Wilma Teubel

Activation of c-MET Induces a Stem-Like Phenotype in Human Prostate Cancer

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway. Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 (‘stem-like signature’). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f. In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.

Gain and loss of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate cancers

Fluorescence in situ hybridization (FISH) with centromere probes was used to investigate numerical aberrations of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate carcinoma (PC) and 11 benign prostatic hyperplasia (BPH) samples. None of the benign specimens showed any chromosomal aberration. Forty-one of 46 PC specimens showed numerical aberrations of one or more chromosomes. All investigated chromosomes showed numerical aberrations in at least 30% of the specimens, gain being more frequent than loss. Comparison of DNA flow cytometry (FCM) and FISH results showed that not only aneuploid tumors but also most diploid tumors harbored numerical chromosome aberrations. Chromosome 10 was the most frequently gained (65%), and Y the most frequently lost chromosome (14%). Nonmetastatic and metastatic tumors differed significantly (P < .05) in the number of copies for chromosomes 7, 8, and 10, but not for 1, 18, and Y. These results suggest strongly that gains of chromosomes 7, 8, and 10 are involved in PC progression.

Specific cytogenetic aberrations in two novel human prostatic cell lines immortalized by human papillomavirus type 18 DNA

Using chromosome banding and fluorescence in situ hybridization (FISH) with painting probes, sequential cytogenetic analysis was performed of two novel prostate cell lines, PZ-HPV-7 and CA-HPV-10, established by human papillomavirus (HPV) 18 DNA transformation. PZ-HPV-7 originates from a normal diploid prostate epithelial cell strain. PZ-HPV-7 progressed from an initial diploid to a hypertetraploid chromosome number with a relative gain of chromosomes 5 and 20 (7 to 8 copies each). Structural changes were limited; 3p- (2 copies), 3q- (1 copy), and possibly a der(16p;12q). CA-HPV-10 originates from an epithelial cell strain derived from a high-grade human prostate cancer specimen, which showed several karyotypic abnormalities including an extra Y chromosome and double minutes (dmin). In early passage the karyotype of CA-HPV-10 appeared unstable with a decreasing number of cells exhibiting dmin. In late passage the dmin were replaced by a large homogeneously staining region (hsr) on 9p+ marker. The hsr was shown by FISH to be of chromosome 1 origin. The modal number was mainly hypertriploid (72, range 69 to 75). Loss of Y was remarkable (0 to 1 copy). Consistent markers included two copies each of del(1)(q12q31) and der(9)t(1;9)(?;p22), and one der(11)t(4;11) (?;q21). HPV type 18 genomic integration sites were identified on 1p for PZ-HPV-7 and on the 9p+ marker for CA-HPV-10. In conclusion, both PZ-HPV-7 and CA-HPV-10 showed clonal cytogenetic changes. These two cell lines constitute a novel in vitro model to study the mechanisms involved in human prostate carcino-genesis.

Cytogenetic Analysis of 39 Prostate Carcinomas and Evaluation of Short Term Tissue Culture Techniques

Karyotypic analysis was performed on 102 prostate cancer specimens which were obtained through radical prostatectomy, transurethral resection, or regional lymph node dissection. Short term tissue culture was applied in all cases. Of the media and growth factors evaluated, F12/DMEM, supplemented with 2% fetal calf serum, insulin, epidermal growth factor, hydrocortisone, and cholera toxin produced the largest increase of in vitro proliferation. Such in vitro cultured cells were all phenotypically acinar epithelial cells, the supposed targets for neoplastic transformation. Stromal cell growth appeared to be completely suppressed. Of the three culture techniques investigated, the method developed in Lund, Sweden, was the most successful: 11/15 cultures yielded metaphases and, in three of these, clonal aberrations were identified. All 39 karyotypes obtained essentially had a 46,XY karyotype with clonal aberrations (eight cases) and/or nonclonal aberrations (30 cases). Clonal structural aberrations involved 2p, 3q, 11p, 17p, and 21q. The clonal numerical aberrations found were: + 8, + dmin, and -Y. The most frequently observed nonclonal aberrations were 8p deletions (five cases) and loss of 6, 7, 8, 15, 17, 18, 21, and/or Y (> or = five cases). In summary, clonal aberrations were observed in 20% of the evaluable PC cell cultures, and nonclonal aberrations in 77%. So, although diploid cells without clonal abnormalities still had a growth advantage, under optimal conditions PC cells were able to proliferate in primary in vitro culture.

