Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics.

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration.

BACKGROUND: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. RESULTS: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. CONCLUSIONS: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

Npm1 haploinsufficiency in collaboration with MEIS1 is sufficient to induce AML in mice.

NPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML). Mutations in the NPM1 gene result in the increased export of NPM1 to the cytoplasm (NPM1c) and are associated with multiple transforming events including the aberrant upregulation of MEIS1 that maintains stem cell and cell cycle-associated pathways in NPM1c AML. However, another consequence of the NPM1c mutation is the inadequate levels of NPM1 wild-type in the nucleus and nucleolus, caused by the loss of one wild-type allele in addition to enforced NPM1 nuclear export. The contribution of NPM1 haploinsufficiency independently of the NPM1 mutation to AML development and its relationship with MEIS1 function is poorly understood. Using mouse models, our study shows that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce a fully penetrant AML in mice that transcriptionally resembles human NPM1c AML. NPM1 haploinsufficiency alters MEIS1-binding occupancies such that it binds the promoter of the oncogene structural maintenance of chromosome protein 4 (SMC4) in NPM1 haploinsufficient AML cells but not in NPM1 wild-type-harboring Hoxa9/Meis1-transformed cells. SMC4 is higher expressed in haploinsufficient and NPM1c+ AML cells, which are more vulnerable to the disruption of the MEIS1-SMC4 axis compared with AML cells with nonmutated NPM1. Taken together, our study underlines that NPM1 haploinsufficiency on its own is a key factor of myeloid leukemogenesis and characterizes the MEIS1-SMC4 axis as a potential therapeutic target in this AML subtype.

The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression.

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.

[Organoids as a milestone on the way to personalized treatment of urothelial carcinoma: a systematic review]. / Organoide als Meilenstein auf dem Weg zur personalisierten Therapie des Urothelkarzinoms: ein systematischer Review.

BACKGROUND: The treatment options for locally advanced and metastatic urothelial carcinoma (UC) are currently limited to established chemotherapy and immunotherapy protocols. Targeted treatment is so far restricted to a small subgroup of patients. Urothelial organoid systems could make a decisive contribution in establishing effective personalized treatment options by enabling drug response prediction through testing the sensitivity of individual patients. The aim of this article is to describe the state of the science of clinically applicable organoid systems for UC. METHODOLOGY: A systematic literature search was conducted in several medical databases (Medline, Cochrane Library) and study registers (ClinicalTrials.gov, the EU Clinical Trials Register and the WHO International Clinical Trials Registry). The search terms and the search strategy were adapted to the databases used. RESULTS: Overall, 7 studies met the inclusion criteria on the topic of UC organoids. These studies describe the fundamental workflow in establishing organoid systems in patients with tumors of the urinary bladder or the renal pelvis. The success rates in generating organoids from non-muscle-invasive bladder cancer were 70-77% and for muscle-invasive bladder cancer 42%. For patient organoids systematic drug testing was carried out. CONCLUSION: The generation of UC organoids is feasible and the ex vivo testing of individual treatment forms is possible. Due to the lack of a standardized methodology, their implementation remains experimental at the moment. The methodology has a high potential to provide a personalized treatment concept to patients with urothelial cancer.

A Comparative Assessment of Replication Stress Markers in the Context of Telomerase.

Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers-p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)-are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RASG12V (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.

Organoids at the PUB: The Porcine Urinary Bladder Serves as a Pancreatic Niche for Advanced Cancer Modeling.

Despite intensive research and progress in personalized medicine, pancreatic ductal adenocarcinoma remains one of the deadliest cancer entities. Pancreatic duct-like organoids (PDLOs) derived from human pluripotent stem cells (PSCs) or pancreatic cancer patient-derived organoids (PDOs) provide unique tools to study early and late stage dysplasia and to foster personalized medicine. However, such advanced systems are neither rapidly nor easily accessible and require an in vivo niche to study tumor formation and interaction with the stroma. Here, the establishment of the porcine urinary bladder (PUB) is revealed as an advanced organ culture model for shaping an ex vivo pancreatic niche. This model allows pancreatic progenitor cells to enter the ductal and endocrine lineages, while PDLOs further mature into duct-like tissue. Accordingly, the PUB offers an ex vivo platform for earliest pancreatic dysplasia and cancer if PDLOs feature KRASG12D mutations. Finally, it is demonstrated that PDOs-on-PUB i) resemble primary pancreatic cancer, ii) preserve cancer subtypes, iii) enable the study of niche epithelial crosstalk by spiking in pancreatic stellate and immune cells into the grafts, and finally iv) allow drug testing. In summary, the PUB advances the existing pancreatic cancer models by adding feasibility, complexity, and customization at low cost and high flexibility.

Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells.

Ex vivo organ culture can be a useful alternative to in vivo models, which can be time-, labor-, and cost-intensive. Here we describe a step-by-step protocol to use de-epithelialized porcine urinary bladders as scaffolds in air-liquid interface in vitro culture systems for a variety of pluripotent stem-cell-derived and patient-derived pancreatic cells and organoids. The scaffold can trigger cell maturation and enable cell-cell interaction and invasion capacity studies. However, this model is limited by the lack of functional vasculature. For complete details on the use and execution of this protocol, please refer to Melzer et al. (2022),1 Breunig et al. (2021),2 and Breunig et al. (2021).3.

Shorter Leukocyte Telomere Length Is Associated with Worse Survival of Patients with Bladder Cancer and Renal Cell Carcinoma.

BACKGROUND: Telomeres are protein-DNA complexes at the tips of linear chromosomes. They protect the DNA from end-to-end fusion and exonucleolytic degradation. Shortening of telomeric DNA during aging can generate dysfunctional telomeres, promoting tumorigenesis. More recent data indicate that both short and long telomeres of peripheral blood leukocyte (PBL) cells can serve as prognostic biomarkers for cancer risk and may be associated with survival of patients with solid cancers. Telomere length in PBL cells could also be a potential prognostic biomarker for survival in bladder cancer (BC) or renal cell carcinoma (RCC). METHODS: The relative telomere length (RTL) of PBL cells was assessed in patients with BC (n = 144) and RCC (n = 144) by using qPCR. A control population of patients without malignant disease (NC, n = 73) was included for comparison. The correlation and association of RTL with histopathological parameters and overall survival (OS) were evaluated. RESULTS: Patients with BC and RCC had significantly shorter telomeres compared to patients without malignant disease. Within the cancer cohorts, multivariate analysis revealed that short RTL is an independent predictor of worse survival in BC (p = 0.039) and RCC (p = 0.041). CONCLUSION: Patients with BC and RCC had significantly shorter telomeres compared to the normal population. Shorter RTL in BC and RCC was an independent predictor of reduced survival.

Telomerase and Pluripotency Factors Jointly Regulate Stemness in Pancreatic Cancer Stem Cells.

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the "stemness" maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion.

BACKGROUND: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion. OBJECTIVE: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. METHODS: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. RESULTS: We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV. CONCLUSIONS: Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.

Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells.

Invasive urothelial carcinomas of the bladder (UCB) characteristically show a loss of differentiation markers. The transcription factor Grainyhead-like 3 (GRHL3) plays an important role in the development and differentiation of normal urothelium. The contribution to UCB progression is still elusive. Differential expression of GRHL3 was assessed in normal human urothelium and in non-invasive and invasive bladder cancer cell lines. The contribution of GRHL3 to cell proliferation, viability and invasion in UCB cell lines was determined by gain- and loss-of-function assays in vitro and in an organ culture model using de-epithelialized porcine bladders. GRHL3 expression was detectable in normal human urothelial cells and showed significantly higher mRNA and protein levels in well-differentiated, non-invasive RT4 urothelial carcinoma cells compared to moderately differentiated RT112 cells. GRHL3 expression was absent in anaplastic and invasive T24 cells. Ectopic de novo expression of GRHL3 in T24 cells significantly impaired their migration and invasion properties in vitro and in organ culture. Its downregulation improved the invasive capacity of RT4 cells. The results indicate that GRHL3 may play a role in progression and metastasis in UCB. In addition, this work demonstrates that de-epithelialized porcine bladder organ culture can be a useful, standardized tool to assess the invasive capacity of cancer cells.

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells.

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma.

Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 µM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.

Downregulation of ORP3 Correlates with Reduced Survival of Colon Cancer Patients with Advanced Nodal Metastasis and of Female Patients with Grade 3 Colon Cancer.

