Accumulating Progenitor Cells in the Luminal Epithelial Cell Layer Are Candidate Tumor Initiating Cells in a Pten Knockout Mouse Prostate Cancer Model

The PSA-Cre;Pten-loxP/loxP mouse prostate cancer model displays clearly defined stages of hyperplasia and cancer. Here, the initial stages of hyperplasia development are studied. Immunohistochemical staining showed that accumulated pAkt+ hyperplastic cells overexpress luminal epithelial cell marker CK8, and progenitor cell markers CK19 and Sca-1, but not basal epithelial cell markers. By expression profiling we identified novel hyperplastic cell markers, including Tacstd2 and Clu. Further we showed that at young age prostates of targeted Pten knockout mice contained in the luminal epithelial cell layer single pAkt+ cells, which overexpressed CK8, Sca-1, Tacstd2 and Clu; basal epithelial cells were always pAkt−. Importantly, in the luminal epithelial cell layer of normal prostates we detected rare Clu+Tacstd2+Sca-1+ progenitor cells. These novel cells are candidate tumor initiating cells in Pten knockout mice. Remarkably, all luminal epithelial cells in the proximal region of normal prostates were Clu+Tacstd2+Sca-1+. However, in PSA-Cre;Pten-loxP/loxP mice, the proximal prostate does not contain hyperplastic foci. Small hyperplastic foci in prostates of PSA-Cre;Pten-loxP/+ mice found at old age, showed complete Pten inactivation and a progenitor marker profile. Finally, we present a novel model of prostate development and renewal, including lineage-specific luminal epithelial progenitor cells. It is proposed that Pten deficiency induces a shift in the balance of differentiation to proliferation in these cells

Trp53 inactivation leads to earlier phaeochromocytoma formation in pten knockout mice

textabstractPhaeochromocytomas (PCCs) are benign neuroendocrine tumours of the adrenal medulla. Approximately 10% of PCC patients develop metastases, but this frequency is much higher in specific subtypes of patients. The reliable diagnosis of malignant PCC can only be made after identification of a metastasis. To study the effect of Trp53 inactivation on PCC pathogenesis in Pten KO mice, we investigated the adrenals of a large cohort of mice with conditional monoallelic and biallelic inactivation of Trp53 and Pten. The adrenal weights were determined for all mice, and in a proportion of these mice, immunohistochemistry for tyrosine hydroxylase and dopamine β-hydroxylase was performed on the adrenals and corresponding lungs. Finally, comparative genomic hybridization (CGH) was performed. The histological and immunohistochemical results confirmed that the adrenal tumours were PCCs. Inactivation of one or both alleles of Trp53 resulted in earlier tumour occurrence in the PtenloxP/loxP mice as well as in the Pten loxP/+ mice. In addition, lung metastases were found in up to 67% of mice. The CGH results showed that the most frequent genomic alterations were loss of chromosome 19 (86%) and gain of chromosome 15 (71%). In this study, we have shown that Pten/Trp53 KO mice showed metastatic PCC at high frequency and primary tumours occurred at younger ages in mice with Trp53 inactivation. Therefore, the present model appears to be a suitable model that might allow the preclinical study of new therapeutics for these tumours

Increased expression of markers associated with inflammatory response in prostate tumors of targeted <i>Pten</i> knockout mice.

<p>(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 <i>Grp</i> and <i>A2m</i> expression in NP (n = 3), HP (n = 3) and prostate tumors (n = 9). Expression of <i>Hprt</i> 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 <i>CD45</i> and <i>F4/80</i> 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 <i>Hprt</i>. 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). * <i>p</i> < 0.05 by Mann-Whitney two tailed test.</p

Characterization of hyperplastic prostates and prostate tumors of targeted <i>Pten</i> knockout mice.

<p>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.</p

Differential expression of markers associated with senescence, proliferation, angiogenesis and apoptosis in TC1 and TC2 prostate tumors as compared to HP/NP in targeted <i>Pten</i> knockout mice.

