BCL9-2 and its function in development and tumorigenesis

1.3.1 BCL9-2 encodes a member of BCL9 protein family

BCL9-2 is a member of the novel BCL9 protein family in vertebrates. Its human homologue BCL9 was initially identified as the product of the B cell lymphoma 9 gene, which was translocated and overexpressed in B-cell malignancies with chro-mosomal translocations (87). The function of BCL9 was disclosed in a genetic screen by discovery of the Drosophila orthologue Legless as a component of Wg/Wnt sig-naling (see below). Legless was identified as an essential co-factor of Wnt/ -catenin signaling during Drosophila development, which binds to -catenin. Legless was functionally replaced by human BCL9 in rescue experiments (57, 88). BCL9-2 was identified as another BCL9 related binding partner of -catenin in a yeast-two-hybrid screen of a mouse embryo cDNA library (54).

The overall sequence identity of the proteins of BCL9 family is relatively low (ap-proximately 35 %). The similarities are comprised within the seven highly conserved homology domains (HDs). These short sequences contain a Pygopus binding main, -catenin binding domain and a nuclear localization signal (NLS) coding do-main (HD1, HD2 and HD3, respectively) in the N-terminus. All proteins of BCL9 family share an additional N-terminal domain, which encodes for a putative sumoy-lation site and, but only in case of BCL9-2 a further NLS. C-terminus of BCL9 pro-teins includes three additional homology domains termed C-HD 1-3, which are less conserved in Drosophila (Figure 3) (54, 57, 89).

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Figure 3. Schematic view of the domain structure of the BCL9 protein family (48).

NHD: N-terminal homology domain, containing a second nuclear localization signal (NLS) in BCL9-2 sequence. PyBD: Pygopus binding domain (HD1), catBD: -catenin binding domain (HDBCL9-2), NLS:

classic nuclear localization signal containing domain (HD3). C-HD1, C-HD2 and C-HD3, C-terminal homology domains-1, -2 and -3, respectively.

The analyses of domain functions revealed some unique features of BCL9-2. In con-trast to Legless/BCL9, BCL9-2 is a nuclear protein (54, 89). Deletion of the second NLS from the N-terminal homology domain (NHD) of BCL9-2 sequence resulted in cytoplasmic localization of the mutant protein (54, 89, 90).

Binding to Pygo2 is crucial for Legless/BCL9 to promote its co-activator function in Wnt/ -catenin dependent manner in Drosophila and vertebrates. In contrast, BCL9-2 does not require interaction with Pygo2 to co-activate Wnt/ -catenin dependent tran-scription in vertebrates (54, 89, 90). Moreover, it was suggested that BCL9-2 trans-locates -catenin to the nucleus thereby regulating -catenin’s adhesion and tran-scriptional functions. Phosphorylation of tyrosine 142 of -catenin promotes BCL9-2 binding, which in turn enhances -catenin transcriptional activity (54).

The function of the C-terminal domains is not yet completely understood. However, they seem to be important for promotion of Wnt/ -catenin signaling, since a deletion of a C-terminus in BCL9 abolished the Wnt/ -catenin dependent expression of a reporter construct in cultured cells (91).

1.3.2 The role of BCL9-2 in normal development and cancer

Considerable number of studies in the last decade described the requirement of BCL9 and BCL9-2 for the Wnt/ -catenin in normal cells and in tumors. However,

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BCL9 was reported to present its transcriptional co-activator function only in particu-lar type of cells and tissues (e.g. in lymphoid or muscle cells), which are in part dis-tinct from BCL9-2 (91-93). Together, these findings demonstrate disdis-tinct, cellular context dependent functions of two proteins believed to fulfill similar tasks.

In vivo studies provided insights into the function of the proteins. In a recent study by Matsuura et al., 2011 a conventional knock-out of BCL9-2 was used to analyze the role of Wnt/ -catenin signaling in the regulation of the GCM1/syncytin pathway in vivo. They reported that BCL9-2^+/- mice were healthy and fertile. In contrast, BCL9-2^-/- mice died at approximately embryonic day (E) 10.5. However, embryonic lethali-ty was rather due to maternal placental defects (94). Studies on inducible BCL9/BCL9-2 double knockout mice and on K19-BCL9-2 transgenics showed that deregulation of BCL9 proteins is dispensable for normal homeostasis in the intestine (92, 93, 95).

BCL9/BCL9-2 double mutant mice showed altered expression of several genes spe-cifically expressed in intestinal stem cells including Lgr5. Moreover, the regenera-tion capacity of BCL9/BCL9-2 intestinal epithelium was diminished after an induc-tion of colitis by treatment of mice with DSS (dextran sulfate sodium). Furthermore, double mutant mice showed a reduced expression of EMT (epithelial-mesenchymal transition) markers in the intestinal adenoma and reduced size of colon tumors (95).

The limiting condition of the double knock out study was the lack of evidence that both proteins share precisely the same function. So, it is not sure, if the resulting phenotype was caused by deregulation of both proteins or if the mutation of one was sufficient. However, the results of the study by Deka et al., 2010 are consistent with our previous findings on BCL9-2 function in normal and cancer cell. Overexpression of BCL9-2 promoted EMT in normal epithelial cells and reconverted a mesenchymal phenotype of colon cancer cells into more epithelial (54). Furthermore, BCL9-2 en-hanced local invasion of the APC^Min/+ adenoma. In addition, overexpression of BCL9-2 induced intestinal tumorigenesis in transgenic K19-BCL9-2 mice (93). Con-sequently, the role of BCL9 proteins rather under pathological conditions became more evident.

While an oncogenic role of BCL9 was implicated for B- and T-cell malignancies, BCL9-2 was associated with epithelial cancers. High BCL9-2 levels were found in

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colon cancer cell lines and HeLa (54, 89, 93, 96). We recently described, that in co-lon cancer cells BCL9-2 co-regulated only a subset of Wnt/ -catenin dependent genes. In addition, for the first time we demonstrated that BCL9-2 also controls the expression of the EphB3/B4 receptors and EphrinB1/B2/B3 ligands independently of -catenin (93). These BCL9-2 specific target are implicated in intestinal cancer (97).

The examination of human colorectal neoplasia and invasive cancers revealed a cor-relation of high BCL9-2 protein level and progressive tumor stages (93, 98).

The expression of BCL9-2 was studied in a single study on human breast cancer samples (99). Here BCL9-2 was described to be higher expressed in breast cancer tissues than in the normal breast. In ductal carcinoma in situ (DCIS), the immuno-histochemical BCL9-2 expression was significantly associated with the nuclear grade and the expression of HER2, c-myc and p53. Moreover the expression of BCL9-2 tended to correlated with -catenin and ER. Similarly, in invasive ductal carcinoma (IDC) BCL9-2 expression correlated significantly with nuclear grade and the expres-sion of HER2.

Although the implication of BCL9-2 in development and progression of colon can-cers is clear, the mechanisms of its deregulation are still not known. The mechanisms and factors, which regulate the expression of BCL9 proteins, are poorly analyzed.

However, recently a first hint in this regard was published. BCL9 was found to be regulated by miRNAs in ovarian cancer (100). Moreover, the potential new functions of C-terminal domains remain to be discovered. Since it is now evident that BCL9-2 can regulate genes independently of -catenin (93), a detection of candidate path-ways, which may interact with BCL9-2, could provide new insights into additional functions of BCL9-2 and contribute to understanding of its oncogenic mechanism.

Aims of the study

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