This is the first study to report regional differences in the levels of PGE2 and 15-PGDH in human colorectal tumours. This was made possible by employing a strict protocol for rapid and uniform processing of orientated tumour tissue ex vivo. Herein, we report that PGE2 levels are higher towards the centre of CRCLM compared with more peripheral cancer tissue. Paradoxically, this was associated with increased levels of 15-PGDH protein at the centre of CRCLM. However, we demonstrated that the 15-PGDH activity level in the centre of CRCLM is reduced and is associated with low NAD+/NADH levels. In vitro studies confirmed that NAD+ availability drives 15-PGDH activity in human CRC cells. We believe that consideration of regional differences in PGE2 metabolism and micro-environmental influences on PGE2 metabolism related to enzyme co-factor availability and/or hypoxia is a paradigm shift in the field of eicosanoid cancer research and is consistent with latest understanding of genetic and epigenetic intra-tumoral heterogeneity [14, 40]. Consideration of intra-tumoral differences in PGE2 metabolism is essential for development of optimal anti-CRC therapy aimed at the COX-PGE2-15-PGDH axis.
Our data highlight significant differences between findings in human cancer tissue ex vivo and experimental observations using CRC cells in vitro. Although we propose that differences in 15-PGDH activity in cancer tissue compared with cultured CRC cells may account for the contrasting relationship between 15-PGDH expression and PGE2 levels in CRCLM tissue versus cell-conditioned medium, we cannot completely rule out that inadvertent stimulation of PGE2 synthesis ex vivo occurred. Avoidance of possible artefactual changes in tissue eicosanoid levels ex vivo will only be possible with other techniques such as MALDI-MS for measurement of PG distribution in frozen tissue sections .
The tissue microarray comparison of regional differences in 15-PGDH immunoreactivity between CRCLM and the paired primary CRC suggests that 15-PGDH expression, and hence PGE2 metabolism, in CRCLM differs from that in the primary CRC, from which the CRCLM were derived. This finding is consistent with recent data describing significant genetic differences between primary CRC and synchronous liver metastasis . Local factors specific to CRCLM may, at least partly, explain regional 15-PGDH expression in CRCLM and the contrast with observations from previous studies of 15-PGDH expression in primary CRCs .
NAD+ and NADH levels were both significantly lower in central rather than peripheral CRCLM tissue, compatible with depletion of the cellular NAD(H) pool. The NAD+/NADH ratios that we observed in human CRCLM tissue are similar to previous studies that have measured tissue NAD(H) levels by the same cycling assay . However, absolute levels of NAD+ and NADH were low compared with other tissues . One testable hypothesis is that the NAD(H) pool is depleted because of increased NAD-consuming enzyme activity in CRC cells. Consistent with this notion, sirtuins such as SIRT1 and poly-(ADP ribose) polymerase expression and activity are increased in cancer tissue . In particular, SIRT1 expression and activity are increased in human hepatoma and fibrosarcoma cells in vitro.
One weakness of our study is that we do not have direct evidence that the central area of CRCLMs that we studied were hypoxic. However, there is substantial indirect evidence that regional hypoxia exists in tumours including CRCLMs [19–21]. Importantly, the regional difference in functional 15-PGDH protein levels in CRCLMs was not mirrored in primary CRC. Central tumour necrosis is more common in CRCLMs than primary CRC tumours and implies greater degrees of hypoxia in the central regions of CRCLMs, which could account for differential 15-PGDH expression in metastatic tumours. This observation, and the fact that elevated 15-PGDH in CRC cells in the centre of CRCLMs is likely inactive secondary to NAD+ deficiency, help to reconcile our data with the existing literature, which, in general (but not exclusively), implies that 15-PGDH has tumour suppressor activity .
Roberts et al. have reported that acute hypoxia (16 hours) did not alter 15-PGDH protein expression in HT-29 human CRC cells, despite an increase in PGE2 levels believed to be secondary to COX-2 induction . It is possible that CRC cell line-specific differences in hypoxia-induced gene expression and NAD+ availability explain the experimental variability in in vitro models. Nevertheless, our data highlight that it is crucial to confirm the relevance of in vitro observations in tissue expression studies, which take into account potential micro-environmental influences.
TGFβ-induced attachment and spreading of LIM1863 human CRC cell colonies allowed us to develop a novel semi-quantitative measure of EMT based on an established model . Using this assay, we have provided support for previous observations that PGE2 drives EMT of CRC and other human cancer cells in vitro, which were based on down-regulation of E-cadherin expression, light-microscopic phenotype changes in adherent cells and cell motility assays [5, 36, 44, 45].
We have contributed to emerging evidence that hypoxia drives EMT . Interestingly, we observed that 15-PGDH expression was maintained in hypoxic TGFβ-induced LIM1863 human CRC cell colonies in vitro and CRC cells in the centre of CRCLMs that had an ‘EMT (E-cadherin-low) phenotype’. This is consistent with our observations that hypoxia induces 15-PGDH in other CRC cell lines in vitro and that 15-PGDH levels are higher in the centre rather than the periphery of CRCLMs. One testable hypothesis is that hypoxia inhibits β-catenin-related signaling , which could lead to de-repression of 15-PGDH . Further studies are required to understand the rather counter-intuitive finding that the main rate-limiting catabolic enzyme for PGE2 inactivation is elevated in a tumour microenvironment, in which cell survival would be potentiated by PGE2. These studies should always take into account NAD+ co-factor availability and measure levels of other lipid mediators, which have anti-proliferative activity, that are also potential substrates for 15-PGDH such as lipoxins .
Previous in vitro studies have demonstrated that Snail, one of the key transcription factors in EMT, represses 15-PGDH expression in CRC cells via direct binding to conserved E-box elements in the 15-PGDH promoter region . However, to our knowledge, the effect of hypoxia on human 15-PGDH gene expression has not been formally assessed. The human 15-PGDH gene promoter contains multiple ETS, AP-1 and CREB binding sites , although no hypoxia-responsive elements for direct hypoxia-inducible factor binding are evident. Therefore, a valid, testable hypothesis is that 15-PGDH is a hypoxia-inducible gene in CRC via ETS-dependent transcriptional up-regulation, which is recognised for several hypoxia-inducible genes .