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Prostate cancer remains the second most common cancer in men, with diverse courses from indolent cases to aggressive diseases. Among the key factors implicated in its pathogenesis are genomic alterations such as the TMPRSS2-ERG and related fusion oncogenes, loss of tumor suppressor PTEN, p53 or NKX3.1, inflammation, enhanced DNA damage, and chromosomal instability. Men with prostate cancer who carry BRCA1/2 mutations are at more risk of worse disease and poor prognosis. Cancer cells with mutant BRCA1 or BRCA2 repair genes with defects in homologous recombination are vulnerable to PARP inhibitors that target the genetic phenomenon known as synthetic lethality to exploit faulty DNA repair mechanisms. With relevance to prostate cancer, other features of cancer cells may also sensitize to PARP inhibitors, including aberrant transcription due to the androgen-driven fusion oncogene TMPRSS2-ERG or PTEN loss. Several models of synthetic lethality and potential biomarkers suggested up to date are also discussed. The chapter also highlights the importance of genetic screening of men with BRCA and shows diagnostic utility of plasma-derived circulating tumor DNA.

Extracellular matrix (ECM) is a complex three-dimensional network that provides structure, strength, and contextual information for cellular growth, communication, differentiation, survival, adhesion, and migration. ECM basic proteins resist compressive forces and/or allow rapid diffusion, others strengthen the matrix, and give resilience or modulate cell-matrix interactions. ECM undergoes turnover and remodeling physiologically and during inflammation, wound repair and tumor invasion. Remodeling of the ECM is an integral component of the angiogenic process and depends on the composition of matrix molecules, soluble pro-angiogenic and anti-angiogenic factors, and their spatial regulation. This review will focus on the myriad roles of those molecules and will emphasize their involvement in critical points of angiogenesis.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.

Background Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. Methods Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. Results Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. Conclusions From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa.

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.

Sphingosine 1-phosphate (S1P) is a bioactive lipid metabolite associated with cancer cell proliferation, survival, migration and regulation of tumor angiogenesis in various cellular and animal models. Sphingosine kinase-1 (SphK1) and S1P lyase are the main enzymes that respectively control the synthesis and degradation of S1P. The present study analyzed the prognostic and predictive value of SphK1 and S1P lyase expression in patients with non-small cell lung cancer (NSCLC), treated with either surgery alone or in combination with adjuvant carboplatin and navelbine. Formalin-fixed, paraffin-embedded tissue samples from 176 patients with NSCLC were stained immunohistochemically using antibodies against SphK1 and S1P lyase, and their expression was correlated with all available clinicopathological factors. Increased expression of SphK1 was significantly associated with shorter overall and disease free survival in patients treated with adjuvant platinum-based chemotherapy. No prognostic relevance for S1P lyase expression was observed. Collectively, the results suggest that the immunohistochemical detection of SphK1 may be a promising predictive marker in NSCLC patients treated with adjuvant platinum-based chemotherapy Refereed/Peer-reviewed

Skp2 is a crucial component of SCFSkp2 E3 ubiquitin ligase and is often overexpressed in various types of cancer, including prostate cancer (PCa). The epithelial-to-mesenchymal transition (EMT) is involved in PCa progression. The acquisition of a mesenchymal phenotype that results in a cancer stem cell (CSC) phenotype in PCa was described. Therefore, we aimed to investigate the expression and localization of Skp2 in clinical samples from patients with PCa, the association of Skp2 with EMT status, and the role of Skp2 in prostate CSC. We found that nuclear expression of Skp2 was increased in patients with PCa compared to those with benign hyperplasia, and correlated with high Gleason score in PCa patients. Increased Skp2 expression was observed in PCa cell lines with mesenchymal and CSC-like phenotype compared to their epithelial counterparts. Conversely, the CSC-like phenotype was diminished in cells in which SKP2 expression was silenced. Furthermore, we observed that Skp2 downregulation led to the decrease in subpopulation of CD44+CD24− cancer stem-like cells. Finally, we showed that high expression levels of both CD24 and CD44 were associated with favorable recurrence-free survival for PCa patients. This study uncovered the Skp2-mediated CSC-like phenotype with oncogenic functions in PCa.

