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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.

Supernatants of UROtsa cells failed to induce endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by UROtsa cells.

S1: Schematic overview of the potential cross talk between bladder cancer cells, the endothelium and the coagulation system. S2: Expression of procoagulant factors and ability of T24 cells to produce thrombin. S3: UCB cells trigger angiogenesis. S4: VWF mediated blood vessel occlusion in peritumoral tissue. Supplementary Table 1. Correlation of the overall patients' survival with the expression of the indicated genes. Supplementary Video 1. Supernatants of UROtsa cells failed to induce endothelial activation.Supplementary Video 2. Supernatants of RT4 cells induced endothelial activation.

Background Bladder cancer cells orchestrate tumour progression by pro-inflammatory cytokines. Cytokines modulate the local tumour microenvironment and increase the susceptibility of tumour distant tissues for metastasis. Here, we investigated the impact of human bladder cancer cell derived factors on the ability to modulate and activate human vascular endothelial cells. Methods The pro-inflammatory and pro-coagulatory potential of four different bladder cancer cell lines was accessed by qRT-PCR arrays and ELISA. Modulation and activation of endothelial cells was studied in microfluidic devices. Clinical relevance of our findings was confirmed by immune histology in tissue samples of bladder cancer patients and public transcriptome data. Results The unbalanced ratio between interleukin (IL)-1 and IL-1 receptor antagonist (IL-1ra) in the secretome of bladder cancer cells converted the quiescent vascular endothelium into a pro-adhesive, pro-inflammatory, and pro-coagulatory surface. Microfluidic experiments showed that tumour cell induced endothelial cell activation promoted leukocyte recruitment and platelet adhesion. Human bladder cancer tissue analysis confirmed that loss of IL-1ra and elevated IL-1 expression was associated with enhanced cancer progression. Conclusions Our data indicate that IL-1 and IL-1ra were dysregulated in bladder cancer and could facilitate tumour dissemination through endothelial cell activation. Targeting the IL-1/IL-1ra axis might attenuate tumour-mediated inflammation and metastasis formation.

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.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.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.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.HINTERGRUND: Die Therapieoptionen beim lokal fortgeschrittenen und metastasierten Urothelkarzinom beschränken sich aktuell auf etablierte Chemo- sowie Immuntherapien. Zielgerichtete Therapien sind bisher nur wenigen Patienten vorbehalten. Urotheliale Organoidsysteme könnten durch patientenindividuelle Testung und Vorhersagen zum Therapieansprechen einen entscheidenden Beitrag für eine effektivere personalisierte Therapie darstellen. Das Ziel der vorliegenden Arbeit ist die Beschreibung des Standes der Wissenschaft bei klinisch einsetzbaren Organoidsystemen für das Urothelkarzinom.Es erfolgte eine systematische Literaturrecherche in mehreren Datenbanken (Medline, Cochrane library) sowie Studienregistern (ClinicalTrials.gov, das EU-Register für klinische Studien und das WHO International Clinical Trials Registry).Insgesamt erfüllten 7 Studien zur Thematik der Urothelkarzinomorganoide die Einschlusskriterien. Diese beschreiben die Etablierung von Organoidsystemen bei Patienten mit Tumoren der Harnblase oder des Nierenbeckens. Die Erfolgsrate zur Etablierung von Organoiden betrug bei nicht-muskelinvasiven Tumoren 70–77 % und bei muskelinvasiven Tumoren etwa 42 %. Bei den Patientenorganoiden wurden systematische Testungen von Medikamenten durchgeführt. Dabei zeigte sich ein individuelles Therapieansprechen der Organoide, vergleichbar mit einem individuellen Therapieansprechen bei Patienten.Bei Urothelkarzinomen ist die Generierung von Organoiden und die Ex-vivo-Testung individueller Therapien möglich. Durch das Fehlen einer standardisierten Methodik bleibt deren Einsatz zum aktuellen Zeitpunkt experimentell. Es besteht durch die Methodik ein hohes Potenzial, Patienten mit einem Urothelkarzinom personalisierte Therapiekonzepte anzubieten.

Background : Various imaging modalities can be used in addition to white light (WL) to improve detection of bladder cancer (BC). Objective : To use real-time multispectral imaging (rMSI) during urethrocystoscopy to combine different imaging modalities to achieve multiparametric cystoscopy (MPC). Design, setting, and participants : The rMSI system consisted of a camera with a spectral filter, a multi-LED light source, a microcontroller, and a computer for display and data acquisition. MSI with this system was achieved via temporal multiplexing. Surgical procedure : MPC was performed in ten patients with a diagnosed bladder tumor. Measurements : We gathered evidence to prove the feasibility of our approach. In addition, experienced urologists performed post-interventional evaluation of images of individual lesions. Images were independently rated in a semiquantitative manner for each modality. A statistical model was built for pairwise comparisons across modalities. Results and limitations : Overall, 31 lesions were detected using the rMSI set-up. Histopathology revealed malignancy in 27 lesions. All lesions could be visualized simultaneously in five modalities: WL, enhanced vascular contrast (EVC), blue light fluorescence, protoporphyrin IX fluorescence, and autofluorescence. EVC and photodynamic diagnosis images were merged in real time into one MP image. Using the recorded images, two observers identified all malignant lesions via MPC, whereas the single modalities did not arouse substantial suspicion for some lesions. The MP images of malignant lesions were rated significantly more suspicious than the images from single imaging modalities. Conclusions : We demonstrated for the first time the application of rMSI in endourology and we established MPC for detection of BC. This approach allows existing imaging modalities to be combined, and it may significantly improve the detection of bladder cancer. Patient summary : Real-time multispectral imaging was successfully used to combine different imaging aids for more comprehensive illustration of bladder tumors for surgeons. In the future, this technique may allow better detection of bladder tumors and more complete endoscopic resection in cases of cancer.

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.

Simple Summary Intrinsic telomere shortening promotes tumorigenesis in cells with impaired DNA damage repair mechanisms, as dysfunctional telomeres lead to chromosomal instability. More recent data show that the telomere length of peripheral blood leukocyte (PBL) cells can be a prognostic marker for survival of patients with solid tumors. However, reports on bladder cancer (BC) and renal cell carcinoma (RCC) are not consistent and partly contradictory. Our results show, first, that telomere length is shorter in patients with BC or RCC compared to patients without malignant disease. More importantly, the relative telomere length (RTL) of PBL cells is associated with survival of patients with BC and RCC. Thus, telomere length in PBL cells could be an auxiliary prognostic marker in BC and RCC. Abstract 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.

Personalized invitro 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 invitro. 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 invitro and invivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

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.

To date, it is still unclear how cancer stem cells (CSCs) regulate their stemness properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. In this study, we investigate how telomerase regulation affects cancer stem cell biology in pancreatic ductal adenocarcinoma (PDAC), and delineate the mechanisms by which telomerase activity and CSC properties are linked. Using 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 TERT-knockdown or pharmacological inhibitor (BIBR1532) resulted in CSC marker depletion in vitro, and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (KLF4, SOX2, OCT3/4, NANOG) and telomerase, which is essential for the self-renewal of pancreatic CSCs. Disruption the balance between telomerase activity and stemness factors, eliminates CSCs via induction of DNA damage and apoptosis, opening future perspectives to avoid CSC driven therapy resistance and tumor relapse in PDAC patients.

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.

Background and aimsAlcoholic 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.MethodsIn this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies.ResultsA 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.ConclusionsDue 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.

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.

Telomerase activity imparts eukaryotic cells with unlimited proliferation capacity, one of the cancer hallmarks. Over 90% of human urothelial carcinoma of the bladder (UCB) tumours are positive for telomerase activity. Telomerase activation can occur through several mechanisms. Mutations in the core promoter region of the human telomerase reverse transcriptase gene (TERT) cause telomerase reactivation in 60-80% of UCBs, whereas the prevalence of these mutations is lower in urothelial cancers of other origins. TERT promoter mutations are the most frequent genetic alteration across all stages of UCB, indicating a strong selection pressure during neoplastic transformation. TERT promoter mutations could arise during regeneration of normal urothelium and, owing to consequential telomerase reactivation, might be the basis of UCB initiation, which represents a new model of urothelial cancer origination. In the future, TERT promoter mutations and telomerase activity might have diagnostic and therapeutic applications in UCB.

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., publishedVersion

// Markus Eckstein 1, * , Ralph M. Wirtz 2, 3, * , Carolin Pfannstil 1, * , Sven Wach 4, * , Robert Stoehr 1, * , Johannes Breyer 6, * , Franziska Erlmeier 7, * , Cagatay Gunes 8, * , Katja Nitschke 5, * , Wilko Weichert 7, * , Wolfgang Otto 6, * , Bastian Keck 4, * , Sebastian Eidt 3, * , Maximilian Burger 6, * , Helge Taubert 4, * , Bernd Wullich 4, * , Christian Bolenz 8, * , Arndt Hartmann 1, * and Philipp Erben 5, * 1 Institute of Pathology, University of Erlangen-Nuremberg, Erlangen, Germany 2 STRATIFYER Molecular Pathology GmbH, Cologne, Germany 3 Institute of Pathology at The St. Elisabeth Hospital Koln-Hohenlind, Cologne, Germany 4 Department of Urology, University of Erlangen-Nuremberg, Erlangen, Germany 5 Department of Urology Mannheim, University of Heidelberg, Mannheim, Germany 6 Department of Urology, University of Regensburg, Regensburg, Germany 7 Institute of Pathology, Technical University Munich, Munich, Germany 8 Department of Urology, University of Ulm, Ulm, Germany * On behalf of the BRIDGE-Consortium Germany Correspondence to: Markus Eckstein, email: markus.eckstein@uk-erlangen.de Keywords: bladder cancer; PD-L1; checkpoint inhibitors; molecular therapy stratification; immunohistochemistry Received: October 26, 2017 Accepted: February 10, 2018 Epub: February 19, 2018 Published: March 13, 2018 ABSTRACT Background: Immunohistochemical PD-L1 assessment is currently used to identify responders towards checkpoint inhibitors although it is limited by inter-observer effects. Here, we conducted a multi-center round robin test to prove the possibility of assessing the PD-L1 status by gene expression to avoid inter-observer effects. Patients and methods: Gene expression of PD-L1 was analyzed in a total of 294 samples (14 cases non-muscle invasive and muscle-invasive bladder cancer; MIBC) in seven centers by a RT-qPCR kit and compared with immunohistochemical scoring of three pathologists (DAKO, 22c3). Both assays were compared towards prognosis prediction in a cohort of 88 patients with MIBC. Results: PD-L1 gene expression revealed very high inter center correlation (centrally extracted RNA: r = 0.68–0.98, p ≤ 0.0076; locally extracted RNA: r = 0.81–0.98, p ≤ 0.0014). IHC Inter-observer concordance was moderate to substantial for immune cells (IC), fair for combined IC/ tumor cell (TC) (IC: κ = 0.50–0.61; IC + TC: κ = 0.50), and fair for TC scoring (κ = 0.26–0.35). Gene expression assessment resulted in more positive cases (9/14 cases positive vs. 6/14 cases [IHC]) which could be validated in the independent cohort. Positive mRNA status was associated with significantly better overall and disease-specific survival (5-year OS: 50% vs. 26%, p = 0.0042, HR = 0.48; 5 year DSS: 65% vs. 40%, p = 0.012, HR = 0.49). The 1% IHC IC cut-off also revealed significant better OS (5 year OS: 58% vs. 31%, p = 0.036, HR = 0.62). Conclusion: Gene expression showed very high inter-center agreement. Gene expression assessment also resulted in more positive cases and revealed better prognosis prediction. PD-L1 mRNA expression seems to be a reproducible and robust tool for PD-L1 assessment.

Telomere shortening as a consequence of cell divisions during aging and chronic diseases associates with an increased cancer risk. Experimental data revealed that telomere shortening results in telomere dysfunction, which in turn affects tumorigenesis in two ways. First, telomere dysfunction suppresses tumor progression by the activation of DNA damage checkpoints, which induce cell cycle arrest (senescence) or apoptosis, as well as by inducing metabolic compromise and activation of immune responses directed against senescent cells. Second, telomere dysfunction promotes tumorigenesis by inducing chromosomal instability in tumor initiating cells, by inhibiting proliferative competition of non-transformed cells, and possibly, also by influencing tumor cell plasticity. The tumor promoting effects of telomere dysfunction are context dependent and require the loss of p53-dependent DNA damage checkpoints or other genetic modifiers that attenuate DNA damage responses possibly involving complex interactions of different genes. The activation of telomere stabilizing mechanisms appears as a subsequent step, which is required to enable immortal grotwh of emerging cancer cells. Here, we conceptually discuss our current knowledge and new, unpublished experimental data on telomere dependent influences on tumor initiation and progression.

BACKGROUND: Emerging imaging technologies such as real-time multispectral imaging (rMSI) hold great potential for simultaneous visualization of multiple target structures using fluorophores on various tumours including bladder cancer (BC). These technologies, however, require a multi-step preclinical evaluation process, including mouse models. OBJECTIVE: To demonstrate the suitability of the new rMSI technology for the detection of premalignant lesions and malignant BC in a preclinical mouse model using contrast agents. METHODS: Tumours were induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), which is known to induce BC in rodent models. In total, 30 mice (C57BL/6) were fed with 0.1% BBN ad libitum in drinking water for up to 5 months. Bladders were excised at 3 (n = 6) and 5 months (n = 24) of treatment and incubated ex vivo with Hexaminolevulinat (HAL, Hexvix®), CD47-FITC, CD90.2-FITC or a combination of CD90.2-FITC/CD47-FITC and HAL. The bladders were analyzed by an endoscopic rMSI prototype system equipped with a spectral filter (Chroma), a 4 mm endoscope (Karl Storz) with 30° optic, a LED light source and a PC with a microcontroller board. RESULTS: 5-month treatment of mice with 0.1% BBN led to the formation of squamous carcinoma (46%, n = 11) while urothelial carcinoma was observed only in one mouse (4%, n = 1). Carcinoma in situ (CIS) was detectable in twelve out of twenty-four mice (50%, n = 12) treated for 5 month and in three out of six mice (50%, n = 3) treated for 3 months.The metabolite of HAL, protoporphyrin IX (PpIX), could be reliably and specifically detected in all of mouse bladder tumours and CIS. However, detection of the CD90.2 surface marker was less reliable, potentially due to species- or tumour-subtype specificity. CONCLUSIONS: This model offers the potential for preclinical imaging studies with combined fluorescence targets, e.g. HAL, in combination with BC-specific antibodies.

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.

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.

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.

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.

n this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged., publishedVersion

Objectives: To measure and to modulate the invasive potential of urothelial carcinoma of the bladder (UCB) cells in a standardized preclinical setting using broad-spectrum matrix-metalloproteinase (MMPs) inhibitors and specific targeting of MMP7. Materials and Methods: MMP expression levels in UCB cells were determined by quantitative real-time PCR (qRT-PCR) and gel zymographies of cell supernatants (MMP9, MMP2 and MMP1) and cell lysates (MMP7). The invasiveness of human UCB cells (HT1197 and T24/83) and human benign urothelial cells (UROtsa) was modulated by a broad-spectrum MMP inhibitor (4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic acid; AHA) and by MMP7 specific siRNAs. MMP7 knockdown efficiency was assessed by qRT-PCR and western blot. Invasive potential of UCB cells was measured by a standardized trans-epithelial electrical resistance (TEER) assay. Results: Different MMP secretion profiles were measured in UCB cells. The active form of MMP7 was exclusively detected in HT1197 cells. Characteristic TEER breakdown patterns were observed in UCB cells when compared to benign cells. Invasive potentials were significantly higher in HT1197 cells than in T24/83 and in UROtsa cells [14.8±5.75 vs. 1.5±0.56 and 1.2±0.15, respectively; p

Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.

Telomere shortening impairs proliferation of transformed cells but also leads to cancer initiation by inducing chromosomal instability. Here, we discuss recent developments in our understanding of the role of telomeres in replication stress and how telomerase expression in somatic stem cells may affect genome integrity control and carcinogenesis.

High telomerase activity is detected in nearly all human cancers but most human cells are devoid of telomerase activity. There is well-documented evidence that reactivation of telomerase occurs during cellular transformation. In humans, tumors can rely in reactivation of telomerase or originate in a telomerase positive stem/progenitor cell, or rely in alternative lengthening of telomeres, a telomerase-independent telomere-length maintenance mechanism. In this review, we will focus on the telomerase positive tumors. In this context, the recent findings that telomerase reverse transcriptase (TERT) promoter mutations represent the most common non-coding mutations in human cancer have flared up the long-standing discussion whether cancer originates from telomerase positive stem cells or telomerase reactivation is a final step in cellular transformation. Here, we will discuss the pros and cons of both concepts in the context of telomere length-dependent and telomere length-independent functions of telomerase. Together, these observations may provoke a re-evaluation of telomere and telomerase based therapies, both in telomerase inhibition for cancer therapy and telomerase activation for tissue regeneration and anti-ageing strategies.

Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-β expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-β activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-β to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-β are involved in TERT gene regulation during carcinogenesis.

Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient-derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis-associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. Conclusion: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease. (HEPATOLOGY 2011;)

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Telomeres form protective caps at the ends of linear chromosomes to prevent nucleolytic degradation, end-to-end fusion, irregular recombination, and chromosomal instability. Telomeres are composed of repetitive DNA sequences (TTAGGG)n in humans, that are bound by specialized telomere binding proteins. Telomeres lose capping function in response to telomere shortening, which occurs during each division of cells that lack telomerase activity-the enzyme that can synthesize telomeres de novo. Telomeres have a dual role in cancer: telomere shortening can lead to induction of chromosomal instability and to the initiation of tumors, however, initiated tumors need to reactivate telomerase in order to stabilize chromosomes and to gain immortal growth capacity. In this review, we summarize current knowledge on the role of telomeres in the maintenance of chromosomal stability and carcinogenesis.

Telomerase activity is repressed in most human somatic tissues during differentiation processes but strongly up-regulated in most human tumors. Regulation of human telomerase activity primarily occurs at the level of transcriptional initiation of the TERT gene, which encodes the catalytic subunit of telomerase. We have generated a novel transgenic mouse model to study the regulation of the human TERT gene promoter in an in vivo system. For this purpose, we have cloned an 8.0-kbp human TERT promoter fragment in front of the bacterial lacZ reporter gene (hTERTp-lacZ), which encodes the β-galactosidase enzyme. Expression of the reporter gene was monitored by reverse transcription–PCR analysis, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining of whole mount preparations, and histologic sections. We find that the activity of the human TERT promoter in most normal mouse tissues recapitulates the expression of the hTERT gene in normal human tissues and is under tighter control when compared with the endogenous mouse TERT gene expression. In testis, where highest lacZ expression was observed, the expression of the reporter gene was restricted to the spermatogonial stem cells and the spermatocytes. Intriguingly, we find increased levels of lacZ expression in mammary tumors of hTERTp-lacZ × p53+/− bitransgenic mouse mammary tumor model. Thus, this transgenic mouse model provides a suitable in vivo system to analyze the expression of the human TERT gene under physiologic conditions and during tumorigenesis.

The enzymatic activity of telomerase counters erosion of telomeric DNA that occurs as a biochemical consequence during replication. This primary function of telomerase resolves the ‘end-replication problem’ and prevents telomere dysfunction and replicative senescence ensuring cellular immortality. The vast majority of human cells are devoid of telomerase activity but reactivation of telomerase occurs in more than 80% of human cancers. Moreover, telomerase activity is a pre-requisite of sustained organ maintenance under physiological hyper-proliferative conditions such as liver regeneration and for the proliferation of activated B- and T- cells. Increasing evidence indicates at telomere length independent functions of telomerase, including DNA-damage repair, mitochondrial function and stem cell activity. Mechanisms of how telomerase can fulfill such diverse functions remain yet to be elucidated. Our recent findings showing that telomerase activates ribosomal biogenesis under oncogenic and regenerative conditions can shed a light to some of these questions.

Telomeres form protective caps at the ends of linear chromosomes to prevent nucleolytic degradation, end-to-end fusion, irregular recombination, and chromosomal instability. Telomeres are composed of repetitive DNA sequences (TTAGGG)n in humans, that are bound by specialized telomere binding proteins. Telomeres lose capping function in response to telomere shortening, which occurs during each division of cells that lack telomerase activity—the enzyme that can synthesize telomeres de novo. Telomeres have a dual role in cancer: telomere shortening can lead to induction of chromosomal instability and to the initiation of tumors, however, initiated tumors need to reactivate telomerase in order to stabilize chromosomes and to gain immortal growth capacity. In this review, we summarize current knowledge on the role of telomeres in the maintenance of chromosomal stability and carcinogenesis.

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy-controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb-dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase-deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy-induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.

DNA damage and telomere dysfunction shorten organismal lifespan. Here we show that oral glucose administration at advanced age increases health and lifespan of telomere dysfunctional mice. The study reveals that energy consumption increases in telomere dysfunctional cells resulting in enhanced glucose metabolism both in glycolysis and in the tricarboxylic acid cycle at organismal level. In ageing telomere dysfunctional mice, normal diet provides insufficient amounts of glucose thus leading to impaired energy homeostasis, catabolism, suppression of IGF-1/mTOR signalling, suppression of mitochondrial biogenesis and tissue atrophy. A glucose-enriched diet reverts these defects by activating glycolysis, mitochondrial biogenesis and oxidative glucose metabolism. The beneficial effects of glucose substitution on mitochondrial function and glucose metabolism are blocked by mTOR inhibition but mimicked by IGF-1 application. Together, these results provide the first experimental evidence that telomere dysfunction enhances the requirement of glucose substitution for the maintenance of energy homeostasis and IGF-1/mTOR-dependent mitochondrial biogenesis in ageing tissues., Shortened telomeres and reduced mitochondrial biogenesis are cellular hallmarks of ageing. Here, Missios et al. show that old mice with telomere dysfunction have an increased energetic demand that cannot be met unless mice are fed a glucose-rich diet, which improves energy metabolism and extends lifespan.

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions This study offers a novel view on the role of telomere attrition in human tumours, providing evidence for tumour suppressor activity resulting from telomere dysfunction-induced DNA damage responses., In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.

International audience; BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.

Adiyaman University, Medical Faculty, Medical Genetic Department, diyaman, Turkey TUBITAK MRC Genetic Engineering and Biotechnology Institute GEBI), Kocaeli, Turkey Institute for Molecular Medicine and Max-Planck 2169 Research roup on Stem Cell Aging, University of Ulm, Albert-Einstein-Allee 11, lm, Germany Departmentof Biochemistry, Faculty ofVeterinaryMedicine,Mehmet kif Ersoy University, Burdur, Turkey Erciyes University, Medical Faculty, Medical Genetic Department, ayseri, Turkey Bulgarian Academy of Sciences, Institute of Molecular Biology, 21 cad. G. Bonchev str.1113 Sofia, Bulgaria