New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma.

Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.

Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells.

Introduction: A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Methods: Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Results: Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Conclusions: Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.

Tumorigenic effects of human mesenchymal stromal cells and fibroblasts on bladder cancer cells.

Background: Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells. Methods: We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays. Results: Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFß-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities. Conclusions: The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy.

Epigenetic Priming and Development of New Combination Therapy Approaches.

Muscle-invasive urothelial carcinoma of the bladder (MIBC) has been treated with cisplatin-based chemotherapy for over 30 years. With the advent of immune checkpoint inhibitors, antibody drug conjugates and FGFR3 inhibitors new therapeutic options have been approved for patients with urothelial carcinoma (UC) and are still under investigation regarding association between patients' response and recently defined molecular subtypes. Unfortunately, similar to chemotherapy, only a fraction of UC patients responds to these new treatment approaches. Thus, either further new efficacious therapeutic options for treatment of individual subtypes or new approaches to overcome treatment resistance and to increase patients' response to standard of care treatment are needed.Epigenetic modifications of DNA and chromatin are known to mediate cellular plasticity or treatment resistance, and the responsible epigenetic regulators are frequently mutated or aberrantly expressed in UC. Thus, these enzymes provide targets for novel drug combination therapies to "episensitize" toward approved standard therapies by epigenetic priming. In general, these epigenetic regulators comprise writers and erasers like DNA methyltransferases and DNA demethylases (for DNA methylation), histone methyltransferases and histone demethylases (for histone methylation), as well as acetyl transferases and histone deacetylases (for histone and nonhistone acetylation). Such modifications, e.g., acetyl groups, are recognized by further epigenetic reader proteins, e.g., like the bromodomain and extra-terminal domain (BET) family proteins that often interact in multi-protein complexes and finally regulate chromatin conformation and transcriptional activity.Concurringly, epigenetic regulators target a plethora of cellular functions. Their pharmaceutical inhibitors often inhibit enzymatic activity of more than one isoenzyme or may have further noncanonical cytotoxic effects. Thus, analysis of their functions in UC pathogenesis as well as of the antineoplastic capacity of corresponding inhibitors alone or in combination with other approved drugs should follow a multidimensional approach. Here, we present our standard approach to analyze cellular effects of new epigenetic inhibitors on UC cells alone to define their potency and to conclude on putative reasonable combination therapy partners. We further describe our approach to identify efficacious synergistic combination therapies (e.g., with cisplatin or PARP inhibitors) that may have reduced normal toxicity through dose reduction, which can then be further analyzed in animal experiments. This approach may also serve as prototype for the preclinical evaluation of other epigenetic treatment approaches.

Predictive value of molecular subtypes and APOBEC3G for adjuvant chemotherapy in urothelial bladder cancer.

OBJECTIVE: Although targeted approaches have become available in second- and third-line settings, platinum-based chemotherapy remains the standard first-line treatment for advanced muscle-invasive bladder cancer (MIBC). Therefore, the prediction of platinum resistance is of utmost clinical importance. METHODS: In this study, we established a routine compatible method for the molecular classification of MIBC samples according to various classification systems and applied this method to evaluate the impact of subtypes on survival after adjuvant chemotherapy. This retrospective study included 191 patients with advanced MIBC (pT≥3 or pN+) who underwent radical cystectomy, with or without adjuvant chemotherapy. A 48-gene panel and classifier rule set were established to determine molecular subtypes according to TCGA, MDA, LundTax, and Consensus classifications. Additionally, 12 single platinum-predictive candidate genes were assessed. The results were correlated with patients' clinicopathological and follow-up data and were validated using independent data sets. RESULTS: Our final evaluation of 159 patients demonstrated better survival in the luminal groups for those who received chemotherapy compared with those who did not. In contrast, no such differences were observed in basal subtypes. The use of chemotherapy was associated with better survival in patients with high APOBEC3G expression (p < 0.002). This association was confirmed using an independent data set of patients who received neoadjuvant platinum therapy. CONCLUSIONS: The proposed method robustly replicates the most commonly used transcriptome-based subtype classifications from paraffin-embedded tissue samples. The luminal, but not basal, molecular subtypes had the greatest benefit from adjuvant platinum therapy. We identified and validated APOBEC3G as a novel predictive marker for platinum-treated patients.

Differential DNA Methylation of THOR and hTAPAS in the Regulation of hTERT and the Diagnosis of Cancer.

BACKGROUND: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5' of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). METHODS: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. RESULTS: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. CONCLUSIONS: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided.

Establishment and Evaluation of Dual HDAC/BET Inhibitors as Therapeutic Options for Germ Cell Tumors and Other Urological Malignancies.

Urological malignancies represent major challenges for clinicians, with annually rising incidences. In addition, cisplatin treatment induced long-term toxicities and the development of therapy resistance emphasize the need for novel therapeutics. In this study, we analyzed the effects of novel histone deacetylase (HDAC) and bromodomain and extraterminal domain-containing (BET) inhibitors to combine them into a potent HDAC-BET-fusion molecule and to understand their molecular mode-of-action. Treatment of (cisplatin-resistant) germ cell tumors (GCT), urothelial, renal, and prostate carcinoma cells with the HDAC, BET, and dual inhibitors decreased cell viability, induced apoptosis, and affected the cell cycle. Furthermore, a dual inhibitor considerably decreased tumor burden in GCT xenograft models. On a molecular level, correlating RNA- to ATAC-sequencing data indicated a considerable induction of gene expression, accompanied by site-specific changes of chromatin accessibility after HDAC inhibitor application. Upregulated genes could be linked to intra- and extra-cellular trafficking, cellular organization, and neuronal processes, including neuroendocrine differentiation. Regarding chromatin accessibility on a global level, an equal distribution of active or repressed DNA accessibility has been detected after HDAC inhibitor treatment, questioning the current understanding of HDAC inhibitor function. In summary, our HDAC, BET, and dual inhibitors represent a new treatment alternative for urological malignancies. Furthermore, we shed light on new molecular and epigenetic mechanisms of the tested epi-drugs, allowing for a better understanding of the underlying modes-of-action and risk assessment for the patient.

Proteomic and transcriptomic profiles of human urothelial cancer cells with histone deacetylase 5 overexpression.

Urothelial carcinoma (UC) of the urinary bladder is a prevalent cancer worldwide. Because histone deacetylases (HDACs) are important factors in cancer, targeting these epigenetic regulators is considered an attractive strategy to develop novel anticancer drugs. Whereas HDAC1 and HDAC2 promote UC, HDAC5 is often downregulated and only weakly expressed in UC cell lines, suggesting a tumor-suppressive function. We studied the effect of stable lentiviral-mediated HDAC5 overexpression in four UC cell lines with different phenotypes (RT112, VM-Cub-1, SW1710, and UM-UC-3, each with vector controls). In particular, comprehensive proteomics and RNA-seq transcriptomics analyses were performed on the four cell line pairs, which are described here. For comparison, the immortalized benign urothelial cell line HBLAK was included. These datasets will be a useful resource for researchers studying UC, and especially the influence of HDAC5 on epithelial-mesenchymal transition (EMT). Moreover, these data will inform studies on HDAC5 as a less studied member of the HDAC family in other cell types and diseases, especially fibrosis.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas.

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

A quantitative polymerase chain reaction based method for molecular subtype classification of urinary bladder cancer-Stromal gene expressions show higher prognostic values than intrinsic tumor genes.

Transcriptome-based molecular subtypes of muscle-invasive bladder cancer (MIBC) have been shown to be both prognostic and predictive, but are not used in routine clinical practice. We aimed to develop a feasible, reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based method for molecular subtyping. First, we defined a 68-gene set covering tumor intrinsic (luminal, basal, squamous, neuronal, epithelial-to-mesenchymal, in situ carcinoma) and stromal (immune, extracellular matrix, p53-like) signatures. Then, classifier methods with this 68-gene panel were developed in silico and validated on public data sets with available subtype class information (MD Anderson [MDA], The Cancer Genome Atlas [TCGA], Lund, Consensus). Finally, expression of the selected 68 genes was determined in 104 frozen tissue samples of our MIBC cohort by RT-qPCR using the TaqMan Array Card platform and samples were classified by our newly developed classifiers. The prognostic value of each subtype classification system and molecular signature scores were assessed. We found that the reduced marker set combined with the developed classifiers were able to reproduce the TCGA II, MDA, Lund and Consensus subtype classification systems with an overlap of 79%, 76%, 69% and 64%, respectively. Importantly, we could successfully classify 96% (100/104) of our MIBC samples by using RT-qPCR. Neuronal and luminal subtypes and low stromal gene expressions were associated with poor survival. In conclusion, we developed a robust and feasible method for the molecular subtyping according to the TCGA II, MDA, Lund and Consensus classifications. Our results suggest that stromal signatures have a superior prognostic value compared to tumor intrinsic signatures and therefore underline the importance of tumor-stroma interaction during the progression of MIBC.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

Background: Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches. Methods: Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing. Results: Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC. Conclusion: Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma.

