The present studies were undertaken to focus on several fundamental questions that remain concerning the functional role of PPARβ/δ in colon carcinogenesis. The first question addressed was whether expression of PPARβ/δ is altered in human and/or rodent colon tumors. It was originally hypothesized that expression of PPARβ/δ is directly up-regulated by the APC/β-CATENIN/TCF4 pathway, similar to that observed for CYCLIN D1 and c-MYC [18
]. While increased expression of PPARβ/δ in colon tumors has also been reported by other laboratories, the weight of evidence indicating that PPARβ/δ expression is not up-regulated by the APC/β-CATENIN/TCF4 pathway is increasing (reviewed in [2
]). For example, expression of PPARβ/δ is not increased in human colon cancer cell lines with gain-of-function mutations in the APC/β-CATENIN/TCF4 pathway, despite clear up-regulation of expression of CYCLIN D1 and/or c-MYC [19
]. To date, no studies have quantitatively examined expression of PPARβ/δ protein from cohorts of tumors and corresponding control tissue from human colon cancer patients. Thus, results from the present study are the first to demonstrate that expression of PPARβ/δ protein is lower in nineteen human colon adenocarcinomas as compared to matched control colon tissue. It is also important to note that this decrease in colon adenocarcinoma PPARβ/δ was associated with increased expression of CYCLIN D1. The decrease in expression of PPARβ/δ was specific for colon adenocarcinomas as similar changes were not found in a cohort of human rectal tumors. The observed decrease in PPARβ/δ expression in nine colon tumors from Apc+/Min-FCCC
mice as compared to matched control colon mucosa is also highly consistent with the changes observed in human colon adenocarcinomas, as decreased expression of PPARβ/δ was also associated with markedly higher expression of CYCLIN D1. While previous work showed no change in expression of PPARβ/δ expression in small intestine tumors from Apc+/Min
], it is important to note that the present study examined expression from colon tumors from Apc+/Min
mice and that whole cell lysates were used rather than nuclear fractions. Collectively, this is the most robust data set published to date that definitively demonstrates that expression of PPARβ/δ protein is decreased, not increased, during colon tumorigenesis.
Most studies to date examining expression of PPARβ/δ during colon carcinogenesis have focused primarily on PPARβ/δ
mRNA expression. Previous studies suggesting that expression of PPARβ/δ is increased during colon tumorigenesis are often limited because expression of protein is not compared with that of mRNA (reviewed in [2
]). Additionally, some studies suggesting increased expression of PPARβ/δ during colon tumorigenesis are limited to immunohistochemical analysis [34
]. This is problematic because immunohistochemical analysis of PPARβ/δ expression is unreliable due to considerable non-specific immunoreactivity of PPARβ/δ antibodies. For example, while one study suggested that expression of nuclear PPARβ/δ is higher in mouse colon tumors based on immunohistochemical analysis [34
], subsequent western blot analysis using samples from the same study revealed no changes in nuclear expression of PPARβ/δ [19
]. The hypothesis that expression of PPARβ/δ is increased during colon tumorigenesis as suggested by some (reviewed in [2
]) is also at odds with the findings that colon and small intestine exhibit the highest expression of PPARβ/δ in mice [6
]. Further, recent evidence from antibody proteomic analysis indicates that while expression of PPARβ/δ is strong in human colon cells, expression of PPARβ/δ is weak to negligible in human colorectal cancer [4
]. Since there is considerable debate whether expression of PPARβ/δ is either increased or decreased during colon tumorigenesis, it is surprising that many reports fail to point out findings reporting that PPARβ/δ is not increased during colon carcinogenesis. Based on the definitive findings from the present work, it is clear that future studies should rigorously examine expression of PPARβ/δ protein to confirm changes in mRNA expression.
