Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named ‘scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absen

Predictors of a positive surgical margin and its impact on the long-term oncological outcome after transperitoneal laparoscopic radical prostatectomy

Heutzutage ist die laparoskopische radikale Prostatektomie (LRP) ein chirurgisches Standardverfahren zur kurativen Behandlung des lokalisierten Prostatakarzinoms. Neben einer Darstellung von Epidemiologie, Pathogenese, diagnostischen Möglichkeiten und verschiedenen therapeutischen Ansätzen ist es das vorrangige Ziel der vorliegenden Arbeit im Follow-Up eines großen Patientenkollektivs von Männern, die sich einer LRP unterzogen haben, zu ermitteln, inwiefern eine positive Korrelation zwischen Schnittrandergebnissen und biochemisch fassbarer Rezidivrate vorliegt. Des Weiteren galt als Anliegen dieser Arbeit, ob unabhängige Prädiktoren für einen positiven Schnittrandstatus und in Folge eines biochemischen Rezidives (BCR) herauszufinden sind, um damit schlussendlich eine Korrelation zur rezidivfreien Überlebensrate nachzuvollziehen. Die hierfür untersuchte Kohorte bestand aus 1.845 Männern, die zwischen 1999 und 2007 laparoskopisch operiert wurden. Der mediane postoperative Follow-Up betrug 56 Monate. Alle wesentlichen prä-, intra- und postoperativen Variablen wurden retrospektiv ermittelt. Ein BCR wurde als PSA - Anstieg > 0,2 ng/ml mindestens sechs Wochen nach erfolgter LRP definiert. Prädiktoren eines positiven Schnittrandstatus (PSM) und eines BCR wurden durch Nutzung der univariaten und multivariablen Logistik bzw. Cox Regressionsanalyse identifiziert. In der aktuellen Kohorte hatten 70,9% der Patienten einen negativen Schnittrandstatus (NSM) und 29,1% einen PSM. Multivariable Regression identifizierte cT2 (OR 1,66, p = 0,001), Biopsie Gleason Score > 7 (OR 1,84, p = 0,031), einen PSA von 10-20 ng/ml (OR 1,58, p = 0,010) und > 20 ng/ml (OR 6,82, p < 0,0001) als unabhängige Prädiktoren für PSM. Die mediane Operationszeit war signifikant länger bei Patienten mit PSM (225 vs. 245 min, p < 0,0001). Prostatavolumen und PSM waren invers assoziiert (p = 0,002). Alter, BMI und die Erhaltung der Gefäßnervenbündel zeigten keine signifikante Korrelation mit dem PSM (alle p > 0,05). Das RFS nach 10 Jahren betrug 82,9% bei NSM bzw. 59,2% bei PSM. Bei Patienten mit NSM war das RFS etwa 20 Monate länger im Vergleich zu Patienten mit PSM (117 versus 97 Monate). Das mediane RFS korrelierte mit dem pathologischem Tumorstadium und dem Schnittrandstatus: 120 Monate (122,0 vs. 111,4), 98,2 (100,5 vs. 92,5), 71,1 (85,4 vs. 61,2) und 58,7 (68,4 vs. 38,5) für organbegrenzt, extraprostatische Ausdehnung, Samenblaseninfiltration und Lymphknotenbefall. Die Multivariable Regression identifizierte einen LRP Gleason Score von 7 (HR 2,45, p 7 (HR 4,76, p < 0,0001), fortgeschrittene Krankheitsstadien (p < 0,0001), ein PSA von 10-20 ng/ml (HR 1,46, p = 0,004) und den PSM (HR 1,49, p = 0,003) als unabhängige Prädiktoren eines BCR. Diese Studie verdeutlicht die Bedeutung des PSM als relevanten unabhängigen Prädiktor für ein BCR und unterstreicht die Notwendigkeit, die Technik der LRP weiter zu verbessern, um hohe Raten an R0 - Resektionen zu erzielen. Der PSM ist durch eine Reihe von Co - Variablen beeinflusst, die hilfreich für die Wahl der Operationstechnik und möglicher alternativer sowie adjuvanter Behandlungsstrategien sind.To date, laparoscopic radical prostatectomy (LRP) is a standard surgical procedure for curative treatment of localized prostate cancer. The aim of the present study was to evaluate the prognostic significance of the surgical margin status and other important factors for the prediction of biochemical recurrence (BCR) and recurrence-free survival (RFS). The cohort consisted of 1.845 men who underwent LRP between 1999 and 2007. Median postoperative follow-up was 56 months. All significant pre-, intra- and postoperative variables were collected retrospectively. BCR was defined as PSA increase > 0.2 ng/ml. Predictors of positive surgical margin (PSM) and BCR were identified utilizing univariate and multivariable logistic and Cox regression analyses, respectively. In the current cohort, 70.9% of patients had negative surgical margin (NSM) and 29.1% had PSM. Multivariable regression identified cT2 (OR 1.66, p = 0.001), biopsy Gleason score (GS) > 7 (OR 1.84, p = 0.031), PSA of 10-20 ng/ml (OR 1.58, p = 0.010) and > 20 ng/ml (OR 6.82, p < 0.0001) as independent predictors of PSM. Mean operative time was significantly longer in patients with PSM (225 vs. 245 min, p < 0.0001). Prostate volume was associated inversely with PSM (p = 0.002). Age, BMI and preservation of the neurovascular bundle showed no significant correlation with PSM (all p > 0.05). RFS after 10 years was 59.2% and 82.9% in the PSM and NSM group, respectively. Overall, RFS in patients with NSM was 20 months longer compared to patients with PSM (117 vs. 97 months). Median RFS correlated well with pathological tumor stage and surgical margin status: 120 months (122.0 vs. 111.4), 98.2 (100.5 vs. 92.5), 71.1 (85.4 vs. 61.2) and 58.7 (68.4 vs. 38.5) for organ confined disease, extraprostatic extension, seminal vesicle infiltration and lymph node invasion, respec-tively. Multivariable regression identified a LRP GS of 7 and > 7 (HR 2.45 and 4.76, p < 0.0001), advanced pathological stage (p < 0.0001), a PSA of 10-20 ng/ml (HR 1.46, p = 0.004) and a PSM (HR 1.49, p = 0.003) as independent predictors of BCR. This study clarifies the importance of PSM as a relevant independent predictor of BCR and underlines the necessity to further improve LRP technique to achieve high rates of R0 resections. PSM is influenced by a number of co-variables, which are helpful for the choice of surgical technique and potential alternative or adjuvant treatment strategies

