Our analysis allows a detailed deconstruction the molecular genetics underlying cancer progression in the prostate and the assessment of the key relevance of Pten and subtle variations in its dose in controlling this process. Based on our findings, we can now attribute distinct preneoplastic or malignant pathological entities to distinct molecular states. Furthermore, this new knowledge allows the reclassification, on the basis of their true molecular nature, of pathological lesions that at a superficial analysis appeared very similar ().
Pten Dose Affects Prostate Tumor Progression
In this supposedly linear and multistep process, which separates two extremely different anatomical and pathological entities, a normal prostatic epithelium from an invasive CaP, there are, in between, a discrete number of anatomically distinct intermediary steps, such as prostate hyperplasia > displasia/low-grade PIN > high-grade PIN > locally invasive CaP > diffused CaP. These entities have been recognized before on the basis of their pathological features. The fact that we are now able to correlate these anatomical stages with specific molecular events, such as the level of expression of a single tumor suppressor gene, not only represents an integration of anatomical, descriptive findings with biological data, but also offers the opportunity of therapeutic interventions.
We show in this study that inactivation of one Pten
allele in the mouse may lead to prostate epithelial hyperplasia, as complete inactivation of p27Kip1
in the mouse leads to what resembles human benign prostate hyperplasia (BPH). However, these two lesions are very different in nature and outcome: the first, in fact, impacts only on epithelial elements and over time evolves to low-grade PIN, while the second impacts on both epithelial and stroma elements, representing hyperplasia of the whole organ, but does not evolve toward malignancy (; Cordon-Cardo et al. 1998
; Di Cristofano et al. 2001a
). This obviously does not exclude that loss of p27Kip1
expression when accompanied by additional genetic events (e.g., loss of PTEN) may lead to a completely different outcome, as is also supported by our previous in vivo analysis in the mouse (; Di Cristofano et al. 2001a
Moreover, our data demonstrate that a further reduction in the Pten dose, as observed in the Ptenhy/− mutants, accelerates tumor progression dramatically, eventually resulting in high-grade PIN and locally invasive carcinoma (). This fact has very important therapeutic implications. As a large number of CaP patients (more than 80%) display at presentation loss of one PTEN allele, but do retain the other normal PTEN allele, it could be proposed that high-grade PIN or locally invasive prostate carcinomas could be prevented or treated by pharmacologically modulating the expression of the remaining PTEN allele or by antagonizing the downstream consequences of PTEN downregulation or partial inactivation (e.g., PI3K/mTOR activation; ).
While the massive prostate hyperplasia/dysplasia observed in the Ptenhy/− mutants is not, as expected, accompanied by complete loss of Pten expression (see E), the low penetrance of the invasive CaP in these mutants strongly suggest that additional events have to occur for this pathological transition to occur. Nevertheless, it is important to underscore that Pten+/− mice, unlike Ptenhy/− mutants, do not develop invasive CaP, but only low-grade PIN lesions (). Thus, a further reduction of the Pten dose seems to be essential for this process. Several possibilities, not mutually exclusive, could be entertained: (i) a more severe reduction in the Pten dose could facilitate cooperative tumorigenesis by rendering the cells permissive and sensitive to the activation of additional oncogenic pathways; (ii) the massive prostate hyperplasia could facilitate the accumulation of additional genetic hits (including complete Pten loss) by simply expanding the pool of actively dividing cells; (iii) a more severe reduction in the Pten dose could protect cells from apoptotic programs that are normally triggered by the occurrence of damaging genetics events.
