RalB directly triggers invasion downstream Ras by mobilizing the Wave complex
One of the most frequent oncogenic events in human cancers is the activation by constitutive mutations of Ras GTPases: K-Ras , H-Ras , N-Ras . Roughly 30% of all human tumors carry Ras genetic alterations; the Ras mutation frequency is particularly high for pancreas ( 91% ) , lung ( 33% ) and colon ( 51% ) cancers ( cbioportal ) ( Gao et al . , 2013; Simanshu et al . , 2017 ) . Up-to-date no effective targeted therapies can be offered to patients with tumors carrying Ras mutations . Since Ras proteins are still considered undruggable ( Cox et al . , 2014 ) , the focus for anti-Ras drug discovery moved downstream . Ras activates three major downstream pathways: the MAP kinases , the PI3 kinases and the Ral GTPases . Despite the considerable translational research efforts on the downstream kinases , the results are very deceiving . For example , MAPK pathway inhibition with MEK inhibitor therapy turned out to be largely ineffective ( Jänne et al . , 2013 ) . On the other hand , the targeting of Ral GTPases has been a much less exploited strategy ( Yan et al . , 2014; Yan and Theodorescu , 2018 ) . The two human Ral proteins ( RalA and RalB ) are activated by six RalGEFs ( Guanine Nucleotide Exchange Factors ) ( Neel et al . , 2011 ) . Among the six , four RalGEFs ( RalGDS , Rgl1 , Rgl2 , Rgl3 ) have a Ras-association ( RA ) domain and are direct effectors of Ras . Activated GTP-bound Ras recruits these RalGEFs at the plasma-membrane triggering the activation of Ral by GDP to GTP exchange . The two remaining RALGEFs ( RalGPS1 and RalGPS2 ) do not bind Ras; their specific activators are still unknown , but they do contain a pleckstrin homology ( PH ) domain ( Rebhun et al . , 2000 ) , responsible for membrane targeting , and a proline-rich motif which binds to SH3-containing signaling proteins ( Ceriani et al . , 2007 ) . Even though , in some cellular contexts , RalA and RalB seem to have overlapping effects , several evidences pointed out a distinct role for RalB in supporting the invasiveness of transformed cells . In the case of migration of the UMUC3 human bladder cancer cell line ( K-Ras mutated ) , RalA seems to even antagonize the pro-migratory function of RalB ( Oxford et al . , 2005 ) . The requirement of RalB for invasion in vitro ( by Transwell Invasion assay ) was shown by shRNA knock-down approach in seven out of nine K-Ras mutated human pancreatic cancer cell lines ( Lim et al . , 2006 ) . Consistently , another study showed that RalB , but not RalA , plays a role in invadopodia formation in human pancreatic cancer cell lines ( Neel et al . , 2012 ) . RalB , but not RalA , is required for the contractility-driven invasion of lung cancer cells ( A549 , K-Ras mutated ) ( Biondini et al . , 2015 ) . Moreover , in vivo metastasis assays in mice ( tail vein injection ) ( Ward et al . , 2001; Lim et al . , 2006 ) and in hamsters ( subcutaneous injection ) ( Rybko et al . , 2011 ) supported a function of RalB pathway in the formation of tumor metastasis , both in Ras-mutated and Rous sarcoma virus-transformed cells . Besides the cancer context , we previously found that RalB , but not RalA , controls the mesenchymal migration of normal cells ( NRK , HEK-HT , wild-type for Ras ) by mobilizing its effector exocyst ( Rossé et al . , 2006; Parrini et al . , 2011; Biondini et al . , 2016 ) , which is an octameric protein complex involved in the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion ( Wu and Guo , 2015 ) . All these works allowed to recognize the relevance of RalB pathway for motility , invasion and metastasis , but the underlying molecular mechanisms remain elusive ( Neel et al . , 2012 ) . We reasoned that the emerging optogenetics technology ( Toettcher et al . , 2013; Tischer and Weiner , 2014 ) might help to overcome this limitation , because of its capacity to dissect the cause-effect relationships linking the activity of a specific protein of interest and the consequent cell behaviors , in time and space . To date , various light-gated protein-interaction modules have been introduced to perturb intracellular protein activities . The one we exploited is based on the interaction between two proteins from Arabidopsis Thaliana: CIB1 and cryptochrome 2 ( CRY2 ) ( Kennedy et al . , 2010 ) . Blue-light illumination induces the heterodimerization of CRY2 with the N-terminus of CIB1 ( CIBN ) . This reaction is reversible and rapid , with response times in the order of seconds ( few seconds for dimerization and ~5 min for dissociation after cessation of blue illumination ) , and does not require exogenous cofactors . In this work , we applied the CRY2-CIBN light dimerization system to selectively activate Ral cascade and to study the primordial phenotypic effect of this activation . By using this novel optogenetics approach , we precisely established the molecular mechanism underlying the capacity of RalB to drive invasion . This mechanism involves the exocyst-dependent recruitment at the leading edge of the Wave Regulatory Complex ( WRC ) , a five-subunit protein complex involved in the formation of the actin cytoskeleton through interaction with the Arp2/3 complex ( Alekhina et al . , 2017; Chen et al . , 2014 ) , but unexpectedly independently of the small GTPase Rac1 , a well-established WRC activator and master regulator of protrusions ( Ridley et al . , 1992; Ridley , 2006 ) . We also found that RalB pathway contribution might be much more relevant than MAPK and PI3K contributions to drive Ras-dependent invasion , as ascertained by using a genetically controlled cell model: the isogenic pair HEK-HT and HEK-HT-H-RasV12 ( Hahn et al . , 1999; O'Hayer and Counter , 2006 ) . Light-induced Ral activation was instructive in promoting cell invasion of the non-transformed HEK-HT cells . Finally , we analyzed Ral proteins’ expression in a cohort of breast cancer samples , pointing out for the first time a potential role of RalB in the invasiveness and metastatic spread of human breast cancers .
