Angelique Ziel-van der Made

Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.

The PTEN tumor suppressor gene is frequently inactivated in human tumors, including prostate cancer. Based on the Cre/loxP system, we generated a novel mouse prostate cancer model by targeted inactivation of the Pten gene. In this model, Cre recombinase was expressed under the control of the prostate-specific antigen (PSA) promoter. Conditional biallelic and monoallelic Pten knock-out mice were viable and Pten recombination was prostate-specific. Mouse cohorts were systematically characterized at 4 to 5, 7 to 9, and 10 to 14 months. A slightly increased proliferation rate of epithelial cells was observed in all prostate lobes of monoallelic Pten knock-out mice (PSA-Cre;Pten-loxP/+), but minimal pathologic changes were detected. All homozygous knock-out mice (PSA-Cre;Pten-loxP/loxP) showed an increased size of the luminal epithelial cells, large areas of hyperplasia, focal prostate intraepithelial neoplasia lesions and an increased prostate weight at 4 to 5 months. More extensive prostate intraepithelial neoplasia and focal microinvasion occurred at 7 to 9 months; invasive prostate carcinoma was detected in all male PSA-Cre;Pten-loxP/loxP mice at 10 to 14 months. At 15 to 16 months, a rare lymph node metastasis was found. In hyperplastic cells and in tumor cells, the expression of phospho-AKT was up-regulated. In hyperplastic and tumor cells, expression of luminal epithelial cell cytokeratins was up-regulated; tumor cells were negative for basal epithelial cell cytokeratins. Androgen receptor expression remained detectable at all stages of tumor development. The up-regulation of phospho-AKT correlated with an increased proliferation rate of the epithelial cells, but not with a reduced apoptosis.

Broadened ligand responsiveness of androgen receptor mutants obtained by random amino acid substitution of H874 and mutation hot spot T877 in prostate cancer

In a subset of endocrine therapy‐resistant prostate cancers, amino acid substitutions H874Y, T877A and T877S, which broaden ligand specificity of the ligand binding domain (LBD) of the androgen receptor (AR), have been detected. To increase our knowledge of the role of amino acid substitutions at these specific positions in prostate cancer, codons 874 and 877 were subjected to random mutagenesis. AR mutants were screened in a yeast readout system for responsiveness to 5α‐dihydrotestosterone, progesterone and dehydroepiandrosterone. At position 874, only the histidine to tyrosine substitution could broaden AR ligand specificity. At position 877, 4 ligand specificity broadening substitutions were found: T877A, T877S, T877C and T877G. The latter 2 were not found in prostate cancer. The AR mutants were tested in mammalian (Hep3B) cells for responsiveness to 13 different ligands. All mutants displayed their own ligand specificity spectrum. Importantly, AR(H874Y) and AR(T877A) could be activated by cortisol. According to the 3‐dimensional structure of the AR LBD, T877 interacts directly with the 17β‐hydroxyl group of androgens. All amino acid substitutions identified at position 877 had smaller side chains than the threonine in the wild‐type receptor, indicating that increased space in the ligand binding pocket is important in broadened ligand specificity. Because H874 does not interact directly with the ligand, its substitution by a tyrosine is expected to change the ligand binding pocket conformation indirectly. For T877C and T877G substitutions, 2‐point mutations are required, and for H874Y, T877A and T877S substitutions, only a 1‐point mutation is sufficient. This most likely explains that the latter 3 have been found in prostate cancer.

Truncated ETV1, Fused to Novel Tissue-Specific Genes, and Full-Length ETV1 in Prostate Cancer

In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin beta3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed.

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.

