Ann C. Williams

The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment

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.

Interaction between β-catenin and HIF-1 promotes cellular adaptation to hypoxia

Aberrant activation of β-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 β-catenin signalling triggered by hypoxia. We show that hypoxia inhibits β-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-1α (HIF-1α) competes with TCF-4 for direct binding to β-catenin. DNA–protein interaction studies reveal that β-catenin–HIF-1α interaction occurs at the promoter region of HIF-1 target genes. Furthermore, rigorous analyses indicate that β-catenin can enhance HIF-1-mediated transcription, thereby promoting cell survival and adaptation to hypoxia. These findings demonstrate a dynamic role for β-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.

Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxia-inducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia.

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)-1alpha induction. Oligonucleotide pull-down and chromatin immunoprecipitation assays reveal that HIF-1alpha 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.

Hedgehog signalling in colorectal tumour cells: Induction of apoptosis with cyclopamine treatment

Hedgehog (Hh) signalling controls many aspects of development. It also regulates cell growth and differentiation in adult tissues and is activated in a number of human malignancies. Hh and Wnt signalling frequently act together in controlling cell growth and tissue morphogenesis. Despite the fact that the majority of colorectal tumours have a constitutively activated canonical Wnt pathway, few previous studies have investigated the expression of Hh signalling components in colorectal tumours. We describe here epithelial cell lines derived from both nonmalignant colorectal adenomas and colorectal adenocarcinomas that express both Sonic and Indian Hh. Interestingly, these cells also express the Hh receptor Patched and the downstream signalling components Smoothened and Gli1, suggesting autocrine Hh signalling in these cells. To test whether autocrine Hh signalling contributes to cell survival, we treated colorectal tumour cells with cyclopamine, a known inhibitor of Hh signalling. Cyclopamine treatment induced apoptosis in both adenoma‐ and carcinoma‐derived cell lines, which could be partially rescued by further stimulation of Hh signalling. These data suggest that autocrine Hh signalling can increase aberrant cell survival in colorectal tumour cells and may be a novel target for colon cancer therapy using drugs such as cyclopamine.

An acidic environment leads to p53 dependent induction of apoptosis in human adenoma and carcinoma cell lines: implications for clonal selection during colorectal carcinogenesis.

As tumours are known to acidify their microenvironment and fluctuations 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 acidification 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.

Increased EP4 receptor expression in colorectal cancer progression promotes cell growth and anchorage independence.

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 micromol/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 micromol/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 micromol/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.

Autolysosomal β-catenin degradation regulates Wnt-autophagy-p62 crosstalk

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

The cannabinoid δ9-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival signalling and induces BAD-mediated apoptosis in colorectal cancer cells

Deregulation of cell survival pathways and resistance to apoptosis are widely accepted to be fundamental aspects of tumorigenesis. As in many tumours, the aberrant growth and survival of colorectal tumour cells is dependent upon a small number of highly activated signalling pathways, the inhibition of which elicits potent growth inhibitory or apoptotic responses in tumour cells. Accordingly, there is considerable interest in therapeutics that can modulate survival signalling pathways and target cancer cells for death. There is emerging evidence that cannabinoids, especially Δ9‐tetrahydrocannabinol (THC), may represent novel anticancer agents, due to their ability to regulate signalling pathways critical for cell growth and survival. Here, we report that CB1 and CB2 cannabinoid receptors are expressed in human colorectal adenoma and carcinoma cells, and show for the first time that THC induces apoptosis in colorectal cancer cells. THC‐induced apoptosis was rescued by pharmacological blockade of the CB1, but not CB2, cannabinoid receptor. Importantly, THC treatment resulted in CB1‐mediated inhibition of both RAS‐MAPK/ERK and PI3K‐AKT survival signalling cascades; two key cell survival pathways frequently deregulated in colorectal tumours. The inhibition of ERK and AKT activity by THC was accompanied by activation of the proapoptotic BCL‐2 family member BAD. Reduction of BAD protein expression by RNA interference rescued colorectal cancer cells from THC‐induced apoptosis. These data suggest an important role for CB1 receptors and BAD in the regulation of apoptosis in colorectal cancer cells. The use of THC, or selective targeting of the CB1 receptor, may represent a novel strategy for colorectal cancer therapy.

