Function, regulation and pathological roles of the Gab/DOS docking proteins

Since their discovery a little more than a decade ago, the docking proteins of the Gab/DOS family have emerged as important signalling elements in metazoans. Gab/DOS proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine and antigen receptors as well as cell adhesion molecules. They also contribute to signal diversification by channelling the information from activated receptors into signalling pathways with distinct biological functions. Recent approaches in protein biochemistry and systems biology have revealed that Gab proteins are subject to complex regulation by feed-forward and feedback phosphorylation events as well as protein-protein interactions. Thus, Gab/DOS docking proteins are at the centre of entire signalling subsystems and fulfil an important if not essential role in many physiological processes. Furthermore, aberrant signalling by Gab proteins has been increasingly linked to human diseases from various forms of neoplasia to Alzheimer's disease

A novel MCF-10A line allowing conditional oncogene expression in 3D culture

Introduction Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance

Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAFV600E and wild‐type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

This study shows that MAPK signalling is robust against protein level changes due to a strong negative feedback from Erk to Raf. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days

EGFR-targeted TRAIL and a Smac mimetic synergize to overcome apoptosis resistance in KRAS mutant colorectal cancer cells

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed DbαEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of DbαEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward DbαEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of DbαEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing DbαEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects DbαEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-RasG12V. In the presence of doxycycline, these cells showed increased resistance to DbαEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the DbαEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between DbαEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that DbαEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase

The atypical kinase RIOK1 promotes tumor growth and invasive behavior

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers

BRAFΔβ3−αC^{Δβ3-αC} in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF$^{Δβ3-αC}$ oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF$^{Δβ3-αC}$ oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF$^{ΔLNVTAP>F}$ and two novel mutants, BRAF$^{delinsFS}$ and BRAF$^{ΔLNVT>F}$, and compare them with other BRAF$^{Δβ3-αC}$ oncoproteins. We show that BRAF$^{Δβ3-αC}$ oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAF$^{Δβ3-αC}$ oncoproteins, e.g., BRAF$^{ΔLNVTAP>F}$, increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAF$^{Δβ3-αC}$ mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds