blocking the interaction of pi3-kinase p110 with ras as well as total loss of p110 or inhibition of its lipid kinase activity are shown to be beneficial in the treatment of established kras mutant lung cancer . because preventing ras binding to p110 has similar effects on kras mutant tumor regression to total removal of p110 , the interaction between ras and p110 has central importance in maintaining pi3-kinase signaling in ras - driven cancers . blocking this could provide a less toxic approach to targeting these tumors than total pi3-kinase enzymatic inhibition . furthermore , simultaneous inhibition of another major ras effector pathway , raf / mek / erk , together with blockade of either p110 or all type i pi3-kinases , causes major tumor regression . activating point mutations in the genes encoding the ras subfamily of small gtp binding proteins drive the formation of a large proportion of human tumors . ras proteins control cell growth through several direct effector enzyme families , the best studied of which are raf kinases , type i phosphoinositide ( pi)3-kinases , and ral - guanine nucleotide exchange factors ( ral - gefs ) ( downward , 2003 ; pylayeva - gupta et  al . , 2011 ) . of these , braf and pik3ca , the genes encoding braf and the p110 pi3-kinase catalytic subunit , respectively , are frequently activated by somatic mutation in human cancer ( weir et  al . , 2004 ) , with overall mutation frequencies around 10% for each . pi3-kinase activity is further implicated in carcinogenesis by the frequent inactivation of the inositol lipid 3phosphatase pten ( cully et  al . , the importance of the interaction of mutant ras with endogenous p110 in tumor development was established by the findings that mice with mutations in the ras binding domain ( rbd ) of p110 were highly resistant to mutant kras - induced lung cancer formation and mutant hras - induced skin cancer formation ( gupta et  al . , 2007 ) . however , it has been speculated that the signaling pathway requirements for tumor cells to survive ( tumor maintenance ) may be considerably relaxed relative to the signaling pathway activity needed to convert a normal cell into a tumor cell ( tumor development ) ( lim and counter , 2005 ) . as tumor maintenance is more relevant than tumor initiation to the treatment of preexisting human cancers , the question of whether blocking the interaction of ras with p110 will affect the maintenance of an existing ras mutant tumor is of considerable importance . inhibition of pi3-kinase activity has been reported to have a significant impact on ras - induced tumors in some settings , especially when combined with inhibition of the raf / mitogen - activated protein kinase extracellular signal - regulated kinase kinase ( mek)/extracellular signal - regulated kinase ( erk ) pathway ( engelman et  al . , 2008 ; halilovic et  al . , 2010 ; she et  al . , 2010 ; 2009 ) . however , it has been unclear whether pi3-kinase signaling is more important for ras mutant tumors compared to tumors with other oncogenic drivers or to normal cells . also , where such pi3-kinase dependency exists , it is not known whether this is because of acute ras - induced activation of pi3-kinase through direct interaction with the rbd of p110 or more indirect and long - term mechanisms , such as transcriptional upregulation of ligands of growth factor receptor tyrosine kinases . here , we have set out to address the issue of the importance , or otherwise , of the direct interaction of ras with the rbd of pi3-kinase p110 in the maintenance of ras - induced tumors . in order to investigate the role played by the direct interaction of p110 with ras in tumor maintenance , we used a previously generated mouse model in which the interaction of p110 with ras was disrupted by the introduction of two point mutations , t208d and k227a , into the endogenous pik3ca gene ( pik3ca ) ( gupta et  al . , 2007 ) . wild - type ( pik3ca ) and pik3ca mice were bred with mice containing a floxed pik3ca allele ( zhao et  al . , 2006 ) and a mouse carrying a conditional cre recombinase ( cre - ert2 ) allele targeted to the ubiquitously expressed rosa26 locus . , 2001 ) so that they spontaneously developed lung adenocarcinomas ( figure  1a ) . by feeding these mice with a tamoxifen diet for 2  weeks , we were able to efficiently remove the floxed pik3ca allele ( figure  s1a available online ) , leaving only one pik3ca or pik3ca allele expressed in these mice . initially , we set out to determine the effect of removing the ability of endogenous p110 to interact with endogenous ras on ras - driven tumors at the earliest possible stages , attempting to mimic as closely as possible the constitutive pik3ca mutation knockin reported previously ( gupta et  al . , 2007 ) . in this we were limited by the fact that tamoxifen