TI - Discussion . AB - Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers , mainly as a consequence of PTEN/PI3K pathway deregulation . The PTEN/PI3K pathway is probably the frequently mutated pathway in human cancer . Furthermore , tyrosine kinase membrane receptors are also mutated or activated ( ie : ErbB2 , EGFR ) contributing to AKT deregulation in mammary tumors . Different works analyze the necessity of the AKT signal in mammary tumorigenesis induced by different oncogenic signals ( reviewed in [39] [40][41] ) . However , the enforced AKT activation found in most mammary tumors suggest that there is also a sufficient signal to promote tumorigenesis . However , this point is still controversial since mammary models do not completely recapitulate the expected tumorigenic phenotype [39] . Several hypotheses , however , have been proposed to explain this apparent phenomenological discrepancy . Transgenic expression of activated AKT does not reach appropriate levels to induce tumorigenesis . Either they are too low to activate oncogenic pathway or too high activating senescence . Enforced AKT activation leads to p53 -dependent senescence , like for PTEN loss [29] or p27 -dependent senescence [31] . Finally , It is also possible that transgenic AKT activation does not occurs in the appropriated target cell ( ie : it is possible that MMTV promoter used in most of the transgenic mouse models it is not activated in the cells susceptible to generate a tumor ) . To investigate the mechanistic basis of the AKT -dependent mechanisms of breast tumorigenesis , we have generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland . Mice expressing activated AKT showed an increased percentage of suckling differentiation even in nulliparous females . In humans high parity has generally been associated with low breast cancer risk in several epidemiological studies [42] [43][44] . In mice ablation of AKT1 dramatically delays mammary gland differentiation during pregnancy and lactation and promotes apoptosis and accelerates involution what correlates with defects in Stat5 PHOSphorylation [45] . Maybe the expression of an activated AKT promotes a differentiation of the mammary gland that protects from tumor development . Moreover , we thoroughly analyzed the phenotype of these mice and correlated to the process of tumorigenesis by either preneoplastic or neoplastic lesions . AKT activation increased the number of benign lesions but neither AKT activated levels or number of cells with AKT activation in the mammary duct correlated with lesion number . Only myrAKT multiparous females from almost all transgenic lines showed an incidence of benign lesions , especially premalignant MIN , lower than nulliparous females . These data suggest that the absence of tumors in AKT -activated mice is not a consequence of inappropriate AKT levels in the transgene . We can also exclude that the AKT is not activated in the right target cells ( ie : cancer prone cell ) since we can increase the number of cells per duct with AKT activation in different lines but we can not observe a parallel increase in benign or malignant lesions . Therefore , our results suggest the existence of a tumor suppressor barrier that prevents tumorigenesis once AKT is activated . Clearly , p53 and p27 dependent senescence are mayor mechanisms proposed to achieve this goal . To explore these mechanisms we have analyzed different molecular markers of senescence in the benign and malignant lesions formed in AKT -activated mice . We have found that p19ARF , p21 , p27 , p16 and HP1gamma seems to be activated in MIN but only p16 disappears in tumors , indicating that pRb might constitute the senescent checkpoint which needs to be overcame in these AKT -dependent lesions to produce tumors . Interestingly , half of tumors containing mutant p53 also lost p16 . To further explore the relevance of p27 and p53 checkpoints , we generated transgenes with activated AKT in p27 ( +/- ) , p53 ( +/- ) or p53 (R172H) mutant backgrounds . Our findings revealed that the enhanced AKT -dependent preneoplastic phenotype was not altered by p27 reduction or p53 activity loss . However , p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinomas . However it was not sufficient to promote full penetrance of the tumorigenic phenotype . These data indicated that activated AKT cooperated with p53 mutant increasing the number and size of carcinomas but not the number of premalignant lesions . However , the number of premalignant lesions , 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 . Moreover , AKT activation in