Genome instability is an essential hallmark in tumor development, including colorectal cancer. We have recently identified the oxysterol binding protein-related protein 3 (ORP3), also known as oxysterol binding protein-like 3 (OSBPL3), as a novel ploidy-control gene, whose knock-out leads to aneuploidy induction and promotes tumor formation, indicating that ORP3 is a bona fide tumor suppressor protein. Here we analyzed expression of ORP3 in a cohort (n = 206) of colon cancer patients in relation to patient survival. We show that low ORP3 mRNA levels correlate with reduced survival of patients with advanced nodal metastasis (N2). While patient survival does not associate with grading when the whole cohort is evaluated, importantly, low ORP3 mRNA levels associate with worse survival of female patients with grade 3 colon cancer. Similarly, low ORP3 mRNA levels associate with worse survival of grade 3 colon cancer patients 70 years of age and younger while low ORP3 mRNA levels seem to be beneficial for colon cancer patients with a T2 tumor size. Together, the data show that ORP3 expression is downregulated during colon cancer progression, which correlates with reduced patient survival. Thus, ORP3 mRNA levels may be a prognostic marker for better stratification of colon cancer patients.

Telomeres and Telomerase in the Development of Liver Cancer.

Liver cancer is one of the most common cancer types worldwide and the fourth leading cause of cancer-related death. Liver carcinoma is distinguished by a high heterogeneity in pathogenesis, histopathology and biological behavior. Dysregulated signaling pathways and various gene mutations are frequent in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), which represent the two most common types of liver tumors. Both tumor types are characterized by telomere shortening and reactivation of telomerase during carcinogenesis. Continuous cell proliferation, e.g., by oncogenic mutations, can cause extensive telomere shortening in the absence of sufficient telomerase activity, leading to dysfunctional telomeres and genome instability by breakage-fusion-bridge cycles, which induce senescence or apoptosis as a tumor suppressor mechanism. Telomerase reactivation is required to stabilize telomere functionality and for tumor cell survival, representing a genetic risk factor for the development of liver cirrhosis and liver carcinoma. Therefore, telomeres and telomerase could be useful targets in hepatocarcinogenesis. Here, we review similarities and differences between HCC and iCCA in telomere biology.

Drug targeting CYP2E1 for the treatment of early-stage alcoholic steatohepatitis.

BACKGROUND AND AIMS: Alcoholic steatohepatitis (ASH)-the inflammation of fatty liver-is caused by chronic alcohol consumption and represents one of the leading chronic liver diseases in Western Countries. ASH can lead to organ dysfunction or progress to hepatocellular carcinoma (HCC). Long-term alcohol abstinence reduces this probability and is the prerequisite for liver transplantation-the only effective therapy option at present. Elevated enzymatic activity of cytochrome P450 2E1 (CYP2E1) is known to be critically responsible for the development of ASH due to excessively high levels of reactive oxygen species (ROS) during metabolization of ethanol. Up to now, no rational drug discovery process was successfully initiated to target CYP2E1 for the treatment of ASH. METHODS: In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. RESULTS: A new class of drug candidates was generated successfully. Two of the most promising small compounds named 12-Imidazolyl-1-dodecanol (abbr.: I-ol) and 1-Imidazolyldodecane (abbr.: I-an) were selected at the end of this process of drug discovery and developability. These new ω-imidazolyl-alkyl derivatives act as strong chimeric CYP2E1 inhibitors at a nanomolar range. They restore redox balance, reduce inflammation process as well as the fat content in the liver and rescue the physiological liver architecture of rats consuming continuously a high amount of alcohol. CONCLUSIONS: Due to its oral application and therapeutic superiority over an off-label use of the hepatoprotector ursodeoxycholic acid (UDCA), this new class of inhibitors marks the first rational, pharmaceutical concept in long-term treatment of ASH.

Knockdown of UTX/KDM6A Enriches Precursor Cell Populations in Urothelial Cell Cultures and Cell Lines.

The histone demethylase UTX (gene: KDM6A) directs cell and tissue differentiation during development. Deleterious mutations in KDM6A occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood; plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation; namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14high precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14low cells. UTX knockdown induced apoptosis and enriched KRT14high cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of KDM6A mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.

Urothelial Carcinoma of the Bladder Induces Endothelial Cell Activation and Hypercoagulation.

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell-derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer-derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell-derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A-mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. IMPLICATIONS: We identified the VEGF-A-mediated release of VWF from endothelial cells to be associated with bladder cancer progression.