treatment of rosa26-creert2 mice in utero or prior to weaning has been associated with cre recombinase - mediated genotoxicity ( schmidt - supprian and rajewsky , 2007 ) . we therefore treated 4-week - old mice with tamoxifen ( figure  1a ) , an age at which some small tumors had already formed , thus switching the mice to express pik3ca alone ( pik3ca ) . we could verify an efficient removal of the floxed allele after the 2-week treatment not only in the tumors , but also in several other tissues ( figure  s1a ) . the number of tumors present on the surface of the lungs of these mice was determined at 16  weeks of age . the number of  tumors in pik3ca mice was strikingly reduced when compared to their control counterparts , indicating that suppression of ras interaction with p110 greatly impairs tumor growth and maintenance at this stage ( figure  1b ) . we also observed a decrease in tumor number in pik3ca mice at the end of the experiment when compared to pik3ca mice at 4  weeks of age prior to the beginning of tamoxifen treatment , thus suggesting tumor regression in pik3ca mice and not just slower tumor development . these results were confirmed by histopathological analyses performed on these samples ( figures 1c , 1d , and s1b ) . tumor burden , number , and size were greatly reduced in pik3ca tumors compared to the pik3ca , pik3ca , or pik3ca tumors . we observed some decrease of the tumor burden in pik3ca mice compared to pik3ca mice in  the absence of tamoxifen treatment ( see below ) . there was , however , no difference in tumor burden in the heterozygous pik3ca mice without ( ) and with ( ) tamoxifen treatment , suggesting that pik3ca gene copy number does not have a major effect on tumor growth at this stage . we also determined tumor grade according to standard histopathological characteristics ( jackson et  al . , 2005 ) and found no differences in the existing tumors between pik3ca mice and  pik3ca mice ( figure  s1c ) . immunohistochemistry staining showed a decrease in phospho - erk ( p - erk ) and p - s6 in pik3ca tumors ( figure  s1d ) . furthermore , the levels of cell proliferation and apoptosis were studied in the tumors of these animals . as shown in figure  1e , tunel staining revealed an increase in the number of tumors displaying apoptosis in pik3ca animals when compared to the pik3ca . the proliferative rates in the tumors in the two backgrounds were also different , as measured by phospho - histone h3 staining for mitotic cells . we could see a higher number of cells stained in the pik3ca tumors than in the pik3ca tumors ( figure  1f ) , so it is likely that the difference in the sizes of the tumors in the wild - type and the pi3-kinase mutant backgrounds is at least in part due to both elevated rates of cell death and decreased proliferation in the absence of direct ras binding to p110. previous results in our lab using the constitutive knockin pik3ca mice had shown no differences in tumor formation between wild - type ( wt ) and heterozygous pik3ca mice , suggesting that the pik3ca allele is functionally recessive to  the pik3ca allele . however , our results here ( figure  1c ) showed some difference in overall tumor burden between pik3ca and pik3ca mice . to investigate this further , we studied tumor formation in mice of the same genotype except lacking the rosa26-creert2 allele . in this case , we could not find any significant difference in either tumor number or size , suggesting that the differences found in our pik3ca and pik3ca mice could be due to some degree of cre recombinase activity in rosa26-creert2 mice even in the absence of tamoxifen treatment ( figures s1e and s1f ) . while the previous experiments demonstrate that the interaction between p110 and ras plays a critical role in early - stage tumor development and maintenance , the very small size of lung tumors in 4-week - old kras mice made it impossible to follow single tumors longitudinally , thus making it hard to distinguish between changes in tumor development ( the appearance of new tumors ) and changes in tumor maintenance ( the persistence of preexisting tumors ) . we therefore studied the effect of pik3ca expression in more established tumors in adult mice . to address this , we treated 10-week - old mice with tamoxifen and used micro - computed tomography ( micro - ct ) analysis to monitor the growth of individual tumors over a 6-week period following the start of treatment ( figure  2a ) . efficient removal of the floxed pik3ca allele was observed soon after treatment and at the end of the experiment ( figure  s2a ) . we first monitored tumor growth in the absence of tamoxifen and found that , while  individual tumor growth was variable , both pik3ca and pik3ca tumors consistently increased in volume over this period ( figure  2b ) . in contrast , exclusive expression of pik3ca following tamoxifen treatment led to partial regression of tumors ( figure  2b ) . furthermore , to investigate whether activation of cre recombinase may by itself have an impact on tumor growth , mice that had rosa26-creert2 but no loxp sites flanking pik3ca were also treated with tamoxifen . we found that these tumors continued to grow following treatment , and thus the effects observed above are directly dependent on p110 and are not due to cre recombinase - associated toxicity ( figures 2b , green lines , and s2b ) . as initial tumor regression was evident following tamoxifen treatment in pik3ca mice , we carried out tunel staining on samples generated 10  days after the commencement of tamoxifen treatment . this revealed that cell death occurred in many of the mutant tumors following tamoxifen treatment , although widespread involution of tumors was not evident ( figure  2c ) . the effect of mutant expression on tumors was also evident using histological analysis of lungs ( figures 2d , 2e , and s2c ) . the lungs in pik3ca mice had fewer tumors present and the overall tumor burden in these animals was reduced . in addition , examination of downstream signaling revealed that while the level of akt phosphorylation varied in individual tumors , mutant tumors displayed lower activation of akt than wt tumors ( figure  s2d ) . in order to determine the more long - term consequences of pik3ca expression on tumor development , we followed tumor growth for 1632  weeks after tamoxifen treatment ( figure  3a ) . examination of individual tumor growth revealed that while pik3ca tumors recovered after the initial tamoxifen treatment and showed robust tumor expansion over a 4-month period , the pik3ca tumors remained static and did not progress further ( figures 3b and s3a ) . this was particularly clear in the 3d tomographs of lungs from treated mice ( figure  3c ) . over time , pik3ca tumors accumulated in the lungs , with some tumors merging ; however , tumor multiplicity remained low in the pik3ca mice ( figure  3c , right ) . interestingly , the tumor burdens in both pik3ca and pik3ca mice were lower than in pik3ca and pik3ca mice , indicating that some gene  dosage effects may be occurring ( figure  s3b ) . examination of p110 levels by western blot revealed that a decrease in p110 expression occurs during tamoxifen treatment , which may transiently affect growth in pik3ca tumors ( figure  s3c ) . our results demonstrate that in established kras - driven tumors , the pi3-kinase pathway continues to be an important mediator of tumor progression . indeed , at these later time points , tumor burden was significantly decreased in pik3ca mice versus pik3ca mice and the difference in tumor size became more evident as the pik3ca tumors continued to grow ( figures 3d and s3d ) . furthermore , activation of the akt pathway downstream of pi3-kinase was impaired in pik3ca mice , indicating that akt signaling downstream of ras continues to be disrupted ( figure  3e ) . we also utilized standard histological grading to look at tumor progression ( figure  s3e ) . while overall tumor number was lower in pik3ca mice , the incidence of higher - grade tumors was also reduced in these mice , with few grade 3 and no grade 4 adenocarcinomas detected in mutant mice 16  weeks after treatment ( figure  s3f ) . however , at later stages of the experiment , some regrowth of mutant tumors was observed ( figure  s3 g ) . this indicates that over extended periods of time , a minority of cells that escaped cre - mediated recombination during tamoxifen treatment may proliferate and restore tumor growth in pik3ca mice ( figure  s3 g ) . we were also interested in the impact of pik3ca expression on immune cell infiltration into the lungs . to look at this , we stained lung samples for markers of different immune cell subtypes . while histological staining showed no dramatic effect of pik3ca on neutrophils , b cells , or t  cells 16  weeks after treatment , we did see a marked decrease in the number of f4/80 macrophages in the lungs ( data not shown ; figure  s3h ) . tumor - associated macrophages have been observed previously in mutant kras - induced lung tumors in mice and have broadly been associated with tumor progression ( de palma and lewis , 2013 ; ji et  al . , 2006 ; pollard , 2004 ) . this suggests that in addition to direct inhibition of tumor growth , disruption of ras signaling to p110 may also affect crosstalk between the tumor and nontumor host cells . while the results above indicate a defect in tumor growth and maintenance in pik3ca mice , it is possible that this defect is not entirely tumor cell autonomous . the pik3ca protein is present not only in the tumor cells , but also surrounding host cells , including stromal , endothelial , and