We exploited the CRY2/CIBN light-gated dimerization system ( Kennedy et al . , 2010 ) to induce activation of endogenous RalA and RalB proteins with a spatial and temporal control . We chose to activate Ral at the plasma-membrane because Ral oncogenic signaling emanates at least in part from the plasma-membrane ( Ward et al . , 2001; Hamad et al . , 2002; Lim et al . , 2005 ) . To do so , the GFP-fused CIBN protein was constitutively targeted to the plasma membrane via a K-Ras CAAX motif . The minimal GEF domain of RGL2 ( 1–518 aa ) , which is catalytically active on both RalA and RalB ( Ferro et al . , 2008 ) , was fused to CRY2-mCherry ( RalGEF-CRY2-mCherry ) . We stably expressed these two constructs in HEK-HT cells , which are immortalized but not transformed ( Hahn et al . , 1999; O'Hayer and Counter , 2006 ) , to generate the ‘OptoRal’ cell line ( CIBN-CAAX/RalGEF-CRY2 ) . As control , we generated the ‘OptoControl’ cell line which expresses CRY2-mCherry only , without the RalGEF domain ( Figure 1—figure supplement 1 ) . Upon blue light illumination ( 100 ms pulses every 15 s ) , RalGEF-CRY2-mCherry reversibly translocated to the plasma membrane following its binding to GFP-CIBN-CAAX ( Figure 1A ) , as shown by TIRF microscopy ( Figure 1B and Video 1 ) . Fluorescence quantifications inside the illuminated area showed that RalGEF-CRY2 recruitment starts in less than 15 s , as expected ( Valon et al . , 2015 ) , reaching a threefold increase in few minutes ( Figure 1C ) . In order to assess whether the recruitment of RalGEF-CRY2 was converted in an efficient Ral activation , we used a Ral activity reporter: the iRFP-tagged Ral GTPase Binding Domain of Sec5 ( Sec5GBD ) , which specifically interacts with both RalA and RalB in their GTP loaded forms ( Takaya et al . , 2004 ) . Since the Sec5GBD-iRFP reporter proteins were in large excess with respect to the endogenous Ral proteins , we overexpressed GFP-RalB in OptoRal cells in order to increase the RalB:Sec5GBD stoichiometric ratio and to achieve the recruitment of a major , detectable fraction of Sec5GBD molecules . Upon blue light illumination , Sec5GBD-iRFP fluorescence at plasma membrane increased 1 . 4 fold ( Figure 1D , E and Video 2 ) , indicating an efficient activation of Ral proteins , presumably a combination of exogenous GFP-RalB , and endogenous RalA and RalB . The light-induced RalB activation was confirmed by pull-down assay ( Figure 2—figure supplement 1A ) and by a FRET-based RalB activity biosensor ( Figure 2—figure supplement 1B ) . The activation was selective for Ral without cross activation of Rac1 or Cdc42 , as shown by over-expressing GFP-Rac1 or GFP-Cdc42 together with a Rac1/Cdc42 activity reporter: the iRFP-tagged GTPase Binding Domain of Pak1 ( Pak1GBD ) ( Valon et al . , 2015 ) , which specifically interacts with Rac1-GTP and Cdc42-GTP ( Sells et al . , 1997; Huang et al . , 2013 ) . Upon blue light illumination , Pak1GBD-iRFP fluorescence at plasma membrane did not increase , on the contrary slightly decreased ( Figure 1E ) , indicating the absence of Rac1 or Cdc42 activation upon recruitment of RalGEF at the plasma membrane . In conclusion , we developed an efficient and selective optogenetic OptoRal system to specifically trigger local activation of Ral proteins . Since the optogenetic perturbations act in a timescale ( tens of second ) much faster than the ones of endogenous feedbacks ( minutes ) ( Valon et al . , 2015 ) , this new approach allowed us to investigate the direct consequences of Ral activation , at both phenotypic and molecular levels . A conspicuous phenotypic consequence of local Ral activation was an increase in cell edge dynamics , leading to protrusion formation ( Video 3 ) . By using an automated method ( Paul-Gilloteaux et al . , 2018 ) , we tracked over time the cell contour inside the illuminated area and we generated edge velocity maps ( Figure 2A ) . The velocity maps of OptoRal cells showed an evident stimulation of cell edge dynamics after light stimulation ( Figure 2B ) . This stimulation corresponded , for the majority of the sampled space and time points , to an increase of positive velocity ( i . e . protrusions ) and not of negative velocities ( i . e . retractions ) . In contrast , no changes of cell edge dynamics were observed upon light stimulation of OptoControl cells ( Figure 2C and Video 4 ) , excluding non-specific effects due to illumination or to CRY2 recruitment . Moreover , the alternations of dark and light periods revealed that the velocity dynamics were reversible and exquisitely controllable by the illumination in OptoRal cells ( Figure 2—figure supplement 2A ) but not in the OptoControl cells ( Figure 2—figure supplement 2B ) . The quantifications of edge velocities from several OptoRal cells before and after illumination showed a highly significant increase of membrane protrusion events , but not of retraction events; this increase was not found in OptoControl cells ( Figure 2D ) . To address a possible role of Rac in this phenotype we treated the OptoRal cells with the RacGEF inhibitor NSC23766 . Treatment with NSC23766 ( 100 µM , 1 hr ) substantially reduced ( ~60% ) Rac1-GTP level ( Figure 2—figure supplement 2C ) , but it did not impair a fully efficient protrusion induction by illumination in OptoRal cells ( Figure 2D ) , suggesting that Rac1 have a marginal role in stimulation of edge velocities when Ral proteins are activated . Importantly , blue illumination of OptoRal cells does not activate at all Rac1 , as shown by the Pak1GBD fluorescent reporter ( Figure 1E ) . These results together support the conclusion that Ral triggers protrusion independently of Rac1 . Our OptoRal system is designed to activate both endogenous RalA and RalB . However , RalB-depleted cells , but not RalA depleted cells , were impaired in protrusions formation , proving that light-controlled stimulation of cell protrusions is essentially due to activation of endogenous RalB , rather than RalA ( Figure 2E and Figure 2—figure supplement 2D for depletion validation ) , consistently with the well-established specific role of RalB in the regulation of motility and invasion ( see Introduction ) . Next , we addressed the question of the molecular mechanisms underlying the capacity of RalB to trigger protrusions independently of Rac1 . A previous work in our lab ( Biondini et al . , 2016 ) established the existence of an interaction between the Wave Regulatory Complex ( WRC ) , a crucial regulator of actin polymerization and protrusion formations ( Alekhina et al . , 2017; Chen et al . , 2014 ) , and the exocyst complex ( Wu and Guo , 2015 ) , a major direct effector of Ral , but the functional consequences of this interaction are still unclear . We reasoned that Ral activation might promote the trafficking and recruitment of the exocyst/WRC complex assembly at the leading edge ( Figure 3A ) . To test this hypothesis , we generated OptoRal and OptoControl cell lines stably expressing the iRFP-fused Abi1 subunit of WRC complex . The correct incorporation of iRFP-Abi1 into the WRC complex was verified by size exclusion chromatography: the exogenous iRFP-Abi1 , as well as the endogenous Abi1 , co-eluted with the Cyfip subunit in fractions of approximately 400 kDa corresponding to the size of the whole WRC complex ( Gautreau et al . , 2004 ) ( Figure 3—figure supplement 1A , B ) . We locally activated Ral at cell periphery and followed iRFP-Abi1 recruitment . For convenience of imaging , we limited the inter-cellular morphology variability by subjecting the OptoRal cells to wound healing , leading to the formation of large front lamellipodia where Ral was activated by light . TIRF images showed that Ral activation induced an immediate increase of iRFP-Abi1 signal at the plasma membrane , both at the leading edge and at the ventral side ( Figure 3B snapshots and Video 5 ) . In contrast , OptoControl cells did not show any light-dependent iRFP-Abi1 recruitment ( Figure 3C snapshots and Video 6 ) . WRC edge recruitment was quantified by using an automated method that measures the maximum fluorescent intensity at the edge ( width of 1 . 12 µm ) ( Paul-Gilloteaux et al . , 2018 ) : the resulting heat maps show a dynamic edge fluorescence appearing upon illumination in OptoRal cells ( Figure 3D , left ) but not in OptoControl cells ( Figure 3D , right ) . We could not apply this method of quantification at the edge on all the imaged cells , because the leading edge was often lifted above the ventral plan captured by TIRF and because the cycling dynamics ( protrusion-retraction alternation and on-off Abi1 recruitment ) were very fast . Instead , fluorescence quantifications from several OptoRal and OptoControl cells could be easily done at ventral location inside the illuminated area: iRFP-Abi1 was recruited in OptoRal cells but not in OptoControl cells , with a robust statistical significance ( Figure 3E ) . To demonstrate that WRC translocation is a direct consequence of its association with the exocyst , we exploited a previously characterized loss-of-interaction Abi1 mutant , Abi1Q56A , which is specifically impaired in its binding to the Exo70 subunit of exocyst ( Biondini et al . , 2016 ) . We generated an OptoRal cell line expressing the iRFP-Abi1Q56A mutant and we verified its correct incorporation into WRC by size exclusion chromatography ( Figure 3—figure supplement 1C ) . The analysis of several cells showed that upon Ral activation the recruitment at the ventral plasma membrane of the AbiQ56A mutant was significantly and substantially reduced with respect to wild-type Abi1 ( Figure 3E ) , indicating that the WRC/exocyst association is required for efficient translocation of WRC complex . Moreover , while illumination stimulated edge velocity of OptoRal cells expressing Abi1 wild-type , it was not effective in stimulating edge velocity of OptoRal cells expressing the Abi1Q56A mutant ( Figure 3F ) , indicating that reduction of WRC recruitment correlated with a decrease in protrusion dynamics . In order to functionally assess the impact on invasion of light-controlled