Peroxisome proliferator activated receptor γ (PPARγ) is a transcription factor of the nuclear hormone receptor superfamily. It has an important role in adipose tissue and in adipogenesis but is also expressed at high levels in colonic epithelium. PPARγ is involved in colorectal carcinogenesis and PPARγ-dependent transcription has anti-carcinogenic effects in many experimental colorectal cancer models while in some other instances PPARγ displays cancer promoting effects[ ¹].

PPARγ together with two other PPARs (PPARα and PPARβ/δ) belongs to the orphan ligand sub-family of the nuclear hormone receptor super-family. Human PPARγ gene is located at chromosome 3p25[ ³]. Higher levels of protein expression are displayed in adipose tissue and colonic epithelium but many other tissues such as pancreatic β cells and vascular endothelium also express the transcription factor[ ⁴].

PPARγ molecule has a domain organization similar to all nuclear receptors. In the amino-terminal part of the protein there is a transcription regulation domain, followed by a DNA binding domain and a hinge region, while the carboxy-terminal part of the molecule is occupied by the ligand binding domain.

Ubiquitination, that is the covalent attachment of the protein ubiquitin to a target protein, is a post-translational modification that can result in diverse outcomes. Ubiquitin is a 76 aminoacid protein that contains several lysine residues through which different types of chains can be formed[ ¹⁷]. A chain of at least four ubiquitin molecules attached to each other through a covalent link between lysine 48 of one molecule and the carboxyterminal glycine of the next molecule and finally attached to a target protein identifies this protein for proteasome degradation. Other types of ubiquitin attachments such as attachment of a single ubiquitin molecule to a target protein (mono-ubiquitination) or attachment of a ubiquitin chain to other lysine residues (e.g. Lys68) result in different outcomes and play roles in DNA repair, endocytosis, histone regulation and nuclear export. Attachment of ubiquitin to the target requires the action of three enzymes. Initially an enzyme called E1 or ubiquitin-activating enzyme binds ubiquitin and transfers it to a second enzyme called E2 or ubiquitin conjugating enzyme using energy from the degradation of ATP to ADP. Finally, a third enzyme called ubiquitin ligase or E3 transfers ubiquitin from E2 to the target protein[ ¹⁸].

Given the high expression level of PPARγ in colorectal epithelium, there is a particular interest in defining the role of PPARγ transcription in colorectal cancer. Activation of PPARγ after exposure of colorectal cancer cell lines to the natural ligand 15-S-hydroxy-eicosatetraenoic acid (15S-HETE) and to thiazolidinedione synthetic ligands leads to growth arrest and induction of apoptosis[ ^{20 - 22}]. Re-induction of differentiation related genes suppressed in colorectal cancer, such as cytokeratins 18 and 19 and intestinal alkaline phosphatase, is also noticed after activation of PPARγ in colorectal cancer cells[ ^{20 , 23}]. In contrast, genes that are induced in cancer such as polyamine metabolism enzyme ornithine decarboxylase and metastasis promoting protein laminin binding protein are repressed after PPARγ activation[ ²³].

Mice bearing human colorectal cancer xenografts display decreased rate of tumor growth after feeding with PPARγ ligands[ ²³]. Rats in which preneoplastic colon lesions, aberrant crypt foci, have been induced by exposure to the chemical azoxymethane, also display decreased lesion formation after PPARγ activation[ ^{24 , 25}]. A third in vivo model of PPARγ activation using Min mice, a strain of mice with activation of adenomatous polyposis coli (APC) gene due to germline mutation, has given controversial results with some studies observing increased colonic polyps formation after PPARγ activation[ ^{26 , 27}] while others have observed decreased tumor formation[ ²⁸]. Another mouse strain with APC inactivation, Apc mice, also displayed decreased polyp formation after PPARγ activation[ ²⁹]. Overall, in vivo models support a suppressive role of PPARγ in colorectal carcinogenesis[ ³⁰]. Controversial results with strains bearing disabled APC may relate to the fact that APC regulates the β-catenin transcription program and different residual β-catenin activity may have diverse roles in carcinogenesis[ ³¹]. This residual activity is further modulated by increased PPARγ activity as will be discussed later.

Induction of cdk inhibitors p21, p18 and p27 by PPARγ is involved in cell cycle arrest following PPARγ activation[ ^{12 , 32}] while interaction of PPARγ with Rb further promotes cell cycle arrest by recruiting histone deacetylase 3 and silencing transcription[ ³³]. Phosphatase PTEN is a target of the PPARγ transcription program and its induction inhibits akt kinase activation, promoting apoptosis[ ¹⁴].

Post-translational regulation of PPARγ involves its degradation by the UPS (Table 1)[ ³⁹]. This is an event that follows ligand binding and nuclear receptor transcription activation and represents a negative feed-back regulation, a common theme in most nuclear receptors. This mechanism has been shown to regulate PPARγ heterodimeric partner RXR as well as retinoic acid receptor, estrogen receptor, progesterone receptor and androgen receptor[ ^{40 - 42}]. Interestingly the two other PPAR family members PPARα and PPARβ/δ, although structurally similar to PPARγ, undergo a reverse regulation upon ligand binding and display delayed ubiquitination and proteasome degradation[ ^{43 , 44}]. PPARγ degradation is dependent on ligand binding but independent of transcription per se.