immune cells , any of which could contribute to the observed tumor regression or stasis . in order to investigate this , we derived lung tumor epithelial cells from 7 to 9  months old krasrosa26-creert2 mice that were either pik3ca or pik3ca . these animals had not been exposed to tamoxifen , so the floxed pik3ca allele in these cells remained unrecombined . tumor cells were sorted to remove immune and endothelial cells and were delivered intratracheally into the lungs of nude ( nunu ) mice . between 10 and 16  weeks after transplantation , tumors began to be visible by micro - ct in the lungs of the nude mice , with in all cases just one tumor detectable per lung . when tumors were visible by micro - ct , mice were treated with tamoxifen to delete the floxed pik3ca allele in the tumor cells and the tumor growth was followed over a period of 12  weeks . in mice transplanted with pik3ca tumor cells , tumors continued to grow rapidly after tamoxifen treatment , with a marked decrease in air content available in the lung ( figures 4a and 4b ) . however , in mice bearing pik3ca tumor cells , tumors completely disappeared after tamoxifen treatment and had not regrown by the end of the experiment , with lung air content increasing somewhat over time ( figures 4a and 4b ) . furthermore , these effects led to enhanced survival of mice inoculated with pik3ca tumor cells versus pik3ca tumor cells ( figure  4c ) . histopathology analysis of the lungs of these mice 12  weeks after tamoxifen treatment showed similar results . nunu mice containing pik3ca tumor cells had large tumors , generally just one occupying most of the lobe , although in several of them there were also small tumor foci in the vicinity of the main one . all the tumors presented a dominant papillary architecture and were usually classified as grade 3 . lungs in those mice explanted with pik3ca cells presented no tumors and had a normal tissue structure ( figure  4d ) . these results indicate a dependence on tumor cell - autonomous ras interaction with p110 for tumor maintenance and growth . however , they do not rule out the possibility that the loss of interaction of ras with p110 in host cells might also contribute to the regression of tumors seen in the autochthonous setting . our experiments thus far have revealed the importance of pi3-kinase activation by oncogenic kras for the progression and continued development of kras - dependent lung tumors . while the crucial role of ras - mediated activation of the pi3-kinase pathway during initial tumor development has been clearly demonstrated ( gupta et  al . , 2007 ) , established tumors may utilize both internal oncogenic ras and exogenous growth factors to activate this important pathway for continued tumor progression . we were thus interested to investigate whether complete removal of p110 would provide an additional effect on tumor growth in comparison to p110-rbd expression . to explore this , we treated pik3ca mice at 10  weeks of age with tamoxifen and followed individual tumor growth ( figure  5a ) . tamoxifen treatment resulted in efficient removal of p110 expression in tumors at the protein level ( figure  5b ) . in tamoxifen - treated pik3ca mice , we observed a partial regression of tumors in a manner similar to pik3ca expression ( figure  5c ) . while the effect of complete removal of p110 expression was slightly more robust , the change in tumor volume at the end of the 6-week period was comparable to that seen with pik3ca expression ( figure  5d ) . thus , in these tumors p110 activation appears to be largely dependent upon interaction with ras . histological analysis of pik3ca mice also showed a clear decrease in the number of tumors present and the overall tumor burden , while tumor size was not significantly changed ( figures 5e and s4a ) . furthermore , ablation of p110 expression in lkr13 cells established from lung tumors in the kras mouse model resulted in decreased proliferation of these cells ( figure  s4b ) . in contrast , ablation of p110 had little effect on proliferation or akt signaling , indicating that these cells are primarily dependent on the p110 isoform of pi3-kinase . analysis of the long - term effects on tumor growth after tamoxifen treatment in pik3ca mice also demonstrated that tumor progression was inhibited ( figure  5f ) . however , regrowth of tumors occurred more rapidly in these mice than in pik3ca mice . some residual expression of p110 was found in remaining tumors , indicating that incomplete recombination in some cells may allow re - establishment of pi3-kinase signaling and proliferation in tumors ( figure  s4c ) . while both p110-rbd expression and p110 depletion have a clear effect on oncogenic kras - driven tumor advancement , these two genetic alterations may have different effects on pi3-kinase