Targeting the CLK2/SRSF9 splicing axis in prostate cancer leads to decreased ARV7 expression.

In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Inhibition of the Sterol Regulatory Element Binding Protein SREBF-1 Overcomes Docetaxel Resistance in Advanced Prostate Cancer.

Resistance to antiandrogens and chemotherapy (Cx) limits therapeutic options for patients with metastatic hormone-sensitive (mHSPC) and metastatic castration-resistant (mCRPC) prostate cancer. In this context, up-regulation of the glucocorticoid receptor is identified as a potential bypass mechanism in mCRPC. A combination of docetaxel and mifepristone (Doc + RU-486), an inhibitor of the glucocorticoid receptor, re-sensitizes docetaxel-resistant cell models to Cx. This study was designed to elucidate the molecular mechanisms responsible for this phenomenon. RNA sequencing was performed in docetaxel-resistant prostate cancer cell models after Doc + RU-486 treatment with consecutive functional assays. Expression of selected proteins was verified in prostatic tissue from prostate cancer patients with progressive disease. Treatment with Doc + RU-486 significantly reduced cancer cell viability, and RNA sequencing revealed sterol regulatory element of binding transcription factor 1 (SREBF-1), a transcription factor of cholesterol and lipid biosynthesis, as a significantly down-regulated target. Functional assays confirmed that SREBF-1 down-regulation is partially responsible for this observation. In concordance, SREBF-1 knockdown and pharmacologic sterol regulatory element binding protein inhibition, together with other key enzymes in the cholesterol pathway, showed similar results. Furthermore, SREBF-1 expression is significantly elevated in advanced prostate cancer tissues, showing its potential involvement in tumor progression and emerging therapy resistance. Therefore, specific inhibition of cholesterol and lipid biosynthesis might also target Cx-resistant cancer cells and represents a potential additive future therapeutic option to improve mCRPC therapy.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Glucocorticoid treatment influences prostate cancer cell growth and the tumor microenvironment via altered glucocorticoid receptor signaling in prostate fibroblasts.

Despite significant therapeutic advances in recent years, treatment of metastatic prostate cancer (PCa) remains palliative, owing to the inevitable occurrence of drug resistance. There is increasing evidence that epithelial glucocorticoid receptor (GR) signaling and changes in the tumor-microenvironment (TME) play important roles in this process. Since glucocorticoids (GCs) are used as concomitant medications in the course of PCa treatment, it is essential to investigate the impact of GCs on stromal GR signaling in the TME. Therefore, general GR mRNA and protein expression was assessed in radical prostatectomy specimens and metastatic lesions. Elevated stromal GR signaling after GC treatment resulted in altered GR-target gene, soluble protein expression, and in a morphology change of immortalized and primary isolated cancer-associated fibroblasts (CAFs). Subsequently, these changes affected proliferation, colony formation, and 3D-spheroid growth of multiple epithelial PCa cell models. Altered expression of extra-cellular matrix (ECM) and adhesion-related proteins led to an ECM remodeling. Notably, androgen receptor pathway inhibitor treatments did not affect CAF viability. Our findings demonstrate that GC-mediated elevated GR signaling has a major impact on the CAF secretome and the ECM architecture. GC-treated fibroblasts significantly influence epithelial tumor cell growth and must be considered in future therapeutic strategies.

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization.

BACKGROUND: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. METHODS: We examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. RESULTS: CD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. CONCLUSION: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

A disease-associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer.

BACKGROUND: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years. METHODS: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information. RESULTS: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively). CONCLUSIONS: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.

Antizyme Inhibitor 1 Regulates Matrikine Expression and Enhances the Metastatic Potential of Aggressive Primary Prostate Cancer.

