It is widely accepted that alterations to cyclooxygenase-2 (COX-2) expression and the abundance of its enzymatic product prostaglandin E(2) (PGE(2)) have key roles in influencing the development of colorectal cancer. Deregulation of the COX-2/PGE(2) pathway appears to affect colorectal tumorigenesis via a number of distinct mechanisms: promoting tumour maintenance and progression, encouraging metastatic spread, and perhaps even participating in tumour initiation. Here, we review the role of COX-2/PGE(2) signalling in colorectal tumorigenesis and highlight its ability to influence the hallmarks of cancer--attributes defined by Hanahan and Weinberg as being requisite for tumorigenesis. In addition, we consider components of the COX-prostaglandin pathway emerging as important regulators of tumorigenesis; namely, the prostanoid (EP) receptors, 15-hydroxyprostaglandin dehydrogenase and the prostaglandin transporter. Finally, based on recent findings, we propose a model for the cellular adaptation to the hypoxic tumour microenvironment that encompasses the interplay between COX-2, hypoxia-inducible factor 1 and dynamic switches in beta-catenin function that fine-tune signalling networks to meet the ever-changing demands of a tumour.

Aberrant activation of beta-catenin promotes cell proliferation and initiates colorectal tumorigenesis. However, the expansion of tumours and the inadequacy of their local vasculature results in areas of hypoxia where cell growth is typically constrained. Here, we report a novel diversion in beta-catenin signalling triggered by hypoxia. We show that hypoxia inhibits beta-catenin-T-cell factor-4 (TCF-4) complex formation and transcriptional activity, resulting in a G1 arrest that involves the c-Myc-p21 axis. Additionally, we find that hypoxia inducible factor-1alpha (HIF-1alpha) competes with TCF-4 for direct binding to beta-catenin. DNA-protein interaction studies reveal that beta-catenin-HIF-1alpha interaction occurs at the promoter region of HIF-1 target genes. Furthermore, rigorous analyses indicate that beta-catenin can enhance HIF-1-mediated transcription, thereby promoting cell survival and adaptation to hypoxia. These findings demonstrate a dynamic role for beta-catenin in colorectal tumorigenesis, where a functional switch is instigated to meet the ever-changing needs of the tumour. This study highlights the importance of the microenvironment in transcriptional regulation.

The Wnt/b-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/b-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of b-catenin expression levels in vitro and in vivo revealed that b-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation b-catenin is selectively degraded via the formation of a b-catenin-LC3 complex, attenuating b-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the b-catenin-LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in b-catenin, which is required for interaction with LC3 and non-proteasomal degradation of b-catenin. Thus, Wnt/b-catenin represses autophagy and p62 expression, while b-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place b-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.

Cyclooxygenase (COX)-2, the inducible key enzyme for prostanoid biosynthesis, is overexpressed in most colorectal carcinomas and a subset of colorectal adenomas. Genetic, biochemical, and clinical evidence indicates an important role for COX-2 in colorectal tumorigenesis. Although COX-2 can be induced by aberrant growth factor signaling and oncogene activation during colorectal tumorigenesis, the role of microenvironmental factors such as hypoxia in COX-2 regulation remains to be elucidated. For the first time, we report that under hypoxic conditions COX-2 protein levels increase in colorectal adenoma and carcinoma cells. Rigorous analyses reveal that COX-2 up-regulation is transcriptional and is associated with hypoxia-inducible factor (HIF)-1A induction. Oligonucleotide pull-down and chromatin immunoprecipitation assays reveal that HIF-1A binds a hypoxia-responsive element on the COX-2 promoter. COX-2 up-regulation during hypoxia is accompanied by increased levels of prostaglandin E 2 (PGE 2 ), which promote tumor cell survival under hypoxic conditions. In addition, elevated levels of PGE 2 in hypoxic colorectal tumor cells enhance vascular endothelial growth factor expression and HIF-1 transcriptional activity by activating the mitogen-activated protein kinase pathway, showing a potential positive feedback loop that contributes to COX-2 up-regulation during hypoxia. This study identifies COX-2 as a direct target for HIF-1 in colorectal tumor cells. In addition, COX-2 up-regulation represents a pivotal cellular adaptive response to hypoxia with implication for colorectal tumor cell survival and angiogenesis. We propose that using modified COX-2-selective inhibitors, which are only activated under hypoxic conditions, could potentially be a novel more selective strategy for colorectal cancer prevention and treatment. (Cancer Res 2006; 66(13): 6683-91)

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As tumours are known to acidify their microenvironment and¯uctuations in lumenal pH have been reported in a number of colonic disease conditions, we investigated whether loss of p53 function, commonly associated with the adenoma to carcinoma transition in human colorectal epithelium, was implicated in the cellular response to changes in extracellular pH. Human colonic adenoma and carcinoma derived cell lines were incubated at an inital pH range of 5.5 ± 8.0 and the attached cell yield and apoptotic cell yield determined after 4 days. Exposure of all cell lines to an acidic growth environment was associated with a G1 arrest, down regulation of the retinoblastoma protein (pRb) protein and switch to the hypophosphorylated form of the protein, and increased expression of the p21 protein. However, induction of apoptosis, associated with increased p53 protein expression but not with changes in Bcl-2 expression, was only detected in the adenoma derived BH/C1 and AA/C1 cell lines which express wild type p53 activity. Furthermore, this induction of apoptosis was inhibited in the transfected cell line AA/ 273p53/B, in which the wild type p53 function has been abrogated. These results suggest that acidi®cation of the microenvironment would provide a selective growth advantage for cells that have lost wild type p53 function, leading to clonal expansion of aberrant cell populations.

Cyclooxygenase-2 and prostaglandin E 2 (PGE 2 ) levels are increased in colorectal cancers and a subset of adenomas. PGE 2 signaling through the EP4 receptor has previously been associated with colorectal tumorigenesis. However, changes in EP4 expression during adenoma to carcinoma progression have not been investigated, neither has whether levels of EP4 influence important markers of malignant potential, such as anchorage-independent growth or the tumors growth response to PGE 2 . We report using immunohistochemistry that in vivo EP4 receptor protein expression was increased in colorectal cancers (100%) as well as adenomas (36%) when compared with normal colonic epithelium. EP4 expression was also higher in colorectal carcinoma compared with adenoma cell lines and increased with in vitro models of tumor progression. Adenoma (PC/AA/C1 and RG/C2) and carcinoma cell lines (HT29) were growth stimulated by PGE 2 up to 0.5 Mmol/L. However, although carcinoma and transformed adenoma (PC/AA/C1SB10C, a transformed derivative of PC/AA/C1) cells remain stimulated by higher doses of PGE 2 (10 Mmol/L), the adenoma cell lines were inhibited. Interestingly, enforced expression of EP4 in the adenoma cell line, RG/ C2, resulted in stimulation of growth by 10 Mmol/L PGE 2 and promoted anchorage-independent growth. Both in vivo and in vitro data from this study suggest that increased EP4 receptor expression is important during colorectal carcinogenesis. We propose that high levels of PGE 2 in a tumor microenvironment would select for cells with increased EP4 expression, and that the EP4 receptor may therefore represent an important target for colorectal cancer prevention and treatment. (Cancer Res 2006; 66(6): 3106-13)