It is also of interest to note that expression of PPARγ1, was markedly lower in colon tumors from Apc+/Min-FCCC
mice, but not in human colon or rectal adenocarcinomas. Similar results have also been observed in small intestine polyps from Apc+/Min
]. Additionally, decreased PPARγ mRNA was also reported to occur in colon polyps from azoxymethane-treated mice that correlated with reduced protein expression based on immunohistochemical analysis [36
]. In contrast, other studies reported no change in expression of PPARγ in Apc+/Min
] or even increased expression of PPARγ in colon polyps from azoxymethane-treated rats or Apc+/Min
]. The reason for these differences cannot be determined from the present work. The reason why decreased expression of PPARγ1 was not found in human colon or rectal tumors in the present study is also uncertain. While one study showed no difference in expression of PPARγ mRNA between colonic epithelial cells and tubular adenomas [42
], decreased PPARγ mRNA has been found in colon tumors from acromegalic patients [43
]. Further studies are necessary to determine why colon tumors from Apc+/Min-FCCC
mice exhibit decreased expression of PPARγ1 while human colon and rectal tumors do not.
The second issue addressed by the present study is whether NSAIDs down-regulate expression of PPARβ/δ during colon carcinogenesis, which in turn promotes apoptotic signaling. This notion is based on the hypothesis that PPARβ/δ is anti-apoptotic and prevents NSAID-induced apoptosis by increasing expression of the 14-3-3ε that enhances sequestration of Bad, a pro-apoptotic member of the B-cell CLL/lymphoma 2 (Bcl-2) family [20
]. This hypothesis is based on studies using human colon cancer cell lines (DLD1 and HT29) and endothelial cells treated with NSAIDs (sulindac sulfide, indomethacin) or hydrogen peroxide to induce apoptosis. Thus, the present study used the same human colon cancer cell lines and the same concentrations of NSAIDs to critically examine the hypothesis that NSAID- or hydrogen peroxide-induced apoptosis is mediated by down-regulation of PPARβ/δ due to decreased expression of 14-3-3ε that leads to increased apoptosis. In contrast to several reports [20
], results from the present study demonstrate that NSAIDs (sulindac, sulindac sulfide, indomethacin) do not decrease expression of PPARβ/δ, but rather, expression of PPARβ/δ is either unchanged or increased by these drugs. This observation is consistent with a number of other studies (reviewed in [2
]) including the recent observation that indomethacin increases expression and function of PPARβ/δ in RKO human colon cancer cell lines [19
]. Additionally, in vivo analyses reveal that nimesulide does not alter expression of PPARβ/δ in the mouse colon [45
], and that sulindac does not alter expression of PPARβ/δ in the colon or colon tumors from Apc+/Min-FCCC
mice as shown from the present studies. While expression of 14-3-3ε was increased in RKO cells co-treated with hydrogen peroxide and 10 μM GW0742, this change in expression was not associated with anti-apoptotic activity, and no changes in 14-3-3ε were observed in all other treatment paradigms. Collectively, these observations suggest that NSAIDs do not down-regulate expression of PPARβ/δ in colon cancer models and emphasizes the need to critically examine the hypothesis that PPARβ/δ is anti-apoptotic and prevents NSAID-induced apoptosis by increasing expression of the 14-3-3ε that enhances sequestration of Bad as suggested by others [20
Since NSAIDs do not down-regulate PPARβ/δ expression in either human colon cancer cell lines or colon tumors from Apc+/Min-FCCC
mice, it is not surprising that ligand activation of PPARβ/δ did not attenuate PARP cleavage following treatment with either sulindac, sulindac sulfide, indomethacin or hydrogen peroxide. In fact, the only change observed in PARP cleavage was that co-treatment of NSAIDs with GW0742 enhanced PARP cleavage in DLD1 and RKO cells. These findings demonstrate that ligand activation can promote apoptosis in human colon cancer cells when combined with indomethacin or sulindac sulfide, rather than attenuate apoptosis as suggested by others (reviewed in [2
]). It is thus noteworthy that a dose-dependent decrease in the percentage of DLD1 cells undergoing early apoptosis was observed in response to ligand activation of PPARβ/δ following induction of apoptosis with hydrogen peroxide. This is important to note, because this change was associated with a concomitant increase in the percentage of cells undergoing late apoptosis/necrosis and a decrease in the percentage of viable cells. These observations might explain why others suggest that PPARβ/δ promotes anti-apoptotic activities, when in fact, this change is associated with more cells that have already undergone apoptosis/necrosis, but not with more viable cells. This also illustrates the need for future studies to comprehensively examine the effect of PPARβ/δ on apoptosis, including examination of different stages of apoptosis and cell viability.