Molecular drivers of metastasis in patients with high-risk localized prostate cancer are poorly understood. Therefore, we aim to study molecular drivers of metastatic progression in patients with high-risk prostate cancer. A retrospective matched case-control study of two clinico-pathologically identical groups of patients with high-risk prostate cancer was undertaken. One group developed metastatic recurrence (n = 19) while the other did not (n = 25). The primary index tumor was identified by a uro-pathologist, followed by DNA and RNA extraction for somatic copy-number aberration (SCNA) analysis and whole-transcriptome gene expression analysis. In vitro and in vivo studies included cell line manipulation and xenograft models.The integrative CNA and gene expression analyses identified an increase in Antizyme Inhibitor 1 (AZIN1) gene expression within a focal amplification of 8q22.3, which was associated with metastatic recurrence of patients with high-risk prostate cancer in four independent cohorts. The effects of AZIN1 knockdown were evaluated, due to its therapeutic potential. AZIN1 knockdown effected proliferation and metastatic potential of prostate cancer cells and xenograft models. RNA sequencing after AZIN1 knockdown in prostate cancer cells revealed upregulation of genes coding for collagen subunits. The observed effect on cell migration after AZIN1 knockdown was mimicked when exposing prostate cancer cells to bio-active molecules deriving from COL4A1 and COL4A2. Our integrated CNA and gene expression analysis of primary high-risk prostate cancer identified the AZIN1 gene as a novel driver of metastatic progression, by altering collagen subunit expression. Future research should further investigate its therapeutic potential in preventing metastatic recurrence. IMPLICATIONS: AZIN1 was identified as driver of metastatic progression in high-risk prostate cancer through matrikine regulation.

Small-molecule profiling for steroid receptor activity using a universal steroid receptor reporter assay.

A critical step in the development of novel drug candidates for the treatment of steroid related diseases is ensuring the absence of crosstalk with steroid receptors (SRs). Establishing this SR cross-reactivity profile requires multiple reporter assays as each SR associates with its unique enhancer region, a labor intensive and time-consuming approach. To overcome this need for multi-reporter assays, we established a steroid receptor inducible luciferase reporter assay (SRi-Luc) that allows side-by-side examination of agonistic and antagonistic properties of small-molecules on all steroid receptors. This state-of-the-art SRi-Luc consists of a unique alteration of four distinct keto-steroid- and estrogen response elements. As proof of principle, the SRi-Luc assay was used to profile a set of novel designed steroidal 1,2,3-triazoles. These triazolized steroidal compounds were developed via our in-house triazolization methodology, in which an enolizable ketone is converted into a triazolo-fused or -linked analog by treatment with a primary amine or ammonium salt in the presence of 4-nitrophenyl azide. From these designed steroidal 1,2,3-triazoles, six successfully reduced androgen receptor activity by 40 %. Although opted as antiandrogens, their cross-reactivity with other SRs was apparent in our SRi-Luc assay and rendered them unsuited for further antagonist development and clinical use. Overall, the SRi-Luc overcomes the need of multi-reporter assays for the profiling of small-molecules on all SRs. This not only reduces the risk of introducing biases, it as well accelerates early-stage drug discovery when designing particular SR selective (ant)agonists or characterizing off-target effects of lead molecules acting on any drug target.

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.

Targeting the glucocorticoid receptor signature gene Mono Amine Oxidase-A enhances the efficacy of chemo- and anti-androgen therapy in advanced prostate cancer.

Despite increasing options for treatment of castration-resistant prostate cancer, development of drug resistance is inevitable. The glucocorticoid receptor (GR) is a prime suspect for acquired therapy resistance, as prostate cancer (PCa) cells are able to increase GR signaling during anti-androgen therapy and thereby circumvent androgen receptor (AR)-blockade and cell death. As standard AR-directed therapies fail to block the GR and GR inhibitors might result in intolerable side effects, the identification of GR signature genes, which are better suited for a targeted approach, is of clinical importance. Therefore, the specific epithelial and stromal GR signature was determined in cancer-associated fibroblasts as well as in abiraterone and enzalutamide-resistant cells after glucocorticoid (GC) treatment. Microarray and ChIP analysis identified MAO-A as a directly up-regulated mutual epithelial and stromal GR target, which is induced after GC treatment and during PCa progression. Elevated MAO-A levels were confirmed in in vitro cell models, in primary tissue cultures after GC treatment, and in patients after neoadjuvant chemotherapy with GCs. MAO-A expression correlates with GR/AR activity as well as with a reduced progression-free survival. Pharmacological MAO-A inhibition combined with 2nd generation AR signaling inhibitors or chemotherapeutics results in impaired growth of androgen-dependent, androgen-independent, and long-term anti-androgen-treated cells. In summary, these findings demonstrate that targeting MAO-A represents an innovative therapeutic strategy to synergistically block GR and AR dependent PCa cell growth and thereby overcome therapy resistance.