Identifikation von Hypoxie-spezifischen Krebsmedikamenten mittels eines in vitro Spheroidmodells

Cancer is among the leading causes of morbidity and mortality worldwide and the effectiveness of novel treatment approaches is often limited by the development of drug resistance. Owing to the rapid growth, high metabolic rates and lagging or insufficient neo-angiogenesis, many solid tumors are characterized by large regions of hypoxia. Tumor hypoxia is a main determinant for resistance development and treatment failure and is therefore an important target for cancer therapy. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. Therefore, a tumor spheroid model that mimics regions of severe hypoxia in tumors was used to perform a high-content screen for the identification of hypoxia-sensitizing compounds that specifically induce cell death in hypoxic tumor spheroids. Using this unbiased phenotypic screening system, a set of compounds including the antipsychotic phenothiazine drug fluphenazine were identified as highly hypoxia-selective hits that act via a novel, yet unreported mode of action. It was discovered that fluphenazine functionally inhibits the lysosomal enzyme acid sphingomyelinase and induces cancer cell death in hypoxic tumor spheroids by sphingomyelin accumulation. Moreover, fluphenazine potentiates the transcriptional activity of hypoxia stress response pathways and causes hypoxia specific cell death via the ER-stress response transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress response pathways and thereby promotes their pro-apoptotic tumor suppressor functions to specifically kill cells in hypoxic tumor areas. This study provides further insight into the survival of cancer cells in hypoxic tumor regions and proposes the inhibition of sphingomyelin metabolism as potential novel target approach in hypoxic tumor regions that could lead to the identification of novel drugs to potentiate anti-cancer therapy.Krebserkrankungen gehören weltweit zu den häufigsten Ursachen für Morbidität und Mortalität. Trotz neuartiger Behandlungsansätze wird die Wirkung von Krebstherapeutika häufig durch die Entwicklung von Therapieresistenzen stark vermindert. Auf Grund des schnellen Wachstums, des hohen metabolischen Verbrauchs und der mangelnden oder unzureichenden Neubildung von sauerstoffversorgenden Blutgefäßen zeigen viele Tumore starke hypoxische Regionen auf. Tumorhypoxie ist eine der Hauptursachen für Resistenzentwicklung und Therapieversagen und zählt damit zu einem wichtigen Angriffspunkt für die Krebstherapie. Die häufigsten Therapieansätze für die zielgerichtete Behandlung von hypoxischen Tumorzellen sind auf die Inhibition von Anpassungssignalwegen fokussiert, welche unter starkem Sauerstoffmangel von den Krebszellen aktiviert werden um ihr Überleben zu sichern. Die Anzahl der bisher identifizierten hypoxiespezifischen Therapeutika ist jedoch sehr gering. Daher wurde in dieser Arbeit ein Tumorsphäroid-Model, welches Tumorregionen mit schwerer Hypoxie nachahmt, verwendet, um einen phänotypischen Screen (sog. High-Content-Screen) durchzuführen und chemische Substanzen zu identifizieren, welche spezifisch den Zelltod von hypoxischen Krebszellen induzieren. Mithilfe dieses Screeningsystems war es möglich eine Gruppe von Wirkstoffen zu identifizieren, welche ihre hypoxiespezifische Wirkung über einen neuen, bisher nicht beschriebenen Mechanismus ausüben. Zu dieser Gruppe gehört unter anderem das antipsychotische Phenothiazin-Arzneimittel Fluphenazin. Es wurde herausgefunden, dass Fluphenazin das lysosomale Enzym saure Sphingomyelinase funktionell inhibiert und durch eine starke Akkumulation von Sphingomyelin den Zelltod in hypoxischen Tumorsphäroiden hervorruft. Darüber hinaus verstärkt Fluphenazin die transkriptionelle Aktivität von hypoxischen Stressadaptionssignalwegen und bewirkt so über den ER-Stress Transkriptionsfaktor ATF4 ein hypoxiespezifisches Absterben von Krebszellen. Zusammenfassend lässt sich aus den hier vorgestellten Daten ein neuer, bisher unerforschter Mechanismus ableiten, welcher durch die Induktion von Sphingolipidstress zur Überaktivierung von hypoxischen Stressadaptionssignalwegen führt und so ihre pro-apoptotischen Tumorsupressorfunktionen aktiviert, um spezifisch Krebszellen in hypoxischen Tumorregionen zu töten. Diese Ergebnisse liefern neue wertvolle Einblicke in das Überleben von hypoxischen Tumorzellen und identifizieren den Sphingomyelinmetabolismus als potentiellen neuen Angriffspunkt für die Krebstherapie hypoxischer Tumore, was zur Entdeckung neuer Wirkstoffe beitragen könnte