signaling in nontumorigenic cells . this will be dictated by the extent of ras involvement during growth factor - mediated activation of pi3-kinase signaling . previous studies exploring the effect of p110 deletion / blockade on insulin signaling revealed that this isoform of pi3-kinase is a key mediator of metabolic responses to insulin ( knight et  al . , 2006 ; we performed a glucose tolerance test on untreated mice and tamoxifen - treated pik3ca , pik3ca , and pik3ca mice to investigate whether insulin - mediated removal of glucose from the blood was affected by genetic alteration of pik3ca . we found that deletion of pik3ca led to a greater impairment in glucose removal from the blood , indicating that nontumorigenic functions of p110 may be affected more by removal of p110 compared to expression of p110-rbd ( figure  5 g ) . furthermore , the health of pik3ca mice was more adversely affected than pik3ca mice , with significantly more mortality occurring within 4  weeks of the start of treatment ( figure  s4d ) . these results suggest that disruption of ras and p110 interaction may target tumor cells more specifically and may be accompanied by fewer side effects associated with global inhibition of p110 pi3-kinase function . we have found that expression of p110-rbd caused partial regression in established tumors , with long - term tumor stasis . however , with this mutant , we have targeted only one of the major pathways downstream of ras , and continued activation of other pathways may provide sufficient survival signals to maintain tumor integrity , at least in part . to investigate whether the erk / mitogen - activated protein kinase ( mapk ) pathway was facilitating the survival of pik3ca;kras tumors , we decided to target this signaling pathway in conjunction with disruption of pi3-kinase signaling . we treated adult kras;rosa26-creert2 ; pik3ca or kras;rosa26-creert2 ; pik3ca mice with tamoxifen for 2  weeks and then treated with the mek inhibitor trametinib for a further 4  weeks , using micro - ct imaging to monitor the effect on tumors . treatment with trametinib promoted partial tumor regression in pik3ca mice , but the effects were significantly stronger in pik3ca mice , with complete regression of some tumors ( figures 6a , s5a , and s5b ) . similar results were found when mice were treated with the lung cancer chemotherapy combination carboplatin plus paclitaxel , with pik3ca mice showing a stronger response than pik3ca animals ( figure  s5a ) . we confirmed that trametinib efficiently inhibited mek by assessing the levels of p - erk in the lungs ( figure  s5c ) . histological analysis also revealed a dramatic reduction of tumor burden in these mice ( figures 6b and s5d ) . there was a significant decrease in tumor number as many of the lesions regressed and tumors that remained were small and dispersed unlike their larger more compact counterparts in untreated mice ( figure  s5e ) . indeed , untreated tumors were consistently higher grade than those in the combination treatment ( figure  s5f ) . tumor burden was also reduced with combined tamoxifen and trametinib treatment of pik3ca mice , likely due in some part to the initial impairment of tumor growth that was observed following removal of a single allele of pik3ca ( figures 2b and s5a ) . next , we aimed to compare these results with pharmacological inhibition of pi3-kinase in a similar kras - driven tumorigenesis background . to do so , we treated kras mice with gdc0941 , an orally bioavailable inhibitor of all class i pi3-kinases , byl719 , a p110 isoform specific inhibitor , and the combination of gdc0941 or byl719 and trametinib . we observed a modest decrease in tumor growth in mice treated with gdc0941 or byl719 alone ( figure  6c ) . interestingly , treatment of these mice with gdc0941 or byl719 alone gave similar tumor responses , suggesting that the decrease in tumor growth is mainly achieved by inhibition of p110 isoform . when kras mice were treated with trametinib together with gdc0941 , or with byl719 , a strong tumor regression response was obtained , with an almost total disappearance of the tumors as evaluated by micro - ct scanning ( figure  6c ) . however , the toxicity was higher in the combined treatments , and treatment had to be stopped in the final week of the 5-week course in about half of the mice in these cohorts . the gdc0941 and trametinib combination tumor regression response was significantly stronger than that obtained with carboplatin plus paclitaxel or gdc0941 combined with carboplatin plus paclitaxel ( figure  s5 these results were confirmed in hematoxylin and eosin ( h&e ) staining where we could see an almost complete disappearance of the tumors in lungs from mice treated with the combined trametinib with gdc0941 or with byl719 therapy ( figures 6d