Preclinical Models in Prostate Cancer: Resistance to AR Targeting Therapies in Prostate Cancer.

Prostate cancer is an androgen-driven tumor. Different prostate cancer therapies consequently focus on blocking the androgen receptor pathway. Clinical studies reported tumor resistance mechanisms by reactivating and bypassing the androgen pathway. Preclinical models allowed the identification, confirmation, and thorough study of these pathways. This review looks into the current and future role of preclinical models to understand resistance to androgen receptor-targeted therapies. Increasing knowledge on this resistance will greatly improve insights into tumor pathophysiology and future treatment strategies in prostate cancer.

MYC-Mediated Ribosomal Gene Expression Sensitizes Enzalutamide-resistant Prostate Cancer Cells to EP300/CREBBP Inhibitors.

Patients with advanced prostate cancer are frequently treated with the antiandrogen enzalutamide. However, resistance eventually develops in virtually all patients, and various mechanisms have been associated with this process. The histone acetyltransferases EP300 and CREBBP are involved in regulation of cellular events in advanced prostate cancer. This study investigated the role of EP300/CREBBP inhibitors in enzalutamide-resistant prostate cancer. EP300/CREBBP inhibitors led to the same inhibition of androgen receptor activity in enzalutamide-resistant and -sensitive cells. However, enzalutamide-resistant cells were more sensitive to these inhibitors in viability assays. As indicated by the RNA-sequencing-based pathway analysis, genes related to the ribosome and MYC activity were significantly altered upon EP300/CREBBP inhibitor treatment. EP300/CREBBP inhibitors led to the down-regulation of ribosomal proteins RPL36 and RPL29. High-level ribosomal proteins amplifications and MYC amplifications were observed in castration-resistant prostate cancer samples of the publicly available Stand Up to Cancer data set. An inhibitor of RNA polymerase I-mediated transcription was used to evaluate the functional implications of these findings. The enzalutamide-resistant cell lines were more sensitive to this treatment. In addition, the migration rate of enzalutamide-resistant cells was strongly inhibited by this treatment. Taken together, the current data show that EP300/CREBBP inhibitors affect the MYC/ribosomal protein axis in enzalutamide-resistant cells and may have promising therapeutic implications.

Assessment of STAT5 as a potential therapy target in enzalutamide-resistant prostate cancer.

Despite enzalutamide's efficacy in delaying the progression of metastatic castration-resistant prostate cancer (CRPC), resistance to this anti-androgen inevitably occurs. Several studies have revealed that the signal transducer and activator of transcription (STAT) 5 plays a role in tumour progression and development of drug resistance such as enzalutamide. Data mining revealed heterogeneous expression of STAT5 in enzalutamide-treated mCRPC patients and enzalutamide-resistant prostate cancer (PCa). Isobologram analysis revealed that the STAT5 inhibitor pimozide combined with enzalutamide has? additive and synergistic inhibitory effects on cell viability in the used models. Functional analysis with siRNA-mediated STAT5 knockdown yielded divergent results. The LNCaP-derived cell line MR49F could be resensitised to enzalutamide by siRNA-mediated STAT5b-knock-down. In contrast, neither STAT5a nor STAT5b knockdown resensitised enzalutamide-resistant LAPC4-EnzaR cells to enzalutamide. In conclusion, our results indicate that STAT5 may be a possible target in a subgroup of enzalutamide-resistant PCa. However, based on the data presented here, a general role of STAT5 in enzalutamide-resistance and its potential as a therapeutic target could not be shown.

p300 is upregulated by docetaxel and is a target in chemoresistant prostate cancer.

Administration of the microtubule inhibitor docetaxel is a common treatment for metastatic castration-resistant prostate cancer (mCRPC) and results in prolonged patient overall survival. Usually, after a short period of time chemotherapy resistance emerges and there is urgent need to find new therapeutic targets to overcome therapy resistance. The lysine-acetyltransferase p300 has been correlated to prostate cancer (PCa) progression. Here, we aimed to clarify a possible function of p300 in chemotherapy resistance and verify p300 as a target in chemoresistant PCa. Immunohistochemistry staining of tissue samples revealed significantly higher p300 protein expression in patients who received docetaxel as a neoadjuvant therapy compared to control patients. Elevated p300 expression was confirmed by analysis of publicly available patient data, where significantly higher p300 mRNA expression was found in tissue of mCRPC tumors of docetaxel-treated patients. Consistently, docetaxel-resistant PCa cells showed increased p300 protein expression compared to docetaxel-sensitive counterparts. Docetaxel treatment of PCa cells for 72 h resulted in elevated p300 expression. shRNA-mediated p300 knockdown did not alter colony formation efficiency in docetaxel-sensitive cells, but significantly reduced clonogenic potential of docetaxel-resistant cells. Downregulation of p300 in docetaxel-resistant cells also impaired cell migration and invasion. Taken together, we showed that p300 is upregulated by docetaxel, and our findings suggest that p300 is a possible co-target in treatment of chemoresistant PCa.

