The control of COX-2 expression is a complex regulatory process that requires input from multiple pathways impacting gene expression on both transcriptional and posttranscriptional levels.10
It is generally well accepted that transcriptional activation of the COX-2 gene PTGS2
is an early event in tumorigenesis because evidence demonstrates the presence of COX-2 mRNA in virtually all colorectal adenomas, adenocarcinomas, and colon cancer cells.2,10,14,15
However, the presence of COX-2 mRNA does not necessarily reflect respective protein levels because variable expression of COX-2 protein and prostaglandins is observed in colon cancer tissue and cells.2,8,15,16
These results imply that enhanced expression of COX-2 protein requires loss of posttranscriptional regulation to occur. This is consistent with observations demonstrating that tumors with increased size and invasiveness display elevated COX-2 mRNA and protein levels17–19
and indicate a link between tumor progression and defects in both the regulation of PTGS2
gene transcription and subsequent mRNA decay.
This study set out to examine the basis for the loss of COX-2 posttranscriptional regulation occurring in colorectal tumors. The expression levels of the mRNA stability factor HuR and decay factor TTP were evaluated in normal colonic epithelium compared with colorectal adenomas, adenocarcinomas, and colon cancer cell lines. Normally expressed at low levels and located in the nucleus, HuR overexpression and cytoplasmic localization were observed in tissues obtained from colon adenomas, adenocarcinomas, and metastases ( and Supplementary Figure 1
, and data not shown). Furthermore, in cells displaying elevated HuR, we observed a concurrent loss of TTP expression. Consistent with these observations, overexpression of the ARE-containing gene COX-2 coincided with elevated HuR and loss of TTP expression. Several studies indicate that HuR overexpression and cytoplasmic localization are a marker for elevated cancer-associated gene expression that is correlated with advancing stages of malignancy and poor clinical outcome.11,20–22
The findings presented here are in agreement indicating a role for HuR overexpression in colon cancer development. It is not known whether HuR overexpression is an oncogenic event in colon or other cancers. Using an ubiquitously expressed HuR transgenic mouse model, Levadoux–Martin et al23
demonstrated impaired gametogenesis to occur; however, loss of transgene expression in somatic tissues limited determination of the oncogenic capacity of HuR in vivo. Similarly, an inducible HuR transgene restricted to myeloid cell lineages promoted stabilization of inflammatory mediator mRNAs but did not induce an observable neoplastic phenotype.24
Although these in vivo studies do not indicate a putative role for HuR overexpression as a tumor-initiating event, they suggest a possible function for HuR overexpression in tumorigenesis by serving as a tumor growth promoter or in a tumor maintenance capacity.
The results presented here imply that the accompanied loss of TTP expression is a critical factor for cancer-associated gene overexpression in tumors. The tumor-derived loss of TTP is of significance because low levels of TTP can efficiently suppress COX-2 expression in the presence of elevated HuR () along with attenuating COX-2 overexpression and cell growth in colon cancer cells (). This ability to antagonize HuR-mediated COX-2 mRNA stabilization implies that both
loss-of-TTP and gain-of-HuR function are required events for COX-2 overexpression. Furthermore, the presence of TTP in normal colon epithelium suggests that it serves in a protective capacity by controlling inflammatory mediator expression levels. This is evident in TTP knock-out mice that develop multiple inflammatory syndromes resulting from increased COX-2 and inflammatory factors because of defects in their respective rapid mRNA turnover.25
These aspects, coupled with the observation that TTP can inhibit tumorigenesis of an H-ras
-dependent mast cell model through degradation of ARE-containing IL-3 mRNA,26
suggest that TTP can serve in a tumor suppressor capacity by attenuating ARE-containing gene expression.
The mechanisms allowing for HuR overexpression and TTP loss in colon tumors are largely undefined. The data presented here in colon tumors () are consistent with prior results demonstrating increased HuR and decreased TTP mRNA levels to occur in colon cancer cell lines.2,27
The HuR (ELAVL1
) and TTP (ZFP36
) genes are located on 19p13.2 and 19q13.1, respectively, and do not appear to lie in regions of genomic alterations occurring in colorectal cancer.28
Pertaining to HuR overexpression, an alternative explanation suggests that increased HuR transcription contributes to overexpression in colon tumors. With regard to this, characterization of the human and murine HuR promoter has identified a number of responsive elements associated with cellular signaling pathways commonly altered in colon cancer.29,30
One explanation for the lack of TTP expression observed in tumor tissue may reside in epigenetic silencing of the TTP promoter. Examination of the human TTP promoter (accession No. AY771351) identified a putative CpG island present in the proximal 3′ region of the promoter, and, in colon cancer cell lines that were deficient in TTP expression, the presence of hypermethylation was observed (unpublished observations). Based on this, we hypothesize that epigenetic alterations governed by changes in DNA methylation or altered chromatin structure promote TTP gene silencing in colorectal tumors. Alternatively, TTP has been demonstrated to regulate its own expression through a negative feedback loop by binding an ARE present in its 3’UTR.31
However, we are unable to detect TTP protein in tumor tissue or colon cancer cells, suggesting that this mechanism of TTP autoregulation may be a means to limit TTP levels in nonneoplastic cells under inflammatory conditions.
There is a growing body of evidence suggesting that defects in ARE-mediated mRNA decay play a central role in chronic inflammation and tumorigenesis. It is estimated that 5%–8% of the human transcriptome is composed of ARE-containing mRNAs,32
and recent findings have demonstrated an enrichment of this subset of transcripts to occur during colon tumorigenesis. Gene expression profiling comparing adenomas to late-stage adenocarcinomas shows a 3- to 4-fold enrichment in ARE-containing genes compared with the genome as a whole, and a similar enrichment is observed as early as stage I tumors.33
The findings presented here provide a mechanistic basis for these results and indicate ARE-mediated posttranscriptional gene regulation to be an important regulatory mechanism involved during the early stages of colorectal tumorigenesis.