From Ras to Rap and Back, a Journey of 35 Years

From Ras to Rap and Back, a Journey of 35 Years

Johannes L. Bos

Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht,The Netherlands

Correspondence: [email protected]

Our laboratory has studied Ras and Ras-like proteins since the discovery of the Ras oncogene35 years ago. In this review, I will give an account of what we have done in these 35 years andindicate the main papers that have guided our research. Our efforts started with the earlyanalysis of mutant Ras in human tumors followed by deciphering of the role of Ras in signaltransduction pathways. In an attempt to interfere in Ras signaling we turned to Rap proteins.These proteins are the closest relatives of Ras and were initially identified as Ras antagonists.However, our studies revealed that the Rap signaling network primarily is involved in spa-tiotemporal control of cell adhesion, in part through regulation of the actin cytoskeleton.More recently we returned to Ras, trying to interfere in Ras signaling by combinatorial drugtesting using the organoid technology.

THE BEGINNING

In 1982, the stunning result that a single-pointmutation converted a normal H-Ras gene into

an oncogene was published by the Weinberglaboratory (Parada et al. 1982). The experimen-tal setup was based on the assumption thatcancer is caused by mutations in genes andthat these “oncogenes,” when transfected intonormal cells, should be able to convert thesecells in tumor cells. For this assay, Weinbergand colleagues transferred genomic DNA froma human kidney tumor cell line into NIH-3T3mouse fibroblasts. This resulted in morpholog-ical transformation of NIH-3T3 cells, suggest-ing the presence of an oncogene in the genomicDNA of these tumor cells. However, the identi-fication of such an oncogene was a painstakingprocess that requested multiple rounds of selec-tion, to reduce the amount of human DNA

present in the transformed mouse cell. Thenext step was the separation of the humanDNA from the mouse DNA based on human-specific repetitive sequences, followed by clon-ing and sequence analysis. Many groups fol-lowed a similar path, resulting in the discoveryof the H-ras-related genes K-ras and N-ras (Hallet al. 1983; Shimizu et al. 1983). Many tumorcell lines and tumor tissues were subsequentlyscreened for oncogenes. But the assay remainedextremely labor-intensive and the success ratewas low. At that time, I was working in the lab-oratory of Alex van der Eb on cell transforma-tion by the adenovirus E1 region. To switch tohuman oncogenes would have been an obviouschoice, but jumping on a very labor-intensivebandwagon was not very attractive. I was, how-ever, struck by a paper of Bruce Wallace showinga method to identify specific mutations in sicklecell anemia using synthetic oligonucleotides

Editors: Linda VanAelst, Julian Downward, and Frank McCormick

Additional Perspectives on Ras and Cancer in the 21st Century available at www.perspectivesinmedicine.org

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(Conner et al. 1983). The basic principle is thatthe thermal stability of a 20-mer oligonucleo-tide annealed to genomic DNA is reduced by acouple of degrees when one base pair is mis-matched. As mutations were only found in co-don 12 and 61 in H-, K-, and N-Ras, we neededa limited number of oligonucleotides represent-ing all possible single base pair changes. At thattime oligonucleotide synthesis was possible inonly a few laboratories, one of which was thelaboratory of Jacques van Boom next door.With some help from my thesis supervisorPiet Borst, I convinced him to make the re-quired 72 different oligonucleotides, coveringall possible codon 12 and 61 mutations. Detect-ing mutant Ras with this approach turned outto work quite well for cell lines and for reallypure tumor samples, but it also had its limits ofdetection (Bos et al. 1984). The discovery of thepolymerase chain reaction (PCR) method(Saiki et al. 1985) resolved this limitation andwe were able to identify Ras mutations in a largevariety of tumors by simple dot-blot assays(Verlaan-de Vries et al. 1986). As a PCR ma-chine, we used a robot arm moving betweenwater baths of different temperatures as our firstPCR machine, with homemade Klenow poly-merase to be added after each cycle. One ofthe highlights was the colon study with BertVogelstein showing K-ras mutations to occurin 50% of the colon tumors (Bos et al. 1987),an observation independently made by ManualPerucho and coworkers (Forrester et al. 1987).Interestingly, Ras mutations were also present in50% of benign adenomas. Together with othergenetic alterations detected by Vogelstein, thisled to the postulation of the sequential activa-tion of oncogenes in colorectal cancer (Vogel-stein et al. 1988). Another highlight was thestudy of lung adenocarcinoma together withSjoerd Rodenhuis, revealing 30% K-ras muta-tions (Rodenhuis et al. 1987). However, theclinical relevance/diagnostic value of Ras mu-tations became only apparent with the develop-ment of epidermal growth factor receptor(EGFR) inhibitors like cetuximab and erlotinib.The presence of a Ras mutation is currently anexclusion criterion for these drugs (Eberhardet al. 2005; Karapetis et al. 2008). Ras mutations

were most frequently found in pancreatic can-cer, as first described by Perucho and coworkers(Almoguera et al. 1988).

In these early studies, we already observedgenetic heterogeneity in tumors. For instance,in melanoma we detected two different Ras mu-tations in one primary tumor that separated inthe metastatic lesions (van ’t Veer et al. 1989). Inseminomas, a morphologically very homoge-neous tumor, Ras mutations were found insome parts of the primary tumor, but not inother parts (Mulder et al. 1989). In acutemyeloid leukemias (AMLs), 30% of which carryN-Ras mutations, we observed that in somecases the Ras mutation remained detectable af-ter complete remission. Interestingly, in thesecases, Ras mutations were also present in theapparently unaffected lymphoid lineage, sug-gesting that the Ras mutation is present in anearly hematopoietic stem cell not affected by thetreatment. In other cases of AML, the Ras mu-tation disappeared after treatment, and did notrecur after relapse. We concluded from thesestudies, first, that Ras mutations can contributeto the oncogenic process at different stages,second, that mutant Ras apparently has noeffect on the lymphoid lineage, and third, thattreatment kills all the aggressive blast cells of theleukemia, but not the underlying premalignantcell clone (Yunis et al. 1989). These and otherearly studies have been summarized in Bos(1989).

Ras REGULATION

After identification of mutated Ras as onco-gene, much effort was put in understandingthe function of Ras, a small GTPase thatnormally cycles between an active GTP-boundand an inactive GDP-bound state, but whenoncogenically mutated, is stalled in the activeGTP-bound state. The obvious rationale beingthat understanding the normal function of Raswould provide insight into how to treat tumorsharboring oncogenic Ras. The kinetics of Rascycling between a GTP- and a GDP-bound statein vitro indicated the existence of accessoryproteins to regulate Ras loading. The first regu-latory protein identified was the CDC25 pro-

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tein, a guanine nucleotide exchange protein forRas in yeast (Broek et al. 1987; Robinson et al.1987). Soon after, Frank McCormick discovereda p120 GTPase-activating protein (p120GAP)guided by the observation that the in vivoGTPase activity of Ras was much higher thanthe in vitro activity (Trahey and McCormick1987; Wong et al. 1992).

At that time, GDP/GTP cycling of Ras wasstudied in vitro and a method measuring Rasactivation in vivo was initially lacking. JulianDownward developed a method based on label-ing of cells with radioactive orthophosphatefollowed by immunoprecipitation of Ras pro-teins. GDP and GTP were eluted and separatedby thin layer chromatography. Using this proce-dure, it was first found that T-cell receptor en-gagement was a strong inducer of Ras activity(Downward et al. 1990). We followed by show-ing that insulin could activate Ras, connectingRas to receptor tyrosine kinases (Burgering et al.1991). Interestingly, many other extracellularstimuli were shown to activate Ras, includingstimuli that use G-protein-coupled receptors(van Corven et al. 1993). This showed that Rasactivation was not part of a signaling pathwayinduced by one specific ligand, but contributesto signaling induced by many factors control-ling cellular responses. Subsequent geneticstudies by others revealed several mammaliancounterparts to the yeast CDC25 protein thatmediate Ras activation, such as the Sos proteins,all having a characteristic CDC25 homology do-main as catalytic domain. The various Ras gua-nine nucleotide exchange factors (GEFs) andGTPase activating proteins (GAPs) and theirmode of action have been reviewed extensively(Bos et al. 2007).

Ras EFFECTORS

In the early 1990s, we learned from Chris Mar-shall that extracellular regulated kinase (ERK),also known as microtubule-associated proteinkinase (MAPK), was activated in cells transfect-ed with mutant Ras (Leevers and Marshall1992). At that time, we were developing a systempioneered by Marino Zerial to use vaccinia virusto introduce dominant negative Ras (RasN17)

into cells. Indeed, in cells expressing RasN17insulin-induced ERK activation was completelyblocked: This made ERK the first “proven”downstream biochemical effect of Ras inmammalian cells (de Vries-Smits et al. 1992).However, Ras and ERK did not directly interactand subsequently Raf was found to be the effec-tor of Ras that, through MAPK/ERK kinase(MEK), activates ERK (Moodie et al. 1993).

Soon hereafter, multiple Ras effectorswere identified, including phosphatidylinositol3-kinase (PI3K) (Kodaki et al. 1994) and Ralguanine nucleotide dissociation stimulator(RalGDS) (Hofer et al. 1994). The discoveryby Boudewijn Burgering and Paul Coffer inour laboratory that the product of PI3K,phosphatidylinositol 3,4,5, triphosphate, acti-vates protein kinase B (Burgering and Coffer1995) opened a completely new line of research,resulting in the identification of FoxO tran-scription factors as main targets for proteinkinase B–mediated phosphorylation (Kopset al. 1999).

The importance of multiple effectors forRas function was beautifully shown by MichaelWhite using effector domain mutants that se-lectively bind to the various effectors: whentransfected separately they had hardly anytransforming activity, but in combination theydid (White et al. 1995).

In the years thereafter, many aspects ofthe signaling networks in which Ras plays acentral role and the role of Ras in cancer wereelucidated (Cox et al. 2014; Papke and Der2017). The most updated version of the Rassignaling network can be found at the NationalCancer Institute (www.cancer.gov/research/key-initiatives/ras/ras-central/blog/2017/mccormick-ras-pathway-v3).

HOW TO INTERFERE IN MUTANT RasSIGNALING

Knowledge on the regulation of Ras rapidly ac-cumulated but did not yet result in treatmentoptions for cancer patients. Initially, it wasthought that Ras action could be inhibited bydisplacing the GTP moiety by a small molecule,but this idea was rapidly abandoned when it be-

From Ras to Rap and Back, A Journey of 35 Years

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https://www.cancer.gov/research/key-initiatives/ras/ras-central/blog/2017/mccormick-ras-pathway-v3







came clear that the affinityof GTP (and GDP)forRas is within the picomolar range. Instead, mostattention was focused on interfering in the lipidmodification of Ras, particularly the carboxy-terminal farnesylation and later geranyl-gerany-lation. When it became clear that Ras belongsto a superfamily of small GTPases with over ahundred members almost all similarly modified,the enthusiasm for this approach diminished.These studies have recently been discussed byCox and coworkers (Cox et al. 2014). Nowadaysmost attention is focused on interfering in sig-naling downstream of Ras (see below).

Rap PROTEINS

Rap proteins were first identified by VeroniquePizon and Pierre Chardin and, based on theirsimilarity to Ras, the authors suggested thatthey may interact with the same effectors asRas (Pizon et al. 1988). Subsequently, Nodaand coworkers identified K-ras revertant 1 (K-rev1), whichwas identical to Rap1, in a screen forgenes that can revert morphological transfor-mation of mutant K-ras cells (Kitayama et al.1989). This ledto the hypothesisthatRap1 mightbe a decoy that traps Ras effectors in an inactivecomplex and could be a possible way to interferein mutant Ras tumors? We became interested inRap after our finding that in certain cell typescyclic adenosine monophosphate (cAMP) caninhibit growth-factor-induced (and Ras-medi-ated) activation of ERK (Burgering et al. 1991).Combined with the observation of DanielAltschuler that cAMP could activate Rap1(Altschuler et al. 1995), this suggested a role forRap1 in inhibiting Ras following cAMP stimu-lation of cells. However, despite the fact that invitro Rap1 can bind to the Ras-binding domainof Raf1, we were unable to show a direct involve-ment of Ras effector signaling (Zwartkruis et al.1998).

A function of Rap proteins distinct from Rasis further supported by the notion that Ras andRap were already present as separate proteins inthe hypothetical last eukaryotic common ances-tor (van Dam et al. 2011). Later in evolution atthe opisthokonta stage the Rap branch sepa-rated in Rap1 and Rap2 and at the vertebrata

stage in Rap1A and B and Rap2A, B, and C(Fig. 1). Research on Rap orthologs in manyspecies has provided pivotal knowledge abouthuman Rap proteins (Frische and Zwartkruis2010), but especially the analysis of the singleRap protein in budding yeast, Rsr1, has led to aframework for the regulation and function ofthese small GTPases. Budding always occurs ad-jacent to a scar of a previous bud. This bud siteselection is however abolished in cells mutatedfor Rsr1 (Bud1), for a GEF (Bud5) or for a GAP(Bud2) for Rsr1. It was found that these pro-teins form a complex that recognizes speciallandmark proteins in the rim of the bud scar.The signal is further propagated to the CDC42complex, which facilitates the local recruitmentof the actin cytoskeleton (Bi and Park 2012). Inaddition, in Drosophila, Rap proteins play a rolein cell migration and the proper localization ofadherence junctions (Asha et al. 1999; Knox andBrown 2002). This led to the hypothesis thatalso mammalian Rap proteins are involvedin spatial control of cell adhesion, most likelythrough actin-driven processes.

REGULATION OF Rap

Our initial studies were devoted to find stimulithat can activate Rap1 and we used platelets asthey abundantly express Rap1. To measure Rapactivity, we developed a nonradioactive pull-down assay, in which active GTP-bound Rap1is precipitated with a GST-coupled Ras-bindingdomain (Franke et al. 1997). Such pull-downassays have subsequently been developed forRas and many other GTPases and are now themost widely used assay to measure activity of avariety of small GTPases, including those fromthe Rho family. We found that all stimuli thatresulted in a rapid activation of platelets alsoactivated Rap1. As one of the hallmarks of plate-let activation is increased integrin-mediatedadhesion and spreading, we postulated thatRap1 may control integrin activation (Frankeet al. 1997). Subsequent studies revealed that alarge variety of stimuli are able to activate Rapproteins in all cell types tested.

The observation that, similar to Ras, Rap1 isactivated by many stimuli opened the quest for

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guanine nucleotide exchange factors (GEFs)and GAPs that could regulate this activity. Thefirst Rap-specific GEF, C3G, was identified byMichiyuki Matsuda and coworkers (Gotoh et al.1995). Among others, it mediates signals fromreceptor tyrosine kinases through the adaptorprotein Crk. Currently at least nine Rap-selec-tive GEFs have been identified (Fig. 2), whichhave, similar to Ras GEFs, a CDC25 homologydomain. However, both in vivo and in vitrothere is a strong specificity for Rap, with Cal-DAGGEF3 (RasGrp3) as an exception as itacts both on Ras and Rap. In addition, almostall these proteins have protein–protein orprotein–lipid interaction domains, suggestingmultiple interaction partners. RapGAPs have adifferent structure and a different mechanism of

catalysis compared to RasGAPs; whereas Ras-GAPs provide a critical arginine (argininefinger) for hydrolysis of GTP, RapGAPs providean aspartate (aspartate thumb). Again, despitesimilarities between Ras and Rap, the GAPs arespecific for their cognate GTPase (Bos et al.2007). Exceptions are the GAP1(IP4BP) familymembers a Rap-like GAP, which upon mem-brane binding adopts activity toward Ras (Sotet al. 2013).

EXCHANGE PROTEIN DIRECTLY ACTIVATEDBY cAMP (Epac)

At one time, it was a dogma that all mammaliancAMP effects were mediated by protein kinaseA. However, we noted that inhibition of protein

Rap1ARap1BRap2ARap2BRap2CN-RasH-RasK-RasE-RasM-Ras

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Figure 1. Evolutionary timeline of the Ras family, Ras, Rap, and Ral proteins. The last eukaryotic commonancestor (LECA) likely contained already representatives of these proteins. Later duplications are responsible forthe different members in mammalian cells. (From van Dam et al. 2011; adapted, with permission, from theauthors.)



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kinase A did not affect cAMP-induced Rap1activity. In a subsequent search in publicly avail-able databases of the human genome project, wefound cAMP-binding domains in close vicinityof CDC25 homology domains. Using purifiedprotein encoded by the corresponding comple-mentary DNA (cDNA), we could show a Rap-specific GEF with activity that was completelydependent on the presence of cAMP. Wecalled this protein exchange protein directly ac-tivated by cAMP (Epac) (de Rooij et al. 1998).Two human Epac proteins were identified: onewith a single cAMP-binding domain (Epac1)and one with two cAMP-binding domains(Epac2). Subsequent structural analysis, in col-laboration with Fred Wittinghofer, revealed adetailed mechanism on how Epac is regulated(Fig. 3A). In the absence of cAMP, Epac is in aclosed conformation with the cAMP-bindingdomain occluding the Rap-binding site. WhencAMP binds, Epac adopts an open conforma-tion, where Rap can bind and to become acti-vated (Rehmann et al. 2006, 2008). In addition,our studies for the first time revealed how in-genuously cAMP acts: the cyclic phosphategroup of cAMP releases a brake that allows amajor conformational change that is furtherstabilized by interactions with the adenosinegroup (Rehmann et al. 2003). Simultaneously,

cAMP also induces the translocation of Epac1(but not Epac2) from the cytosol to the plasmamembrane (Ponsioen et al. 2009). This isfacilitated by a cAMP-induced conformationalchange in the Dishevelled, EGL-10, and pleck-strin (DEP) domain (Li et al. 2011), resultingin an increased affinity for membrane-boundphosphatidic acid (Consonni et al. 2012).Several additional membrane anchors havebeen identified for Epac1, including activatedEzrin, showing that, dependent on the condi-tions, Epac1 can localize differently (Gloerichet al. 2010).

DEVELOPMENT OF 007

Further studies on the function of Epac1 werehampered by the fact that cAMP activates bothprotein kinase A (PKA) and Epac. However,Stein Døskeland pointed out to us a peculiarityin the cAMP-binding domain, where a highlyconserved glutamate that forms hydrogenbounds with the 20-hydroxyl of the ribose ofcAMP was absent in the cAMP-binding do-main of Epac1 and the high-affinity, secondbinding site of Epac2. This suggested that thesehydrogen bonds, and thus the 20OH of the ri-bose may be required specifically for cAMP tointeract with PKA, but not with Epac. Together

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Figure 2. Rap guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Indicated arethe domain structures of Rap-specific GEFs and GAPs. (From Bos et al. 2007; adapted, with permission, fromthe authors.)

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with Frank Schwede and Hans-GottfriedGenieser, we started to modify cAMP, resultingin a highly Epac-selective compound 8-CPT-20OMe-cAMP (Fig. 3B), which we called 007(Enserink et al. 2002). Using this compound,we demonstrated that cAMP through Epac isable to modify integrin-mediated cell-substra-tum adhesion and E-cadherin-mediated cell–cell adhesion (Rangarajan et al. 2003; Priceet al. 2004). Subsequently, many studies havebeen published using this compound therebyshowing the versatile role Epac proteins play incAMP signaling, including insulin secretion,endothelial barrier function, memory, andheart rhythm (for further details, see Gloerichand Bos 2010).

DOWNSTREAM TARGETS OF Rap1PROTEINS

007 did not induce activation of ERK, indicatingthat B-raf is not a downstream target of Rap1(Enserink et al. 2002). In this respect, a claimthat B-raf is a downstream target of Rap1 isincorrect. The first genuine effector for a Rapprotein identified was the Drosophila Canoeprotein (Boettner et al. 2003). This protein issimilar to the mammalian junctional proteinAF6 with ubiquitin-like Ras association (RA)domains to bind active Rap1. Subsequently,AF6 was found to be one of the effectors ofRap1 that mediate some, but not all, responsesof Rap1 activation. Indeed, of the many RA-

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Figure 3. (A) Crystal structure of Epac 2 in the closed (left) and open (right) conformation. The gray area (right)represents the first cyclic adenosine monophosphate (cAMP) domain and Dishevelled, EGL-10, and pleckstrin(DEP) domain visualized by cryoelectron microscopy (Rehmann et al. 2006, 2008). (B) Structure of 8CPT-20OMe-cAMP (007).



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domain-containing proteins multiple have beenimplicated to transduce Rap1 signaling. Theseeffectors, together with the existence of multipleRap1-GEFs and GAPs, prompted us to extendthe hypothesis based on the yeast studies. Weproposed that a cell can have multiple indepen-dent Rap modules consisting of a Rap protein, aGEF, and probably a GAP, each recognizingdifferent landmarks (spatial cues) to regulatelocalized recruitment of the actin cytoskeletonin time and in space using a variety of effectorsystems. The presence of additional regulatoryand interaction domains, particularly in thevarious GEFs, provide ample opportunities forspecific landmark recognition and regulation.The consequence of this hypothesis was thatwe had to search for a specific Rap signalingmodule in a defined model system. Althoughwe explored Rap1-induced integrin-mediatedcell adhesion and Rap1-induced cell spreadingin epithelial cells, we particularly focused ourattention on endothelial barrier function. En-dothelial cells are highly responsive to 007 asmeasured by, for instance, an increased electri-cal resistance of the monolayer. This increasedbarrier function is accompanied by a shift that isobserved from radial stress fibers to circumfer-ential actin, which could explain the increasedtightness of endothelial cell–cell junctions.

Using small-interfering RNA (siRNA) screens,we found that Rap1 interacted with a complexof two related RA-domain-containing proteins,Rasip1 and Radil. Interestingly, both Radiland Rasip1 are homologous to Canoe, the firsteffector found for Rap1 in Drosophila. Impor-tantly, Radil directly interacts with ArhGAP29,a Rho-specific GAP, and we could show thatArhGAP29 mediates 007-induced decrease inradial stress fibers and increased barrier func-tion (Fig. 4, left) (Post et al. 2013, 2015). In-triguingly, multiple Rap1 modules may controlbarrier function simultaneously. For instance,Mochizuki and coworkers discovered that cir-cumferential actin formation is mediated by theCDC42-GEF FGD5 (Ando et al. 2013). HowFGD5 is activated is still unclear, but, in over-expression studies, we observe that FGD5 alsoforms a complex with Radil, suggesting that theRadil/Rasip complex regulates both pathways.Also, the Rap1 effector AF6 is involved in thisprocess (Birukova et al. 2013), but how thesedifferent effectors cooperate is still unclear.Surprisingly, Krit1, previously identified as aRap1 effector in a similar assay in Huvec cells(Glading et al. 2007), did not affect barrier func-tion in our experiments (Pannekoek et al. 2011).

