Final Report Summary - TGFB IN CRC (TGF-beta signaling in colorectal cancers)
With our research project, we proposed to deepen our understanding of the regulation of the Smad tumour-suppressor proteins through ubiquitination mechanisms and to test the relevance of the enzymes regulating Smad ubiquitination in animal models of intestinal tumourigenesis.
The specific project objectives were the following:
1) To test the impact of Ectodermin depletion on the development and progression of intestinal tumourigenesis caused by genetic inactivation of APC or by chemical carcinogenesis. For the first time, we are in the position to test the relevance for cancer of Smad inhibition, and not of Smad activity. To this end, we will take advantage of the conditional Ectodermin knockout mice.
2) To develop a new mouse model for Usp9x, by germ-line and conditional knock-out. This should reveal the relevance of Smad4 ubiquitination in mammalian tissues.
For objective 1, we raised mice bearing both constitutive and inducible recombination of Ectodermin in the intestinal epithelium, in combination with deletion of the tumour-suppressor APC (used here as a genetic driver of intestinal neoplasia). We checked for efficient recombination of the loci in animals, and controlled that Ectodermin protein levels were depleted in recombinant tissues. We performed histo- / pathological analyses of the tumours in contro (APC deleted) and experimental (Ecto; APC deleted) mice, in terms of tumours per mice, dimension of tumours, proliferative/Ki67 index, and histological grading. Similar analyses were carried out also on mice (wild-type and Ecto deleted) where intestinal tumourigenesis was induced by chemical treatment with an AOM/DSS regime. Results indicate that Ectodermin is not a relevant pro-oncogenic gene in the context of APC-driven intestinal tumourigenesis.
We also decided to extend our observations to another genetic system where Smad4 plays important tumour-suppressive functions (and thus, Ecto should behave as pro-oncogenic molecule), by using pancreas inactivation of Ecto in combination with expression of an activated oncogenic Kras*. Unexpectedly, we found here that Ectodermin plays powerful tumour-suppressive roles, and that these are independent from Smad4 (by analysing mice bearing compound deletion of Ecto and Smad4). This suggests that, during pancreas tumourigenesis, Ectodermin opposes tumour progression, likely by inhibiting some yet-to-be-identified oncogenic pathway.
We also extended our previous findings on the regulation of Smad4 by Ectodermin. We discovered that Smad4 ubiquitination is regulated in cells and vertebrate embryos by two important molecules involved in cell polarity and non-canonical Wnt signalling, Dishevelled (Dvl) and Par1b (Mamidi et al., 2012). Dvl and Par1b form a complex that precludes Smad4 from interacting with Ecto, thus keeping Smad4 active. This represents a new layer of cross-talk with the TGF-beta pathway that takes place by influencing Smad4 ubiquitination and activity, rather than by regulating receptor activity and R-Smad phosphorylation.
For objective 2, we produced ES cells bearing conditional / inactivatable USP9X, we raised mice from these ES and established a new strain, and subsequently carried out preliminary analyses on the allele. We could successfully induce deletion of the floxed allele, and this resulted in efficient depletion of the endogenous protein in mouse tissues. Mice with inactivation of USP9X are early embryonic lethal, and the characterisation of these phenotypes is under way in the lab. We also started the derivation of mice bearing both constitutive and inducible recombination of USP9X in the intestinal epithelium, in combination with deletion of the tumour-suppressor APC.
Potential impact:
This work contributes key new elements to our understanding of TGFbeta signalling in vivo. It shows that Smad4 ubiquitination by Ectodermin (and deubiquitination by USP9X) is differentially used in embryonic and adult tissues, such that early embryos are strictly dependent on negative Smad4 regulation by Ectodermin (Morsut et al., 2010), whereas this activity appears dispensable in the adult intestinal and pancreas epithelium. This work also shows that Ectodermin is endowed with potent tumour suppressive activities, opening new avenues of research aimed at understanding which is the relevant molecular target for this latter activity. Finally, our findings shed new light on the cross-talk between tissue polarity cues, such as non canonical Wnt and Par1b, and the TGF-beta pathway (Mamidi et al., 2012).