The stem cell inhibitor salinomycin decreases colony formation potential and tumor-initiating population in docetaxel-sensitive and docetaxel-resistant prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) is one of the most frequently diagnosed tumors in men. In general, therapies for localized PCa are curative. However, treatment of advanced PCa is considered palliative since development of therapy resistance occurs rapidly. It has been shown that tumor-initiating cells are likely involved in therapy resistance. They are not eliminated by conventional therapies and thereby lead to tumor progression and relapse. The aim of this study was to evaluate the effects of the known stem cell inhibitor salinomycin on this critical subpopulation of cells. METHODS: Expression of the cell surface markers CD24 and CD44 was assessed by immunofluorescence and fluorescence-activated cell sorting. Colony formation efficiency and classification of colony types with varying tumor-initiating potential (holoclones, meroclones, and paraclones) were analyzed in an automated way by the newly developed CATCH-colonies software in the absence or presence of salinomycin. RESULTS: Automated high-resolution colony formation analysis consistently identified the various colony types in a broad range of PCa cell lines. Serial clonogenic assays confirmed that holoclones show the highest colony formation potential and maintain their tumor-initiating capacity over multiple rounds. Furthermore, holoclones showed high expression of CD44, while CD24 was not expressed in these clones, thus representing the well-described tumor-initiating CD24- /CD44high population. Salinomycin decreased the CD24- /CD44high population in both docetaxel-sensitive PC3 and docetaxel-resistant (DR) PC3-DR. Moreover, treatment of PC3, DU145, PC3-DR, and DU145-DR with salinomycin led to a significant reduction in the colony formation potential by targeting the colonies with high tumor-initiating potential. CONCLUSIONS: Taken together, we demonstrated that salinomycin specifically targets the tumor-initiating cell population in docetaxel-sensitive and docetaxel-resistant PCa cells and may represent a potential therapeutic approach for the treatment of advanced PCa.

Drivers of AR indifferent anti-androgen resistance in prostate cancer cells.

Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.

The STAT3 Inhibitor Galiellalactone Reduces IL6-Mediated AR Activity in Benign and Malignant Prostate Models.

IL6/STAT3 signaling is associated with endocrine therapy resistance in prostate cancer, but therapies targeting this pathway in prostate cancer were unsuccessful in clinical trials so far. The mechanistic explanation for this phenomenon is currently unclear; however, IL6 has pleiotropic effects on a number of signaling pathways, including the androgen receptor (AR). Therefore, we investigated IL6-mediated AR activation in prostate cancer cell lines and ex vivo primary prostate tissue cultures in order to gain a better understanding on how to inhibit this process for future clinical trials. IL6 significantly increased androgen-dependent AR activity in LNCaP cells but importantly did not influence AR activity at castrate androgen levels. To identify the underlying mechanism, we investigated several signaling pathways but only found IL6-dependent changes in STAT3 signaling. Biochemical inhibition of STAT3 with the small-molecule inhibitor galiellalactone significantly reduced AR activity in several prostate and breast cancer cell lines. We confirmed the efficacy of galiellalactone in primary tissue slice cultures from radical prostatectomy samples. Galiellalactone significantly reduced the expression of the AR target genes PSA (P < 0.001), TMPRSS2 (P < 0.001), and FKBP5 (P = 0.003) in benign tissue cultures (n = 24). However, a high heterogeneity in the response of the malignant samples was discovered, and only a subset of tissue samples (4 out of 10) had decreased PSA expression upon galiellalactone treatment. Taken together, this finding demonstrates that targeting the IL6/STAT3 pathway with galiellalactone is a viable option to decrease AR activity in prostate tissue that may be applied in a personalized medicine approach.