Several other effectors for Rap1 have beenidentified, such as the RA-domain-containing

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Figure 4. Graphical summary of (A) Epac1-mediated regulation of radial stress fibers through the Radil-Rasip1-ArgGAP29 complex, and of (B) Rap2A-mediated intestinal brush borders formation (for explanation see text).

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protein Riam, which plays an important role inintegrin-mediated cell adhesion (Lafuente et al.2004).

Rap2

The early deviation of Rap1 and Rap2 in evolu-tion suggests that they may have different func-tions and indeed, although Rap2 is regulated bythe same GEFs and GAPs as Rap1, it is clearlydifferent as Rap2(-GTP) binds to a differentset of effector proteins lacking an RA domain:the Traf2 and Nck-interacting kinases (TNIKs)(Taira et al. 2004). We stumbled on Rap2A inthe analysis of brush-border formation in W4intestinal epithelial cells. In these cells, brush-border formation is induced by doxycycline-in-duced activation of the LKB–Strd1a complex(Baas et al. 2004). When we tested the involve-ment of Rap proteins in this process, we foundthat siRNA mediated depletion of Rap2A, butthe other Rap proteins abolished this process.Subsequent studies revealed a pathway, whichis induced by PIP2 enriched apical membraneformation. PIP2 serves as anchor for phospho-lipase D, which produces phosphatidic acid.Phosphatidic acid is then the anchor for theRapGEF PDZ-GEF. As a consequence, PDZ-GEF is recruited to the apical membrane,activates Rap2A, which in turn recruits andactivates the serine-threonine kinase TNIK.TNIK activates the serine-threonine kinaseMst4, which induces the phosphorylation andactivation of Ezrin. This activation of Ezrin is acritical step as active Ezrin can rescue Rap2depletion. Ezrin recruits the actin cytoskeletonfor brush-border formation (Fig. 4, right)(Gloerich et al. 2012). The similarity with theyeast model is evident: A landmark is recog-nized in two steps by a GEF for Rap followedby effector binding, which in several steps leadsto the recruitment of actin.

ARE WE FINISHED WITH Ras?

Although our analysis and those of many otherresearch groups have provided much insightinto the role Rap proteins play in cellular re-sponses, the initial idea that Rap1 counteracts

mutant Ras signaling and could be used asa route to interfere in mutant Ras signalingin cancers turned out to be too naı̈ve. However,despite huge efforts by many groups and manypharmaceutical industries, the alternative ap-proach to interfere in the Ras pathway by selec-tive inhibitors has not worked either as we arestill unable to treat Ras-mutant cancers withtargeted drugs (Stephen et al. 2014). Ras itselfis still largely “undruggable,” and the alternativeto inhibit downstream targets of mutant Ras,most notably Raf, MEK, ERK, and PI3K, havethus far failed in the clinic, especially whensingle drugs are used. For such an approach tobe successful, it is essential that mutant Ras-containing tumors cells do have a specificsensitivity for these pathway drugs. Indeed, ge-netic and drug screens in mutant cell lines dididentify several possible targets for mutant Rastumor cell lines, but these have not been trans-lated into clinical targets (Downward 2015).Perhaps the commonly used mutant Ras-con-taining cell lines identify sensitivities that do notoccur in tumors. We therefore shifted to thetumor organoid model system developed byHans Clevers (Sato et al. 2009; van de Weteringet al. 2015). The advantage of this model systemis that tumor organoids can be rapidly estab-lished from almost all individual (epithelial)tumors and thus can capture the genetic diver-sity of these tumors. Moreover, drug responsescan be compared with organoids from normaltissue to determine the therapeutic window.Initial studies using a number of colon tumororganoids already indicated the power of thistechnology (van de Wetering et al. 2015). As afirst proof-of-concept, we tested various target-ed drugs that are currently being used in clinicaltrials for inhibiting mutant Ras-containingcolorectal cancer, most notably pan-EGFR in-hibitors in combination with MEK inhibitors(Sun et al. 2014). We found that these combi-nations gave a clear cell-cycle arrest of mutantK-ras-containing organoids, supporting theobservation that this combination has an effecton mutant Ras tumor cells. However, organoidswith nonmutated K-Ras remain far more sensi-tive for this drug combination. Furthermore, wenoted that organoids from normal tissue were