The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2

Background and aims: Cyclooxygenase 2 (COX-2) is upregulated in most colorectal cancers and is responsible for metabolism of the endogenous cannabinoid, anandamide, into prostaglandin-ethanolamides (PG-EAs). The aims of this study were to determine whether anandamide and PG-EAs induce cell death in colorectal carcinoma (CRC) cells, and whether high levels of COX-2 in CRC cells could be utilised for their specific targeting for cell death by anandamide. Methods: We determined the effect of anandamide on human CRC cell growth by measuring cell growth and cell death, whether this was dependent on COX-2 protein expression or enzyme activity, and the potential involvement of PG-EAs in induction of cell death. Results: Anandamide inhibited the growth of CRC cell lines HT29 and HCA7/C29 (moderate and high COX-2 expressors, respectively) but had little effect on the very low COX-2 expressing CRC cell line, SW480. Induction of cell death in HT29 and HCA7/C29 cell lines was partially rescued by the COX-2 selective inhibitor NS398. Cell death induced by anandamide was neither apoptosis nor necrosis. Furthermore, inhibition of fatty acid amide hydrolase potentiated the non-apoptotic cell death, indicating that anandamide induced cell death was mediated via metabolism of anandamide by COX-2, rather than its degradation into arachidonic acid and ethanolamine. Interestingly, both PGE2-EA and PGD2-EA induced classical apoptosis. Conclusions: These findings suggest anandamide may be a useful chemopreventive/therapeutic agent for colorectal cancer as it targets cells that are high expressors of COX-2, and may also be used in the eradication of tumour cells that have become resistant to apoptosis.

Apoptosis and disease: a life or death decision

The European Tissue Culture Society Conference and Workshop on ‘Apoptosis and Disease’ was held at the University of Bristol, between 26 and 27 November 2003, and was organized by A. Hague and C. Paraskeva. ![][1] Apoptosis is an evolutionarily conserved form of cell death that was first described by Kerr and colleagues in 1972 (Kerr et al , 1972). It is essential for successful embryonic development and maintains normal cellular homeostasis in adult organisms. Gain‐ and loss‐of‐function models of genes in the core apoptotic pathway suggest that perturbation of cellular homeostasis can be a primary pathological event that results in disease. There is now compelling evidence that insufficient apoptosis can result in cancer or autoimmunity, whereas accelerated cell death is evident in degenerative diseases, immunodeficiency and infertility. Not surprisingly, a huge endeavour aimed at unravelling this fundamental biological process has led to major advances in our understanding of the apoptotic process during the past 20 years. The primary aim of this symposium was to provide “an overview of the regulation, signalling and execution of apoptosis in model systems relevant to human diseases”. In this report, we highlight recent findings from several laboratories that outline crucial control points in the apoptotic pathway, some of which could provide rational targets for therapeutic intervention. ### Initiating the demolition phase of apoptosis During apoptosis, the controlled destruction of the cell is coordinated, from within, by the caspase family of cysteine proteases. This meeting focused on two of the major apoptotic pathways: one initiated by the activation of death receptors and the other by stress‐inducing stimuli (Fig 1; reviewed in Adams, 2003; Danial & Korsmeyer, 2004). Triggering of cell surface death receptors of the tumour necrosis factor (TNF) receptor superfamily, including TNF‐R1, CD95 and TNF‐related apoptosis‐inducing ligand (TRAIL)‐R1 and ‐R2, results in the rapid activation of the ‘initiator’, caspase 8, after it … [1]: /embed/graphic-1.gif