Ral activation , we performed a Transwell invasion assay coupled with optogenetic illumination . For this purpose , we designed and built a customized 12-well array LED to deliver optical stimuli to the cells inside the Transwells ( Figure 4—figure supplement 1 ) . Blue light was administrated continuously from the bottom of the Transwell plate for the whole duration of the invasion assay ( 6 hr ) . Using this device , we compared the invasive capabilities of OptoControl and OptoRal cells , as well of OptoControl iRFP-Abi1 wild-type , OptoRal iRFP-Abi1 wild-type , and OptoRal iRFP-Abi1Q56A , in both dark and light stimulation conditions . Illumination of OptoRal cells , but not of OptoControl cells , was sufficient to induce a substantial increase of invasive capabilities ( Figure 4A ) . Similar results were obtained upon expression of the wild-type Abi1 subunit of WRC complex , whereas OptoRal cells expressing Abi1Q56A ( the allele specifically impaired in its binding to exocyst [Biondini et al . , 2016] ) failed to invade upon light stimulation ( Figure 4B ) . These results strongly support the conclusion that activation of Ral drives cell invasion by recruiting WRC via the exocyst . To evaluate the contribution of Ral activation downstream oncogenic Ras in cell invasion , we took advantage of the fact that HEK-HT cells , non-transformed , develop a tumorigenic , invasive and metastatic phenotype in vivo upon expression of oncogenic H-RasV12 ( Hahn et al . , 1999; O'Hayer and Counter , 2006 ) . We compared this isogenic pair , HEK-HT and HEK-HTRasV12 , by using two different in vitro invasion assays: the widely used ‘Transwell Invasion assay’ ( invasion through a thin matrigel layer ) and the ‘Inverted Invasion assay’ with a more 3D setting ( invasion through a thick collagen gel ) . In both assays , we found as expected that HEK-HT cells did not invade at all , while HEK-HT-RasV12 displayed a strong invasive capacity ( Figure 5A , B ) . Interestingly , the HEK-HT-RasV12 cells invaded as multi-cellular clusters in the 3D collagen gel ( Figure 5B , photos below ) . In HEK-HTRasV12 cells , the silencing of RalB impaired Transwell invasion of approximatively 60% , the silencing of RalA had not effect , and the silencing of both RalB and RalA reached up to 90% invasion inhibition ( Figure 5C ) ( see Figure 5—figure supplement 1A for depletion efficiencies ) , indicating that Ras-dependent RalB activation substantially contributes to the invasive phenotype of Ras-mutated cells , and that RalA is dispensable , but it might partially compensate when RalB is absent . The specific role of RalB , with respect to RalA , for cell motility and invasion is in perfect agreement with several previous works ( Oxford et al . , 2005; Rossé et al . , 2006; Lim et al . , 2006; Rybko et al . , 2011 ) . The RalB requirement for in vitro invasion of HEK-HTRasV12 cells was confirmed using the Inverted Invasion assay with collagen ( Figure 5D ) ( see Figure 5—figure supplement 1B for depletion efficiency up to 5 days post-siRNA transfection ) . The double mutant H-RasV12G37 , which is completely impaired in stimulating MAP kinase activity but maintains Ral activation ( White et al . , 1995; Bettoun et al . , 2016 ) ( Figure 5—figure supplement 1C ) , showed the same invasive capabilities by Transwell invasion assay as compared with H-RasV12 ( Figure 5E , center ) , indicating that Ras-dependent MAPK hyper-activation is dispensable to drive efficient invasion , in this genetically controlled cell model . Since HEK-HT-RasV12G37 cells retained a residual Ras-dependent activation of PI3K pathway activation , as assessed by AKT phosphorylation ( Lim et al . , 2005; Bettoun et al . , 2016 ) , we used a PI3K inhibitor ( PIK90 ) , which targets all PI3K isoforms , to completely inhibit PI3K pathway ( Figure 5—figure supplement 1D ) . PIK90-treated HEK-HT-RasV12G37 cells are still capable to invade as efficiently as untreated HEK-HT-RasV12G37 and HEK-HT-RasV12 cells ( Figure 5E , right ) , indicating that also Ras-dependent PI3K hyper-activation is dispensable to drive efficient Transwell invasion . These unexpected results were confirmed with HEK-HT-RasV12 cells by a purely pharmacological approach using the MEK inhibitor trametinib and the PI3K inhibitor PIK90 , alone or in combination . Despite a nearly complete inhibition of Erk or Akt phosphorylation , trametinib-treated or PIK90-treated HEK-HT-RasV12 cells were still able to invade to the same extent as control vehicle-treated cells; only the combined MAPK and PI3K blockage displayed an inhibitory tendency , although not statistically significant ( Figure 5F ) . Taken together these results showed that the Ras-RalGEF-RalB signaling axis is necessary to promote invasion in Ras-transformed cells , while MAPK and PI3K pathways appear to be dispensable , at least in our experimental context . Next , we aimed at identifying the missing molecular link between Ras and RalB , that is the specific RalGEFs implicated in Ras-dependent cell invasion . We thus silenced each of the six RalGEFs by two independent siRNA in HEK-HT-RasV12 cells . The depletion of RGL1 and RGL2 substantially reduced invasion . RLGL3 , RalGDS and RalGPS1 were clearly not required for invasion , while RalGPS2 silencing showed a potential inhibitory effect , even though statistically not significant ( Figure 6A ) . RGL1 and RGL2 have a Ras-association domain ( RA ) . We therefore concluded that RGL1 and RGL2 are the molecular actors that promote invasion down-stream oncogenic Ras by activating RalB . By immunofluorescence staining , we found that endogenous RGL2 ( Figure 6B ) and RalB ( Figure 6C ) were more recruited at cell edges in HEK-HTRasV12 cells with respect to normal HEK-HT cells , consistent with the model in which plasma-membrane Ras-GTP binds and recruits RGL2 , which in turn binds and activates its substrate RalB , promoting invasiveness via exocyst-mediated WRC recruitment ( Figure 6D ) . Noteworthy , both RGL2 and RalB localize also at endomembranes , where local signaling might drive additional outputs . While Ral proteins have been involved in several cancers frequently carrying Ras mutations , such as pancreas , lung , colon , bladder , melanoma ( Yan and Theodorescu , 2018 ) , their roles have not been explored in breast cancers , in which the Ras mutation frequency is rather low ( 2 . 3% ) ( cbioportal ) ( Gao et al . , 2013 ) . We investigated by immunohistocytochemistry ( IHC ) the protein levels of RalA and RalB in 502 invasive ductal carcinoma representative of the four main molecular subtypes ( luminal A , luminal B , HER2+ , triple-negative ) ( see Figure 7—figure supplement 1A for intensity scores ) . In invasive ductal carcinoma , RalB protein expression was slightly higher in luminal A and B than in HER2 +and triple-negative ( TN ) tumors ( Figure 7—figure supplement 1B ) , while RalA protein expression was slightly lower in TN tumors as compared to the other subtypes ( Figure 7—figure supplement 1C ) . More interestingly , both RalB and RalA expression was higher in tumor cells than in normal juxtatumoral cells ( Figure 7A , B ) . We also relatively compared the Ral staining at in situ and invasive compartments of invasive ductal carcinoma , and at lymph node metastasis . Strikingly , RalB expression significantly increased in a manner consistent with disease progression: the median H-score was = 0 . 5 in normal juxtatumoral cells , =1 in tumor cells of in situ compartment , =1 . 5 in tumor cells of invasive compartment , and = 2 in tumor cells at lymph node metastasis ( Figure 7A , C ) . On the contrary , RalA expression did not change in tumor cells regardless of the in situ , invasive or metastatic localization ( Figure 7B ) . With the limitation that RalB amount does not necessarily reflect RalB activity , these results suggest that RalB might have a role in human breast cancer invasion and metastasis . Consistent with this hypothesis , RalB silencing was able to impair invasion ( by Transwell invasion assay ) of two triple-negative breast cancer cell lines: MDA-MB-231 ( carrying the KRasG13D mutation ) and BT549 ( Ras wild-type ) ( Figure 7D and Figure 7—figure supplement 1D ) .
Collectively our findings established the utter relevance of the activation of RalB to promote invasion . By exploiting optogenetics to study causality , we showed that not only RalB was permissive for protrusion formation and invasion , but it was instructive in absence of any oncogenic mutation . Moreover , we provide a detailed molecular model of how the Ras-RalB signaling axis governs cell invasion ( Figure 6D ) : active Ras binds and recruits the two RalGEFs RGL1 and RGL2 , which activate RalB; activated RalB binds to the exocyst complex , promoting its assembly ( Moskalenko et al . , 2003 ) and recruitment to the leading edge ( Rossé et al . , 2006 ) ; by its direct association with the WRC complex ( Biondini et al . , 2016 ) , exocyst drives WRC to the leading edge , where WRC stimulates actin polymerization , protrusion formation , motility and invasion . Unexpectedly , using the model of isogenic cell lines HEK-HT and HEK-HT-H-RasV12 , we found that hyper-activations of MAP kinase and PI3 kinase pathways were not required for Ras-driven invasion . A huge previous literature had undoubtedly shown that MAPK and PI3K are involved in migration and invasion of various normal and cancer cell models ( Keely et al . , 1997; Klemke et al . , 1997; Ward et al . , 2001; Janda et al . , 2002; Campbell et al . , 2007 ) . However we speculate that this could not be the case in specific , relevant cellular contexts , such as the HEK-HT-H-RasV12 cells in which the RalB pathway appears to be the dominant driver of invasion down-stream oncogenic Ras . In support of this hypothesis , in a panel of pancreatic cell lines , Ral pathway was found to be more commonly activated as compared to MAPK and PI3K , since abnormally high Ral-GTP levels were much more frequent than abnormally high levels of phosphorylated ERK1/2 or phosphorylated AKT ( Lim et al . , 2005 ) . In human cancer contexts , the contribution of Ral pathway activation is likely still under-estimated . Even though genetic alterations in Ral genes are rare events in human cancers , occurring in only 1 . 