Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Inhibition of Cyclin-Dependent Kinase 8/Cyclin-Dependent Kinase 19 Suppresses Its Pro-Oncogenic Effects in Prostate Cancer.

Progression of prostate cancer (PCa) is characterized by metastasis and castration resistance after response to androgen deprivation. Therapeutic options are limited, causing high morbidity and lethality. Recent work reported pro-oncogenic implications of the Mediator subunits cyclin-dependent kinase (CDK) 8 and 19 for the progression of PCa. The current study explored the underlying molecular mechanisms of CDK8/CDK19 and tested effects of novel CDK8/CDK19 inhibitors. PC3, DU145, LNCaP, and androgen-independent LNCaP Abl were used for in vitro experiments. Two inhibitors and CDK19 overexpression were used to modify CDK8/CDK19 activity. MTT assay, propidium iodide staining, wound healing assay, Boyden chamber assay, and adhesion assay were used to investigate cell viability, cell cycle, migration, and adhesion, respectively. Peptide-kinase screen using the PamGene platform was conducted to identify phosphorylated targets. Combining CDK8/CDK19 inhibitors with anti-androgens led to synergistic antiproliferative effects and sensitized androgen-independent cells to bicalutamide. CDK8/CDK19 inhibition resulted in reduced migration and increased collagen I-dependent adhesion. Phosphorylation of multiple peptides linked to cancer progression was identified to be dependent on CDK8/CDK19. In summary, this study substantially supports recent findings on CDK8/CDK19 in PCa progression. These findings contribute to a better understanding of underlying pro-oncogenic effects, which is needed to develop CDK8/CDK19 as a therapeutic target in PCa.

The alternative renin-angiotensin-system (RAS) signalling pathway in prostate cancer and its link to the current COVID-19 pandemic.

BACKGROUND: The renin-angiotensin system is known to maintain blood pressure and body fluids. However, it has been found to consist of at least two major constituents, the classic and the alternative pathway, balancing and supporting each other's signalling in a very intricate way. Current research has shown that the renin-angiotensin system is involved in a broad range of biological processes and diseases, such as cancer and infectious diseases. METHODS AND RESULTS: We conducted a literature review on the interaction of the renin-angiotensin system and prostate cancer and explored the research on the possible impact of the SARS-CoV-2 virus in this context. This review provides an update on contemporary knowledge into the alternative renin-angiotensin system, its role in cancer, specifically prostate cancer, and the implications of the current COVID-19 pandemic on cancer and cancer care. CONCLUSION: In this work, we aim to demonstrate how shifting the RAS signalling pathway from the classic to the alternative axis seems to be a viable option in supporting treatment of specific cancers and at the same time demonstrating beneficial properties in supportive care. It however seems to be the case that the infection with SARS-CoV-2 and subsequent impairment of the renin-angiotensin-system could exhibit serious deleterious long-term effects even in oncology.

The Role of Urine and Washing Cytology in Primary Transurethral Resection of Bladder Tumours.

INTRODUCTION: Urine cytology (UC) is a recommended tool for the diagnosis of urothelial malignancies. Thus far, no specific recommendations regarding the role of washing cytology (WC) have been included in the guidelines. The goal of our study was to analyse the relationship between the histology of transurethrally (transurethral resection of the bladder [TURBT]) resected bladder tumours (BCa) and intraoperative UC or WC findings. MATERIALS AND METHODS: Five hundred consecutive primary TURBT cases conducted between November 2010 and 2015 at our department of the University Hospital Luebeck were retrospectively analysed. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of UC and WC were evaluated to detect BCa. Multivariate logistic regression models were fit to further examine associations between patient- and tumour-related factors and a bladder UC or WC positive for BCa. RESULTS: UC was performed in 297 patients, WC in 294 patients, and both in 261 patients. Sensitivity was 50.7% in UC, 58.1% in WC, and 62.1% for both tests combined. Specificity was 97.8% for UC, 98.0% for WC, and 96.4% for the combined tests. PPV was 98.0% for UC, 98.1% for WC, and 97.2% for combined tests. NPV was 47.8% for UC, 54.5% for WC, and 55.9% for the combined tests. The multivariate analyses revealed no association between positive UC or WC results and subsequent radical cystectomy (UC OR 1.35, 95% CI: 0.3-5.7; WC OR 2.0, 95% CI: 0.4-11.4). Neither UC nor WC was significantly correlated with local recurrence. CONCLUSIONS: Cytologic testing is an important diagnostic tool in BCa detection, showing acceptable sensitivity of around 60% and excellent specificity of over 90%. UC and WC present similar sensitivity. Our results advocate, however, against cytologic testing during primary TURBT, especially with regard to the lack of value in assessing the risk of recurrence. The clinical benefit of taking both types of samples at once is minimal. Furthermore, intraoperative WC collection does not reliably predict subsequent cystectomies.