<p>(A) Relative mRNA expression levels of senescence markers <i>Cdkn1a</i>, <i>Trp53</i>, <i>Cdkna2a</i> and <i>Dec1</i> 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 <i>Ki67</i> and <i>Pcna</i> 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 <i>CD31</i> and <i>Tie2</i> in NP, HP, TC1 and TC2. (D) Relative mRNA expression of pro-apoptotic markers <i>Bax</i> and <i>Bak1</i> in NP, HP, TC1 and TC2. The indicated gene expressions were plotted relative to the housekeeping gene <i>Hprt</i>. Sample size NP, HP and TC1 (n = 3) and TC2 (n = 6). Data are given as mean +/- SE. * <i>p</i> < 0.05 by Mann-Whitney two tailed test.</p

Differential expression of epithelial and mesenchymal markers in TC1 and TC2 tumors.

<p>(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 <i>CK8</i>, <i>E-cadherin</i>, <i>Snail</i> and <i>Fibronectin</i> 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 <i>Hprt</i>. (C) Representative Q-PCR analysis of <i>Snail</i> and <i>E-cadherin</i> expression. Expression of <i>Hprt</i> was used as Q-PCR reference. Data are presented as mean +/- SE. * <i>p</i> < 0.05, by Mann-Whitney two tailed test.</p

A reported 20-gene expression signature to predict lymph node-positive disease at radical cystectomy for muscle-invasive bladder cancer is clinically not applicable

<div><p>Background</p><p>Neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) provides a small but significant survival benefit. Nevertheless, controversies on applying NAC remain because the limited benefit must be weight against chemotherapy-related toxicity and the delay of definitive local treatment. Therefore, there is a clear clinical need for tools to guide treatment decisions on NAC in MIBC. Here, we aimed to validate a previously reported 20-gene expression signature that predicted lymph node-positive disease at radical cystectomy in clinically node-negative MIBC patients, which would be a justification for upfront chemotherapy.</p><p>Methods</p><p>We studied diagnostic transurethral resection of bladder tumors (dTURBT) of 150 MIBC patients (urothelial carcinoma) who were subsequently treated by radical cystectomy and pelvic lymph node dissection. RNA was isolated and the expression level of the 20 genes was determined on a qRT-PCR platform. Normalized Ct values were used to calculate a risk score to predict the presence of node-positive disease. The Cancer Genome Atlas (TCGA) RNA expression data was analyzed to subsequently validate the results.</p><p>Results</p><p>In a univariate regression analysis, none of the 20 genes significantly correlated with node-positive disease. The area under the curve of the risk score calculated by the 20-gene expression signature was 0.54 (95% Confidence Interval: 0.44-0.65) versus 0.67 for the model published by Smith <i>et al</i>. Node-negative patients had a significantly lower tumor grade at TURBT (p = 0.03), a lower pT stage (p<0.01) and less frequent lymphovascular invasion (13% versus 38%, p<0.01) at radical cystectomy than node-positive patients. In addition, in the TCGA data, none of the 20 genes was differentially expressed in node-negative versus node-positive patients.</p><p>Conclusions</p><p>We conclude that a 20-gene expression signature developed for nodal staging of MIBC at radical cystectomy could not be validated on a qRT-PCR platform in a large cohort of dTURBT specimens.</p></div

Univariate regression analysis of the ΔCt-values of the 20 single genes predicting node-positive disease at radical cystectomy (n = 139).

<p>Univariate regression analysis of the ΔCt-values of the 20 single genes predicting node-positive disease at radical cystectomy (n = 139).</p

Patient and tumor characteristics of all included patients with lymph node-negative or lymph node-positive disease (n = 139).

<p>The p-value indicates the (non)significant differences between both groups.</p

Beeswarm plots for BST2 and IF127 showing the distribution of ΔCt values for node-negative versus node-positive patient samples.

<p>The box indicates the upper and lower quartiles of distribution. The solid line indicates the median and the dotted line indicates the mean ΔCt value.</p