Urothelial carcinoma (UC) is the most frequent histological type of cancer in the urinary bladder. Genomic changes in UC activate MAPK and PI3K/AKT signal transduction pathways, which increase cell proliferation and survival, interfere with cell cycle and checkpoint control, and prevent senescence. A more recently discovered additional category of genetic changes in UC affects chromatin regulators, including histone-modifying enzymes (KMT2C, KMT2D, KDM6A, EZH2), transcription cofactors (CREBBP, EP300), and components of the chromatin remodeling complex SWI/SNF (ARID1A, SMARCA4). It is not yet well understood how these changes contribute to the development and progression of UC. Therefore, we review here the emerging knowledge on genomic and gene expression alterations of chromatin regulators and their consequences for cell differentiation, cellular plasticity, and clonal expansion during UC pathogenesis. Our analysis identifies additional relevant chromatin regulators and suggests a model for urothelial carcinogenesis as a basis for further mechanistic studies and targeted therapy development.

CTLA4 promoter hypomethylation is a negative prognostic biomarker at initial diagnosis but predicts response and favorable outcome to anti-PD-1 based immunotherapy in clear cell renal cell carcinoma.

BACKGROUND: In metastatic clear cell renal cell carcinoma (ccRCC), different combination therapies, each including anti-PD-1 immune checkpoint blockade (ICB), are applied as first-line treatment. Robust predictive biomarkers for rational upfront therapy decisions are lacking, although they are urgently needed. Recently, we showed that CTLA4 promoter methylation predicts response to ICB in melanoma. Here, we aimed to investigate CTLA4 methylation in ccRCC and its utility to serve as a predictive biomarker for anti-PD-1 based ICB in metastatic ccRCC. METHODS: CTLA4 methylation was analyzed with regard to transcriptional gene activity (mRNA expression), intratumoral immune cell composition, and clinical course in two ccRCC cohorts obtained from The Cancer Genome Atlas (TCGA cohort, n=533) and the University Hospital Bonn (UHB Non-ICB Cohort, n=116). In addition, CTLA4 methylation as well as CD8+ T cell infiltrates and PD-L1 expression were evaluated in pre-treatment samples from a multicenter cohort (RCC-ICB Cohort, n=71). Patients included in the RCC-ICB Cohort were treated with either first line anti-PD-1 based combination therapy (n=25) or monotherapy post-tyrosine kinase inhibition in second line or later. Analyses were performed with regard to treatment response according to RECIST, progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) following treatment initiation. RESULTS: CTLA4 promoter hypomethylation was significantly correlated with CTLA4 mRNA expression, lymphocyte infiltration, and poor OS in both primary ccRCC cohorts (TCGA: HR 0.30 (95% CI 0.18 to 0.49), p<0.001; UHB Non-ICB: HR 0.35 (95% CI 0.16 to 0.75), p=0.007). In contrast, CTLA4 promoter hypomethylation predicted response and, accordingly, favorable outcomes (PFS and OS) in patients with ICB-treated ccRCC, overcompensating the negative prognostic value of CTLA4 hypomethylation at initial diagnosis. Moreover, in multivariable Cox regression, CTLA4 promoter hypomethylation remained an independent predictor of improved outcome in ICB-treated ccRCC after co-adjustment of the International Metastatic Renal Cell Carcinoma Database Consortium score (HR 3.00 (95% CI 1.47 to 6.28), p=0.003). CONCLUSIONS: Our study suggests CTLA4 methylation as a powerful predictive biomarker for immunotherapy response in metastatic RCC.

Epigenetic Treatment of Urothelial Carcinoma Cells Sensitizes to Cisplatin Chemotherapy and PARP Inhibitor Treatment.