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similarly sensitive for this combination thantumor organoids with nonmutated K-ras, andthus much more sensitive than mutant K-rasorganoids, indicating that for this combinationthe therapeutic window is limited (Verissimoet al. 2016).

It is now more than 35 years ago that Raswas identified as an oncogene in human cancersand we still have not solved the problem ofhow to target mutant Ras in cancer. As a con-sequence, patients carrying mutant Ras in theirtumors are excluded from therapies targetingthe EGFR signaling cascade. Moreover, whenpatients carrying normal Ras in their tumorsare treated, the appearance of mutant Ras is afrequent resistance mechanism. To make per-sonalized medicine a success, we have to comeup with a solution. New model systems liketumor organoids may be helpful in the identi-fication of new drug combinations, but also infinding those patients that may uniquely re-spond to such combinations.

ACKNOWLEDGMENTS

I thank all members of my laboratory in pastyears for the tremendous contributions theyhave made and are still making to understandRas-like small GTPases and hopefully to find adrug combination that works for mutant Ras-containing tumors. I also thank my colleaguesFried Zwartkruis, Willem Jan Pannekoek, andBoudewijn Burgering for critically reading thismanuscript.

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August 4, 20172018; doi: 10.1101/cshperspect.a031468 originally published onlineCold Spring Harb Perspect Med

Johannes L. Bos From Ras to Rap and Back, a Journey of 35 Years

Subject Collection Ras and Cancer in the 21st Century

Targeting Ras with MacromoleculesDehua Pei, Kuangyu Chen and Hui Liao RAS Family

MRAS: A Close but Understudied Member of the

Lucy C. Young and Pablo Rodriguez-Viciana

Structures, Mechanisms, and Interactions−−Ras-Specific GTPase-Activating Proteins

Klaus Scheffzek and Giridhar ShivalingaiahSon-of-Sevenless and RasThe Interdependent Activation of

KuriyanPradeep Bandaru, Yasushi Kondo and John

KinasesRas-Mediated Activation of the Raf Family

Elizabeth M. Terrell and Deborah K. MorrisonCancersTargeting the MAPK Pathway in RAS Mutant

Sarah G. Hymowitz and Shiva MalekPosttranslational Modifications of RAS Proteins

Ian Ahearn, Mo Zhou and Mark R. Philips RelationshipRas and the Plasma Membrane: A Complicated

Gorfe, et al.Yong Zhou, Priyanka Prakash, Alemayehu A.

Kras in OrganoidsDerek Cheng and David Tuveson

Kras and Tumor Immunity: Friend or Foe?Jane Cullis, Shipra Das and Dafna Bar-Sagi

for Pancreatic CancerKRAS: The Critical Driver and Therapeutic Target

Andrew M. Waters and Channing J. DerCancers

-MutantKRASSynthetic Lethal Vulnerabilities in

Andrew J. Aguirre and William C. Hahn

Targeting Oncogenic MutantsConformational Preferences and Implications for The K-Ras, N-Ras, and H-Ras Isoforms: Unique

Jillian A. Parker and Carla Mattos

Efforts to Develop KRAS InhibitorsMatthew Holderfield

PI3K: A Crucial Piece in the RAS Signaling PuzzleAgata Adelajda Krygowska and Esther Castellano

Validation of Therapeutic TargetsK-Ras-Driven Lung and Pancreatic Tumors: Genetically Engineered Mouse Models of

BarbacidMatthias Drosten, Carmen Guerra and Mariano

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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From Ras to Rap and Back, a Journey of 35 Years

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