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

Abstract

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.

E2F4 (E2F transcription factor 4, p107/p130-binding)

“…Although E2F4 has been particularly described as a repressor of both transcription and cell cycle progression (Vairo et al, 1995;Muller et al, 1997;Rayman et al, 2002), several studies have reported other roles such as 1-its binding to E2F-responsive elements as a pocket protein-free E2F during S phase, 2-its capacity to induce E2F target genes and 3-its implication in proliferation. Hence, these studies suggest that E2F4 can also act as a transcriptional activator (Verona et al, 1997;Wells et al, 1997;Ross et al, 1999;Lang et al, 2001;Garneau et al, 2009;van Amerongen et al, 2009). For example, studies carried out by Lo et al, 2011 demonstrated that the majority of E2F4 binding sites are located proximal to transcription start sites.…”

“…Accordingly, forced expression of nuclear E2F4 promotes S-phase entry into cardiomyocytes (Ebelt et al, 2005;van Amerongen et al, 2009). Moreover, nuclear E2F4 expression is associated with proliferation of rapid renewing tissues such as bone marrow (Kinross et al, 2006;Zhang et al, 2010), digestive tract (Rempel et al, 2000;Garneau et al, 2009) and skin (Wang et al, 2000;Wang et al, 2001). Many in vivo and in vitro studies have led to the identification of numerous roles of E2F4 in different cellular processes such as nervous system development, intestinal homeostasis, bone development, myogenesis, adipogenesis and erythropoiesis, to name a few.…”

“…E2F4 also appears to play a critical role in rapid renewing tissues. In the gut, E2F4 is highly and preferentially expressed in the nucleus of proliferative cells (Dagnino et al, 1997b;Deschenes et al, 2004;Garneau et al, 2009). Loss of E2F4 in the small intestine results in a significant decline in proliferative zones (crypts) and a shortening and a reduction in the number of intestinal villi (Rempel et al, 2000).…”

“…Loss of E2F4 in the small intestine results in a significant decline in proliferative zones (crypts) and a shortening and a reduction in the number of intestinal villi (Rempel et al, 2000). The role of E2F4 in maintaining intestinal homeostasis is also reinforced by the fact that it is overexpressed in the nucleus of colorectal cancer cells, contributing to hyperproliferation (Mady et al, 2002;Garneau et al, 2007;Garneau et al, 2009). In human and mouse epidermis, E2F4 is expressed in the basal and the immediately suprabasal cells, fading in upper cell layers (Dagnino et al, 1997b;Paramio et al, 2000;Wang et al, 2000;D'Souza et al, 2001).…”

“…Note Summary: Mutation (AGC repeat) (Yoshitaka et al, 1996;Souza et al, 1997;Ikeda et al, 1998;Moriyama et al, 2002); Increased expression (Mady et al, 2002;Garneau et al, 2009). Many studies have reported the presence of mutations in E2F4 AGC trinucleotide repeats in colorectal cancer bearing microsatellite instability (MSI).…”