5% to 2% of patient cases considering a variety of cancer types , alterations in genes coding for Ral regulators in human tumors are not uncommon . Notably , RGL1 and RalGPS1 are found amplified in 11% of breast cancer patients , meanwhile RalGAPA1 , RalGAPA2 and RalGAPB are altered in 14% , 8% and 6% of lung cancer patients , respectively ( cbioportal bioinformatics platform ) ( Gao et al . , 2013 ) , consistent with the notion that dysregulation of Ral might be important for oncogenesis or tumor progression . In almost all cancer types examined ( pancreas , colon , lung , bladder , prostate , melanoma ) , increased overexpression and/or activation of both RalA and RalB have been observed in patient tumor samples compared with normal tissues , regardless of their Ras mutation status ( Yan and Theodorescu , 2018 ) . Moreover , Ral-GTP level were found elevated in various tumor-derived cell lines harboring different Ras status , including pancreas ( Lim et al . , 2005 ) , colon ( Martin et al . , 2011 ) , bladder ( Saito et al . , 2013 ) , liver ( Ezzeldin et al . , 2014 ) , lung ( Male et al . , 2012 ) and brain ( Ginn et al . , 2016 ) . The only exception so far is squamous cell carcinoma ( SCC ) , where RalA was found to suppress rather than promote tumor progression ( Sowalsky et al . , 2010 ) . In this work we examined Ral protein expression in patient breast tumor samples and we found overexpression of both RalA and RalB as compared to normal breast tissues , therefore adding breast cancers to the list of human cancers with potential overstimulation of Ral pathway . Very interestingly , RalB ( but not RalA ) protein expression increased in a manner consistent with disease progression ( normal < in situ < invasive < metastatic tissues ) , supporting once more a crucial role for RalB in cancer invasion and metastasis . In conclusion , we propose that the pharmacological inhibition of the here established Ras-RGL1/2-RalB-exocyst-WRC pathway holds promises as anticancer strategy and definitely warrants further investigations .
Cells were grown in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 2 mM glutamine , penicillin , streptomycin , 10% fetal bovine serum . All cell lines were systematically tested to exclude mycoplasma contamination using a qPCR-based method ( VenorGem Classic , BioValley ) and authenticated by SRT profiling ( GenePrint 10 system , Promega ) . See Supplementary file 1 for detailed information and selection antibiotics . Transient DNA transfections were performed with Lipofectamine Plus reagents ( Invitrogen ) or JetPRIME ( Polyplus ) . Transient double siRNA and DNA transfections were performed with RNAiMAX ( Invitrogen ) and JetPEI ( Polyplus ) . See Supplementary file 2 and Supplementary file 3 for plasmid and siRNA sequences used in this work . All cells lines stably expressing the optogenetic dimerizing system were generated from HEK-HT cell lines via infection using lentiviruses . For viral production , 293 T cells were transfected with Lipofectamine 2000 ( Invitrogen ) with pHR-CRY2 or pLVX viral vectors along with lentiviral packaging plasmid ( psPAX2 ) and VSVG expression vector ( pCMV-VSV-G ) . Viral supernatant was harvested at 72 hr post-transfection , filtered , and added to the recipient cell lines for 24 hr with 6 μg/ml polybrene ( # 107689 , Sigma-Aldrich ) . Cells were sorted for co-expression of the fluorescent constructs by cell sorting on a BD FacsAria II ( BD Bioscience , Flow Cytometry Core Facility Institut Curie ) , and cell populations were used in bulk . For imaging cells were plated on 25 mm glass coverslips , pre-coated with 120 µg/mL of collagen type I of rat-tail ( Institut de Biotechnologie Jacques Boy ) , and placed in an observation chamber ( Pecon , Meyer Instruments ) . Imaging was performed at 37°C and 5% CO2 in a heating chamber ( Pecon , Meyer Instruments ) on an inverted microscope Olympus IX71 equipped with a 100X objective with NA 1 . 45 . The microscope was controlled with the software Metamorph ( Molecular Devices ) . Differential interference contrast ( DIC ) imaging was performed with a far-red filter ( cutoff 550 nm , BLP01-635R-25 , Semrock ) in the illumination path to avoid CRY2 activation . Total internal reflection fluorescence ( TIRF ) images were acquired using an azimuthal TIRF module ( ilas2 , Roper Scientific ) . Localized stimulation was performed with the FRAP system at low laser power ( ~5% ) with both 491 nm and 401 nm wavelength light lasers ( Stradus ) , 2–3 100 ms pulses , every 10–15 s . Imaging of Cherry and iRFP was done with 561 nm and 642 nm laser ( Stradus ) , respectively . For image processing and data analysis , after background subtraction , the mean fluorescence over the time inside the blue light activated region ( ROI ) was measured using Metamorph software . The mCherry and iRFP recruitment curves obtained from TIRF images were expressed as fold increase of fluorescence: the value of fluorescence for each time point was normalized by the pre-illumination fluorescence ( mean of the first frames without light activation ) . For cell edge morphodynamics and recruitment analysis , a custom-built ImageJ plugin , named ‘Recruitment Edge Dynamics’ , was used ( Paul-Gilloteaux et al . , 2018 ) . The Transwell insert consisted in a porous membrane ( 8 µm pore size ) with on top a thin layer of matrigel matrix that mimics the extracellular matrix ( #354483 , BD Biosciences ) . Prior to seeding in the Transwell chambers , cells were starved overnight in DMEM with 0 . 2% serum . 100 , 000 cells per well were seeded on the top of the insert in 0 . 2% serum medium , while 10% serum medium was placed in the well below , to create a chemoattractant gradient . After 6 hr of incubation at 37°C and 5% CO2 , the insert was washed with PBS , non-invasive cells on the top of the insert were removed with a cotton swab . The cells that did invade on the bottom were fixed with 4% PFA , permeabilized with 0 . 