and s5h  s5j ) . finally , we checked the efficiency of the gdc0941 and trametinib treatments in hitting their targets by assessing p - erk and p - akt levels in tumors of mice treated daily by these drugs . both akt and erk phosphorylation were inhibited in tumors treated with their respective pathway inhibitors , either alone or in combination ( figure  s5k ) . in these studies , we have attempted to assess the ongoing requirements of preexisting mutant kras - induced lung tumors for direct interaction of ras with p110 and to determine whether they differ from the previously established almost absolute requirement for this interaction in the de novo development of kras - driven lung tumors ( gupta et  al . , 2007 ) . we show here that genetically blocking the interaction of ras with p110 causes partial regression of tumors , followed by long - term tumor stasis , although by itself it is not sufficient to cause complete regression of established tumors in the autochthonous setting . this suggests that eradication of existing tumors is harder to achieve than prevention of the formation of new tumors , with the requirements for tumor maintenance being less stringent than for tumor development . however , combination targeting of ras s interaction with p110 with inhibition of the raf / erk pathway at mek causes much improved tumor regressions . while ras - induced activation of pi3-kinase is clearly important in the continued growth of ras mutant lung cancer in this system , we did not see a clear - cut pathway dependency for tumor maintenance as has been reported by others ( lim and counter , 2005 ) , but rather contributions of both pi3-kinase and raf / mek / erk pathways . although the oncogenic role of ras in the promotion of lung cancer has been known for many years , treatment strategies that effectively target ras mutant tumors have yet to be established . in contrast to the progress observed with epidermal growth factor receptor ( egfr ) or activin receptor - like kinase ( alk ) inhibitors in lung cancers with alterations in  those driver oncogenes , specific inhibition of ras mutant cancers has been difficult to achieve ( maemondo et  al . , 2010 ; shaw et  al . , our results demonstrate that ras - induced pi3-kinase activation is clearly important in these tumors in a mouse model of kras - driven lung adenocarcinoma . the pi3-kinase pathway thus provides an attractive therapeutic target , with combination of pi3-kinase inhibition with parallel pathway inhibitors or chemotherapeutic drugs likely to provide the greatest benefit . the value of testing the therapeutic response of kras mutant lung cancers in mice to the treatment of human disease is clearly highly dependent on how accurately the genetically engineered mouse model used reflects human kras mutant lung cancer in the clinic . several mouse models of kras - driven lung cancer have been developed over the past two decades ( kim et  al . , 2005 ) . the model that we use in this study , kras ( johnson et  al . , 2001 ) , is designed to have sporadic expression in single cells of g12d mutant kras off its own promoter triggered by  spontaneous intrachromosomal recombination . while the mutant kras protein is expressed in a few cells in all tissues , tumor development is seen only in the lung at high penetrance , with occasional thymic lymphomas also occurring . in another expression of mutant kras is induced by local introduction of cre recombinase activity in the lung using an adenoviral vector , with the number of cells expressing mutant kras being dependent on the viral titer applied . the kras model recapitulates many of the features of human kras mutant lung cancer , and this model has played an important role in extending our knowledge of this disease ( hatley et  al . , 2010 ; hollander et  al . , 2011 ; takahashi et  al . , 2010 ) . pathological progression of tumors in these mice from hyperplasia to adenoma and then adenocarcinoma over time phenocopies the progression of human lung tumors and permits establishment of a tumor microenvironment in the lungs . furthermore , the partial response seen here of lung tumors in kras mice to the current standard of care chemotherapies utilized against human lung cancer suggests that this model does to some degree reflect the therapeutic response of human kras mutant lung cancers . extending the observations described in our study to more complex kras / p53 or kras / lbk1 mouse models at a later stage may also provide additional insights into human kras mutant lung cancer biology and treatment . the earliest point at which pik3ca gene recombination could be induced without nonspecific cre recombinase toxicity was postweaning , at 4  weeks of age , at which time small lung tumors are readily detectable in kras mice by histology or visual examination