NOTCH3DR · Investigating a novel role of Notch3 as a dependence receptor and its relevance in vivo

Investigating a novel role of Notch3 as a dependence receptor and its relevance in vivo

Cells of multicellular organisms interact with each-other and with their environment thanks to proteins called receptors present on their membrane. These receptors are activated following specific interaction with one or several proteins soluble in the environment or expressed by neighbour cells, called ligands. The receptors have long been seen as inactive when the ligands are absent. However, it is now known that certain receptors can have different functions depending on the availability of the ligands. Some such receptor induces cell death in absence of signal and cell survival when the signal is activated. Due to this dual function, they have been called dependence receptors. Indeed, cells that do express such receptors are dependent on a specific signal for their survival. These receptors have shown to be implicated in development and cancers. Indeed, cancer cells have evolved to avoid dependence on these receptors. Some cancer cells have lost the expression of the receptor and can therefore survive even in absence of the signal. Other cancer cells gained the ability to express the signal themselves. As these receptors are very important for survival of the cancer cells, they became a putative target to develop therapeutic strategies. It is therefore of great interest to find and describe the molecular mechanisms of these receptors. The NOTCH3DR project characterised a new dependence receptor. Notch3 belongs to a family (Notch1 to 4) of genes implicated in development of muticellular organisms and implicated more recently in pathology of cancers. We demonstrated that over-expression of Notch3 induced cell death of cancer cells from different origins. Furthermore when cancer cells expressing Notch3 were cultured in presence of cells expressing ligands, cell death induced by Notch3 was abrogated. These in vitro expreriments clearly showed that Notch3 behaves like a dependence receptor. We next studied the molecular mechanisms of Notch3-induced cell death. We observed that cell death induced following overexpression of Notch3 was dependent on caspases, enzymes implicated in a specific cell death mechanism named apoptosis. More particularly, caspase-9 was shown to be necessary for cell death induced by Notch3. This shows that Notch3 behaves like other known dependence receptor. We next showed that Notch3 was cleaved by caspases during the process of induction of apoptosis. We are now evaluated whether this process is required for induction of apoptosis by identifying mutants that are not cleaved by caspases anymore. As we showed that Notch3 behaves like a dependence receptor in vitro, we next studied the involvement of Notch3 in cancers. We first studied expression of Notch3 ligands in panels of lung cancer cell lines and breast cancer cell lines. We observed an important correlation between Notch3 expression and expression of Dll-4, ligand of Notch3 in lung cancer cell lines. We observed as well a correlation with Jagged-2, another ligand of Notch3 in breast cancer cell lines. Interestingly, we observed a very significant increase in Jagged-1 in primary sample of patients suffering from renal cell carcinomas. Furthermore Jagged-1 inhibition in renal cell carcinomas cell in culture induced cell death. We therefore hypothesise that Notch3 dependence receptor function could be involved in several type of cancers. Although more investigation in vivo are needed this project demonstrated that Notch3 may have a new function as a dependence receptor. This could prove to be of particular importance in cancer therapy. We therefore began to investigate ways to induce Notch3 dependence receptor activity in cancer cells. We are testing interfering agents to inhibit the interaction between Notch3 and its ligands. Particularly, we are aiming at identifying agents that would be specific of Notch3 and that would not target other Notch receptors and would therefore be more specific and less toxic.

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