Muscle-invasive urothelial carcinoma (UC) is treated with cisplatin-based chemotherapy, which is only moderately efficient, mostly due to development of resistance. New therapy approaches are therefore urgently needed. Epigenetic alterations due to frequent mutations in epigenetic regulators contribute to development of the disease and to treatment resistance, and provide targets for novel drug combination therapies. Here, we determined the cytotoxic impact of the second-generation bromodomain protein inhibitor (BETi) PLX51107 on UC cell lines (UCC) and normal HBLAK control cells. PLX51107 inhibited proliferation, induced apoptosis, and acted synergistically with the histone deacetylase inhibitor romidepsin. While PLX51107 caused significant DNA damage, DNA damage signaling and DNA repair were impeded, a state defined as BRCAness. Accordingly, the drug strongly synergized with cisplatin more efficiently than romidepsin, and with the PARP inhibitor talazoparib to inhibit proliferation and induce cell death in UCC. Thus, a BETi can be used to "episensitize" UC cells to cytotoxic chemotherapy and inhibitors of DNA repair by inducing BRCAness in non BRCA1/2 mutated cancers. In clinical applications, the synergy between PLX51107 and other drugs should permit significant dosage reductions to minimize effects on normal tissues.

Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment.

Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC50 values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.

Downregulation of Cell Cycle and Checkpoint Genes by Class I HDAC Inhibitors Limits Synergism with G2/M Checkpoint Inhibitor MK-1775 in Bladder Cancer Cells.

Since genes encoding epigenetic regulators are often mutated or deregulated in urothelial carcinoma (UC), they represent promising therapeutic targets. Specifically, inhibition of Class-I histone deacetylase (HDAC) isoenzymes induces cell death in UC cell lines (UCC) and, in contrast to other cancer types, cell cycle arrest in G2/M. Here, we investigated whether mutations in cell cycle genes contribute to G2/M rather than G1 arrest, identified the precise point of arrest and clarified the function of individual HDAC Class-I isoenzymes. Database analyses of UC tissues and cell lines revealed mutations in G1/S, but not G2/M checkpoint regulators. Using class I-specific HDAC inhibitors (HDACi) with different isoenzyme specificity (Romidepsin, Entinostat, RGFP966), cell cycle arrest was shown to occur at the G2/M transition and to depend on inhibition of HDAC1/2 rather than HDAC3. Since HDAC1/2 inhibition caused cell-type-specific downregulation of genes encoding G2/M regulators, the WEE1 inhibitor MK-1775 could not overcome G2/M checkpoint arrest and therefore did not synergize with Romidepsin inhibiting HDAC1/2. Instead, since DNA damage was induced by inhibition of HDAC1/2, but not of HDAC3, combinations between inhibitors of HDAC1/2 and of DNA repair should be attempted.

Evaluation of Chemotherapeutic Drugs for Treatment of (Cisplatin-Resistant) Germ Cell Cancer Cell Lines.

Cisplatin resistance still remains a major obstacle in the standard chemotherapeutic approach in late-stage and metastatic testicular germ cell cancer (GCC) patients. This multifactorial and complex phenomenon arises (concomitantly) on several levels due to impaired transport, decreased adduct formation, increased DNA-repair, decreased apoptosis, or compensating pathways. Evaluation of novel therapeutic approaches and pharmacological inhibitors still remains necessary to treat cisplatin-resistant GCCs. In this chapter, we present in vitro techniques to measure cytotoxic impacts of chemotherapeutic drugs on GCC cell lines. Specifically, we will discuss the measurement of relative cell viability by XTT assay, as well as cell cycle distribution and apoptosis assay by Nicoletti- and Annexin V/PI apoptosis assay with subsequent flow cytometry, respectively, to evaluate the effects of cytotoxic treatment in GCC cell lines.

Many Different LINE-1 Retroelements Are Activated in Bladder Cancer.

Human genomes contain about 100,000 LINE-1 (L1) retroelements, of which more than 100 are intact. L1s are normally tightly controlled by epigenetic mechanisms, which often fail in cancer. In bladder urothelial carcinoma (UC), particularly, L1s become DNA-hypomethylated, expressed and contribute to genomic instability and tumor growth. It is, however, unknown which individual L1s are activated. Following RNA-immunoprecipitation with a L1-specific antibody, third generation nanopore sequencing detected transcripts of 90 individual elements in the VM-Cub-1 UC line with high overall L1 expression. In total, 10 L1s accounted for >60% of the reads. Analysis of five specific L1s by RT-qPCR revealed generally increased expression in UC tissues and cell lines over normal controls, but variable expression among tumor cell lines from bladder, prostate and testicular cancer. Chromatin immunoprecipitation demonstrated active histone marks at L1 sequences with increased expression in VM-Cub-1, but not in a different UC cell line with low L1 expression. We conclude that many L1 elements are epigenetically activated in bladder cancer in a varied pattern. Our findings indicate that expression of individual L1s is highly heterogeneous between and among cancer types.