5% NP-40 and incubated with 5 mg/ml DAPI to stain the nuclei . The porous membranes were cut out and mounted on slides using ProLong Gold antifade reagent ( Invitrogen ) . Cell nuclei were imaged using an epifluorescence a Zeiss Axioplan microscope ( 10X objective ) , images were acquired with a Coolsnap HQ2 camera ( Roper Scientific ) and counted using ImageJ plugin ‘Cell Counter’ . The Invasion index is calculated from the mean of all the replicates per each condition , normalized on the mean of the control per each experiment . For Transwell coupled with optogenetic , each well was illuminated with an independent LED using a custom-made illumination device consisting of an array of 5 mm blue LEDs ( 480 nm , 12 cd , 30° ) driven by an Arduino Due micro-controller and delivering 3 . 5 mW each . Blue light was administered continuously from the bottom of the Transwell plate for the whole duration of the experiment ( 6 hr ) . Inverse invasion assays were performed accordingly to a previously published protocol ( Kajiho et al . , 2016 ) , but using a collagen gel instead of Matrigel . An ice-cold gel preparation of bovine collagen type I ( #5005-B , Advanced Biomatrix ) at a final concentration of 2 . 3 mg/mL , in MEM medium containing 0 . 28% NaHCO3 ( pH = 8 ) , was incubated 2 . 5 hr at 4°C ( nucleation step ) , pipetted into 12-well 8-μm-pore-diameter transwells ( #353182 , Corning ) ( 230 μm gel preparation per insert ) , inserted into a 12-well tissue culture plate , and incubated at 37°C for 1 hr ( polymerization step ) . The transwells were then inverted , 4 × 104 cells were seeded on the underside of the porous membrane , and placed in 6-well plate for 2 hr ( cell attachment step ) . The transwells were inverted back , washed 3 times with 1 ml of serum-free medium , and placed in 1 . 5 ml of serum-free medium inside a 12-well plate ( lower chamber ) ; 230 µl of 10% FBS-DMEM supplemented with 25 ng/ml EGF were added inside the transwell ( upper chamber ) . The cells were then allowed to invade upward into the bovine collagen and toward the gradient of serum/EGF for 4 days at 37°C and 5% CO2 . Cells were live stained with CellTrace yellow ( Thermofisher ) for 1 hr at 37°C and rapidly imaged by confocal microscopy ( Zeiss LSM880 , 10x objective , excitation at 545 nm and emission at 605 nm ) . Optical sections were captured at 5 µm intervals , starting from the underside of the transwell membrane and moving upward in the direction of cell invasion . The resulting fluorescence images were quantified using ImageJ software . For each independent experiment , data were generated from two duplicate transwells , and optical sections were acquired from two areas of each transwell . For immunoblotting , cells were lysed in RIPA buffer ( 150 mM NaCl , 2 mM MgCl , 2 mM CaCl2 , 0 . 5% NaDOC , 1% NP40 , 0 . 1% SDS , 10% Glycerol , 50 mM Tris-HCL pH 8 . 0 ) containing 2 mM Na3VO4 , 10 mM NaF , 1 mM DTT and a protease inhibitor mixture ( #0589291001 , Roche ) . Equal amounts of protein were diluted in 4x Laemmli buffer and resolved by SDS-PAGE . Proteins were transferred to 0 . 45 μm nitrocellulose membranes ( Whatman ) by wet transfer and blocked with 3% BSA in TBS/0 . 05% Tween-20 for 30 min . Primary antibodies were: mouse anti-RalA ( #610222 , BD Transduction Laboratories , dilution 1:1000 ) ; rabbit anti-RalB ( #3523 , Cell Signaling , dilution 1:500 ) ; mouse anti AKT ( pan ) ( #2920 , Cell Signaling , dilution 1:1000 ) ; rabbit anti p-AKT ( Ser437 ) ( #9271 , Cell Signaling , dilution 1:1000 ) . Protein levels were detected using LICOR Odyssey Infrared Imaging System ( LI-COR Biosciences ) upon incubation with IRDye secondary antibodies for 1 hr at room temperature . For immunofluorescence , cells were cultured on coverslips , fixed with 4% paraformaldehyde , quenched with 1M Glycine solution , permeabilized with 0 . 1% of Triton-100X , incubated with 4% FBS +1% BSA blocking solution , then with primary and secondary antibodies , every step being in PBS buffer . Primary antibodies were: mouse anti-RalB ( #WH0005899M4 , clone 4D1 , Sigma-Aldrich , dilution 1:200 ) , mouse anti-RGL2 ( #H00005863-M02 , clone 4D10 , Novus biologicals , dilution 1:200 ) . For RT-qPCR analysis , total RNA was extracted using RNaeasy Plus Mini kit ( Quigen ) . Retrotranscription and amplification were obtained using the iScript cDNA synthesis kit ( BioRad ) , the SYBR Green Master Mix kit or TaqMan ( Applied Biosystems ) on the ABI Prism7900 SequenceDetection System ( Perkin-Elmer Applied Biosystems ) . See Supplementary file 4 for the RT-qPCR primers . Rac1 activity was measured using the ‘Rac1 Pull-down Activation Assay Biochem Kit’ ( # BK035 , Cytoskeleton ) , which is based on beads-coupled GST-Pak1GBD . A similar pull-down assay was used for RalB activity but using home-made beads-coupled GST-Sec5GBD . For RalB activity measurement by FRET , cells were transfected with a validated FRET-based RalB biosensor ( Martin et al . , 2014 ) and imaged with an Olympus IX71 microscope equipped with a FRET filter set ( 86002v1JP4 , Chroma Technology ) and a 63x oil-immersion objective . YFP/CFP images were generated using Metamorph and ImageJ software . Cells were washed in ice‐cold PBS and lysed in 30 mM Tris ( pH 7 . 