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Comparative Study
. 2016 Jan 25;11(1):e0147500.
doi: 10.1371/journal.pone.0147500. eCollection 2016.

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice

Hanneke Korsten  1 , Angelique C J Ziel-van der Made  1 , Wytske M van Weerden  2 , Theo van der Kwast  1 , Jan Trapman  1 , Petra W Van Duijn  1   2
Affiliations
Comparative Study

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice

Hanneke Korsten et al. PLoS One. .

Abstract

Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m) developed at older age (>10m) into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC), adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK), and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7-8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1) and tumor class 2 (TC2). TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma/intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor model, histopathological, molecular and biological heterogeneity occurred during later stages of tumor development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of hyperplastic prostates and prostate tumors of targeted Pten knockout mice.
The prostate histology (HE staining) of the anterior lobe of hyperplastic prostates (4-5m) and different growth patterns (IDC, adenocarcinoma, undifferentiated carcinoma and carcinosarcoma) in prostate tumors (> 10m) are shown (Magnification: 200x). Normal prostate (NP) is included as a reference. A more detailed picture of the nuclear structures in these prostates is shown at higher magnifications. To characterize the prostate cells consecutive slides stained for P63 and Cytokeratin are shown for each growth pattern.
Fig 2
Fig 2. Two molecular subclasses of prostate tumors in PSA-Cre targeted Pten knockout mice match major prostate tumor growth patterns (A) Unsupervised hierarchical clustering of global gene expression data of thirteen tumor samples. Yellow and blue boxes indicated TC1 and TC2 tumors respectively. Where TC1 tumors mainly consisted of carcinomas, in TC2 tumors carcinosarcoma was the dominant growth pattern. More heterogeneous tumors are indicated by the dual blue/yellow colored boxes. Top-20 list of genes higher (B) and lower (C) expressed in TC1 (n = 3) tumors as compared to TC2 (n = 6) tumors as identified by SAM.
Green indicates lower gene expression and red indicates higher expression. The expression of Tff2 (indicated by the red dot) and Ptn (indicated by the green dot) was confirmed by Q-PCR analysis. Expression Hprt was used as Q-PCR reference. * p < 0.05, by Mann-Whitney two tailed test.
Fig 3
Fig 3. Differential expression of epithelial and mesenchymal markers in TC1 and TC2 tumors.
(A) Unsupervised hierarchical clustering of global gene expression data of normal prostates (NP), hyperplastic prostates (HP) and TC1 and TC2 prostate tumors. (B) The relative mRNA expression levels of CK8, E-cadherin, Snail and Fibronectin in NP (n = 3), HP (n = 3), TC1 (n = 3) and TC2 (n = 6) as detected in the expression arrays. Indicated gene expressions were plotted relative to the housekeeping gene Hprt. (C) Representative Q-PCR analysis of Snail and E-cadherin expression. Expression of Hprt was used as Q-PCR reference. Data are presented as mean +/- SE. * p < 0.05, by Mann-Whitney two tailed test.
Fig 4
Fig 4. Increased expression of markers associated with inflammatory response in prostate tumors of targeted Pten knockout mice.
(A) Top-20 list of genes higher expressed in prostate tumors compared to HP as identified by SAM. Green indicates lower expression and red indicates higher expression. (B) Q-PCR analysis of Grp and A2m expression in NP (n = 3), HP (n = 3) and prostate tumors (n = 9). Expression of Hprt was used as Q-PCR reference. Data are given as mean +/- SE. (C) Top-5 processes annotated by Ingenuity analysis based on genes differentially expressed between HP and prostate tumors. (D) Relative RNA expression analysis of CD45 and F4/80 in NP (n = 3), HP (n = 3) and TC1 (n = 3) and TC2 (n = 6) prostate tumors. Indicated CD45 and F4/80 expression was plotted relative to the housekeeping gene Hprt. Data are presented as mean +/- SE. (E) CD45 and F4/80 staining of immune cells in NP, HP and tumor (Magnification: 125x and 200x respectively). * p < 0.05 by Mann-Whitney two tailed test.
Fig 5
Fig 5. Differential expression of markers associated with senescence, proliferation, angiogenesis and apoptosis in TC1 and TC2 prostate tumors as compared to HP/NP in targeted Pten knockout mice.
(A) Relative mRNA expression levels of senescence markers Cdkn1a, Trp53, Cdkna2a and Dec1 in NP, HP, TC1 and TC2 (left panel). Representative p21 immunostaining in tissue slides of the anterior prostate lobe of NP, HP, TC1 and TC2 (right panel). Magnification: 200x. (B) Relative mRNA expression levels of proliferation markers Ki67 and Pcna and representative pictures of BrdU+ cells in anterior prostate lobes of NP, HP, TC1 and TC2 (Magnification: 200x). (C) Relative mRNA expression of angiogenesis markers CD31 and Tie2 in NP, HP, TC1 and TC2. (D) Relative mRNA expression of pro-apoptotic markers Bax and Bak1 in NP, HP, TC1 and TC2. The indicated gene expressions were plotted relative to the housekeeping gene Hprt. Sample size NP, HP and TC1 (n = 3) and TC2 (n = 6). Data are given as mean +/- SE. * p < 0.05 by Mann-Whitney two tailed test.
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The research leading to these results has received support from: The Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115188, resources of which are composed of financial contribution from the European Union's 7th Framework Programme (2007‐2013) and EFPIA companies' in kind contribution. www.predect.eu. Dutch Cancer Society KWF (Grant number:EMCR 2006-3592, www.kwfkankerbestrijding.nl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.