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).
