Bos Group

Ras proteins in cancer

Many genes mutated in tumors encode for proteins involved in signal transduction. The focus of the Bos lab is to understand the signaling networks that lead to cancer, particularly the networks that involve Ras and Ras-like GTPases. This research topic originated from earlier work revealing that Ras is mutated in at least 15% of all cancers and if gene products are included that are linked to the Ras-signalling pathway the mutation rate may be well over 50%. Interestingly, a very close relative of Ras, Rap1, can counteract the oncogenic properties of mutated Ras. We have discovered that one of the mechanisms of Ras reversion by Rap1 is the induction of integrin-mediated cell adhesion and E-cadherin-mediated cell junction formation.

Rap proteins are a family of five different proteins, Rap1A and B, Rap2A, B and C. Although these proteins are very similar to Ras proteins, Rap proteins were already present in the hypothetical first eukaryotic cell (LECA). Their function is first eluded in yeast where an ortholog of Rap proteins, Bud1 or Rsr1, is involved in the recognition of polarity cue to recruit establisment factors for bud formation. Our lab has developed this model further in mammalian cells. Recent results from Anneke Post and Willem Jan Pannekoek showed that Rap1 directly inhibits Rho signalling through the adaptor proteins Rasip1 and Radil. These proteins medaites the translocation of ArhGAP29 to the plasma membrane where Rap1 is located. ArhGAP29 is a GTPase activating protein for Rho. As a consequnce Rho at the plasma membrane is inactivated, and radial stress fibers are resolved. The biological consequence is increased cell spreading and for endothelial cells increased tightening of the junctions and as such increased barrier function. 

Epac: a multi-domain regulator of Rap under direct control of cAMP

Structure Epac2

In the analysis of the Rap1 signaling network Johan de Rooij and Fried Zwartkruis discovered a guanine nucleotide exchange factor for Rap1, Epac. Epac is an exchange protein directly activated by cAMP. To study this cAMP effector pathway in further detail we developed of a cAMP analog specific for Epac, 8-CPT-2’OMe-cAMP or 007This analog is currently a key tool in assigning functions to Epac, and thus Rap, which include the regulation of cell adhesion and cell junction formation. We are also interested to understand the molecular details of how Epac is spatially and temporally regulated. An highlight was the determination of the crystal structure of the full length Epac protein in the active and inactive conformation by Holger Rehmann (figure). In addition, Bas Ponsioen and Martijn Gloerich showed that cAMP also regulates Epac spatially, as cAMP induces the translocation of Epac1 to the plasma membrane. This process is mediated by a conformational change in the DEP domain after activation, allowing Epac1 to tether to phosphatidic acid in the plasma membrane. 

Regulation of apical polarity

In analysing intestinal epithelial cells Martijn Gloerich found an critical role for the RapGEF, PDZ-GEF, and Rap2A in the spatial and temporal control of brushborder formation (figure).
Recently Lucas Bruurs did the exiting finding that in these cells "singularity" (only one brushborder per cell) is tightly controled by a disease related lipid flippase and CDC42
 
  
Targetting mutant Ras in patient-derived tumor organoids

More recently, together with Hugo Snippert, we have developed microscopic tools to monitor patient-derived tumor organoids in response to combination therapies. We could confirm the exquisite resistance of mutant Ras-containing tumor organoids from the colon for pan-EGFR inhibitors. Further combinations are tested to find the Achilles heel of these mutant Ras-containing tumor organoids

Technologies applied

We are using a variety of different technologies.

  1. High-end microscopy, which includes a confocal laser scanning and Delta-vision microscope and real time imaging. We have access to Tirf microscopy and EM.
  2. Biochemical and Biophysical approaches, including monitoring junction formation by Electrical Cell-substrate Impedance Sensing (ECIS).
  3. Functional Genomics using siRNAs and Crispr-Cas9.
  4. Organoid technology