Alexander Greenhough

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.

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.

Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma–carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription–polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=−0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=−0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma–carcinoma transition.

LGR5 promotes survival in human colorectal adenoma cells and is upregulated by PGE2: implications for targeting adenoma stem cells with NSAIDs

Cyclooxygenase-2 is overexpressed in the majority of colorectal tumours leading to elevated levels of prostaglandin E2 (PGE2), promoting many hallmarks of cancer. Importantly, PGE2 is reported to enhance Wnt/β-catenin signalling in colorectal carcinoma cells and in normal haematopoietic stem cells where it promotes stem cell function. Although Wnt signalling plays a crucial role in intestinal stem cells, the relationship between PGE2 and intestinal stem cells is unclear. Given that the key intestinal cancer stem cell marker LGR5 (leucine-rich G-protein coupled receptor 5) is a Wnt target and PGE2 enhances Wnt signalling, the focus of this study was to investigate whether PGE2 regulated LGR5 expression in colorectal adenoma cells and whether LGR5 was important for tumour cell survival. PGE2 upregulated LGR5 protein in adenoma (RG/C2) and carcinoma (DLD-1) cell lines. LGR5 knockdown induced cell death in RG/C2 and AA/C1 adenoma cells, suggesting that LGR5 has an important survival-promoting role in adenoma cells. Indeed, we detected LGR5 protein expression in 4 of 4 human adenoma cell lines. Furthermore, LGR5 small interfering RNA inhibited the survival-promoting effects of PGE2 in RG/C2, suggesting that PGE2 promotes adenoma cell survival, at least in part, by increasing LGR5 expression. These studies, therefore, show the first link between PGE2 and LGR5 in human colorectal adenoma and carcinoma cells and demonstrate a survival-promoting role of LGR5. As non-steroidal anti-inflammatory drugs (NSAIDs) cause adenomas to regress in FAP patients, these studies could have important implications for the mechanism by which NSAIDs are chemopreventive, as lowering PGE2 levels could reduce LGR5 expression and survival of LGR5(+) adenoma stem cells.

HGF/Met signalling promotes PGE2 biogenesis via regulation of COX-2 and 15-PGDH expression in colorectal cancer cells

Evidence points towards a pivotal role for cyclooxygenase (COX)-2 in promoting colorectal tumorigenesis through increasing prostaglandin E(2) (PGE(2)) levels. PGE(2) signalling is closely associated with the survival, proliferation and invasion of colorectal cancer cells. Recently, a reduction in PGE(2) inactivation, a process mediated by the nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), has also been shown to promote tumoral PGE(2) accumulation. The hepatocyte growth factor (HGF) receptor, Met, is frequently over-expressed in colorectal tumours and promotes cancer growth, metastasis and resistance to therapy, although the mechanisms for this have not been fully elucidated. Here, we report that HGF/Met signalling can promote PGE(2) biogenesis in colorectal cancer cells via COX-2 up-regulation and 15-PGDH down-regulation at the protein and messenger RNA level. Pharmacological inhibition of MEK and PI3K suggested that both extracellular signal-regulated kinase (ERK) and AKT signalling are required for COX-2 protein up-regulation and 15-PGDH down-regulation downstream of Met. Notably, inhibition of Met with the small molecule inhibitor SU11274 reduced COX-2 expression and increased 15-PGDH expression in high Met-expressing cells. We also show that hypoxia potentiated HGF-driven COX-2 expression and enhanced PGE(2) release. Furthermore, inhibition of COX-2 impeded the growth-promoting effects of HGF, suggesting that the COX-2/PGE(2) pathway is an important mediator of HGF/Met signalling. These data reveal a critical role for HGF/Met signalling in promoting PGE(2) biogenesis in colorectal cancer cells. Targeting the crosstalk between these two important pathways may be useful for therapeutic treatment of colorectal cancer.

