Christos Paraskeva

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.

Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a ‘field defect’ involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells

Focal adhesion kinase (pp125FAK) is present at sites of cell/extracellular matrix adhesion and has been implicated in the control of cell behaviour. In particular, as a key component of integrin-stimulated signal transduction pathways, pp125FAK is involved in cellular processes such as spreading, motility, growth and survival. In addition, a number of reports have indicated that pp125FAK may be up-regulated in human tumour cells of diverse origin, and consequently, a role has been proposed for pp125FAK in the development of invasive cancers. However, to date the mechanisms that lead to elevated pp125FAK expression in tumour cells have not been determined. Here we used in situ hybridization to confirm chromosome 8q as the genomic location of the human fak gene and report that elevation of pp125FAK protein in cell lines derived from invasive squamous cell carcinomas is accompanied by gains in copy number of the fak gene in all cases examined. In addition, we observed increased fak copy number in frozen sections of squamous cell carcinomas. Furthermore, increased dosage of the fak gene was also observed in many cell lines derived from human tumours of lung, breast and colon, including two cell lines Calu3 and HT29, in which fak was amplified. In addition, in an in vitro model for human colon cancer progression there was a copy number gain of the fak gene during conversion from adenoma to carcinoma, which was associated with increased pp125FAK protein expression. Thus, we show for the first time that many cell lines derived from invasive epithelial tumours have increased dosage of the fak gene, which may contribute to the elevated protein expression commonly observed. Although other genes near the fak locus are co-amplified or increased in copy number, including the proto-oncogene c-myc, the biological properties of pp125FAK in controlling the growth, survival and invasiveness of tumour cells, suggest that it may contribute to the selection pressure for maintaining increased dosage of the region of chromosome 8q that encodes these genes.

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.

VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF165 and antiangiogenic VEGF165b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGFxxxb, but there was a variable upregulation of VEGFxxx and downregulation of VEGFxxxb in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGFxxxb, whereas colonic carcinoma cells expressed predominantly VEGFxxx. However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGFxxxb to predominantly VEGFxxx. VEGF165b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF165b bound to bevacizumab with similar affinity as VEGF165. However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF165, it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF165b. Both bevacizumab and anti-VEGF165b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF165b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

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.

A role for epidermal growth factor receptor, c-Src and focal adhesion kinase in an in vitro model for the progression of colon cancer

We have examined the function of the epidermal growth factor (EGF) receptor, c-Src and focal adhesion kinase (FAK) in the progression of colon cancer using an in vitro progression model. A non-tumorigenic cell line was derived from a premalignant colonic adenoma (PC/AA) from which a clonogenic variant was established (AA/C1). Following sequential treatment with sodium butyrate and the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine an anchorage-independent line was isolated which, with time in culture, became tumorigenic when injected into athymic nude mice (AA/C1/SB10). We have shown that both EGF receptor and FAK protein levels were elevated in the carcinoma cells as compared to the adenoma cells, while the expression and activity of c-Src were unaltered during the adenoma to carcinoma transition. EGF induced the movement of the carcinoma cells into a reconstituted basement membrane which was not seen with the premalignant adenoma cells. This increased motility was accompanied by an EGF-induced increase in c-Src kinase activity, relocalisation of c-Src to the cell periphery and phosphorylation of FAK in the carcinoma cells but not in the adenoma cells. This suggests that c-Src plays a role in the biological behaviour of colonic carcinoma cells induced by migratory factors such as EGF, perhaps acting in conjunction with FAK to regulate focal adhesion turnover and tumour cell motility. Furthermore, although c-Src has been implicated in colonic tumour progression, we demonstrate here that in the adenoma to carcinoma in vitro model c-Src is not the driving force for this progression but co-operates with other molecules in carcinoma development.

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.

bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis

Butyrate, a short chain fatty acid produced in the colon as a result of fermentation of dietary fibre by symbiotic bacteria, induces apoptosis in colonic tumour cell lines. Three human colonic adenoma cell lines (AA/C1, RG/C2 and BH/C1) and one carcinoma cell line (S/KS/FI) were used to determine the effects of butyrate on the expression of bcl‐2, bax and bak to examine the possible role of these proteins in the induction of apoptosis. RG/C2 and BH/C1 cells express p‐26‐bcl‐2 and butyrate treatment decreased p26‐bcl‐2 levels in association with apoptosis, whereas bax and bak levels remained constant. AA/C1 and S/KS/FI cells have no detectable p26‐bcl‐2. In S/KS/FI cells, bax or bak levels did not change in response to butyrate. However, in AA/C1 cells, butyrate‐induced apoptosis was associated with increased bak levels. Therefore, in AA/C1 cells butyrate‐induced apoptosis appears to be mediated through bak. Furthermore, butyrate also induced apoptosis and increased bak levels in AA/C1 cells transfected with a bcl‐2 expression vector which expressed high levels of p26‐bcl‐2. For S/KS/FI cells, two bcl‐2 transfectants gave different results. bcl‐2 protected against apoptosis in one transfectant in which bak levels were not elevated in response to butyrate, whereas it did not protect in the other transfectant in which bak levels were increased after butyrate treatment. The results suggest that expression of constitutively high levels of p26‐bcl‐2 only conferred protection against apoptosis when bak levels were not elevated in response to butyrate and that expression of constitutively high levels of p26‐bcl‐2 does not counter the effects of bak. Different mechanisms appear to be involved in cell death signalling in different tumours since butyrate may induce apoptosis via elevated levels of bak or reduced levels of p26‐bcl‐2. Int. J. Cancer 72:898–905, 1997.