of the lung surface , although not by micro - ct scanning . although these tumors can not be followed longitudinally , it appears that they undergo more profound regressions than larger tumors treated at 10  weeks . it is possible that larger tumors acquire additional mechanisms in conjunction with direct input from ras for maintaining pi3-kinase pathway activity . there thus appears to be some evidence for a progressive decrease in the degree of dependency on direct interaction of ras with p110 as tumors become more established . one concern with the approach used here to study the role of the ras binding domain of p110 in  vivo is the effect of changing gene dosage of pik3ca following cre recombinase - mediated deletion of a heterozygous floxed allele . for example , pik3ca expression and reduction of the wt level may both contribute to the tumor regression observed in pik3ca mice following tamoxifen treatment . however , this has been controlled for in all experiments with comparisons made between a single rbd mutant and a single wt allele of pik3ca , and we consistently observed enhanced antitumor effects with pik3ca expression . when comparison was made between mice in which wt pik3ca gene dosage was changed from two to one , some decrease in tumor growth rate , but never actual tumor regression , was seen . in order to distinguish tumor cell - intrinsic requirements for ras s interaction with p110 from effects involving nontumor cells of the host , such as stroma , endothelial , and immune cells , we carried out orthotopic transplantation experiments using tumor cells from engineered mice introduced into the lungs of animals with normal pi3-kinase signaling . these showed that a major component of the inhibitory effect of removing the ras and p110 interaction on tumor fate could be recapitulated when the interaction is prevented in the tumor cells alone . we can not rule out the possibility that changes in host cell signaling also contribute , but there is clearly a major tumor cell - autonomous effect . the fact that tumor regression occurs in nude mice indicates that this can occur without the adaptive immune system , although again it is not possible to exclude a contributing role . it is also apparent that transplanted tumors actually show much more profound regressions than autochthonous tumors when the ras and p110 interaction is blocked . one might speculate that the tumor niche established on transplantation provides a less robustly trophic environment for the tumor cells than the niche that evolves during the extended evolution of an autochthonous tumor . comparison of the effect on kras - driven autochthonous tumors of total deletion of p110 with the removal of the ability of p110 to interact with ras reveals similar levels of tumor regression and stasis . this indicates that in this model of lung tumors driven by activated kras , the direct binding of ras to p110 is a critical component of the regulation of this pi3-kinase isoform . however , it is clear that ablating only the ras interaction with p110 has less systemic toxicity for the mice than total p110 loss , as exemplified by less disruption of glucose homeostasis and also better general health status . it is noticeable , however , that ubiquitous loss of p110 in adult mice does not lead to  very major effects on mouse viability when compared with p110 loss or inactivation in developing embryos ( bi et  al . , 1999 ; 2006 ) , possibly reflecting some redundancy between pi3-kinase isoforms , as well as inevitably less than 100% deletion of pik3ca on cre activation . it is possible that a pharmacological strategy that targeted ras interaction with p110 may have fewer adverse effects than targeting p110 lipid kinase activity , but it may still be effective in opposing tumor progression . recently , some progress has been made in identifying small molecules that bind directly to ras ( maurer et  al . , 2012 ; shima et  al . , 2013 ; sun et  al . , 2012 ) , and these might eventually lead to drugs that would be able to block the interaction of ras with downstream effector enzymes . at present , however , the relatively low potency of the compounds identified , which is low m at best , has precluded assessment of their efficacy in the kras lung cancer mouse model . another likely advantage of direct ras effector interaction site targeted drugs would be that they would also be expected to inhibit other ras effector pathways such as raf / mek / erk , which are activated from the same region of ras . it has long been clear that inhibition of mek and pi3-kinase together promotes death of ras mutant cancer cells ( engelman et  al . , 2008 ) , although this approach has significant toxicities and may not show enhanced selectivity for the ras mutant phenotype relative to cells with wild - type ras ( molina - arcas et  al . , 2013 ) . combining pharmacological inhibition of mek together with genetic blockade of ras interaction with p110 also considerably improved tumor clearance in the model system used here , as did drug - mediated inhibition of both mek and type i pi3-kinases . use of a p110 isoform - specific pi3-kinase inhibitor had similar effects to a pan type i pi3-kinase inhibitor , reinforcing the view that p110 is the critical pi3-kinase isoform in this setting . we now have evidence that ras does not control p110 through direct interaction ( fritsch et  al . , 2013 ) , so it is unlikely that inhibition of this ubiquitous isoform will be helpful in ras - driven cancer . the other type i pi3-kinase isoforms , p110 and p110 , are unlikely to be expressed at major levels in the epithelial tumor cells studied here , although their inhibition could play an important role in the interaction of the tumor with host cells , especially those of the innate and adaptive immune systems . achieving effective targeting of the multiple signaling pathways driven by ras through direct blockade of the effector - binding region of ras remains a tantalizing , if distant , goal in cancer drug discovery . the possibility that drugs might be made that are specific for mutant , rather than wild - type , ras proteins could also add greatly to their therapeutic potential . the results reported here suggest that such an approach could show great promise in the treatment of ras mutant cancers if the challenge of finding effective ras - effector protein - protein interaction inhibitors could be met . the final mouse model was generated by breeding of four different mouse strains : pik3ca ( gupta et  al . , 2007 ) , kras ( johnson et  al . , 2001 ) , c57bl/6-gt(rosa)26sor ( taconic artemis gmbh ; referred to here as rosa26-creert2 ) , and pik3ca mice ( zhao et  al . , 2006 ) . all animal experiments were approved by the cruk london research institute animal ethics committee and conformed with uk home office regulations under the animals ( scientific procedures ) act 1986 including amendment regulations 2012 . total erk , p - erk ( thr202/tyr204 -e10 ) monoclonal , total akt , pakt ( ser473 ) , p - s6 , p110 , and p110 antibodies were all purchased from cell signaling technology . tamoxifen diet ( 400  mg / kg ) was obtained from harlan ( teklad lab animal diets ) . mice were treated with tamoxifen food pellets for 2  weeks and returned to normal diet for the duration of the experiment . to confirm that cre - mediated excision of the floxed allele occurred , dna was extracted from tumors , lung , and other tissue samples using extract - n - amp tissue pcr kit ( sigma ) . genotyping pcrs for the pik3ca and pik3ca alleles was then carried out using the following primers : forward 5-ctgtgtagcctagtttagagcaaccatcta-3 , reverse 5-cctctctgaacagttcatgtttgatggtga-3. for therapeutic drug treatments , drugs were given by oral gavage daily with vehicle , trametinib ( 3  mg / kg / day ) , gdc0941 ( 100  mg / kg / day ) , or byl719 ( 50  mg / kg / day ) for 5  weeks . trametinib and gdc0941 were dissolved as described previously ( gilmartin et  al . , 2011 ) . paclitaxel ( 10  mg / kg / day ) and carboplatin ( 50  mg / kg / day ) were administered by intraperitoneal injections twice a week , and both drugs were dissolved in pbs . mice were sacrificed and the lung tissue was immediately removed and fixed overnight in 10% neutral buffered formalin . tissue sections were cut at 4  m and were stained with h&e or were immunostained with various antibodies . pictures of each lung lobe were taken on a nikon eclipse 90i microscope with the 1 objective . lung and tumor area were measured using nis elements 3.0 sp7 software ( nikon instruments b.v . tumor grading was carried out by a pathologist and tumors were classified according to standard histopathological grading ( jackson et  al . , 2005 ) . significance was determined with graphpad prism 5 software using the student s t test unless stated otherwise .
summaryras proteins directly activate pi3-kinases . 
 mice bearing a germline mutation in the ras binding domain of the p110 subunit of pi3-kinse are resistant to the development of ras - driven tumors . 
 however , it is unknown whether interaction of ras with pi3-kinase is required in established tumors . 
 the need for ras interaction with p110 in the maintenance of mutant kras - driven lung tumors was explored using an inducible mouse model . in established tumors , 
 removal of the ability of p110 to interact with ras causes long - term tumor stasis and partial regression . 
 this is a tumor cell - autonomous effect , which is improved significantly by combination with mek inhibition . 
 total removal of p110 expression or activity has comparable effects , albeit with greater toxicities .