E2F family of transcription factors regulates various cellular functions related to cell cycle and apoptosis. Its individual members have traditionally been classified into activators and repressors, based on in vitro studies. However their contribution in human cancer is more complicated and difficult to predict. We review current knowledge on the expression of E2Fs in digestive system malignancies and its clinical implications for patient prognosis and treatment. E2F1, the most extensively studied member and the only one with prognostic value, exhibits a tumor-suppressing activity in esophageal, gastric and colorectal adenocarcinoma, and in hepatocellular carcinoma (HCC), whereas in pancreatic ductal adenocarcinoma and esophageal squamous cell carcinoma may function as a tumor-promoter. In the latter malignancies, E2F1 immunohistochemical expression has been correlated with higher tumor grade and worse patient survival, whereas in esophageal, gastric and colorectal adenocarcinomas is a marker of increased patient survival. E2F2 has only been studied in colorectal cancer, where its role is not considered significant. E2F4’s role in colorectal, gastric and hepatic carcinogenesis is tumor-promoting. E2F8 is strongly upregulated in human HCC, thus possibly contributing to hepatocarcinogenesis. Adenoviral transfer of E2F as gene therapy to sensitize pancreatic cancer cells for chemotherapeutic agents has been used in experimental studies. Other therapeutic strategies are yet to be developed, but it appears that targeted approaches using E2F-agonists or antagonists should take into account the tissue-dependent function of each E2F member. Further understanding of E2Fs’ contribution in cellular functions in vivo would help clarify their role in carcinogenesis.

The E2F family of transcription factors is involved in a vast number of cellular functions related to cell cycle and apoptosis. The study of E2F began in the mid 1980s when it was identified as a transcription activator of the adenoviral E2 gene promoter[1,2]. The role of the prototype member, E2F1, in cancer was identified in the early 1990s through its ability to bind to and regulate the retinoblastoma protein pRb[3-7] with several members to follow either through their homology to E2F1 or through their association with pRb-related proteins (pocket proteins)[8-16].

Early in vitro studies rose expectations that, through the traditional classification of the E2F family members into activators and repressors, accurate predictions about their contribution in human carcinogenesis could be possible, with oncogenic behavior expected for the former and tumor-suppressing function for the latter group. Nevertheless, in vivo studies have sunk the initial enthusiasm as their role appears to be by far more complicated[17].

E2F1 is the most thoroughly investigated member of the E2F family in human malignancies. Other members have also been studied but in lesser extent. In non-small cell lung carcinoma, increased E2F1 and E2F3 expression has been associated with worse patient prognosis[18-21]. In breast cancer, enhanced E2F1 or E2F4 expression have been proposed as poor prognostic indicators, whereas increased E2F5 expression has been reported in certain histological subtypes[22-26]. In ovarian cancer, E2F1-5, E2F7 and E2F8 expression is reportedly increased. In addition, increased E2F4 and E2F7 expression has been related to better overall or disease-free survival, respectively, whereas that of E2F8 was linked to worse overall survival[27-30]. In prostate cancer, E2F2 and E2F3 expression increases, while E2F1 is absent[25,31]. In urothelial carcinomas of the bladder, E2F3 expression is enhanced, while that of E2F1 depends on the presence of invasion[25,32-37]. Increased E2F1 expression has also been observed in thyroid cancer, small cell lung carcinoma, glioblastoma and lymph node metastases from malignant melanoma[38-42].

The aim of the current review is to summarize the collective knowledge on the role of various E2F family members in digestive system malignancies and to identify possible clinical implications for patients’ diagnosis and prognosis and for future treatment strategy design.

To date, eight members of the mammalian E2F family have been recognized and characterized[36,43], though the most recently identified, namely E2F7[44,45] and E2F8[46,47], bear little homology to their traditional counterparts (Figure 1). Two E2F3 proteins, E2F3a and E2F3b, have been identified, which are produced from the same gene through the use of alternate promoters[48,49]. E2F7 also has two isoforms, E2F7a and E2F7b, which are produced through by alternative splicing of the primary transcript[50].