Expression of the Androgen-Regulated Fusion Gene TMPRSS2-ERG Does Not Predict Response to Endocrine Treatment in Hormone-Naïve, Node-Positive Prostate Cancer

BACKGROUND Fusion of the androgen-regulated gene transmembrane protease, serine 2, TMPRSS2, to the v-ets erythroblastosis virus E26 oncogene homolog (avian), ERG, of the erythroblast transformation-specific (ETS) family is the most common genetic alteration in prostate cancer (PCa). OBJECTIVE To determine whether expression of androgen-regulated TMPRSS2-ERG predicts response to endocrine treatment in hormone-naïve, node-positive PCa. DESIGN, SETTING, AND PARTICIPANTS Eighty-five patients with histologically confirmed, node-positive PCa who were without treatment at the moment of lymph node dissection were analysed. RNA was isolated from the paraffin-embedded lymph node metastases and complementary DNA (cDNA) was made. The quality of cDNA was tested by polymerase chain reaction (PCR) analysis of the expression of the housekeeping gene hydroxymethylbilane synthase, HMBS (formerly PBGD). TMPRSS2-ERG expression was analysed by PCR using a forward primer in TMPRSS2 exon 1 and a reverse primer in ERG exon 4. MEASUREMENTS The primary end point was time from start of endocrine therapy to the occurrence of three consecutive rises in prostate-specific antigen (PSA) that were at least 2 wk apart and resulted in two 50% increases over the PSA nadir. Secondary end points were time to PSA nadir after start of endocrine treatment and cancer-specific and overall survival. RESULTS AND LIMITATIONS TMPRSS2-ERG was expressed in 59% of the 71 patients who could be analysed. Median duration of response to endocrine therapy was 20.9 mo versus 24.1 mo for gene fusion-positive versus gene fusion-negative patients (95% confidence intervals: 18.6-23.1 vs 18.9-29.4, p=0.70). Furthermore, no significant differences were seen between the two groups for the secondary end points. CONCLUSIONS Expression of TMPRSS2-ERG is frequent in lymph node metastases of patients with untreated PCa; however, expression of this androgen-regulated fusion gene did not correspond with duration of response to endocrine therapy. Our results suggest that expression of TMPRSS2-ERG is not a candidate marker to select for metastatic PCa patients who will benefit more from endocrine treatment.

Validation of a DNA Methylation-Mutation Urine Assay to Select Patients with Hematuria for Cystoscopy

Purpose: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay. Materials and Methods: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer. Results: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92–0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy. Conclusions: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.

Conditional Pten knock‐out mice: a model for metastatic phaeochromocytoma

Phaeochromocytomas (PCCs) are neuro‐endocrine tumours of the adrenal medulla that are usually benign, but approximately 10% of patients develop metastases. Malignant PCCs can only be diagnosed with certainty if metastases are present. Here we describe adrenal tumours generated in a Pten conditional knock‐out (KO) mouse model. We characterized the molecular alterations in these tumours and compared them with human PCC. Thirty‐two of 41 (78%) male Psa‐Cre;Pten‐loxP/loxP mice presented adrenal tumours that were shown to be PCC by histology and by immunohistochemical staining for enzymes in the catecholamine biosynthetic pathway. In 6 of 17 investigated mice, histological and immunohistochemical evidence was obtained for the presence of PCC lung metastases. Array comparative genomic hybridization (CGH) analysis of the primary tumours showed loss of chromosomes 6 and 19, which are syntenic to human 3p and 11q. Another frequent alteration found was gain of chromosome 15, which is syntenic to human chromosome 5. The molecular aberrations in the mouse model corresponded to the alterations found in a subtype of human PCC, suggesting that the PCC of the Pten KO mice might be representative of human PCC. The mouse model should allow further studies into the pathogenesis of human malignant PCCs and into therapeutic strategies for these tumours. Copyright

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

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.

Trp53 inactivation leads to earlier phaeochromocytoma formation in pten knockout mice

Phaeochromocytomas (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 Pten(loxP/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.

Tumor heterogeneity, aggressiveness, and immune cell composition in a novel syngeneic PSA-targeted Pten knockout mouse prostate cancer (MuCaP) model

Prostate cancer is recognized as a heterogeneous disease demanding appropriate preclinical models that reflect tumor complexity. Previously, we established the PSA‐Cre;PtenLoxP/LoxP genetic engineered mouse model (GEMM) for prostate cancer reflecting the various stages of tumor development. Prostate tumors in this Pten KO model slowly develop, requiring more than 10 months. In order to enhance its practical utility, we established a syngeneic panel of cell lines derived from PSA‐Cre targeted Pten KO tumors, designated the mouse prostate cancer (MuCap) model.