2 ) , 150 mM NaCl and 1% CHAPS . Lysates were clarified by centrifugation at 40 000 r . p . m . for 30 min . Protein was fractionated on a Superose 6 10/300 GL Increase ( GE Healthcare Life Sciences ) connected to an ÄKTA pure chromatography system ( GE Healthcare Life Sciences , Protein Expression and Purification Core Facility , Institut Curie ) . The column was eluted with 30 mM Tris ( pH 7 . 2 ) , 150 mM NaCl and 1% CHAPS at 0 . 5 ml/min and 0 . 5 ml fractions were collected . Prior to SDS-PAGE analysis , proteins were concentrated with 20% Trichloroacetic Acid Protein ( TCA ) , washed in Acetone and resuspended in Laemmli buffer . Primary tumors and lymph nodes were surgically removed before any radiation , hormonal or chemo-therapy , using a cohort of patients ( n = 649 ) treated at the Institut Curie from 2005 to 2006 . In this cohort , 502 invasive ductal carcinomas were selected for Ral detection; after discarding the samples with staining problems , the final numbers of analyzed cases were n = 466 for RalA and n = 448 for RalB . Tissue microarrays ( TMA ) consisted of replicated tumor cores ( 1 mm diameter ) selected from whole-tumor tissue sections and a matched tissue core from adjacent non-tumoral breast epithelium ( referred to as normal breast tissue ) . Immunoistochemistry ( IHC ) staining was performed using a Dako Autostainer Plus according to previously published protocols ( Lodillinsky et al . , 2016 ) . Antibodies were: anti-RalB ( #WH0005899M4 , clone 4D1 , Sigma-Aldrich ) , anti-RalA ( #610222 , BD Transduction Laboratories ) . H-scores were calculated using the following formula: stained cell percentage x stain intensity/100 . Analysis of human samples was performed in accordance with the French Bioethics Law 2004–800 , the French National Institute of Cancer ( INCa ) Ethics Charter , and after approval by the Institut Curie review board and ethics committee ( Comité de Pilotage du Groupe Sein ) that waived the need for written informed consent from the participants . Women were informed of the research use of their tissues . Data were analyzed anonymously . Results are shown as mean ± standard deviation ( SD ) or standard error of the mean ( SEM ) . Statistical analysis was performed using Graphpad Prism ( v5 . 0 ) and R Software v . 3 . 3 . 2 ( R Core Team , 2016 ) . Comparisons between two groups were assessed using Student t-test . Comparisons between more than two groups were assessed using one-way ANOVA test . Comparisons between paired data were assessed using Wilcoxon signed-rank test . For breast cancer H-score analysis , Shapiro-Wilk normality test and non-parametric Mann-Whitney tests and Wilcoxon signed-rank test for paired data were used . p values less than 0 . 05 were considered significant .
The two Ral GTPases , RalA and RalB , have crucial roles downstream Ras oncoproteins in human cancers; in particular , RalB is involved in invasion and metastasis . However , therapies targeting Ral signalling are not available yet . By a novel optogenetic approach , we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex ( WRC ) via its effector exocyst , with consequent induction of protrusions and invasion . We show that active Ras signals to RalB via two RalGEFs ( Guanine nucleotide Exchange Factors ) , RGL1 and RGL2 , to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways . Moreover , on the clinical side , we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis . This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies .
Cancers develop when cells in the body divide rapidly in an uncontroled manner . It is generally possible to cure cancers that remain contained within a small area . However , if the tumor cells start to move , the cancer may spread in the body and become life threatening . Currently , most of the anti-cancer treatments act to reduce the multiplication of these cells , but not their ability to migrate . A signal protein called Ras stimulates human cells to grow and move around . In healthy cells , the activity of Ras is tightly controled to ensure cells only divide and migrate at particular times , but in roughly 30% of all human cancers , Ras is abnormally active . Ras switches on another protein , named RalB , which is also involved in inappropriate cell migration . Yet , it is not clear how RalB is capable to help Ras trigger the migration of cells . Zago et al . used an approach called optogenetics to specifically activate the RalB protein in human cells using a laser that produces blue light . When activated , the light-controlled RalB started abnormal cell migration; this was used to dissect which molecules and mechanisms were involved in the process . Taken together , the experiments showed that , first , Ras ‘turns on’ RalB by changing the location of two proteins that control RalB . Then , the activated RalB regulates the exocyst , a group of proteins that travel within the cell . In turn , the exocyst recruits another group of proteins , named the Wave complex , which is part of the molecular motor required for cells to migrate . Zago et al . also found that , in patients , the RalB protein was present at abnormally high levels in samples of breast cancer cells that had migrated to another part of the body . Overall , these findings indicate that the role of RalB protein in human cancers is larger than previously thought , and they highlight a new pathway that could be a target for new anti-cancer drugs .