Profiling of antibody production against xenograft-released proteins by protein microarrays discovers prostate cancer markers.

This study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of serum markers for prostate cancer. By immunizing immuno-competent mice with serum from nude mice bearing prostate cancer xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several prostate cancer-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing prostate cancer xenografts and prevalidated in human serum samples of prostate cancer patients. Among the discovered and validated proteins were the members of the TAM receptor family (TYRO3, AXL, and MERTK), ACY1, and PSMA1. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons.

Characterization of chromosome 8 aberrations in the prostate cancer cell line LNCaP-FGC and sublines

Abstract In two androgen-dependent (FGC and P70) and two androgen-independent (LNO and R) sublines of the prostate cancer model LNCaP numerical and structural aberrations of chromosome 8 were investigated in detail. The techniques used were whole chromosome paint (WCP) and fluorescence in situ hybridization (FISH) with three cosmid probes mapping to different parts of the p-arm (D8S7 (8p23.3), LPL (8p22) and PLAT (8p11.1)). By WCP all four cell lines showed four copies of chromosome 8 in most cells. However, FISH demonstrated that in all sublines deletions in the 8p region were present. The majority of both FGC and P70 had two copies of cosmids D8S7 and LPL. The cosmid PLAT showed a broader distribution (1–4 copies), especially in P70. Compared with FGC and P70, both LNO and R showed a larger number of copies (3 or 4) of all three cosmid loci. It is discussed that this difference is probably the result of nondisjunction as a reaction to loss of other sequences on 8p, possibly the tumor suppressor gene (TSG) mapping to 8p21. The fact that both sublines LNO and R are androgen-independent raises the possibility of a link between TSG loss on 8p and androgen independence.

Abstract C97: Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling

Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

27 Kruisresistentie tussen taxanen en de nieuwe hormonale middelen abiraterone en enzalutamide heeft potentiële implicaties voor de volgorde van behandeling van het gemetastaseerd castratieresistent prostaatcarcinoom

SamenvattingDe behandelmogelijkheden voor patiënten met gemetastaseerd castratieresistent prostaatcarcinoom (mCRPC) zijn de laatste jaren sterk toegenomen door de introductie van nieuwe middelen als cabazitaxel, abiraterone en enzalutamide.

22 Nieuw in-vivo-progressiemodel voor castratieresistent prostaatcarcinoom

SamenvattingCastratieresistent prostaatcarcinoom (CRPC) kan worden behandeld met de hormoonsynthese (CYP17A1) remmer abiraterone, of het antiandrogeen enzalutamide.

1 Castratieresistente prostaatkankergroei berust op omzetting van bijnierandrogenen naar DHT en niet op intrinsieke hormoonsynthese

Introductie Castratieresistente prostaatkanker (CRPC) kenmerkt zich onder andere door een stijgend PSA, wat duidt op activiteit van de androgeenreceptor (AR), ondanks lage serumtestosteronwaarden. Twee mogelijke verklaringen voor deze AR-activatie zijn: 1) de-novohormoonsynthese en 2) conversie van bijnierandrogenen naar DHT in prostaatkanker (PC) cellen. We hebben in een in-vitrostudie deze hypothesen getest door de groei in hormoonnaïeve (HNPC-) en CRPC-cellijnen te stimuleren met de substraten voor de-novosynthese (de hormoonprecursors progesteron en pregnenolon) of met de bijnierandrogenen DHEA en androsteendion. Daarnaast hebben we een cocultuurmodel ontwikkeld om groeistimulatie van PC door de bijnier en behandeling met de bijnierandrogeen-syntheseremmer orteronel te testen.