Asporin has been reported as a tumor suppressor in breast cancer, while asporin-activated invasion has been described in gastric cancer. According to our in silico search, high asporin expresion associates with significantly better relapse free survival (RFS) in patients with low-grade tumors but RFS is significantly worse in patients with grade 3 tumors. In line with other studies, we have confirmed asporin expression by RNA scope in situ hybridization in cancer associated fibroblasts. We have also found asporin expression in the Hs578T breast cancer cell line which we confirmed by quantitative RT-PCR and western blotting. From multiple testing, we found that asporin can be downregulated by bone morphogenetic protein 4 while upregulation may be facilited by serum-free cultivation or by three dimensional growth in stiff Alvetex scaffold. Downregulation by shRNA inhibited invasion of Hs578T as well as of CAFs and T47D cells. Invasion of asporin-negative MDA-MB-231 and BT549 breast cancer cells through collagen type I was enhanced by recombinant asporin. Besides other investigations, large scale analysis of aspartic acid repeat polymorphism will be needed for clarification of the asporin dual role in progression of breast cancer.

// Eva Slabakova 1, 2 , Gvantsa Kharaishvili 3 , Monika Smějova 1, 4 , Zuzana Pernicova 1, 2 , Tereza Suchankova 1 , Jan Remsik 1, 2, 5 , Stanislav Lerch 1, 5 , Nicol Strakova 1, 2 , Jan Bouchal 3 , Milan Kral 6 , Zoran Culig 2, 7 , Alois Kozubik 1, 5 , Karel Soucek 1, 2, 5 1 Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic 2 Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne’s University Hospital Brno, Brno, Czech Republic 3 Department of Clinical and Molecular Pathology and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic 4 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic 5 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 6 Department of Urology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic 7 Division of Experimental Urology, Department of Urology, Medical University of Innsbruck, Austria Correspondence to: Karel Soucek, e-mail: ksoucek@ibp.cz Eva Slabakova, e-mail: slabakova@ibp.cz Keywords: epithelial-mesenchymal transition, MDM2/MDMX, SNAI2/SLUG, TWIST, prostate/breast cancer Received: July 28, 2015 Accepted: September 15, 2015 Published: September 25, 2015 ABSTRACT Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.

Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action.

Background: Breast cancer (BC), the most frequent malignancy in women worldwide, is currently diagnosed in about 1.4 million female patients annually. Approximately 10-20% of BC is represented by triple negative breast cancer (TNBC) which is aggressive, the prognosis is poor and patients cannot benefit from targeted treatment based on hormonal or HER2 receptors. For this reason, search for markers that can predict the efficacy of chemotherapy in TNBC is a priority. Methods and Results: This review focuses on BCL2 protein as a prognostic marker in TNBC and its potential as a predictor of sensitivity to chemotherapy. Conclusion: BCL2 protein expression is a positive prognostic factor in BC. Better survival of patients with BCL2 positivity (BCL2+) has been explained by the correlation with estrogen receptor positive (ER+) status. BCL2+ is however not simply a surrogate marker for ER+. Moreover, BCL2 protein expression is also a positive prognostic marker in the TNBC subgroup. We and others show, that low BCL2 expression was associated with good outcome of TNBC patients treated with both adjuvant and neoadjuvant anthracycline-based chemotherapy. On the other hand, recent studies have shown that a subset of TNBC patients may benefit from the classical adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) regimen. Given the heterogeneity of TNBC there is an urgent need to find and validate the sensitivity predictors to these regimens making them usable in clinical practice. BCL2 enrichment has been described in the mesenchymal stem-like (MSL) TNBC subgroup.

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.

Recently, miR-23b has emerged as a promising new cancer biomarker but its role in lung cancer has not been established yet. Patients still do not respond well to available treatments, probably due to expression of multidrug resistance (MDR) proteins, such as P-gp, MRP and LRP/MVP. The aim of this study was to determine the role of miR-23b in non-small cell lung cancer (NSCLC) and its relationship to the patient outcome together with MDR transporter proteins. We immunohistochemically evaluated expression of P-gp, MRP and LRP/MVP and quantified the relative levels of miR-23b in 62 NSCLC patients´ samples. The prognostic significance of miR-23b and MDR proteins was tested by Kaplan-Meier and Cox-regression analysis. Our results showed that miR-23b is mostly downregulated in NSCLC samples (57/62) and that its upregulation in tumors is connected with longer progression-free survival (PFS; P = 0.065) and overall survival (OS; P = 0.048). The Cox proportional hazard model revealed that the risk of death or relapse in NSCLC patients with miR-23b downregulation increases together with LRP/MVP expression and both risks decrease with miR-23b upregulation (HRPFS = 4.342, PPFS = 0.022; HROS = 4.408, POS = 0.015). Our findings indicate that miR-23b, especially in combination with LRP/MVP expression, might serve as a suitable prognostic biomarker for NSCLC patients.