TRIM21 Expression as a Prognostic Biomarker for Progression-Free Survival in HNSCC.

Patients with head and neck squamous cell carcinoma (HNSCC) continue to have a rather poor prognosis. Treatment-related comorbidities have negative impacts on their quality of life. TRIM21 is a cytosolic E3 ubiquitin ligase that was initially described as an autoantigen in autoimmune diseases and later associated with the intracellular antiviral response. Here, we investigated the role of TRIM21 as a biomarker candidate for HNSCC in predicting tumor progression and patient survival. We analyzed TRIM21 expression and its association with clinical-pathological parameters in our HNSCC cohort using immunohistochemistry. Our HNSCC cohort included samples from 419 patients consisting of primary tumors (n = 337), lymph node metastases (n = 156), recurrent tumors (n = 54) and distant metastases (n = 16). We found that cytoplasmic TRIM21 expression was associated with the infiltration of immune cells into primary tumors. In addition, TRIM21 expression was significantly higher in primary tumors than in lymph node metastases, and increased TRIM21 expression was correlated with shorter progression-free survival in HNSCC patients. These results suggest that TRIM21 could be a new biomarker for progression-free survival.

Targeting cyclin-dependent kinase 7-association between CDK7 and pMED1 expression in prostate cancer tissue.

Cyclin-dependent kinase (CDK) 7-mediated phosphorylation of Mediator-complex subunit 1 (MED1) enhances androgen receptor (AR) activity in prostate cancer (PCa). Hyperactive AR-signalling plays a key role for the development of castration resistance. Several CDK7 inhibitors are currently under investigation in Phase I/II trials addressing solid tumours, including PCa. Aim of this study was to characterize the CDK7/phospho-(p)MED1 axis in human tissue. Immunohistochemistry was performed on 595 PCa samples including 394 primary tumour foci obtained by radical prostatectomy (RP), 64 advanced or recurrent tumours obtained by palliative transurethral resection of the prostate (pTUR), 65 lymph node metastases (LNM), 35 distant metastases (DM) and 36 benign samples. CDK7 is expressed in 79.3% of PCa tissues and protein levels are significantly higher in LNM, pTUR and DM and lower in benign tissues compared to primary tumours. CDK7 and pMED1 expression show strong positive correlation. High expression of CDK7 associated with shorter 5-year biochemical recurrence-free-survival (63.0% vs. 85.0%) and reduced survival persists when adjusted for T-Stage, nodal status, resection boundaries, grade group and pre-operative prostate-specific antigen in multivariate Cox-regression (hazard ratio 4.30; 95% CI, 1.43 to 12,40, P = 0.007). High CDK7 and pMED1 levels correlate with nuclear AR expression. CDK7 positive tumours harbour higher Ki67 expression indices and show more frequently positive ERG (ETS-related gene)-status. In conclusion, CDK7 is frequently expressed in human PCa and predicts disease recurrence after RP. Therapeutical inhibition of CDK7 might be a promising approach in treatment of advanced PCa.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer.

BACKGROUND: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer. METHODS: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes. RESULTS: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1. CONCLUSIONS: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells.

The Gene Expression Landscape of Prostate Cancer BM Reveals Close Interaction with the Bone Microenvironment.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed "Manually Annotated and Curated Nanostring-data Platform". In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

Increased mediator complex subunit CDK19 expression associates with aggressive prostate cancer.

The Mediator complex is a transcriptional regulator interacting with transcription factors and RNA-polymerase-II. Recently, we identified its subunit CDK19 to be specifically expressed in prostate cancer (PCa) and to be functionally implicated in PCa aggressiveness. Aim of our study was to comprehensively characterize the protein expression of CDK19 and its paralog CDK8 in PCa. We performed immunohistochemistry (IHC) for CDK19/CDK8 on a large cohort including needle biopsies from 202 patients, 799 primary tumor foci of radical prostatectomy specimens from 415 patients, 120 locally advanced tumor foci obtained by palliative transurethral resection, 140 lymph node metastases, 67 distant metastases and 82 benigns. Primary tumors were stained for the proliferation marker Ki67, androgen receptor (AR) and ERG. For 376 patients, clinic-pathologic data were available. Primary endpoint was disease-recurrence-free survival (DFS). Nuclear CDK19 and CDK8 expression increases during progression showing the highest intensity in metastatic and castration-resistant tumors. High CDK19 expression on primary tumors correlates with DFS independently from Gleason grade and PSA. Five-year-DFS rates of patients with primary tumors expressing no, moderate and high CDK19 are 73.7, 56.9 and 30.4%, respectively. CDK19 correlates with Gleason grade, T-stage, Ki67 proliferation-index, nuclear AR expression and ERG-status. Therapeutic options for metastatic and castration-resistant PCa remain limited. In the current study, we confirmed an important role of the Mediator subunit CDK19 in advanced PCa supporting current developments to target CDK19 and its paralog CDK8. Furthermore, CDK19 protein expression has the potential to predict disease recurrence independently from established biomarkers thus contributing to individual management for PCa patients.

EVI1 as a Marker for Lymph Node Metastasis in HNSCC.

BACKGROUND: HNSCC is the sixth most common cancer in humans and has still a very poor prognosis. The treatment methods so far are very often associated with mutilation and impairment in the quality of life. Except for p16 expression, there are no reliable prognostic markers in HNSCC so far. Ecotropic Viral Integration Site 1 (EVI1) is a well-described prognostic marker in leukemia and different types of solid cancers. In these, a high EVI1 expression is associated with a poor prognosis. In HNSCC, it is not known so far if EVI1 has any prognostic relevance. MATERIALS AND METHODS: We used our representative tissue cohort of 389 primary HNSCCs, of which 57.2% had one or more lymph node metastases. Here EVI1 expression was analyzed via immunohistochemistry and correlated with the clinical characteristics of these patients. RESULTS: Although in HNSCC EVI1 expression does not predict poor survival, a high EVI1 expression in the primary tumor correlates with a lymph node metastatic disease. CONCLUSION: Consequently, EVI1 may serve as a biomarker to predict an occult lymph node metastasis in a clinical nodal negative (cN0) HNSCC.

CDK19 as a Potential HPV-Independent Biomarker for Recurrent Disease in HNSCC.

The Mediator complex is a central integrator of transcription and a hub for the regulation of gene expression. Cyclin dependent kinase (CDK) 19 and its paralog CDK8 are part of its kinase domain and contribute to cancer progression in different cancer entities. STAT1 is an important immune modulator and a downstream substrate of CDK8/CDK19 mediated phosphorylation. So far, little is known about CDK19's role in head and neck squamous cell carcinoma (HNSCC) progression, its link to STAT1 activity, and related immune modulation. Immunohistochemistry for CDK19, activated pSTAT1, and PD-L1, known to be affected by STAT1, was conducted on samples of 130 primary tumors, 71 local recurrences, 32 lymph node metastases, and 25 distant metastases of HNSCC. Compared to primary tumors, CDK19 is overexpressed in local recurrences and distant metastases as well as in primary tumors that developed local recurrence after initial therapy. Patients with high-CDK19-expressing primary tumors have a significantly shorter disease-free survival. CDK19 expression correlates with pSTAT1 expression in primary tumors associated with recurrent disease, local recurrent tumors, lymph node metastases, and distant metastases. pSTAT1 expression correlates with PD-L1 expression in recurrent tumors. Our findings identify CDK19 as a potential biomarker in HNSCC to predict recurrent disease and support recent developments to target CDK19 and its paralog CDK8 in advanced cancer.