The fact that Ptenhy/−
mutants develop massive prostate hyperplasia not accompanied by an overall increase in body weight and size underscores the fact that different tissues and organs are differentially sensitive to Pten dose variations, the prostatic epithelium being one of the most sensitive. In agreement with this notion, MEFs from Ptenhy/−
mutants do not display proliferative advantage in standard culture condition over wild-type MEFs. In this respect, it is also important to remember that, unlike p27Kip1−/−
mice, which display hyperplasia of both stromal and parenchymal components in the prostate (as well as an increase in body mass; Fero et al. 1996
; Kiyokawa et al. 1996
; Nakayama et al. 1996
), in Ptenhy/−
mutants it is specifically the prostatic epithelium that actively proliferates, leading to prostate hyperplasia (). Interestingly, this does not parallel what is observed in other model systems, such as Drosophila
, where dose variations in the expression of Pten
or downstream signaling components (e.g., TOR) do result in variations in body size and mass (reviewed in Oldham and Hafen 2003
While we and others had previously implicated Pten
heterozygous loss in prostate tumorigenesis when in cooperation with additional oncogenic events (such as loss of p27Kip1
or the expression of the large T oncogene; Di Cristofano et al. 2001a
; Kwabi-Addo et al. 2001
; Kim et al. 2002
; Abate-Shen et al. 2003
), we demonstrate in this article that complete inactivation of Pten
alone in the prostate has already catastrophic consequences leading to invasive, diffuse, and highly aggressive malignancies (). Although it cannot be formally excluded that complete Pten
loss would still cooperate with additional oncogenic events toward full-blown transformation, this finding underscores once more the exquisite sensitivity of the prostatic epithelium to loss of Pten
. This information should be factored in when tailoring the treatment of CaP or high-grade PIN lesions that have completely lost PTEN
function. These lesions may be extremely sensitive to PI3K or mTOR inhibitors, while modulation of PTEN
expression would not be a therapeutic option in these cases (). On the basis of these findings, it would seem therefore of paramount importance to routinely assess the status of the PTEN
gene and its expression in human CaPs and precancerous lesions as a key biomarker to opt for the most appropriate therapeutic or chemopreventive intervention modalities. It remains to be seen whether complete Pten
inactivation in the mouse prostate also influences the metastatic potential of these invasive CaPs alone or in combination with additional oncogenic events. In this respect, while MRI and postmortem analyses have indeed revealed the presence of lung neoplastic lesions in our conditional Pten
mutants, morphological and molecular analyses have so far excluded the metastatic nature of these tumors (M. Niki et al., unpublished data).
The stepwise reduction in the Pten dose results in clear progressively quantitative
changes in the prostate epithelial cells prior to tumor development. Increased cellular proliferation correlates with increased phosphorylation of Akt. In Pten
null epithelial cells, both in the preneoplastic stage or in overt CaPs, phospho-Akt is found to accumulate almost exclusively at the plasma membrane (see C). Whether this represents the extreme consequence of Akt superactivation or rather reflects a genuine qualitative change in Akt biology and function in Pten
null prostate epithelial cells remains to be determined. It also remains to be resolved whether the reduction in p27Kip1
expression is also associated with its cytoplasmic relocalization, as previously reported in human breast cancer cells suffering AKT hyperactivation (Liang et al. 2002
; Shin et al. 2002
; Viglietto et al. 2002
). Taken together, this analysis supports a Pten dose-dependent model with progressive
changes at the molecular level, arguing against a Pten threshold model for tumor suppression.
By contrast, clear qualitative morphological changes were observed in the prostate epithelium when analyzing the Pten hypomorphic series. While Pten+/− mice never display massive prostatic enlargement, further reduction to Ptenhy/− levels leads to a sharp increase in prostate mass. In addition, Pten+/− mice never develop invasive CaP, whereas Ptenhy/− mice do (). These data clearly support a threshold model for Pten tumor suppression at the physiopathological level.
Similarly, qualitative morphological changes are also caused by the number of cells suffering complete Pten inactivation, as observed when comparing Ptenpc1 versus Ptenpc2 mice. In Ptenpc2 mice, signs of cellular dysplasia and irregular glandular formations were frequently observed, but they were mostly absent from Ptenpc1 mutants. Along the same tenet, profound mass increase was always observed at early timepoints in Ptenpc2 mice, but not in Ptenpc1 mutants. This is likely due to the marked difference in the number of Pten null cells present in the parenchyma of these two models at puberty, reflecting the different levels of Creexpression in the two models. These observations are also consistent with a threshold model for Pten tumor suppression as a function of the number of Pten null cells in a given organ.
From a biological standpoint, it will be challenging to determine in the future whether this “field effect” would also play a role in the natural history of human CaP. On the one hand, it could be speculated that human CaP likely arises from a single transformed cell and that therefore that the field effects observed in mouse models will not be critical determinants in the natural history of the human disease. On the other hand, the multistep nature of the neoplastic process (and hence the genetic heterogeneity of the lesion) and the fact that the tumor will eventually grow to form “fields” of neoplastic cells may suggest that these effects, as well as stromal and parenchymal interactions, are key aspects of the human disease that the mouse model can recapitulate.
Taken together, our findings demonstrate the key importance of Pten in CaP tumor suppression and the dramatic impact that subtle changes in Pten levels and extent of Pten inactivation may have on tumor initiation and progression in the prostate.