β-catenin represses expression of the tumour suppressor 15-prostaglandin dehydrogenase in the normal intestinal epithelium and colorectal tumour cells

Background Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE2). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/β-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether β-catenin represses 15-PGDH expression. Methods The effect of modulating Wnt/β-catenin signalling (using β-catenin siRNA, mutant TCF4, Wnt3A or GSK3 inhibition) on 15-PGDH mRNA, protein expression and promoter activity was determined in colorectal cell lines by immunoblotting, qRT-PCR and reporter assays. The effect of β-catenin deletion in vivo was addressed by 15-PGDH immunostaining of β-catenin−/lox-villin-creERT2 mouse tissue. 15-PGDH promoter occupancy was determined using chromatin immunoprecipitation and PGE2 levels by ELISA. Results The study shows for the first time that β-catenin knockdown upregulates 15-PGDH in colorectal adenoma and carcinoma cells without affecting COX-2 protein levels. A dominant negative mutant form of TCF4 (dnTCF4), unable to bind β-catenin, also upregulated 15-PGDH; conversely, increasing β-catenin activity using Wnt3A or GSK3 inhibition downregulated 15-PGDH. Importantly, inducible β-catenin deletion in vivo also upregulated intestinal epithelial 15-PGDH. 15-PGDH regulation occurred at the protein, mRNA and promoter activity levels and chromatin immunoprecipitation indicated β-catenin/TCF4 binding to the 15-PGDH promoter. β-catenin knockdown decreased PGE2 levels, and this was significantly rescued by 15-PGDH siRNA. Conclusion These data suggest a novel role for β-catenin in promoting colorectal tumorigenesis through very early 15-PGDH suppression leading to increased PGE2 levels, possibly even before COX-2 upregulation.

The proapoptotic BH3-only protein Bim is downregulated in a subset of colorectal cancers and is repressed by antiapoptotic COX-2/PGE 2 signalling in colorectal adenoma cells

Overexpression of cyclooxygenase-2 (COX-2) and elevated levels of its enzymatic product prostaglandin E2 (PGE2) occur in the majority of colorectal cancers and have important roles in colorectal tumorigenesis. However, despite the established prosurvival role of PGE2 in cancer, the underlying mechanisms are not fully understood. Here, we have shown that PGE2 suppresses apoptosis via repression of the proapoptotic BH3-only protein Bim in human colorectal adenoma cells. Repression of Bim expression was dependent upon PGE2-mediated activation of the Raf-MEK-ERK1/2 pathway, which promoted Bim phosphorylation and proteasomal degradation. Reduction of Bim expression using RNA interference reduced spontaneous apoptosis in adenoma cells and abrogated PGE2-dependent apoptosis suppression. Treatment of COX-2-expressing colorectal carcinoma cells with COX-2-selective NSAIDs-induced Bim expression, suggesting that Bim repression via PGE2 signalling may be opposed by COX-2 inhibition. Examination of Bim expression in two established in vitro models of the adenoma–carcinoma sequence revealed that downregulation of Bim expression was associated with tumour progression towards an anchorage-independent phenotype. Finally, immunohistochemical analyses revealed that Bim expression is markedly reduced in approximately 40% of human colorectal carcinomas in vivo. These observations highlight the COX-2/PGE2 pathway as an important negative regulator of Bim expression in colorectal tumours and suggest that Bim repression may be an important step during colorectal cancer tumorigenesis.

Cannabinoids and cancer: potential for colorectal cancer therapy

Despite extensive research into the biology of CRC (colorectal cancer), and recent advances in surgical techniques and chemotherapy, CRC continues to be a major cause of death throughout the world. Therefore it is important to develop novel chemopreventive/chemotherapeutic agents for CRC. Cannabinoids are a class of compounds that are currently used in the treatment of chemotherapy-induced nausea and vomiting, and in the stimulation of appetite. However, there is accumulating evidence that they could also be useful for the inhibition of tumour cell growth by modulating key survival signalling pathways. The chemotherapeutic potential for plant-derived and endogenous cannabinoids in CRC therapy is reviewed.