NR2F6 as a Prognostic Biomarker in HNSCC.

Head and neck squamous cell carcinoma (HNSCC)is the 6th most common cancer in humans worldwide and is associated with a poor prognosis for patients. NR2F6 has been identified as an immune checkpoint molecule in tumor-infiltrating T lymphocytes and is associated with a poor prognostic outcome in various cancers. The prognostic value of NR2F6 in HNSCC has not been described yet. We used a large, representative and clinically well-characterized cohort of 383 HNSCC patients, of which 22.4% developed a local recurrence. The NR2F6 expression was analyzed by using immunohistochemistry and was afterward correlated with clinical characteristics and clinicopathological features of HNSCC patients. Primary tumors from patients who develop a local recurrence have a higher NR2F6 expression than primary tumors which do not develop a local recurrence. Furthermore, a high NR2F6 expression is associated with poorer recurrence-free survival, although there is no correlation with overall survival. NR2F6 expression is independent of the T stage and UICC stage. NR2F6 might be a new prognostic biomarker for the early detection of local recurrences in HNSCC patients. Therefore, it may help to improve the recognition of patients who would benefit from more frequent follow-up examinations.

Clinical and molecular implications of MED15 in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) progression depends on various dysregulated pathways. Regulation of diverse pathways is mediated by the mediator complex. The mediator subunit MED15 is essential for transforming growth factor (TGF)-ß signaling and involved in breast and prostate cancers. We investigated the implication of MED15 in HNSCC. IHC for MED15 was performed on 324 tissue samples, and TGF-ß assessed the use of Ki-67 and pSMAD3 as markers. MED15 knockdown followed by proliferation and migration assays, as well as TGF-ß1 treatment followed by MED15 analysis, was also performed. MED15 was overexpressed in 35% of primary tumors, 30% of lymph node metastases, and 70% of recurrences in contrast to no or low expression in benign tumors. MED15 overexpression in primary tumors from patients who developed recurrences was associated with higher mortality rates and occurred at highest frequency in oral cavity or oropharyngeal tumors. Furthermore, MED15 expression correlated between primary tumors and corresponding lymph node metastases. MED15 correlated with proliferation in tissues, and MED15 knockdown reduced proliferation and migration. We observed an association between MED15 and TGF-ß activity in tissues because TGF-ß activation led to increased MED15 expression and reduced pSMAD3 on MED15 knockdown. Taken together, our results implicate MED15 in HNSCC and hint that MED15 overexpression is a clonal event during HNSCC progression. MED15 may serve as a prognostic marker for recurrence and as a therapeutic target.

MED15, encoding a subunit of the mediator complex, is overexpressed at high frequency in castration-resistant prostate cancer.

The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-ß3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-ß signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-ß-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model.

Somatic copy number alterations by whole-exome sequencing implicates YWHAZ and PTK2 in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.

[Therapy sequences and duration in mCRPC: a retrospective review of the Lübeck mCRPC cohort]. / Therapiesequenzen und ­dauer beim mCRPC: Eine retrospektive Aufarbeitung der Lübecker mCRPC-Kohorte.