Due to the urgent need for new prostate cancer (PCa) therapies, the role of androgen receptor (AR)-interacting proteins should be investigated. In this study we aimed to address whether the AR coactivator nuclear receptor coactivator 1 (NCOA1) is involved in PCa progression. Therefore, we tested the effect of long-term NCOA1 knockdown on processes relevant to metastasis formation. [(3)H]-thymidine incorporation assays revealed a reduced proliferation rate in AR-positive MDA PCa 2b and LNCaP cells upon knockdown of NCOA1, whereas AR-negative PC3 cells were not affected. Furthermore, Boyden chamber assays showed a strong decrease in migration and invasion upon NCOA1 knockdown, independently of the cell line's AR status. In order to understand the mechanistic reasons for these changes, transcriptome analysis using cDNA microarrays was performed. Protein kinase D1 (PRKD1) was found to be prominently up-regulated by NCOA1 knockdown in MDA PCa 2b, but not in PC3 cells. Inhibition of PRKD1 reverted the reduced migratory potential caused by NCOA1 knockdown. Furthermore, PRKD1 was negatively regulated by AR. Immunohistochemical staining of PCa patient samples revealed a strong increase in NCOA1 expression in primary tumors compared with normal prostate tissue, while no final conclusion could be drawn for PRKD1 expression in tumor specimens. Thus, our findings directly associate the AR/NCOA1 complex with PRKD1 regulation and cellular migration and support the concept of therapeutic inhibition of NCOA1 in PCa.

Background: Small leucine rich proteoglycans (SLRPs), major non-collagen components of the extracellular matrix (ECM), have multiple biological roles with diverse effects. Asporin, a member of the SLRPs class I, competes with other molecules in binding to collagen and affects its mineralization. Its role in cancer is only now being elucidated. Methods: The PubMed online database was used to search relevant reviews and original articles. Furthermore, altered asporin expression was analysed in publicly available genome-wide expression data at the Gene Expression Omnibus database. Results: Polymorphisms in the N-terminal polyaspartate domain, which binds calcium, are associated with osteoarthritis and prostate cancer. Asporin also promotes the progression of scirrhous gastric cancer where it is required for coordinated invasion by cancer associated fibroblasts and cancer cells. Besides the enhanced expression of asporin observed in multiple cancer types, such as breast, prostate, gastric, pancreas and colon cancer, tumour suppressive effects of asporin were described in triple-negative breast cancer. We also discuss a number of factors modulating asporin expression in different cell types relevant for alterations toing the tumour microenvironment. Conclusion: The apparent contradicting tumour promoting and suppressive effects of asporin require further investigation. Deciphering the role of asporin and other SLRPs in tumour-stroma interactions is needed for a better understanding of cancer progression and potentially also for novel tumour microenvironment based therapies.

An actin-binding protein filamin A connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Recently, it has been reported that filamin A is required for efficient regulation of early stages of DNA repair process. Moreover, some in vitro studies showed that the overexpression of filamin A determines resistance to various cytotoxic drugs, including cisplatin. We aimed to analyse the expression of filamin A protein in resected NSCLC (Non Small Cell Lung Cancer) specimens, to investigate the association of the level of filamin A protein expression and other clinicopathological features, and possible relationship between the expression of filamin A and survival outcome in NSCLC patients, treated with platinum-based combination chemotherapy. We performed filamin A protein immunohistochemistry on formalin-fixed and paraffin-embedded (FFPE) tissue sections from 135 NSCLC patients, using EP2405Y antibody against C-terminus of filamin A. Cytoplasmic, membranous and nuclear positivity of filamin A was evaluated semi-quantitatively and correlated with available clinicopathological data. Patients were divided into two groups for survival analysis (I group - patients treated with adjuvant platinum-based chemotherapy, II group - patients with surgical treatment only). We found significant positive correlation between filamin A protein expression and NSCLC stage (r=0.249; p

Background Collagen triple helix repeat containing 1 (CTHRC1) affects Wnt signalling, collagen deposition and bone formation. It is an extracellular matrix protein which is also abnormally expressed in the tumour microenvironment. CTHRC1 has not been studied in breast cancer by immunohistochemistry. Aims To examine expression of CTHRC1 together with periostin and versican in breast cancer patients and investigate its association with clinicopathological characteristics. Methods The formalin-fixed paraffin-embedded tissues of 173 invasive carcinomas (classified into WHO histotypes and luminal, triple negative and Her2 subtypes), as well as normal tissues, precursor lesions and metastatic lymph nodes were stained by relevant antibodies, assessed semiquantitatively by histoscore and statistically evaluated. Results Expression of CTHRC1, versican and periostin was significantly higher in breast cancer than in normal tissue or precursor lesions. CTHRC1 stromal expression was enhanced in triple negative cases and also in patients with bone metastasis. Periostin expression was high in primary tumours, in particular triple negative ones, and also in their lymph node metastases. Cox regression analysis showed that in patients with high periostin, the risk of bone metastases increased with increased CTHRC1 expression. Conclusions CTHRC1 and periostin play important roles in breast cancer progression. These preliminary results show that combined evaluation of CTHRC1 and periostin could serve as a potential marker for breast cancer bone metastasis; the other observations contribute to the description of the tumour microenvironment, with implications for lymph node and bone metastasis.

Introduction: The Skp2 F-box protein is the substrate recruiting component of the SCF (Skp1-Cullin 1-F-box) type of E3 ubiquitin-ligase complexes. Skp2 plays an important role in prostate tumorigenesis (e.g., via recently reported stabilization of EZH2 or Twist1), which needs further elucidation. Patients and Methods: Prostate cancer patient cohort (N=101) was analyzed by immunohistochemistry for the following proteins: Skp2, AR, Ki-67, Slug, E-cadherin, MdmX, Mdm2, and p53. The indicated proteins were also analyzed in seminal vesicles and lymph node metastases (SV=35 and LN=18 patients, respectively), as well as in benign prostatic hyperplasia (BPH=34). Colocalization analysis was performed using Perkin Elmer Opal Multiplex kit, Vectra 3.0 imaging system, and confocal microscope Carl Zeiss LSM 780. Independent prostate cancer cohort (N=30) was evaluated for TMPRSS2-ERG fusion by a triple-color deletion assay and assessed for Skp2, ERG, and NQO1 protein expression. Cox regression analysis and Kaplan-Meier curves were used to assess importance of Skp2, Slug, EZH2, and Twist1 for biochemical relapse in the TCGA prostate cancer dataset (N=488, RNA seq). Results: Expression of E-cadherin was lower in primary prostate cancer and seminal vesicles compared to BPH and LN, which may be linked to epithelial-mesenchymal transition. Opposite expression profile was observed for Skp2, MdmX, and Slug. High Gleason score (≥8, N=30) was significantly associated with higher Skp2 and lower E-cadherin expression (p Conclusion: Immunohistochemistry and colocalization studies revealed association between Skp2 and Slug in aggressive prostate cancer. Other factors such as TMPRSS2-ERG fusion or oxidative stress may contribute to prostate cancer progression. Citation Format: Jan Bouchal, Gvantsa Kharaishvili, Alena Mickova, Mariam Gachechiladze, Alena Burdova, Martin Mistrik, Milan Kral, Balazs Gyorffy, Zdenek Kolar. Skp2 associates with Slug and androgen receptor in aggressive 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 A006.

Dissemination of cancer cells to distant organs has fatal consequences to most of the patients with malignant tumors. Patients with prostate and breast cancer show apparent overlap of the most common sites of cancer metastasis, suggesting that breast and prostate tumor cells share common mechanisms of dissemination and colonization. During each step of prostate and breast cancer metastasis, malignant cells display phenotypic plasticity that is associated with the manifestation of epithelial and mesenchymal properties or an epithelial-to-mesenchymal transition (EMT). One of the molecules that most likely interlink processes of pathologic plasticity of cancer cells, their dissemination capability, and response to microenvironmental factors is Tumor-Associated Calcium Signal TransDucer 2 (Trop-2, TACSTD2). Trop-2 is a type-I transmembrane glycoprotein encoded by TACSTD2 gene often associated with tumorigenesis and cancer progression, but the data remain controversial. Trop-2 deregulation has been repeatedly proposed as an event associated with cancer progression and poor patient prognosis. Instead of this simplistic view, our results showed Trop-2 level change as a context-dependent, dynamic event associated with cancer plasticity and dissemination. Using antibody-based surface profiling of selected cancer stem-like cell markers in human and mouse prostate and breast cancer cell lines, we identified subpopulation of Trop-2+ cells within culture of metastatic prostate cell line DU-145 and similarly Trop-2+ subpopulation within mouse mammary cancer cell line 4T1. Gene expression analysis of sorted subpopulations showed significant correlation of Trop-2 with epithelial phenotype, and this finding was further validated in wide panel of human and murine cell lines and independent patient datasets. We further proved that expression of Trop-2 is regulated by EMT transcription factors and DNA methylation. Moreover, immunohistochemical analysis of Trop-2 in pairs of primary prostate tumors and lymph node metastasis showed strong association with E-cadherin and epithelial-mesenchymal plasticity in patient samples. In conclusion, we showed that Trop-2 expression associates with epithelial phenotype and can be suppressed either epigenetically or through the action of EMT master regulators. Acknowledgments: This work was supported by Ministry of Health of the Czech Republic, grant no. 15-33999A and 15-28628A, and by GAČR 15-11707S, HistoPARK (CZ.1.07/2.3.00/20.0185), and by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) and ICRC-ERA-HumanBridge a.k.a. REGPOT (Grant agreement no. 316 345) from the European Regional Development Fund. Citation Format: Ján Remšík, Lucia Binó, Zuzana Kahounová, Gvantsa Kharaishvili, Sarka Simeckova, Radek Fedr, Tereza Nehybova, Eva Slabáková, Lucia Knopfová, Jan Bouchal, Milan Kral, Petr Benes, Karel Soucek. Trop-2 plasticity is driven by epithelial-to-mesenchymal transition in prostate cancer cells [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 B084.

Neither targeted therapies nor predictors for chemotherapy sensitivity are available for triple-negative breast cancer (TNBC). Our study included 187 patients with TNBC, 164 of whom were treated with anthracycline-based adjuvant chemotherapy. Eleven molecular biomarkers were analyzed. BCL2, epidermal growth factor receptor (EGFR), MYC, TOP2A, and Ki-67 protein expression was evaluated by immunohistochemistry. The status of the EGFR, MYC, and TOP2A genes and chromosomes 7, 8, and 17 was assessed using fluorescence in situ hybridization. High BCL2 expression predicted poor relapse-free survival (RFS) in patients treated with anthracycline-based adjuvant chemotherapy (p = 0.035), poor breast cancer-specific survival (BCSS) (p = 0.048), and a trend to poor overall survival (OS) (p = 0.085). High levels of BCL2 expression predicted poor OS in basal-like (BL) TNBC patients treated with adjuvant anthracycline-based regimens (log-rank p = 0.033, hazard ratio (HR) 3.04, 95 % confidence interval (CI) 1.04–8.91) and a trend to poor RFS (log-rank p = 0.079) and poor BCSS (log-rank p = 0.056). Multivariate analysis showed that BCL2 status, tumor size, and nodal status all had independent predictive significance for RFS (p = 0.005, p = 0.091, p = 0.003, respectively; likelihood ratio test for the whole model, p = 0.003), BCSS (p = 0.012, p = 0.077, p = 0.01, respectively; likelihood ratio test for the whole model, p = 0.016), and OS (p = 0.008, p = 0.004, p = 0.004, respectively; likelihood ratio test for the whole model, p = 0.0006). Similarly, multivariate analysis for BL TNBC showed BCL2, tumor size, and nodal status all had independent predictive significance for RFS (likelihood ratio test for the whole model, p = 0.00125), BCSS (p = 0.00035), and OS (p = 0.00063). High EGFR expression was associated with poor BCSS (p = 0.039) in patients treated with anthracycline-based adjuvant chemotherapy. Patients who underwent anthracycline-based adjuvant chemotherapy and exhibited CMYC amplification had a trend to worse BCSS (p = 0.066). In conclusion, high BCL2 expression is a significant independent predictor of poor outcome in TNBC patients treated with anthracycline-based adjuvant chemotherapy, and this is the first study showing the BCL2 prediction in BL TNBC. BCL2 expression analysis could facilitate decision making on adjuvant treatment in TNBC patients.

