Evaluation der Tumormarker TKTL1, Apo10 und GD2 in Rhabdomyosarkom- und Neuroblastom-Tumoren

Das Rhabdomyosarkom (RMS) und das Neuroblastom (NB) gehören zu den häufigsten extrakranialen, soliden Tumoren im Kindesalter. Dabei beruhen Tumorstaging, Diagnose und Verlaufskontrollen bisher überwiegend auf bildgebenden Verfahren und auf histopathologischen Untersuchungen von Gewebebiopsien. In dieser Arbeit wurde evaluiert, ob die Detektion der Tumormarker TKTL1, Apo10 sowie GD2 (in NB) in Makrophagen (MΦ) aus dem Blut von RMS und NB-Patienten mit dem EDIM-Test möglich ist. Dabei sollte außerdem das Potential dieser Methode und der Tumormarker für die Diagnose und das Monitoring der beiden Tumorentitäten überprüft werden. In dem ersten Teil der Arbeit wurden Tumorzelllinien und Gewebematerial der beiden Tumorentitäten als auch in vitro differenzierte (MΦ) auf die Expression der drei Tumormarker untersucht, um die Nutzbarkeit dieser Marker in RMS und NB zu analysieren. In einer prospektiven Studie konnte dann gezeigt werden, dass sich diese Tumormarker in MΦ aus Blut von RMS und NB-Patienten durch den EDIM-Test durchflusszytometrisch detektieren lassen. Die Expressions-scores der Tumormarker in den Tumorpatienten waren im Vergleich zu gesunden Probanden signifikant erhöht. Prä- und postoperative Messungen weisen außerdem auf den Nutzen des EDIM-Tests für das Monitoring von Tumorresektionen. In dem letzten Teil der Arbeit wurde der Hintergrund der erhöhten TKTL1-Expression analysiert. Dabei wurde eine DNA-Hypomethylierung als mögliche Ursache untersucht. Durch die Behandlung von RMS- und NB-Zelllinien mit dem demethylierenden Reagenz Decitabin konnte eine Korrelation zwischen einer erhöhten TKTL1 mRNA Expression und dem Methylierungszustand der DNA beobachtet werden. Durch eine semi quantitative Methylierungs-Analyse von Tumor Gewebeproben konnte eine Hypomethylierung einer CpG-Insel im TKTL1-Promotor aufgezeigt werden. Dabei korreliert die TKTL1 Expression mit dem Grad der Hypomethylierung des TKTL1 Promotors. Die im Rahmen dieser Arbeit erzielten Erkenntnisse zeigen zum ersten Mal die Möglichkeit der Detektion von RMS und NB-Tumoren mithilfe des EDIM-Tests. Die Verwendung dieses Tests könnte die Detektion von RMS- und NB-Tumoren in Kindern verbessern und relevant für die Diagnose dieser Tumore sein

Epitope Detection in Monocytes (EDIM) As a New Method of Liquid Biopsy in Pediatric Rhabdomyosarcoma

Biomarkers allowing characterization of pediatric rhabdomyosarcoma (RMS) are lacking. Epitope detection in monocytes (EDIM) is a novel method focused on detection of the biomarkers TKTL1 (transketolase-like protein 1) and Apo10 (epitope of DNaseX) in activated monocytes (CD14(+)/CD16(+)) from patient’s blood. We investigated the expression of these biomarkers in RMS cell lines, tumor material, and peripheral blood from RMS patients. Expression levels of TKTL1 and DNaseX/Apo10 in RMS cell lines (RH30, RD) and tumor samples were analyzed by RT-PCR and flow cytometry. Blood samples of 29 RMS patients were measured and compared to 27 healthy individuals. The percentages of activated CD14(+)/CD16(+) monocytes harboring TKTL1 and Apo10 were determined. EDIM-TKTL1 and EDIM-Apo10 expression scores were calculated. The relationship between TKTL1 expression and DNA-hypomethylation was evaluated. Both RMS cell lines and tumor samples showed significantly higher expression levels of TKTL1 and DNaseX/Apo10 compared to skeletal muscle cells (SkMC). EDIM-TKTL1 and EDIM-Apo10 scores were positive in 96.5% of patients with RMS. All healthy controls had negative corresponding scores. RMS cell lines show increased expression levels of the biomarkers TKTL1 and DNaseX/Apo10. The sensitivity of the EDIM blood test indicates that this assay might serve as an additional tool in pediatric RMS

Serum and Glucocorticoid Inducible Kinase 1-Sensitive Survival, Proliferation and Migration of Rhabdomyosarcoma Cells

Background/Aims: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. Methods: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. Results: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 – an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. Conclusions: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma

Epitope detection in monocytes (EDIM) for liquid biopsy including identification of GD2 in childhood neuroblastoma-a pilot study

BACKGROUND: Neuroblastoma (NB) is the most common paediatric extracranial solid malignancy. We analysed the role of the epitope detection in monocytes (EDIM) technique for liquid biopsy in NB patients. METHODS: Tumour epitopes transketolase-like 1 (TKTL1), Apo10 (DNaseX) and GD2 were assessed: expression levels in seven NB tumour samples and five NB cell lines were analysed using RT-PCR and flow cytometry. LAN-1 cells were co-cultured with blood and assessed using EDIM. Peripheral blood macrophages of patients with neuroblastoma (n = 38) and healthy individuals (control group, n = 37) were labelled (CD14(+)/CD16(+)) and assessed for TKTL1, Apo10 and GD2 using the EDIM technology. RESULTS: mRNA expression of TKTL1 and DNaseX/Apo10 was elevated in 6/7 NB samples. Spike experiments showed upregulation of TKTL1, Apo10 and GD2 in LAN-1 cells following co-culturing with blood. TKTL1 and Apo10 were present in macrophages of 36/38 patients, and GD2 in 15/19 patients. The 37 control samples were all negative. EDIM expression scores of the three epitopes allowed differentiation between NB patients and healthy individuals. CONCLUSIONS: The EDIM test might serve as a non-invasive tool for liquid biopsy in children suffering from NB. Future studies are necessary for assessing risk stratification, tumour biology, treatment monitoring, and early detection of tumour relapses

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells

Background/Aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development