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Mammalian E2F family of transcription factors. In the context of digestive system malignancies, E2F1 may exhibit a tumor-suppressing role in colorectal, gastric and esophageal adenocarcinoma, as well as in hepatocellular carcinoma. On the contrary, in pancreatic ductal adenocarcinoma and esophageal squamous carcinoma a tumor-promoting role has been shown. A tumor-promoting role is demonstrated for E2F4 in colorectal, gastric and liver carcinogenesis. E2F8 may contribute to hepatocarcinogenesis, as it has been shown to be strongly upregulated in human hepatocellular carcinoma. DBD: DNA binding domain; LZ: Leucine zipper; MB: Marked box.

The greatest homology among the different members of this family is observed in the DNA binding domain. The older members E2F1-6 possess N-terminal DNA binding domain, followed by leucine zipper and marked box domains that mediate heterodimerization. With the exception of E2F6, the established members of the family possess a C-terminal transactivation domain containing the pocket protein binding region[36]. E2F1-3 have nuclear localization signals adjacent to their cyclin A-binding domain. This ensures their movement to the nucleus, thereby modulating E2F activity in a cell cycle-dependent manner[51-53]. E2F1-6 require heterodimerization with one of the dimerization partner (DP) proteins, DP1, DP2 or DP3 in order to form functional transcription factors which can bind DNA with high affinity[54-59]. E2F4 and E2F5 have bipartite nuclear export signals, which mediate their export to the cytoplasmic compartment and they rely on heterodimerization with one of DP proteins for their translocation to the cell nucleus[60,61]. The proteins DP2 and DP3 are thought to be alternatively spliced products of a single gene. The role of DP subunits is not completely elucidated and it appears that the function of E2F-DP heterodimer is dictated by the E2F subunit[56,57].

The transcriptional activity of E2F1-5 is regulated through their binding with the pRb protein or the related pocket proteins p107 and p130[62-64]. E2F1-3 bind to pRb when the latter is found in its hypophosphorylated form[13], E2F4 can bind to any of the pocket proteins, whereas E2F5 associates predominantly with p130[65,66]. Generally, when a pocket protein is hypophosphorylated can associate and block the transcriptional activity of E2F-DP heterodimers by masking the transcription activation domain of E2Fs, rendering them inactive (passive repression)[43]. Furthermore, the pocket protein/E2F-DP complex is guided to E2F binding sites where it can recruit histone deacetylases that suppress transcription by remodelling the nucleosome (active repression)[67-70]. E2F6-8 are presumed to repress E2F-responsive genes independently of pocket proteins[10,11,50,71].

E2F family members have been traditionally classified as activators and repressors. E2F1-3a are often referred to as activators because they transcriptionally activate certain target genes, for example cyclin E. This E2F subclass is expressed in a cell cycle-regulated manner exhibiting highest levels in the late G1 and S phase. In other words, they induce the entrance of quiescent cells in the S phase of cell cycle and overcome arrest mediated by the p16^INK4a cyclin-dependent kinase inhibitor[43,72]. E2F3b-5 comprise the repressor subclass because their main function seems to be the repression of transcription of some E2F target genes when they associate with pocket proteins. This subgroup is expressed constitutively, but transcriptional repression by these factors takes place predominantly in cells which are in quiescence and early G1 phase[43]. E2F6-8 are also considered as repressors but they do so, as previously stated, by a manner independent of pocket protein family members[10,11,16,44-46,71]. The abovementioned classification, which is based on results of in vitro studies, is probably oversimplified and does not accurately reflect the dynamics of E2F-dependent transcriptional control[17,43]. The study of E2Fs’ role in vivo has been challenging because of three main obstacles impending complete understanding of their functions. Firstly, there is a high degree of functional redundancy among activators and repressors. Secondly, there is a functional antagonism between E2F-mediated activation and repression in the regulation of normal cell proliferation. Thirdly, the members of this family have the ability to regulate each others expression, forming complex feedback loops to ensure a balance between activators and repressors in each phase of the cell cycle[17].