Project implications (wider societal implications):
TGFb and BMP are tumour suppressors and metastasis inducers. Identifying a new set of enzymes for their regulation opens the possibility to target this pathway with new small-molecule inhibitors and thus new therapeutic perspectives. Studying in which tissues these enzymes are active is fundamental to understand where and when their inhibition could prove useful.
The specific project objectives were the following:
1) To test the impact of Ectodermin depletion on the development and progression of intestinal tumourigenesis caused by genetic inactivation of APC or by chemical carcinogenesis. For the first time, we are in the position to test the relevance for cancer of Smad inhibition, and not of Smad activity. To this end, we will take advantage of the conditional Ectodermin knockout mice.
2) To develop a new mouse model for Usp9x, by germ-line and conditional knock-out. This should reveal the relevance of Smad4 ubiquitination in mammalian tissues.
For objective 1, we raised mice bearing both constitutive and inducible recombination of Ectodermin in the intestinal epithelium, in combination with deletion of the tumour-suppressor APC (used here as a genetic driver of intestinal neoplasia). We checked for efficient recombination of the loci in animals, and controlled that Ectodermin protein levels were depleted in recombinant tissues. We performed histo- / pathological analyses of the tumours in contro (APC deleted) and experimental (Ecto; APC deleted) mice, in terms of tumours per mice, dimension of tumours, proliferative/Ki67 index, and histological grading. Similar analyses were carried out also on mice (wild-type and Ecto deleted) where intestinal tumourigenesis was induced by chemical treatment with an AOM/DSS regime. Results indicate that Ectodermin is not a relevant pro-oncogenic gene in the context of APC-driven intestinal tumourigenesis.
We also decided to extend our observations to another genetic system where Smad4 plays important tumour-suppressive functions (and thus, Ecto should behave as pro-oncogenic molecule), by using pancreas inactivation of Ecto in combination with expression of an activated oncogenic Kras*. Unexpectedly, we found here that Ectodermin plays powerful tumour-suppressive roles, and that these are independent from Smad4 (by analysing mice bearing compound deletion of Ecto and Smad4). This suggests that, during pancreas tumourigenesis, Ectodermin opposes tumour progression, likely by inhibiting some yet-to-be-identified oncogenic pathway.
We also extended our previous findings on the regulation of Smad4 by Ectodermin. We discovered that Smad4 ubiquitination is regulated in cells and vertebrate embryos by two important molecules involved in cell polarity and non-canonical Wnt signalling, Dishevelled (Dvl) and Par1b (Mamidi et al., 2012). Dvl and Par1b form a complex that precludes Smad4 from interacting with Ecto, thus keeping Smad4 active. This represents a new layer of cross-talk with the TGF-beta pathway that takes place by influencing Smad4 ubiquitination and activity, rather than by regulating receptor activity and R-Smad phosphorylation.
For objective 2, we produced ES cells bearing conditional / inactivatable USP9X, we raised mice from these ES and established a new strain, and subsequently carried out preliminary analyses on the allele. We could successfully induce deletion of the floxed allele, and this resulted in efficient depletion of the endogenous protein in mouse tissues. Mice with inactivation of USP9X are early embryonic lethal, and the characterisation of these phenotypes is under way in the lab. We also started the derivation of mice bearing both constitutive and inducible recombination of USP9X in the intestinal epithelium, in combination with deletion of the tumour-suppressor APC.
Potential impact:
This work contributes key new elements to our understanding of TGFbeta signalling in vivo. It shows that Smad4 ubiquitination by Ectodermin (and deubiquitination by USP9X) is differentially used in embryonic and adult tissues, such that early embryos are strictly dependent on negative Smad4 regulation by Ectodermin (Morsut et al., 2010), whereas this activity appears dispensable in the adult intestinal and pancreas epithelium. This work also shows that Ectodermin is endowed with potent tumour suppressive activities, opening new avenues of research aimed at understanding which is the relevant molecular target for this latter activity. Finally, our findings shed new light on the cross-talk between tissue polarity cues, such as non canonical Wnt and Par1b, and the TGF-beta pathway (Mamidi et al., 2012).
Project implications (wider societal implications):
TGFb and BMP are tumour suppressors and metastasis inducers. Identifying a new set of enzymes for their regulation opens the possibility to target this pathway with new small-molecule inhibitors and thus new therapeutic perspectives. Studying in which tissues these enzymes are active is fundamental to understand where and when their inhibition could prove useful.