Background Tumor heterogeneity and the plasticity of cancer cells present challenges for effective clinical diagnosis and therapy. Such challenges are epitomized by neuroendocrine transdifferentiation (NED) and the emergence of neuroendocrine-like cancer cells in prostate tumors. This phenomenon frequently arises from androgen-depleted prostate adenocarcinoma and is associated with the development of castration-resistant prostate cancer and poor prognosis. Results In this study, we showed that NED was evoked in both androgen receptor (AR)-positive and AR-negative prostate epithelial cell lines by growing the cells to a high density. Androgen depletion and high-density cultivation were both associated with cell cycle arrest and deregulated expression of several cell cycle regulators, such as p27Kip1, members of the cyclin D protein family, and Cdk2. Dual inhibition of Cdk1 and Cdk2 using pharmacological inhibitor or RNAi led to modulation of the cell cycle and promotion of NED. We further demonstrated that the cyclic adenosine 3′, 5′-monophosphate (cAMP)-mediated pathway is activated in the high-density conditions. Importantly, inhibition of cAMP signaling using a specific inhibitor of adenylate cyclase, MDL-12330A, abolished the promotion of NED by high cell density. Conclusions Taken together, our results imply a new relationship between cell cycle attenuation and promotion of NED and suggest high cell density as a trigger for cAMP signaling that can mediate reversible NED in prostate cancer cells.

Background: In an earlier publication we identified asporin as a novel cancer related protein (BMC Cancer 2007, 7:55), also reported among bone metastasis specific genes (Cancer Letters 2009, 276:212-220). Asporin is a member of the class I small leucine-rich repeat proteoglycan family and besides affinity to collagen, it binds calcium and initiates collagen mineralization. Asporin is abundantly expressed in tissues with high remodeling rate (e.g. peridontal ligament) whereas extremely high turnover of matrix proteins is also associated with invasive cancer. Material and methods: Formalin-fixed paraffin-embedded tissues [invasive ductal (101) and lobular (39) carcinomas classified according to hormone receptors and Her-2; 5 bone metastases from autopsies] were immunohistochemically stained with in-house polyclonal peptide antibody against asporin. The antibody was validated on uterus tissue as a positive control together with a commercial antibody (Abcam). Data were evaluated by non-parametric Mann-Whitney U test and Spearman correlation coefficient. Expression of asporin in both tissues and cell lines was also queried in databases Gene Expression Omnibus, ArrayExpress and Oncomine. Expression of asporin was further tested by RT-PCR in selected breast cancer cell lines. Migration and invasion of MDA-MB-231 cells were tested using real-time cell analysis Xcelligence system (Roche). Transwells were coated by solidifying collagen for 6 hours (with or without recombinant asporin). Starved cells were seeded on the collagen in transwells while FBS was used as a chemoattractant in bottom wells. Results: Levels of asporin were significantly higher in invasive lobularcarcinomas than ductal ones (P Conclusion: Asporin is expressed by both invasive breast carcinomas and bone metastases. Mechanistically, asporin may affect extracellular matrix structure and promote invasive growth of breast cancer cells. Further investigation of the role of asporin in metastasizing to bone is additionally justified given its affinity for calcium. Acknowledgements This work was supported by grants NS 9956-4 from the Czech Ministry of Health and MSM 6198959216 from the Czech Ministry of Education. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-18.

Background. The Wnt signaling pathway is crucial for cell fate decisions, stem cell renewal, regulation of cell proliferation and differentiation. Deregulated Wnt signaling is also implicated in a number of hereditary and degenerative diseases and cancer. Methods and results. This review highlights the role of the Wnt pathway in the regulation of stem/progenitor cell renewal and prostate gland development and how this signaling is altered in prostate cancer. Recent evidence suggests that Wnt signaling regulates androgen activity in prostate cancer cells, enhances androgen receptor expression and promotes the growth of prostate cancer even after androgen ablation therapy. There is also strong evidence that Wnt signaling is enhanced in androgen-ablation resistant tumors and bone metastases. Conclusions. Further study of the modulators of this pathway will be of therapeutic relevance as inhibition of Wnt signaling may have the potential to reduce the self-renewal and aggressive behaviour of prostate cancer while Wnt signaling activation might enhance stem cell activity when tissue regeneration is required.

