F UTURE PERSPECTIVES

Several aspects of E6AP function have been analyzed during this work, including the characterization of the novel E6AP interaction partner Herc2 as well as the identification of a new role for E6AP in estrogen receptor signaling. Although these topics have been described in an independent manner, it is quite possible that these two E6AP functions are interconnected. Additionally, E6AP has been associated with various functions, which exceed its role in Angelman Syndrome. Thus this part of the discussion is supposed to put the results obtained during this project into a broader context and to give an indication about the future perspectives of this project.

During this work it was shown that Herc2 influences E6AP ligase activity. Thus, it would be just as interesting to investigate whether Herc2 might have an effect on E6AP mediated modulation of estrogen receptor signaling. In order to address this question, preliminary reporter assays have already been performed in HEK293T cells, and the effect of Herc2 overexpression alone and in combination with E6AP was investigated (Figure 34).

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Figure   34  Herc2   overexpression   represses   Estrogen   receptor   signaling   in   Luciferase   based   Reporter   assays.HEK293T   cells   were   transfected   with   the   indicated   constructs   (   ERE-­‐Reporter   (Rep),   HA-­‐tagged   estrogen   receptor   (ER)   and   HA-­‐tagged   E6AP   variants   (wild   type   E6AP   (E6APwt   and   a   Herc2-­‐binding   site   deficient   E6AP   mutant   (E6APΔ150-­‐200).   Transfection   efficiency   was   determined   by   ß-­‐galactosidase   assay   and   luciferase   activity   was   measured.   Quantification   reflects   the   ratio   of   luciferase   activity   and   ß-­‐

galactosidase  activity.  The  respective  experiment  was  performed  by  Stian  Olsen,  a  former  student,  under   my  supervision.  

Discussion 95

The data obtained indicate that both Herc2 as well as E6AP overexpression negatively affect estrogen receptor response. However, as E6AP overexpression was still able to repress estrogen receptor signaling under Herc2 knockdown conditions and vice versa Herc2 was able to inhibit estrogen receptor response in cells depleted for E6AP (data not shown), it appears that the observed effects are not dependent on each other and that Herc2 and E6AP hold a repressor function independent of the interaction partner.

These findings, however, could probably also explain the overlapping phenotypes of Herc2 mutant and E6AP null mice. Both mouse models display impairments in reproduction as well as motor dysfunctions. Thus, it might be speculated that the observed phenotypes in Herc2 mutant mice are the result of deregulated estradiol signaling. Therefore it may be of future interest to elucidate the mechanisms of Herc2 in estrogen receptor signaling in detail.

E6AP is causally involved in two severe human diseases, Angelman Syndrome and cervical cancer. The potential role of E6AP as a repressor of estrogen receptor signaling in the context of Angelman Syndrome has already been discussed in detail.

However, several publications indicate that estrogen receptor signaling is crucial during cervical carcinogenesis (Brake and Lambert, 2005). Thus, a potential involvement of E6AP in this context will be discussed in the following. Studies with 16E6 transgenic mice support the idea that estradiol contributes to tumor development as well as persistence and malignant progression during cervical carcinogenesis (Brake and Lambert, 2005). A follow-up study, additionally revealed that knockout of ERα in E7 transgenic mice abolishes the development of cervical but not skin cancer in these mice. Thus, the estrogen receptor seems to be required for HPV-mediated cervical carcinogenesis (Chung et al., 2008). In addition a third study clearly demonstrated that E6AP is crucial for cervical carcinogenesis in E6 transgenic mice, as E6AP knockout in these mice completely prevent the development of cervical cancer (Shai et al., 2010). Taken together, E6AP, ERα, and estradiol treatment are essential for cervical carcinogenesis in mice. Furthermore, a connection between the long-term use of oral contraceptives and the development of cervical cancer has already been reported a long time ago (Moodley et al., 2003). Although E6AP, E6 and ERα may operate in different pathways during carcinogenesis, the finding that E6AP regulates estrogen receptor signaling raises the question if and how these findings are connected. Preliminary estrogen receptor reporter assays indicated that expression of E6 proteins represses reporter activity and that co-expression of wild-type E6AP but not of a E6-binding site deficient E6AP mutant or a catalytic inactive E6AP variant enhanced this effect (Figure35). Thus, it can be speculated that although E6AP represents a repressor on its own, E6 and E6AP seem to display synergistic repressor functions. Moreover this synergistic effect seems to depend on the interaction of E6 with E6AP and the E3 ubiquitin ligase activity of E6AP. It should also be mentioned

Discussion 96

that overexpression of E7 did not affect estrogen receptor signaling in these reporter assays (data not shown). Additionally, although low-risk E6 proteins showed repression of reporter activity, the effect was significantly lower as compared to high-risk proteins. Thus, it can be speculated that E6AP together with high-high-risk E6 modulates ER signaling during cervical carcinogenesis. Due to the fact that loss of ERα signaling seems to prevent cervical carcinogenesis, a repressor function of E6AP and E6 seems to be counter-intuitive. However, as discussed earlier, E6AP has been reported to act as a co-activator of estrogen receptor signaling (Nawaz et al., 1999).

This leads to the hypothesis, that although some estrogen responsive genes may be repressed by the presence of E6AP and E6, other genes may show enhanced expression and may hence be involved in progression of tumorigenesis. Thus, the investigation of E6AP function in steroid hormone signaling should not be restricted to Angelman Syndrome but should prospectively also include HPV-associated cervical cancerogenesis.

Several cancer types have been shown to be estrogen responsive, with breast cancer being probably the best-studied hormone dependent cancer. Treatment of breast cancer patients with estrogen receptor antagonists like tamoxifen can help to prevent relapse and metastasis. If symptoms of Angelman Syndrome were indeed the result of

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Figure  35  16E6  overexpression  represses  Estrogen  receptor  signaling  in  Luciferase  based  Reporter  assays.  

H1299  K3  cells  (shRNA  mediated  stable  knockdown  of  endogenous  E6AP  expression)  were  transfected  with   the   indicated   constructs   (   ERE-­‐Reporter   (Rep),   HA-­‐tagged   estrogen   receptor   (ER)   and   HA-­‐tagged   E6AP   variants  (  E6AP  wild  type  (E6APwt),  a  catalytic  inactive  mutant  (E6APina)  and  a  E6-­‐binding  deficients  E6AP   mutant   (deltaE6).   Transfection   efficiency   was   determined   by   ß-­‐galactosidase   assay   and   luciferase   activity   was  measured.  Quantification  reflects  the  ratio  of  luciferase  activity  and  ß-­‐galactosidase  activity.

Discussion 97

deregulated estrogen receptor signaling, one could think about similar treatments.

Angelman Syndrome is regularly diagnosed in early childhood. Thus, it is likely that the phenotypes are established during development and might not be reversible.

However it might still be possible that treatment with estrogen receptor antagonists could alleviate symptoms. Studies with Ube3a-/- mice might therefore be a good model to study the effect of estrogen receptor antagonists on the Angelman Syndrome phenotype.

In summary, future work on Angelman Syndrome should include thorough investigation of the mechanisms of E6AP function in estrogen receptor signaling as these investigations might help to elucidate the detailed molecular basis of this disorder, which in the end could potentially contribute to the development of a therapy for this severe neurological disease.

Literature 98