PPARγ co-activator PGC-1α is also regulated by the UPS[ ⁴⁵]. PGC-1α ubiquitination through a domain at the C-terminal part of the molecule leads to proteasome degradation. This helps in maintaining an optimal level of the co-activator for facilitation of transcription of PPARγ and also of other transcription factors for which PGC-1α functions as a co-activator such as PPARα, PPARβ/δ and estrogen related receptor α (ERRα)[ ⁴⁶]. Proteasome inhibition leads to ubiquitinated PGC-1α accumulation and formation of non-functional aggregates. Thus, the UPS function maintains the optimal levels of PGC-1α in order to perform its co-activator function[ ⁴⁵]. PGC-1α is additionally regulated at the transcriptional level by both PPARγ and ERRα, creating a regulation loop[ ^{47 , 48}].

PPARγ transcription factor interacts with several other factors and pathways in colorectal cancer and the final output is defined by a network of interactions in which the ubiquitin-proteasome system participates at multiple levels. Some of these interactions are outlined in the following paragraphs.

β -catenin

Abundance of β-catenin, a transcription factor activated in most human colorectal cancers, is regulated by at least three parallel pathways that culminate in its ubiquitination and proteasome degradation. The first pathway involves phosphorylation by glycogen synthase kinase 3β (GSK3β), facilitated by a complex in which APC takes part and results in ubiquitination by ubiquitin ligase TrCP[ ^{57 , 58}]. In the second pathway, ubiquitination of β-catenin is performed by ubiquitin ligase Siah-1 which is induced by p53 activation[ ^{59 , 60}]. In a third pathway, a currently unknown ubiquitin ligase mediates PPARγ-activated ubiquitination of β-catenin[ ⁶¹]. Tumorigenic β-catenin displays enhanced transcription activity due to mutation of serine to alanine at position 37 (S37A) rendering it resistant to GSK3β/ APC-mediated phosphorylation and ubiquitination/ proteasome degradation. This molecule can interact with PPARγ and be ubiquitinated through an alternative mechanism in order to be degraded by the proteasome[ ⁶²]. In this way, PPARγ suppresses β-catenin activity through enhanced proteasome-mediated degradation which possibly is a result of induction of molecules involved in the ubiquitination process[ ⁶²]. Conversely when stabilized, β-catenin inhibits PPARγ activity, an effect requiring direct interaction of the two molecules. This interaction involves the TCF binding domain of β-catenin and a catenin binding domain in PPARγ that is close to the ligand binding domain of the molecule[ ⁶²]. Another study, although confirming the interaction of β-catenin with PPARγ in colorectal cancer cells, found transactivation and not repression of PPARγ activity in putative PPREs[ ⁶³]. Nevertheless this study used a highly artificial system in which both genes were transfected in colon cancer cell lines before co-immunoprecipitation and reporter gene experiments[ ⁶³]. Their physiologic relevance is debatable in view of the already mentioned evidence for a reverse interaction. Furthermore, in adipocytes there is a mutual antagonistic relationship between the wnt/β-catenin signalling pathway and PPARγ[ ^{64 - 66}], strongly supporting an analogous relationship in colorectal tissue. A role of the UPS in this interaction is implied by the fact that both transcription factors are regulated by the system, although the exact regulation mechanism of the interaction must be complex.

NF-κ B

Another pathway interacting with both PPARγ and with β-catenin signalling and that is regulated by the UPS is the one culminating in the activation of transcription factors of the NF-κB family. NF-κB is activated in colorectal cancer as it is down-stream of activated K-ras. NF-κB has proliferative and anti-apoptotic effects and is activated in many cancers. Its main regulation is effectuated by phosphorylation and ubiquitination of its inhibitor I-κB. This is then degraded in the proteasome, releasing NF-κB in order to enter the nucleus and begin its transcription program. Additional regulation of NF-κB results from direct interactions with PPARγ and β-catenin which both result in inhibition of NF-κB transcription[ ^{67 - 69}]. PPARγ also trans-represses NF-κB-regulated genes indirectly through binding to promoters and recruiting co-repressors. For this action, SUMOylation [i.e. Small ubiquitin-related modifier (SUMO) binding] of PPARγ is required[ ⁷⁰]. Metastasis mediating chemokine receptor CXCR4 is regulated at the transcription level by NF-κB and PPARγ in a reciprocal way. Thus, the interaction has the potential to regulate metastasis of colorectal cancer cells, NF-κB being a promoter while PPARγ being a suppressor of colorectal cancer metastatic process[ ⁷¹].

Given that all three transcription factors play significant roles in colorectal cancer and all three are regulated by the UPS, it is evident that a complex interaction with UPS is central in determining the final proliferation outcome of the neoplastic cell. NF-κB is also of paramount importance in inflammation and inflammation-induced carcinogenesis. Nuclear receptors in general and PPARγ in particular can antagonize this action, having anti-inflammatory effects[ ⁷²].

Colorectal carcinogenesis involves the acquisition of genetic lesions over time, leading to progression from hyperplasia to adenoma to carcinoma. Most common genetic lesions include APC mutations, activating β-catenin transcription, K-ras mutations and Smad4 (DPC4) mutations. All these pathways and others involved in colorectal carcinogenesis are regulated by the UPS.