Eugenio Zoni

A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response

Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening.

Correction to: Store-Operated Ca2+ Entry as a Prostate Cancer Biomarker — a Riddle with Perspectives

The article Store-Operated Ca2+ Entry as a Prostate Cancer Biomarker — a Riddle with Perspectives, written by Sven Kappel, Ines Joao Marques, Eugenio Zoni, Paulina Stokłosa, Christine Peinelt, Nadia Mercader, Marianna Kruithof-de Julio, and Anna Borgström, was originally published electronically.

Abstract A068: Metabolic signature in lethal vs. nonlethal prostate cancer

Introduction and Aim: Prostate cancer (PCa) is the most frequent malignancy detected in males and the second leading cause of death from cancer in males. Despite the current diagnostic standards, mainly based on PSA detection, histologic grading, and tumor-lymph nodes-metastases (TNM) staging, there is still an urgent need for the identification of new prognostic markers to help in the definition of an individualized and personalized treatment for patients. Metabolites reflect gene expression and protein function and perturbed metabolism is considered a novel “hallmark of cancer.” Therefore, the analysis of the metabolomic profiling of high-risk and lethal PCa might help in the characterization of the molecular characteristics of this subgroup of patients. In this study, we aimed to identify the metabolic signature for lethal prostate cancer, in order to characterize those patients at highest risk of metastatic progression. Experimental Procedure: We employed high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI) to detect and visualize metabolites in formalin-fixed, paraffin-embedded (FFPE) PCa tissue microarrays (TMAs) in a cohort of 211 PCa patients who underwent radical prostatectomy between 1994 and 2001. To calculate the prognostic power of measured metabolites, we separated the patients’ cohort into good and poor survivor groups by the application of intensity cut-offs optimized to the clinical endpoint (e.g., PSA-free survival). Statistical differences in patient survival were measured using the Kaplan-Meier log-rank test and multivariate survival analysis performed using Cox proportional hazards regression models. Results: We employed a metabolic profiling approach to distinguish lethal from nonlethal disease in a subset of lethal PCa patients (PCa-related death within 5 years following radical prostatectomy) vs. nonlethal PCa patients (survival > 10 years following radical prostatectomy, without evidence of recurrence). By virtual microdissection molecular signatures were selected from tumor regions and subsequently statistically compared, leading to 172 small molecules significantly different between lethal and nonlethal PCa patients (p Next, we used metabolite MSI data to address patient survival outcome between lethal and nonlethal PCa cases. Statistical analysis showed that different m/z species significantly correlated with patients’ outcome (e.g., PSA-free survival and disease-free survival). Preliminary pathway enrichment analysis displayed a significant upregulation of purine and pyrimidine metabolism KEGG pathway in lethal vs. nonlethal disease. Strikingly, these networks have been previously associated with PCa progression in RNA-based studies. Conclusion: In this study we identified a metabolic pattern able to discriminate between lethal and nonlethal disease. Further validation of an independent cohort is needed to validate the novel findings of metabolites and might lead to the identification of novel biologic markers involved in PCa progression. Citation Format: Eugenio Zoni, Achim Buck, Annette Feuchtinger, Martin Spahn, Axel Walch, Marianna Kruithof-de Julio. Metabolic signature in lethal vs. nonlethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A068.

