The Pace of Prostatic Intraepithelial Neoplasia Development Is Determined by the Timing of Pten Tumor Suppressor Gene Excision

Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16–20 wks post-tamoxifen exposure, to overtly malignant lesions by ∼1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10–12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development

Morphologic alterations in the prostates of <i>ARR2PBCreER(T2)</i>×<i>Pten^fl/fl</i> mice injected at 6 wks of age, and subjected to histopathological analysis 16–20 wks post-OHT exposure.

1<p>Abnormal cellular morphology refers to nuclear enlargement, prominent nucleoli, hyperchromasia, and deviations in cell size and appearance.</p>2<p>Atypical nuclear morphology refers to nuclei displaying nuclear and nucleolar enlargement and increased number of nucleoli.</p

There is increased cell proliferation in the luminal epithelia of <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice exposed to OHT.

<p>Immunohistochemical analyses revealed increased numbers of proliferating cells within the luminal epithelium of <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice treated with OHT at 6 wks of age as indicated by Ki67 staining, as shown by the dark dots and arrows in (B) and (D). (A) and (C) control <i>Pten^fl/fl</i> at 10 wks and 20 wks post-OHT; (B) <i>ARR2PBCreER(T2)×Pten^fl/fl</i> at 10 wks post-OHT and a (D) a representative PIN lesion in a <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mouse at 20 wks post-OHT. Scale bar: 50 µm. (E) Quantification of the percentage of proliferating cells in control and <i>ARR2PBCreER(T2)×Pten^fl/fl</i> aged for 4–10 wks and 16–20 wks post-OHT injections. There is a significant increase in the percentage of proliferating cells in the PIN regions of 16–20 p.i. experimental animals both over control animals and the normal regions within the experimental animals (p-value<0.001).</p

Morphologic alterations in the prostates of <i>ARR2PBCreER(T2)</i>×<i>Pten^fl/fl</i> mice injected at 2 wks of age, and subjected to histopathological analysis 10–12 wks post-OHT exposure.

1<p>Abnormal cellular morphology refers to nuclear enlargement, prominent nucleoli, hyperchromasia, and deviations in cell size and appearance.</p>2<p>Atypical nuclear morphology refers to nuclei displaying nuclear and nucleolar enlargement and increased number of nucleoli.</p

Timelines of disease progression in the <i>PbCre4×Pten^fl/fl</i> and <i>ARR2PBCreER(T2 )×Pten^fl/fl</i> mice injected with OHT either at two weeks or six weeks.

<p>Timelines of disease progression in the <i>PbCre4×Pten^fl/fl</i> and <i>ARR2PBCreER(T2 )×Pten^fl/fl</i> mice injected with OHT either at two weeks or six weeks.</p

There is increased cell death in the luminal epithelia of <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice exposed to OHT.

<p>There were increased numbers of apoptotic cells within the luminal epithelium of <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice treated with OHT at 2 wks of age and aged for 6 wks (B) and also in <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice treated with OHT and aged for 4–10 wks (D). (E) Quantification of the percentage of apoptotic cells in control and <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice. There was a significant increase in the percentage of apoptotic cells over controls in <i>ARR2PBCreER(T2)×Pten^fl/fl</i> injected with OHT either at 2 wks and 6 wks of age (p-value<0.001). Scale bar: 50 µm.</p

Transition from PIN to invasive carcinoma is seen in <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice exposed to OHT and aged for 42–52 wks.

<p>(i) Widespread high grade PIN lesions were seen in multiple glands within the prostates of experimental animals at 42–52 wks post-OHT, (ii) with abnormal cellular and nuclear morphology and microinvasive lesions (arrows). (iii) Proliferating cells within these lesions were detected by immunostaining with the anti-Ki67 antibody (arrows; arrowhead shows a mitotic figure) and high levels of phospho-S6 expression were also detected (iv). Representative immunohistochemical analysis using an antibody against high molecular cytokeratins 1,5,10, and 14 that stains basal cells: although basal cytokeratin staining was present within normal-appearing glands (v) (black arrows), with progression to invasive carcinoma there was a lack of cytokeratin staining (vi) (red arrows) within the same prostate. Scale bars: (i), 100 µm; (ii–vi), 25 µm.</p

Transition from the earliest stages of transformation to occur progressively in <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice following OHT exposure.

<p>(A) Nuclear enlargement with prominent and multiple nucleoli is seen in the luminal epithelial layer at different times post-OHT; in single cells at 4 wks post-OHT (arrows (i)); becoming more widespread at 8–10 wks post-OHT (arrows (iii)) and in the majority of cells in PIN lesions at 16–20 wks post-OHT (iv). Low grade hyperplasia is seen in the luminal layer at 4 wks post-OHT (rectangle (ii)). At 10 wks post-OHT, hyperplasia became more pronounced and nuclear enlargement with prominent nucleoli and hyperchromasia became more prevalent (arrows (iii)). Early stages of PIN, such as nuclear overlapping and mild tufting are noticeable by 10 wks post-OHT (iii). At 16–20 wks post-OHT more advanced PIN stages were evident (iv), with overt tubular dysplasia while basement membranes remained unbreached. PIN was prominent and most cells within these lesions showed nuclear enlargement, prominent nucleoli, hyperchromasia, and abnormal morphology (iv). (B) High grade PIN lesions were present in mice at 16–20 wks post-OHT. (i) While <i>Pten^fl/fl</i> controls showed normal morphology after OHT treatment, PIN lesions are seen 16–20 wks post-OHT administration in <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice, with different categories of high grade PIN being evident; including as tufting, micropapillary and flat atypia (ii), and cribriform structures (iii) and (iv) were seen in both focal intra-tubular as well as the more widespread lesions in the experimental animals. Scale bars: 50 µm.</p

Histological analysis of <i>ARR2PBCreER(T2)×Pten^fl/fl</i> mice exposed to OHT at 2 wks of age.

<p><i>ARR2PBCreER(T2)×Pten^fl/fl</i> injected at 2 wks of age displayed a wide range of lesions by 10–12 wks post-OHT: (i) hyperplastic lesion, (ii) PIN, (iii) high grade PIN with occasional microinvasive cells (arrows), (iv) high grade PIN lesions with a wide distribution area, stromal invasion of epithelial cells (yellow arrowheads) and displaying stromal thickening (blue arrows), (v) increased proliferation in a positive mouse aged for 6 wks as indicated by Ki67 staining, (vi) positive phospho-S6 staining of a positive animal at 6 wks post-OHT). Scale bars: (i–iii and v–vi), 50 µm; (iv), 100 µm.</p