parous females decrease the incidence of MIN , but in the presence of p53 mutant this protection was not observed . Together with the data of no mutations in the p53 nor 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 . The molecular comparison of tumors from myrAKT , p53 ( R172H ) or double myrAKT ; p53 ( R172H ) mice suggests that myrAKT ; p53 ( R172H ) tumors seem to proceed from p53 ( R172H ) expressing cells and that AKT accelerated the process of tumorigenesis , rather than from cells with activated AKT where the p53 brake has been eliminated . On the other hand , R172H p53 mutant , equivalent to human R175H , is a structure defective mutant whose point mutation determines an important conformational alteration [46] . The presence of conformational mutants has been strongly associated with breast cancer patients [47] [48][49] . Mutant p53 proteins do not represent the mere loss of wild type p53 activity , but gain new additional oncogenic functions which contribute to the development , maintenance and spreading of cancer [46] [50] , [51] [52] . It has been shown that mutant p53 triggers different pathways that represent the molecular basis of the gain of function activity [52] . These scenarios might favor the increase of MIN detected in multiparous double transgenic mice that do no progress to carcinoma due to the senescence induced by AKT independent of p53 . On the other hand , the expression of mutant p53 might contribute to tumor initiation , while active AKT background might contribute to tumor progression through several of the pleiotropic effects induced by AKT activity ( Reviewed in [53] , [54] , contributing to the large increase in size observed in carcinomas . On the other hand , increased proliferation can also contribute to the increased number of carcinomas by increasing carcinoma detection . 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 . This is confirmed by the lost of p16 in half of tumors with mutated p53 . These data fully agree with our hypothesis that pRb might constitute the main senescent checkpoint which needs to be overcome in these AKT -dependent lesions to produce tumors . This hypothesis also correlated with the works of Yu et al [55] , Landis et al [56] and Reddy et al [57] reporting the necessity of CDK4 activity for mammary tumorigenesis . Our data point towards loss of CDK4 inhibitor p16 is the genetic alteration in the hyperplasia to tumor transition in the AKT activated transgene . In fact , previous results from our group showed that activated p110alpha transgenic expression in CDK4 (R24C) mutant background produces an increase in preneoplastic lesions and in tumor formation [26] . In human breast tumors , activation of cyclin D1 is a common event . Amplification of the gene has been detected in about 15% of breast cancers , while overexpression of cyclin D1 at mRNA and protein levels is seen in up to 50% of primary tumors , mostly ER-positive and well-differentiated tumors [58] [59] . Furthermore , joint overexpression of cyclin D and PI3K pathway activation , either directly or indirectly through membrane receptors , is a common event described in human tumors [60] [40] , [61] . Since breast cancer is notable for the high incidence of cyclin D1 aberrations with up to 50% of tumors expressing elevated levels of the protein ( reviewed in [59] [59][62] ) , we can expect to find p16 defects among the remaining tumors . To date , there have been reports suggesting that p16 may be affected by methylation in primary breast cancers with quoted frequencies of 15-30% [63] [64][65][66][67] and there is a relatively high rate of LOH at 9p21 [68] [69][63] . However , due the complex nature of the 9p21 loci containing also the p14ARF and p15INK4b tumor supressors , it is difficult to score the true relevance of p16 in this loci loss . Furthermore , a high number of breast cell lines show abnormalities , including the spontaneously immortalized line MCF10A [65] [70] . In HMEC , p16 silencing by promoter methylation has been reported as one of the initial events occurring to allow cell proliferation , contributing to alleviate senescent checkpoint [71] . In our model , cyclin D1amplification or p16 lost in breast tumors could be equivalent in maintaining CDK4 activity and therefore inactivating pRb checkpoint . 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 . It seems that in this model , pRb pathway is more important checkpoint controlling senescence . The cooperation observed between activated AKT and mutant p53 seems to indicate that AKT enhances proliferation of mutant p53 -induced tumors .