PD33-06 MCAM SUPPORTS THE AGGRESSIVE PHENOTYPE IN HUMAN PROSTATE CANCER

METHODS: Formalin-fixed paraffin embedded (FFPE) primary prostate cancer samples were obtained from the Welsh Cancer Bank. Targeted-NGS was performed using the Life Technologies Ion Torrent: Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion Personal Genome Machine sequencer. The hotspot panel covers ~2800 COSMIC mutations of 50 oncogenes and tumour suppressor genes. Standard IHC techniques were also used concentrating on markers of the Wnt, PI3Kinase (PI3K) and MAP-Kinase (MAPK) oncogenic signalling pathways. RESULTS: 61 primary prostate cancer samples were sequenced, 58 from radical retropubic prostatectomy (RRP) specimens and 3 from transurethral resection of prostate (TURP) sections, with a range of Gleason Scores (GS). 21/61 (34.4%) samples harboured a mutation in a cell cycle pathway gene such as TP53 or RB1 and 3/61 (4.9%) in a DNA repair gene such as ATM. 10/61 (16.5%) of samples harboured a mutation in a gene associated with the Wnt pathway such as APC or CTNNB1. 14/61 (23.0%) of samples analysed had a mutation in a gene commonly associated with the PI3K pathway such as PTEN or AKT1. 5/61 (8.2%) had a mutation in a gene associated with the MAPK pathway such as KRAS. IHC profiles were analysed on 317 prostate samples: 73 normal and 244 cancers. There was greater expression of markers associated with Wnt, PI3K and MAPK signalling pathways in prostate cancer samples when compared to normal samples. There was greater expression in high-risk GSs with some markers associated with biochemical recurrence following RRP. Furthermore, we were able to separate lowand high-risk GS samples based on molecular profiles using markers of the Wnt, PI3K and MAPK and principle components analysis. CONCLUSIONS: Targeted NGS and IHC can identify recurrent mutations and signalling pathway aberrations within primary prostate cancer samples, which have potential to be targeted and used in routine clinical practice. In addition, the molecular signatures of lowand highrisk are different and can be separated using a combination of markers and IHC. This finding could explain the marked difference in the behaviours of these tumours types.

MP44-17 PATIENT DERIVED XENOGRAFTS AS PRECLINICAL MODELS OF UROLOGICAL MALIGNANCIES

INTRODUCTION AND OBJECTIVES: The cross-talk of mammalian target of rapamycin (mTOR) pathway is clinical limitation of mTOR inhibitor for the treatment of urothelial carcinoma (URCa). This study is to search mTOR pathway downstream genes to overcome cross-talk at non muscle invasive low grade (LG)-URCa of the bladder. METHODS: Gene expression patterns, gene ontology, and gene clustering by dual (p70S6K and S6K) siRNAs or rapamycin in 253J and TR4 cell lines were investigated by microarray analysis and we selected mTOR/S6K pathway downstream genes which were suppressed to siRNAs, and rapamycin up-regulated or rapamycin down-regulated. We validated mTOR downstream genes with immunohistochemistry using tissue microarray of 90 non-muscle invasive LG-URCa patients whether genes can predict clinical outcomes, and knockout study to evaluate the synergistic effect with rapamycin. RESULTS: In the microarray analysis, we selected mTOR pathway downstream genes which consisted of 4 rapamycin downregulated (FOXM1, KIF14, MYBL2, and UHRF1), and 4 rapamycin upregulated (GPR87, NBR1, VASH1 and PRIMA1). In tissue microarray, FOXM1, KIF14, and NBR1 were more expressed at T1, and MYBL2 and PRIMA1 were more expressed at tumor size more than 3 cm. In multivariate Cox regression model, KIF14 and NBR1 were significant predictors of recurrence in non-muscle invasive LG-URCa of the bladder. In NBR1 knock out model, rapamycin treatment showed synergistic effect to inhibit cell viability and colony forming ability compared to rapamycin only. CONCLUSIONS: KIF14 and NBR1 were mTOR/S6K pathway downstream genes to predict recurrence in non-muscle invasive LGURCa of the bladder and NBR1 knockout overcome the rapamycin coss-talk.