Background Bioactive sphingolipid sphingosine-1-phosphate (S1P) plays an important role in various aspects of cancer biology, including cell proliferation, transformation, survival and migration. Sphingosine kinase 1 (SphK1) is a key enzyme, responsible for S1P production from sphingosine. On the other hand, S1P is irreversibly degraded by the sphingosine-1-phosphate lyase (SPL). We aimed to investigate the possible importance of the immunohistochemical expression of Sphk1 and S1P lyase in patients with NSCLC. Methods Tumor tissue microarrays (TMA) were constructed from 201 paraffin blocks consisted of representable parts of primary lung cancer. TMA blocks were cut at 4 μm sections and stained for antibodies against Sphk1 and SPL, according to standard immunohistochemical (IHC) protocol. IHC stained slides were evaluated by two independent investigators using histoscore method. Correlations between the levels of both examined proteins and all available clinicopathological features were analyzed using SPSS statistical software. Results Cox-regression analysis showed that in patients with stage III-IV NSCLC, without neo-adjuvant chemotherapy, the expression of SphK1 positively correlates with better disease free survival (DFS); increased SphK1 expression is associated with decreased risk of relapse ( p = 0.020, HR = 0.383). The same analysis also showed that the risk of NSCLC relapse is significantly higher in stage III–IV patients positive for SPL ( p = 0.0003, HR = 1.005), while there is no difference in the SPL negative group. Conclusion According to our study results immunohistochemical expression of Sphk1 and SPL might serve as additional useful prognostic markers for patients with advanced non-small-cell lung cancer; however, more extended study is necessary.

1132 Background: The role of BRCA mutation status as a predictor in TNBC is unclear. Data show an association of high BCL2 expression and resistance to anthracyclines. BCL2, size and nodal status a...

Triple-negative breast cancers (TNBCS) are aggressive with poor prognosis. Patients cannot benefit from targeted treatment. Moreover, little is known of which TNBC patients will benefit from nontargeted adjuvant treatment. The objective was to search for predictors of adjuvant chemotherapy in TNBC.

We have shown that nestin expression is higher in breast carcinoma with a basal phenotype [1] and collagen triple helix repeat containing 1 (CTHRC1) and periostin may predict bone metastasis of breast cancer [2]. Our aim therefore was to examine the simultaneous role of nestin and CTHRC1 in breast cancer progression.

1087 Background: Preclinical data show an association of BCL2 expression and resistance to anthracyclines. The absence of BCL2 expression in prechemotherapy samples is associated with a higher probability of pathological complete response to neoadjuvant doxorubicin-based chemotherapy. The aim of our research is identification of markers predicting sensitivity to adjuvant treatment in TNBC. Here we focus on BCL2 protein as a putative predictor of sensitivity to adjuvant anthracycline-based chemotherapy. The objective was to determine whether BCL2 expression predicts relapse in TNBC patients treated with anthracycline-based regimens. Methods: The study included 187 patients with TNBC, 178 of whom were treated with adjuvant chemotherapy (164 had anthracyclines). BCL2 analysis was performed using IHC, proportion score and intensity score were counted. The data were analysed with software Statistica and R. Results: High BCL2 expression predicts poor relapse free survival (RFS) in patients treated with adjuvant anthracycline-based regimens (logrank p=.035, hazard ratio, HR 2.37, 95%CI 1.04-5.41). High BCL2 predicts trend to poor overall survival (OS) in patients treated with adjuvant anthracycline-based regimens (logrank p=0.075, HR 2.31, 95%CI 0.90-5.97). In univariate analysis of anthracycline treated patients, stage (RFS p=.0004, OS p=.0005), size (RFS p=.003, OS p=.00009) and nodal status (RFS p=0.018, OS p=.028) were associated with outcome, as well. In multivariate analysis of athracycline treated patients, BCL2, size and nodal status had an independent predictive significance for both RFS (p=.005, p=.056, p=.003) (logrank test, p=0.0004) and OS (p=.014, p=.006, p=.012) (logrank test p=0.0007). Conclusions: This study is the first to prove that high BCL2 expression predicts poor outcome in TNBC treated with adjuvant anthracycline-based chemotherapy. BCL2 expression could facilitate decision making on adjuvant treatment in TNBC patients and its assessment should be included in standard diagnostics. In patients with high BCL2 expression other types of adjuvant treatment should be considered. Grants: IGA NS10286,IGA NS10357-3 and Biomedreg CZ.1.05/2.1.00/01.0030.

Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.