Deregulated expression of E2F family of transcription factors is a common phenomenon in human cancer. Little is known though about the magnitude and the nature of their contribution. The prevailing view is that E2F activators and repressors operate in a coordinated manner to achieve proper cell cycle progression and/or apoptosis and that disturbance of this well orchestrated interaction can contribute to carcinogenesis. According to the traditional classification of the different E2F members into activators and repressors, it would be expected that clear predictions about their function in cancer could be made. Unfortunately, this is not the case and this classical approach stemming from in vitro studies is openly challenged in practice[17,43]. To complicate things further, numerous E2F target genes have been identified reflecting the fact that E2Fs participate in cellular processes beyond the cell cycle[123-127].

Regarding digestive system malignancies, E2F1 may exhibit a tumor-suppressing role in colorectal, gastric and esophageal adenocarcinoma, as well as in hepatocellular carcinoma, probably through pro-apoptotic activity[25,78,82-86,96,97,108]. On the contrary, a tumor-promoting role has been attributed to E2F1 in the context of pancreatic ductal adenocarcinoma and esophageal squamous carcinoma[73-76,119]. It is very interesting though that in experimental models involving adenoviral transfer of E2F1 in esophageal squamous and pancreatic cancer cell lines apoptosis was evoked[77,121,122]. The possible role, correlations and prognostic significance of E2F1 expression in digestive system malignancies is summarized in Table 1.

E2F4, a classical “repressor”, seems to play a tumor-promoting role in colorectal, gastric and liver carcinogenesis[90,92,93,97,117]. It is also well documented that AGC repeats in the E2F4 gene are commonly found mutated in various gastrointestinal malignant neoplasms exhibiting microsatellite instability and they are thought to represent one of the targets of DNA mismatch repair deficiency[89-94,105-107,117]. Of note, in colorectal cancer, an inverse relationship between the immunohistochemical expression of E2F1 and E2F4 has been demonstrated when examined in serial histological sections[97]. It would be interesting to investigate if such a relationship can be confirmed in other gastrointestinal cancers as well and, if so, whether there is a common “switch” connecting mechanistically these two opposing transcription factors.

E2F family of transcription factors serves key roles in cell cycle progression, apoptosis, cell differentiation and stress responses. Following their traditional classification into activators and repressors, it has been tempting to assign to them oncogenic or tumor-suppressing functions, thus predicting their role in carcinogenesis. A number of investigators have focused into showing a prognostic value of E2F expression in different kind of digestive tract malignancies[25,74-76,78,88,96,119]. Others have gone a step further and, based on their experimental work, suggest therapeutic strategies involving adenoviral transfer of E2F in a gene therapy context to sensitize cancer cells for conventional chemotherapeutic agents[82,121,122].

However, clinical and experimental studies in mice openly challenge this traditional view, which does not appear to reflect the complexity of E2F function in tumorigenesis. It appears, though, that this function is exerted in a tissue-dependent manner. It is appealing to consider the development of treatment strategies involving E2F-antagonists, that would suppress cell proliferation, and E2F-agonists, that would promote apoptosis[128]. Nevertheless, targeted therapeutic approaches against E2F family members should take into account the tissue-dependent function of each member.

It is quite obvious that, although our knowledge on this intriguing family of transcription factors is seemingly increasing, little is known about their function in vivo. The introduction and use of modern molecular techniques and experimental models have identified numerous targets for these factors, helping us unravel the mystery of their contribution in normal tissues. This can be the necessary step in order to clarify to which extent they exert pivotal roles in cancer development[17].

The authors would like to thank Mrs. Maria Chatzopoulou for her kind contribution in creating Figure 1.