MP99-16 MIR-221 MODULATES TUMOR GROWTH IN VIVO AND IS A REGULATOR OF TGF-? SIGNALING IN HUMAN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Docetaxel (DTX) is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to DTX therapy and their disease will continue to progress. The mechanisms by which resistance develops is still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in DTX resistance in CRPC. METHODS: Total RNA from parental C4-2B prostate cancer cells and DTX resistant C4-2B cells (C4-2B TaxR) was submitted for small RNA deep sequencing. Data was analyzed to ascertain which miRNAs expressions were most altered in C4-2B TaxR cells compared to parental cells. Having identified an increase in miR-181a in resistant cells, its expression was modulated in C4-2B and C4-2B TaxR cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without DTX. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phosphop53 expression was assessed by western blot and apoptosis was measured by ELISA in C4-2B TaxR cells with inhibited miR-181a expression with or without DTX. RESULTS: miR-181a is significantly upregulated in C4-2B TaxR cells compared to parental C4-2B cells as analyzed by small RNA sequencing. Overexpression of miR-181a in C4-2B cells confers DTX and cabazitaxel resistance. Knockdown of miR-181a in C4-2B TaxR cells re-sensitizes them to treatment with both DTX and cabazitaxel. miR-181a knockdown alone induced apoptosis in C4-2B TaxR cells which is further enhanced by DTX. We next assessed if miR-181a altered expression or activity of ABCB1, which is overexpressed/active in C4-2B TaxR cells and promotes resistance to DTX by pumping the drug out of cells. We found that miR-181a does not impact ABCB1 expression or activity. Since we previously demonstrated that phosphop53 can modulate DTX sensitivity, we determined if miR-181a can alter p53 expression in C4-2B TaxR cells. Knockdown of miR-181a in C4-2B TaxR cells induced phospho-p53 expression, suggesting that miR-181a induced resistance to DTX is mediated by inhibition of phospho-p53 expression. CONCLUSIONS: Overexpression of mir-181a in C4-2B TaxR cells contributes to their resistance to DTX and inhibition of mir-181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and induction of apoptosis.

MP41-10 MODELLING PROSTATE CANCER USING PRIMARY AND METASTATIC CANCEROIDS

INTRODUCTION AND OBJECTIVES: A recent study showed that adipose-derived stem cells are able to counteract urethral stricture formation in rats. The aim of this study was to evaluate the feasibility of autologous adipose derived stromal vascular fraction (SVF) transplantation into male urethra stricture walls after direct vision incision of urethra (DVIU). METHODS: A prospective clinical study was undertaken after ethics approval and appropriate patient consent. The inclusion criteria were: male patients older than 18 years, with single short recurrent not-obliterating urethral stricture (<2 cm). The exclusion criteria were: patients not willing to consent, multiple strictures and those not deemed suitable for endoscopic management. Failure was defined as need for further interventions. Preoperative workup included history, examination, retrograde urethrogram (RGU), voiding cystourethrogram (VCUG), urine culture, renal function tests, AUA score, IIEF, PROM. Plastic surgery team performs liposuction to extract 50 mls of fat from the patient’s abdominal wall. 50ml fat-SVF was obtained using a Goog Manufacturing Practice collagenase (Celase , Cytori Therapeutics, San Diego, USA) according to a standard protocol. SVF was diluted in 5 ml saline solution for the injection. A cystoscopy was performed and the stricture evaluated, a glide wire was placed and an urethrotomy performed at 6 o’clock position in bulbar urethra. Gide wire was left in situ. The SVF solution was injected at the site of the stricture and on either side of the stricture. A 12 Fr urinary catheter was placed. The urinary catheter was removed after 24 hrs. Follow up was of 3.5 months. RESULTS: Two patients were included in the study. The main characteristics are reported in table 1. No local or systemic side effects or complications were recorded. No recurrence of urethral stricture was detected in both patients after 3.5 months. Table 1 CONCLUSIONS: This is the first study to demonstrate a successful autologous SVF transplantation in male urethral stricture after DVIU. Further studies are necessary to confirm the efficacy of SVF in preventing urethral stricture recurrence.

Emerging aspects of microRNA interaction with TMPRSS2-ERG and endocrine therapy.