The effect of increasing expression of PPARβ/δ in human colon cancer cells was the last important issue examined by the present study. Whether PPARβ/δ promotes or attenuates colon tumorigenesis remains uncertain. Results from the present study showing markedly lower expression of PPARβ/δ in both human colon adenocarcinomas and colon tumors from Apc+/Min-FCCC
mice suggest that increasing expression of PPARβ/δ will attenuate colon tumorigenesis. Indeed, PPARβ/δ attenuation of colon tumorigenesis has been observed in some null mouse models [45
], but not all [48
]. Similarly, knockdown of PPARβ/δ in HCT116 human colon cancer cells is reported to increase cell proliferation in one model [50
], but prevent xenograft tumorigenesis in another [51
]. Thus, examination of PPARβ/δ over-expression is an alternative approach to those that have been used previously to examine the role of PPARβ/δ expression in colon carcinogenesis. Over-expression of PPARβ/δ in RKO and DLD1 cells increased the efficacy of ligand activation as target gene expression is enhanced in cells over-expressing PPARβ/δ as compared to control cells. Why enhanced target gene expression in HT29 cells over-expressing PPARβ/δ compared to control was not observed cannot be determined from this work, but could be due to differences in the presence of co-activators, co-repressors or other accessory proteins (e.g. RXR), epigenetic differences in the promoter regions of PPARβ/δ target genes, to the fact that HT29 cells have two mutant copies of the APC allele and/or to differences related to site(s) of integration of the retroviral vector. Ligand activation of PPARβ/δ inhibited clonogenicity in RKO cells, but over-expression of PPARβ/δ did not markedly enhance this effect. Ligand activation of PPARβ/δ with GW0742 inhibited clonogenicity in DLD1 cells, but only at a concentration of 10 μM. No change in clonogenicity was found in either Migr1-control DLD1 cells or in DLD1 cells over-expressing PPARβ/δ in response to ligand activation of PPARβ/δ. The lack of an enhanced effect in cells with stable integration of either Migr1 or Migr1-hPPARβ/δ suggests that the Migr1 vector contributes to this phenotype. Interestingly, despite the lack of enhanced efficacy on target gene expression, over-expression of PPARβ/δ caused enhanced inhibition of clonogenicity in HT29 cells as compared to control HT29 cells. The reason why enhanced inhibition of clonogenicity was only observed in HT29 cells that modestly over-expressed PPARβ/δ and co-treated with the highly specific PPARβ/δ GW0742, but not in control HT29 cells treated with GW0742, is unclear. This could be due to differences in the ability of PPARβ/δ to alter gene expression and function in the different cell lines through undefined mechanisms. The inhibition of clonogenicity suggests that the observed decrease in expression of PPARβ/δ found in human colon adenocarcinomas could be causally related to colon tumor progression, and that restoring or activating PPARβ/δ may be a suitable target for preventing colon tumorigenesis. This is consistent with previous work showing PPARβ/δ-dependent inhibition of colon tumorigenicity following ligand activation of PPARβ/δ in mice [45
]. Importantly, in the presence of increased expression of PPARβ/δ, human colon cancer cell line clonogenicity is either unaffected or is inhibited further in response to ligand activation of PPARβ/δ. No increase in clonogenicity was observed in any of the three different models.
Combined, the results from these studies significantly advance the field because they are the first to provide quantitative evidence from both human and mouse models of colon cancer demonstrating that expression of PPARβ/δ is lower during colon tumorigenesis. These findings increase the body of evidence supporting the hypothesis that activating PPARβ/δ prevents colon tumorigenesis. The reason(s) why some studies suggest that PPARβ/δ promotes tumorigenesis through a variety of unconfirmed mechanisms remain unclear. However, given the findings from the present studies, future work should include definitive examination of PPARβ/δ expression, and not rely on past reports to suggest increased expression of PPARβ/δ during colon tumorigenesis. Results from the present study also emphasize that comprehensive analysis of apoptosis including cytometric analysis of viable and apoptotic cells are recommended. Finally, there remains a need for more fundamental research on the role of PPARβ/δ in colon cancer to help resolve conflicting reports in the literature.