additionally inhibition of the proteasome with bortezomib prevented induction of the anti-apoptotic transcription factor NF-κB that results from cell treatment with the topoisomerase I inhibitor CPT-11[START_REF] Cusack | Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: Implications for systemic nuclear factor-κB inhibition[END_REF]. Combined treatment of colorectal cancer cell lines and of colorectal xenograft bearing mice with bortezomib and CPT-11 or its active metabolite, SN-38 resulted in significantly enhanced inhibitory activity compared with each drug alone[START_REF] Cusack | Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: Implications for systemic nuclear factor-κB inhibition[END_REF].Combination of proteasome inhibitors lactacystin and bortezomib with cyclooxygenase (Cox) inhibitor sulindac enhances the cytotoxicity of each drug alone in vitro and in vivo in nude mice bearing colorectal tumor xenografts[START_REF] Choi | Lactacystin augments the sulindac-induced apoptosis in HT-29 cells[END_REF][START_REF] Minami | Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity[END_REF]. These synergistic effects may relate to the fact that Cox inhibitors prevent increased Cox-2 activity resulting from proteasome inhibition-induced up-regulation of Cox-2[START_REF] Chen | Transcriptional regulation of cyclooxygenase-2 in response to proteasome inhibitors involves reactive oxygen species-mediated signaling pathway and recruitment of CCAAT/Enhancer-binding protein δ and CREB-binding protein[END_REF].A C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPT 10 They are, though, at least partially Cox-independent because synergy has been observed in colorectal carcinoma cells not displaying Cox activity[START_REF] Voutsadakis | Combined treatment of colorectal cancer cell lines with aspirin and bortezomib[END_REF][START_REF] Choi | Lactacystin augments the sulindac-induced apoptosis in HT-29 cells[END_REF].Proteasome inhibitors bortezomib and PSI (N-benzyloxycarbonyl-Ile-Glu( O-tertbutyl-Ala-leucinal) have been found to have synergistic inhibitory effects with histone deacetylase inhibitors FK228 and CBHA (m-carboxycinnamic acid bis-hydroxamide) in colorectal cancer cells in vitro[START_REF] Adachi | Synergistic effect of histone deacetylase inhibitors FK228 and m-Carboxycinnamic acid bis-hydroxamide with proteasome inhibitors PSI and PS-341 against gastrointestinal adenocarcinoma cells[END_REF]. Generation of reactive oxygen species and stabilization of the pro-apoptotic bcl-2 family member Bim may at least partially mediate the synergistic effect.Role of the UPS in colorectal tumor inhibition observed with diverse agents not being classic UPS inhibitors

of 1 .

 1 3 mg/m 2 on days 1, 4, 8 and 11 of a twenty one days cycle and sixteen were evaluable for response. No objective responses were seen. Thirteen patients had progressive disease and three patients had stable disease as their best response (Table). The median time to progression was 5.1 weeks and the 6 month progression free survival was 11.4 %. As a result the study was closed before the first 19 planned patients were entered. Nevertheless, this negative study leaves some open questionsA C C E P T E D M A N U S C R I P Tregarding the efficacy of proteasome inhibition with the bortezomib doses used. In accompanying correlative studies no changes in transcription factor NF-κB or its inhibitor I-κB were observed in biopsies obtained from liver metastases before and after treatment. Bortezomib 1.3 mg/m 2 twice weekly as used in this study causes 65%

Figure 1 :

 1 Figure legendsFigure 1: Simplified representations of three major pathways involved in colorectal carcinogenesis and regulated by the UPS. A. The Wnt/β-catenin/ TCF4 pathway. B. K-ras initiating signalling. C. TGF-β pathway. Molecules which are proteasome substrates are represented in star signs. Arrows denote activation, ┴ signs denote inhibition.

Figure 2 :

 2 Figure2: The regulation of the transcription factor HIF-1 in normoxic and hypoxic conditions. HIF-1 is composed of the HIF-1α subunit which is ubiquitinated and degraded in normoxic conditions (A) and of HIF-1β subunit which is not subject to UPS regulation. In hypoxic conditions (B) that are often present in tumor masses HIF-1α is not degraded by the proteasome and is entering the nucleus to transcribe its target genes such as VEGF, erythropoietin and genes encoding enzymes of the anaerobic metabolism. VHL, the E3 ligase mediating HIF-1α ubiquitination is mutated in about 10% of colorectal cancers leading to deregulated HIF activity. HIF target gene VEGF signalling is already clinically targeted in colorectal cancer with the use of the monoclonal antibody bevacizumab.

Figure 3 :

 3 Figure 3: Regulation of the cell cycle and apoptosis by the UPS is mediated by stabilization of CDK inhibitor p27 (A) and of bcl-2 family members Bik, Bid, Baxand Bad (B), respectively. Other important regulators of these processes such as, for example p21, p53, caspases, Smac/Diablo and securin, are also proteasome substrates.

of 23 patients in an intention to treat analysis). This is an impressive stability rate

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