Functional impact of colorectal cancer-associated mutations in the transcription factor E2F4.

The transcription factor E2F4 plays a critical role in cell cycle progression of normal and cancerous intestinal epithelial cells. Contrary to other E2Fs, the coding region of the E2F4 gene contains a longer spacer segment of a CAG trinucleotide repeat sequence encoding 13 consecutive serine residues, which is highly vulnerable to frameshift mutations in situations of genetic instability. Mutations in this region of the E2F4 gene have been observed in colorectal tumors with microsatellite instability. However, the effect of these changes on its function in colorectal cancer cells is currently unknown. We generated E2F4(CAG)12 and E2F4(CAG)14 mutants and compared their activity to the E2F4 wild-type, E2F4(CAG)13. Luciferase assays with the thymidine kinase-luc reporter gene revealed that the mutants were more transcriptionally active than wild-type E2F4. The mechanism of increased activity of E2F4 was primarily related to protein stability, due to a significantly enhanced half-life of E2F4 mutants comparatively to that of wild-type E2F4. However, the association with the pocket protein p130/RBL2 did not account for this increased protein stability. Sequencing analysis of the endogenous E2F4 gene in a series of colorectal cancer cell lines showed that the microsatellite-unstable cell line SW48 exhibited a serine deletion in this gene. Accordingly, E2F4 half-life was much more elevated in SW48 cells in comparison to Caco-2/15, a microsatellite-stable cell line. Notably, in soft-agar assays, both mutants more potently increased anchorage-independent growth in comparison to wild-type E2F4. In conclusion, our data demonstrate that cancer-associated E2F4 mutations enhance the capacity of colorectal cancer cells to grow without anchorage, thereby contributing to tumor progression.

ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells.

BACKGROUND: The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown. RESULTS: Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3ß phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/ß-catenin and MEK/ERK signaling, respectively. CONCLUSIONS: The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cells.

The generation of knock-out mice for E2F4 gene expression has suggested a role for this transcription factor in establishing and/or maintaining the intestinal crypt compartment. Having previously demonstrated that E2F4 is cytoplasmic in quiescent-differentiated cells but nuclear in growth factor-stimulated proliferative cells, the present study was aimed at determining the role of E2F4 in the control of human intestinal epithelial proliferation. Results herein demonstrate that lentiviral infection of an shRNA which specifically knocked-down E2F4 expression slowed down G1/S phase transition and the proliferation rate of normal human intestinal epithelial cells (HIEC) and of colon cancer cells. Protein expression of Cdk2, cyclins D1 and A, Cdc25A and c-myc was markedly down-regulated in shE2F4-expressing cells; by contrast, expression of the cell cycle inhibitors p21(Cip/Waf) and p27(Kip1) was increased. In addition, the expression of many genes involved in DNA synthesis was down-regulated in shE2F4-expressing cells, whereas no modulation in E2F1 expression was observed. A decrease in E2F4 in colon cancer cell lines also resulted in a reduction in soft-agar growth capacity. Immunofluorescence experiments in human fetal intestine revealed that cells expressing high nuclear levels of E2F4 also expressed cyclin A protein. Lastly, E2F4 and its target cyclin A were up-regulated and mostly nuclear in human colorectal tumor cells in comparison to the corresponding benign epithelium. These results indicate that nuclear E2F4 may be determinant in the promotion of proliferation of human intestinal epithelial crypt cells and colorectal cancer cells.

Nuclear expression of E2F4 induces cell death via multiple pathways in normal human intestinal epithelial crypt cells but not in colon cancer cells.

E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between green fluorescent protein (GFP) and wild-type (wt)E2F4 or GFP and nuclear localization signal (NLS)-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies, and activation of caspase 9 and caspase 3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, and phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS compared with wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Downregulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g., p53, Bax loci, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.