TI - P53 Mutant Expression Cooperated with AKT Activation in the Generation of Breast Carcinoma . AB - It has been proposed that AKT activation triggers senescence in primary cells and that this tumor suppressor mechanism might be dependent on the presence of p53 [29] [30] . However , our data from MIN and tumors of myrAKT models suggest that p53 pathway is not involved in the pre to malignant progression . However , other tumor models , such as head and neck , show p53 and AKT cooperation in tumorigenesis [33] , [34] . To further explore whether this mechanism plays a role in our experimental system , we generated mice carrying the activated AKT transgene and the mutant p53 (R172H) in the mammary ductal epithelia by crossing the MMTV-myrAKT with the wap-p53 (R172H) line . Double heterozygous transgenes were analyzed in deep . Neither , total survival nor survival of mice with mammary tumors was altered in these mice ( Figure 4 ) . And the range of preneoplastic lesions did not vary in doubles myrAKT ; p53 (R172H) from p53 mutant mice alone ( Figure 4 ) . We observed that while p53 (R172H) transgenic mice did not show MIN , doubles myrAKT ; p53 (R172H) showed 40% mice carrying MIN , percentage similar to the observed in virgin myrAKT transgenic lines ( Figure 4 ) . The transgenic lines with p53 (R172H) are multiparous and the incidence of MIN in doble transgenic line is equivalent to the virgin myrAKT mice . Identical data was observed when myrAKT transgene was expressed in a p53 ( +/- ) heterozygous background in virgin females ( Figure S5 ) . Therefore , p53 mutant does not alter the MIN to carcinoma transition in myrAKT1 transgenics . Moreover , multiparous females that expressed p53 mutant and myrAKT reached levels of MIN equivalent to virgin females . To further explore the role of p53 in AKT -dependent MIN we compared the behavior of senescent markers p19ARF , p21 or p53 in MIN from myrAKT in wild type or p53 (R172H) mutant backgrounds ( Figure 4D ) , or in p53 ( +/- ) heterozygous background ( Figure S6 ) . P19ARF is increased in all lines . However , it shows different levels among the different lines , being greatly overexpressed in the presence of mutant p53 as expected . p21 was equally increased in all lines while p53 showed clear staining in both lines expressing the mutant p53 (R172H) . Although we did observed a 30% p53 (R172H) mice with carcinomas , the number of mice with carcinoma in doubles myrAKT ; p53 (R172H) increased to 80% ( Figure 5A ) , tumors that showed bigger size . While in p53 (R172H) mice carcinomas showed an average size of less than 0.03 cm3 , in double myrAKT ; p53 (R172H) mice , carcinomas had an average volume of 0.2 cm3 ( Figure 5B and C ) . Both however , showed clear p53 staining ( confirming p53 (R172H) mutant expression ) . Double myrAKT ; p53 (R172H) tumors also showed activated AKT , but in general the levels were lower than in myrAKT only tumors . These data indicated that activated AKT cooperated with p53 mutant increasing the number and size of carcinomas but not the number of premalignant lesions , suggesting a more aggressive phenotype instead of a predisposition to develop a malignant lesion . However , the number of premalignant lesions , especially MIN , do not decrease as consequence of the transition to carcinoma , nor the double myrAKT ; p53 (R172H) transgenic mice showed full penetrance phenotype despite broad expression of transgenes in mammary ducts . Together with the data of no mutations in the p53 or p19ARF in tumors from myrAKT suggest that the constitutive activation of AKT induced a senescent barrier that is independent of p53 . Therefore , the mutant p53-AKT observed cooperation might be due to an increase in proliferation induced by AKT activation in p53 (R172H) -induced carcinomas . To explore this possibility we compared the different tumors arising in the different cohorts . These tumors were analyzed immunohistologically for a set of common mammary tumor markers and evaluated by double blind observations by 2 independent pathologists . Discrete values between 0 ( no expression ) and 3 ( higher levels ) was assigned to each tumor and the correlation between different tumors analyzed by using the function hcluster ( package amap ) of the free statistical software R . The different tumors analyzed showed very high pathological variability across all transgenic lines . Carcinomas and adenocarcinomas with different degrees of mixed , glandular , acinar or solid pathological features were observed in all lines . No specific morphological pattern was assigned to any genetic background ( Figure 5D ) . We observed that while AKT levels were very high (3) in all myrAKT tumors , tumors from double myrAKT ; p53 (R172H) transgenic mice were more heterogeneous but in general showed lower levels of activated AKT ( Figure 5D , Figure S7 ) . Furthermore , ER phosphorylaTION was associated with AKT activation in agreement with previous results indicating the relevance of ER PHOSphorylation by AKT [35] [36][27][26] . All tumors showed clear E-cadherin staining as reflection of their epithelial origin . CK6 , nanog , p63 , ER y PR are expressed in all myrAKT tumors but only in a very small ( aprox 20% ) tumors from double myrAKT ; p53 (R172H) , and mostly low levels ( Figure S7 ) . However it absence in most double myrAKT ; p53 ( R172H ) tumors correlated with the observed in p53 ( R172H ) induced carcinomas ( Figure 5D , Figure S7 ) . Furthermore , p63 , CK6 and nanog levels are homogeneous and higher in myrAKT tumors while tumors with mutant p53 ( from p53 ( R172H ) and myrAKT ; p53 ( R172H ) mouse lines ) are heterogeneous and show lower levels of expression of these proteins . ErbB2 ( Her2 , neu ) overexpression is observed in 20-30% human breast cancer [37] . In mouse models loss of PTEN function lead to elevated erbB2 protein levels [38] . We have tested whether in our model AKT activated transgene induced erbB2 protein . Our data showed a few ErbB2 barely positive tumors which correlated with no expression of ER and PR and high hystopathological grade . Only one tumor , probably outlayer , showed high ErbB2 positivity . The overall picture shows that tumors from double myrAKT ; p53 ( R172H ) transgenic mice are more close to tumors from p53 ( R172H ) , while tumors from myrAKT are different . However , the number of tumors is very small and a much higher number of tumors are necessary to analyze to further support our conclusions . Taken together , our experiments suggest that although tumor proliferation may be blocked by the presence of wild type p53 , p53 -dependent senescence might not be the only tumor suppressor mechanism impeding tumorigenesis in the mammary glands of myrAKT transgenic mice . The cooperation observed between activated AKT and mutant p53 seems to indicate that AKT enhances the proliferation of mutant p53 -induced tumors .