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Unraveling the conceptional novel ADAM17 regulation by PP2A in metastasis formation.

Periodic Reporting for period 1 - UNNAMEd-2 (Unraveling the conceptional novel ADAM17 regulation by PP2A in metastasis formation.)

Reporting period: 2019-01-01 to 2020-12-31

Cancer research has made great progress during the last decades, resulting in significantly improved prognostic tools and treatment options. However, in addition to severe illness many patients experience resistance to therapy. Additionally, almost half of the patients develop metastasis, which are responsible for about 90% of all cancer related death. Cancer development is a multi-step process that requires the orchestration between the cancer cell and the cells in the environment. One factor that is known to influence cancer development and metastasis by releasing signaling molecules from the cancer cell surface is ADAM17. It sits as a pair of molecular scissors in the cell's outer membrane and cleaves other proteins from the cell surface. Studies trying to inhibit ADAM17 and thereby tumor progression however failed due to the low specificity of the drug to ADAM17. Furthermore, ADAM17 produced on other cells of the tumor environment is crucial for tumor growth inhibition and blocking of ADAM17 activity on these cells enhances tumor growth. The activity of ADAM17 is highly regulated as well as the expression of it substrates. Another strategy to inhibit the pro-tumorigenic function of ADAM17 is by targeting molecules known to regulate ADAM17 activity, or by blocking the shedding of specific substrates. The group of our collaborator Prof. Jakob Nielsson identified a novel binding partner of ADAM17, namely the protein phosphatase PP2A. Phosphorylation’s serves often as a molecular switch to activate or switch “ON” enzymes. They can also initiate the destruction of the protein, the binding to other proteins or the translocation to another part of the cells. Phosphatases in turn remove these phosphate groups and therefore switch the protein “OFF”. Prof. Nielssons team identified a novel binding motif for a specific isoform of PP2A, B56, and found that this binding motif is present in the intracellular part of ADAM17. We therefore hypothesized, that PP2A is a negative regulator of ADAM17.
The objectives of your study was therefore to:
- Understand whether and how the function of ADAM17 is regulated by PP2A.
- Determine the molecular mechanisms of the regulation and its impact in cancer development
Expressing a specific PP2A-B56 inhibitor designed by our collaborator Prof. Nielsson, decreased the function of ADAM17 in multiple cancer cells measured by the release of an ADAM17 specific substrates Amphiregulin and TGF-α. Released Amphireguiln and TGF-α binds and activates a receptor called EGFR on the same cell, in this case the cancer cell, or on cells in the surrounding e.g. the tumor environment. Activated EGFR has been shown to induce cancer cell proliferation, migration and invasion and can shape the tumour microenvironment to support tumour growth. We then generated cells so they do not express ADAM17 anymore, using the CRIPSR/Cas9 technology in both human and mouse cancer cell lines and re-introduced fully functional ADAM17 or ADAM17 mutant variants that have an increased or decreased binding ability to PP2A. By mutating the PP2A binding side on ADAM17 we confirmed that PP2A is removing activating phosphate groups from the cytoplasmic part of ADAM17 shown by phosphor-proteomics together with our collaborator Prof. Arminja Kettenbach. In other words, we have confirmed that binding of PP2A to ADAM17 switches the enzymatic activity “OFF”. Our collaborators from the Nielssons lab furthermore showed, that PP2A dephosphorylates all the phosphate groups that are on ADAM17, independent were they are on the intracellular part of ADAM17. Furthermore, we showed that mutations of the PP2A binding side influences ADAM17 function in cancer cells. We found that AREG shedding was impaired when PP2A is strongly bound to ADAM17 while mutations inhibiting the binding of PP2A enhanced ADAM17 function and AREG release. In functional assays we could show, that enhanced PP2A binding led to decreased proliferation and invasion of cancer cells in vitro. Next, we injected 4T1 breast cancer cell lines expressing ADAM17 or PP2A binding mutants into the mammary fad pat of wt Balb/c mice. We showed, that enhanced PP2A binding to ADAM17 significantly decreased tumor growth in these mice.
In summary, we showed for the first time, that activating phosphorylations on ADAM17 can be removed and thereby regulate the enzymatic activity of ADAM17. We furthermore showed that the interaction between ADAM17 and PP2A has an impact on tumour growth in a mouse model. With PP2A activators already under development, we believe that our study could contribute in the development of next generation anti-tumor drugs. The results of the project has been published (Kruse, Gnosa, Nasa, Garvanska et al. EMBO J (2020).
We hope that the findings achieved during this action will also apply for human tumors and thereby, lead to more efficient anti-cancer treatments. Additionally, tools and preliminary results have been created during the project that led to a new research project were we are evaluating the role of ADAM17 on the function of the tumor-microenvironment and we are looking at the interaction between ADAM17 and PP2A from a evolutionary perspective.
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