BACKGROUND: Prostate cancer is one of the most common cancers in men in Europe. Several classes of agents can be considered for the treatment of metastatic prostate carcinoma, and their use is supported by extensive guidelines. In the treatment of metastatic castration-resistant prostate cancer (mCRPC), it is currently unclear which sequence of systemic therapies is most effective. Currently approved system therapies in the castration-resistant setting generally include hormone-manipulating agents, taxane-based chemotherapies, radioactive agents, or inhibitiors of DNA repair mechanisms. This study aims to summarize real world data of mCRPC therapy. METHODS: Retrospectively, 90 mCRPC patients undergoing treatment at the University Hospital Schleswig-Holstein, Lübeck Campus between February 2006 and March 2020 were identified. The patient data were analyzed for their treatment sequence and disease progression. Due to the inclusion period, the mCRPC therapy sequences studied were limited to: Abiraterone, Cabazitaxel, Docetaxel, Enzalutamide, Lutetium-177-PSMA and Radium-223. The analysis includes the therapy sequences and their duration, clinical information of the respective cohort, overall and cancer-specific survival (OS/CSS) as well as time to second-line therapy in relation to the respective first-line therapy. RESULTS: Approximately two-thirds of patients underwent a true therapy sequence (at least two of the drugs listed above), with this proportion halving by the third line.The majority of patients received the sequence (first/second line) abiraterone/docetaxel (n=13), followed by docetaxel/abiraterone (n=12) and abiraterone/enzalutamid (n=10) and docetaxel/docetaxel (n=8).Within the different docetaxel sequences, first-line (mean 4.7 months ± SD 3.1; median 4.0) and rechallenge (mean 5.3 months ± SD 5.9; median 3.0) therapy durations were the longest. The subjective side effect rate of docetaxel was lower in the second line, so that a better tolerability can be assumed here.The abiraterone/docetaxel sequence was used mainly in patients with metachronous metastases. Among the different sequences of abiraterone, first-line (mean 10.8 months ± SD 10.2; median 9.0) and second-line (mean 10.6 months ± SD 9.0; median 7.0) therapy durations were the longest.The sequence abiraterone/enzalutamide was prescribed mainly to older patients with synchronous metastases. Among the different enzalutamide sequences first-line (mean 9.6 months ± SD 7.1; median 7.0) and rechallenge (mean 11.0 ± SD 0.0; median 11.0) therapy durations were the longest.In contrast, the sequence docetaxel/docetaxel was used mainly in younger patients with a high initial PSA.The evaluation shows a trend that both abiraterone and enzalutamide can account for a survival advantage in the first line. CONCLUSION: Ultimately, an optimal treatment sequence cannot be confidently derived from these data.However, it was found that only a small proportion of patients underwent fourth- or even fifth-line treatment at all. Thus, the focus on first- and second-line in this study seems reasonable. It could be shown in a trend that docetaxel as first-line therapy seems to be disadvantegous regarding OS as well as CSS when compared to abiraterone or enzalutamide. However, due to the small number of patients in this study, a clear significance cannot be derived. Moreover, the subjectively better tolerability of docetaxel in the second-line setting could provide an impetus for treatment planning in multimorbid elderly patients in the future. The sequence abiraterone/docetaxel may offer a beneficial option for initial mCRPC therapy.

CEACAM6 Promotes Lung Metastasis via Enhancing Proliferation, Migration and Suppressing Apoptosis of Prostate Cancer Cells.

BACKGROUND/AIM: Metastatic prostate cancer (mPCa) results in high morbidity and mortality. Visceral metastases in particular are associated with a shortened survival. Our aim was to unravel the molecular mechanisms that underly pulmonary spread in mPCa. MATERIALS AND METHODS: We performed a comprehensive transcriptomic analysis of PCa lung metastases, followed by functional validation of candidate genes. Digital gene expression analysis utilizing the NanoString technology was performed on mRNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue from PCa lung metastases. The gene expression data from primary PCa and PCa lung metastases were compared, and several publicly available bioinformatic analysis tools were used to annotate and validate the data. RESULTS: In PCa lung metastases, 234 genes were considerably up-regulated, and 78 genes were significantly down-regulated when compared to primary PCa. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was identified as suitable candidate gene for further functional validation. CEACAM6 as a cell adhesion molecule has been implicated in promoting metastatic disease in several solid tumors, such as colorectal or gastric cancer. We showed that siRNA knockdown of CEACAM6 in PC-3 and LNCaP cells resulted in decreased cell viability and migration as well as enhanced apoptosis. Comprehensive transcriptomic analyses identified several genes of interest that might promote metastatic spread to the lung. CONCLUSION: Functional validation revealed that CEACAM6 might play an important role in fostering metastatic spread to the lung of PCa patients via enhancing proliferation, migration and suppressing apoptosis in PC-3 and LNCaP cells. CEACAM6 might pose an attractive therapeutic target to prevent metastatic disease.

ALDH1A1 drives prostate cancer metastases and radioresistance by interplay with AR- and RAR-dependent transcription.

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro, in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations.