Philip Kienzl

LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

Epithelial splicing regulatory protein 1 and 2 paralogues correlate with splice signatures and favorable outcome in human colorectal cancer

ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression. Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P=0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P=0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15–0.91, P=0.032; ESRP2: HR=0.23, 95%CI 0.08–0.65, P=0.006). Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC.

Prevention of allergy by virus-like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

In high‐risk populations, allergen‐specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus‐like nanoparticles (VNP) expressing surface‐exposed or shielded allergens.

Abstract 4597: Telomere maintenance mechanism and expression in colorectal cancer

Tumors can overcome cellular senescence, one of the main barriers to tumor development and immortality by telomere maintenance mechanism (TMM). 90% of colorectal carcinomas (CRC) use telomerase activity (TA) as TMM. Mechanisms of remaining CRC tumors are not well analyzed yet and may utilize alternative lengthening of telomeres (ALT) or a not defined telomere maintenance mechanism (NDTMM). Both TA and expression of telomeric repeat containing RNA (TERRA) were recently identified to correlate with tumor grade. This led us to evaluate possible correlations of TMM and TERRA levels to clinical data. Tissues from 49 CRC patients (35 grade II, 14 grade III, 2 polyps) and cell lines (N = 7) were assessed. Tumor tissues were compared to matched adjacent non-tumor tissues. Mean telomere length (TL) was measured by real-time PCR and telomere restriction fragment (TRF) length analyses. TMM were evaluated by measuring TA with telomere amplification protocol (TRAP) and by detection of ALT with c-circles. Real-time RT-PCR assays for relative quantity (RQ) of transcript levels for TERRA, telomerase genes hTERT and hTERC were determined in relation to levels of house-keeping genes and normal tissues. RQ values were analyzed for significant differences between groups by Mann-Whitney test and were assessed after log-transformation with the Pearson9s correlation calculation. TL of grade II and grade III tumors were in the range of 5.5 ± 1.3kbp and 5.3 ± 0.5kbp, respectively (mean ± SD), and were comparable with TL of normal tissue and cell lines. TA was detected with mean of 27 TPG units in 90% (34 of 38) and with mean of 4 units in 24% (9 of 38) of tumor and normal tissues, respectively. All cell lines analyzed demonstrate TA with mean of 51 units. A restricted set of tumors consisting of 2 with and 4 without TA was analyzed for ALT. One of the analyzed tumors without detectable TA showed significantly elevated levels of c-circles, but did not reach levels of sarcomas and cell models with ALT, indicating the existence of NDTMM in CRC. Within the transcripts analyzed, TERRA levels showed a moderate negative correlation with TA (Pearson r=−0.45, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4597. doi:1538-7445.AM2012-4597

Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication

Significance Rhinovirus (RV) is the causative agent of the common cold and other respiratory tract infections. Despite the vast prevalence, effective treatment or prevention strategies are lacking. Here, we analyzed metabolic alterations in infected cells and found a pronounced reprogramming of host cell metabolism toward an anabolic state, which involved enhancement of glucose uptake and glycogenolysis. We further demonstrate that these alterations can be reverted by treatment with 2-deoxyglucose, a glycolysis inhibitor, which results in a disruption of RV replication in vitro and in vivo. Thus, we show how the specific metabolic fingerprint of viral infection can be used to generate targets for antiviral therapy. Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.

An intra-individual surgical wound comparison shows that octenidine-based hydrogel wound dressing ameliorates scar appearance following abdominoplasty

Hypertrophic scar formation because of surgical procedures is associated with higher levels of pain, a lower quality of life, and poor cosmetic outcome and requires more resources in follow‐up management. An octenidine‐based hydrogel has been shown to modulate immunological function in an in vitro wound model, suggesting an improved scar formation. In this prospective, randomised, observer‐blinded, and intra‐patient‐controlled study, 45 patients who underwent abdominoplasty or mastectomy with transverse rectus abdominis muscle (TRAM) flap reconstruction were given both a standard postoperative wound dressing on one wound side and an octenidine‐based hydrogel with transparent film dressing, covered with standard postoperative dressing on the other side. Four instances of hypertrophia were reported in the gel side versus 12 in the standard dressing side. Visual Analogue Scale (VAS) pain scores taken during postoperative dressing changes showed reduced scores on the gel side at all time points. Vancouver Scar Scale (VSS) scores showed improvement in the gel side at 3, 6, and 12 months postoperatively. Skin distensibility measured using a cutometer showed significantly improved measures in gel‐treated wounds, similar to measures of healthy skin. Trans‐epidermal water loss (TEWL), measured using a tewameter, showed improved values on the gel side soon after surgery, with both the control and the gel side normalising after approximately 6 months. The octenidine‐based wound dressing demonstrates improved wound healing associated with a lower incidence of hypertrophic scar formation.

Aseptic surgical preparation for upper eyelid blepharoplasty via full-face octenidine antiseptic without antibiotic medication shows effective prophylaxis against post-surgical wound infection: New insights into blepharoplasty outcomes

Blepharoplasty is the third most common plastic surgical procedure in the USA. Due to the emergence of multiresistant bacteria, optimising the antiseptic procedure is crucial. Choice of antiseptics plays an important role as they may cause skin irritation and colouring of disinfected areas. In this study, the use of the aqueous antiseptic octenisept® (octenidine) was evaluated in the outcome of blepharoplasties: incidence of wound dehiscence; haematoma; and infection in correlation with gender, medication, smoking habits and time of year. This retrospective surveillance study included 352 patients (median age 58·3 years). Skin disinfection was performed thrice prior to blepharoplasty. Sutures were removed on day 6. None of the patients suffered from wound infection. The total rate of wound dehiscence was 6·3%, with a higher ratio among male patients. Smokers and patients on anticoagulant medication showed a significantly higher incidence of wound dehiscence. Throughout the year, rates of wound dehiscence were highest in summer. Aseptic surgical preparation for blepharoplasty via full‐face scrub with octenisept® without oral antibiotic prophylaxis is well tolerated, with no report of wound infection, which may improve antibiotic stewardship as well as patient comfort. Elective upper eyelid blepharoplasty may ideally be performed in winter.

Abstract LB-084: A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres

LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells. Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084