Prostate cancer (PCa) is the most common malignancy detected in males and the second most common cause of cancer death in western countries. The development of the prostate gland, is finely regulated by androgens which modulate also its growth and function. Importantly, androgens exert a major role in PCa formation and progression and one of the hypothesized mechanism proposed has been linked to the chromosomal rearrangement of the androgen regulated gene TMPRSS2 with ERG. Androgens have been therefore used as main target for therapies in the past. However, despite the development of endocrine therapies (e.g. androgen ablation), when PCa progress, tumors become resistant to this therapeutic castration and patients develop incurable metastases. A strategy to better understand how patients respond to therapy, in order to achieve a better patient stratification, consists in monitoring the levels of small noncoding RNAs (microRNAs). microRNAs are a class of small molecules that regulate protein abundance and their application as biomarkers to monitor disease progression has been intensely studied in the last years. In this review, we highlight the interactions between microRNAs and endocrine-related aspects of PCa in tissues. We focus on the modulation of TMPRSS2-ERG and Glucocorticoid Receptor (GR) by microRNAs and detail the influence of steroidal hormonal therapies on microRNAs expression.

ALK1Fc Suppresses the Human Prostate Cancer Growth in in Vitro and in Vivo Preclinical Models

Prostate cancer is the second most common cancer in men and lethality is normally associated with the consequences of metastasis rather than the primary tumor. Therefore, targeting the molecular pathways that underlie dissemination of primary tumor cells and the formation of metastases has a great clinical value. Bone morphogenetic proteins (BMPs) play a critical role in tumor progression and this study focuses on the role of BMP9- Activin receptor-Like Kinase 1 and 2 (ALK1 and ALK2) axis in prostate cancer. In order to study the effect of BMP9 in vitro and in vivo on cancer cells and tumor growth, we used a soluble chimeric protein consisting of the ALK1 extracellular domain (ECD) fused to human Fc (ALK1Fc) that prevents binding of BMP9 to its cell surface receptors and thereby blocks its ability to activate downstream signaling. ALK1Fc sequesters BMP9 and the closely related BMP10 while preserving the activation of ALK1 and ALK2 through other ligands. We show that ALK1Fc acts in vitro to decrease BMP9-mediated signaling and proliferation of prostate cancer cells with tumor initiating and metastatic potential. In line with these observations, we demonstrate that ALK1Fc also reduces tumor cell proliferation and tumor growth in vivo in an orthotopic transplantation model, as well as in the human patient derived xenograft BM18. Furthermore, we also provide evidence for crosstalk between BMP9 and NOTCH and find that ALK1Fc inhibits NOTCH signaling in human prostate cancer cells and blocks the induction of the NOTCH target Aldehyde dehydrogenase member ALDH1A1, which is a clinically relevant marker associated with poor survival and advanced-stage prostate cancer. Our study provides the first demonstration that ALK1Fc inhibits prostate cancer progression, identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. Altogether, these findings support the validity of ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

Store-Operated Ca2+ Entry as a Prostate Cancer Biomarker — a Riddle with Perspectives

Purpose of ReviewStore-operated calcium entry (SOCE) is dysregulated in prostate cancer, contributing to increased cellular migration and proliferation and preventing cancer cell apoptosis. We here summarize findings on gene expression levels and functions of SOCE components, stromal interaction molecules (STIM1 and STIM2), and members of the Orai protein family (Orai1, 2, and 3) in prostate cancer. Moreover, we introduce new research models that promise to provide insights into whether dysregulated SOCE signaling has clinically relevant implications in terms of increasing the migration and invasion of prostate cancer cells.Recent FindingsRecent reports on Orai1 and Orai3 expression levels and function were in part controversial probably due to the heterogeneous nature of prostate cancer. Lately, in prostate cancer cells, transient receptor melastatin 4 channel was shown to alter SOCE and play a role in migration and proliferation. We specifically highlight new cancer research models: a subpopulation of cells that show tumor initiation and metastatic potential in mice and zebrafish models.SummaryThis review focuses on SOCE component dysregulation in prostate cancer and analyzes several preclinical, cellular, and animal cancer research models.