Quantitative imaging of bone remodeling in patients with a unicompartmental joint unloading knee implant (ATLAS Knee System)—effect of metal artifacts on a SPECT-CT-based quantification

Background!#!SPECT-CT using radiolabeled phosphonates is considered a standard for assessing bone metabolism (e.g., in patients with osteoarthritis of knee joints). However, SPECT can be influenced by metal artifacts in CT caused by endoprostheses affecting attenuation correction. The current study examined the effects of metal artifacts in CT of a specific endoprosthesis design on quantitative hybrid SPECT-CT imaging. The implant was positioned inside a phantom homogenously filled with activity (955 MBq !##!Results!#!Significant effects caused by CT metal artifacts on attenuation-corrected SPECT were observed for the different slice positions, reconstructed slice thicknesses of CT data, and pitch and CT-reconstruction kernels used (all, p &amp;lt; 0.0001). Based on the optimization, a set of three protocols was identified minimizing the effect of CT metal artifacts on SPECT data. Regarding the reference region, the activity concentration in the anatomically correlated volume was underestimated by 8.9-10.1%. A slight inhomogeneity of the reconstructed activity concentration was detected inside the regions with a median up to 0.81% (p &amp;lt; 0.0001). Using an X-ray tube current of 40 mA showed the best result, balancing quantification and CT exposure.!##!Conclusion!#!The results of this study demonstrate the need for the evaluation of SPECT-CT protocols in prosthesis imaging. Phantom experiments demonstrated the possibility for quantitative SPECT-CT of bone turnover in a specific prosthesis design. Meanwhile, a systematic bias caused by metal implants on quantitative SPECT data has to be considered

Intragenic deletion of TRIM32 in compound heterozygotes with sarcotubular myopathy/LGMD2H

In 2005 the commonality of sarcotubular myopathy (STM) and limb girdle muscular dystrophy type 2H (LGMD2H) was demonstrated, as both are caused by the p D487N missense mutation in TRIM32 originally found in the Manitoba Hutterite population. Recently, three novel homozygous TRIM32 mutations have been described in LGMD patients. Here we describe a three generation Swedish family clinically presenting with limb girdle muscular weakness and histological features of a microvacuolar myopathy. The two index patients were compound heterozygotes for a frameshift mutation in TRIM32 (c.1560delC ) and a 30 kb intragenic deletion, encompassing parts of intron 1 and the entire exon 2 of TRIM32. In these patients, no full-length or truncated TRIM32 could be detected. Interestingly, heterozygous family members carrying only one mutation showed mild clinical symptoms and vacuolar changes in muscle. In our family, the phenotype encompasses additionally a mild demyelinating polyneuropathic syndrome. Thus STM and LGMD2H are the result of loss of function mutations that can be either deletions or missense mutations