Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public “The Cancer Genome Atlas” (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other non-endocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong “Warburg effect”, glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.

Increased cFLIP expression in thymic epithelial tumors blocks autophagy via NF-κB signalling Increased cFLIP expression in thymic epithelial tumors blocks autophagy via NF-κB signalling

Background: WNT4-driven non-canonical signaling is crucial for homeostasis and age-related involution of the thymus. Abnormal WNT signaling is important in many cancers, but the role of WNT signaling in thymic tumors is largely unknown.Materials & Methods: Expression and function of WNT4 and FZD6 were analyzed using qRT–PCR, Western blot, ELISA, in biopsies of non-neoplastic thymi (NT), thymoma and thymic carcinomas. ShRNA techniques and functional assays were used in primary thymic epithelial cells (pTECs) and TC cell line 1889c. Cells were conventionally (2D) grown and in three-dimensional (3D) spheroids.Results: In biopsy, WHO classified B3 thymomas and TCs showed increased WNT4 expression compared with NTs. During short-term 2D culture, WNT4 expression and secretion declined in neoplastic pTECs but not in 3D spheroids or medium supplemented with recombinant WNT4 cultures. Under the latter condition, the growth of pTECs was accompanied by increased expression of non-canonical targets RAC1 and JNK. Down-regulation of WNT4 by shRNA induced cell death in pTECs derived from B3 thymomas and led to decreased RAC1, but not JNK protein phosphorylation. Pharmacological inhibition of NF-κB decreased both RAC1 and JNK phosphorylation in neoplastic pTECs.Conclusions: Lack of the age-related decline of non-canonical WNT4 expression in TETs and restoration of declining WNT4 expression through exogeneous WNT4 or 3D culture of pTECs hints at an oncogenic role of WNT4 in TETs and is compatible with the WNT4 autocrine loop model. Crosstalk between WNT4 and NF-κB signaling may present a promising target for combined interventions in TETs.

Background: Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Thymic squamous cell carcinomas are rare thoracic cancers and because of their relative rarity and close histologic similarity to pulmonary squamous cell carcinomas, thymic carcinomas might be misdiagnosed as lung carcinomas involving the mediastinum. Therefore, it is important to differentiate thymic SCC from lung SCC with mediastinal extension. In this effort, comprehension of the molecular alterations is urgently needed in both malignancies in order to identify molecular marker, which could be helpful for accurate novel diagnosis and therapy of thymic and lung carcinomas. Methods: Gene expression profiling was achieved with affimetrix microarray. Six biopsies of thymic SCC and 9 lung SCCs were studied. Differential gene expression was analysed by ANOVA (JMP Genomics, version 4; SAS, Cary, NC, USA). Array-based gene expression in tumors was re-evaluated by qRT-PCR in two independent groups of each tumor cohort. The differential expressed genes were analysed on protein level using western blot and /or immunohistochemistry. Their regulation by microRNA 204 (miR204) was investigated using miR 204 mimitic in primary thymic carcinoma cells and in 1889c cell line. Results: Ten pathways were described in this study. All two tumor cohorts were differentially enriched by all pair-wise comparisons for pathways of focal adhesion, ECM receptor interaction, and regulation of actin cytoskeleton which was significant enriched in thymic carcinomas. The transcriptional misregulation, NF-κB signaling and apoptosis pathways were more dominant in pulmonary squamous cell carcinomas. Among the differential expressed genes stem cells related gene signature PAX1, PBX3, SPIC1 and FOXG1 were significant overexpressed in thymic carcinomas, while miR204 is rather downregulated and is associated with the negative regulation of the following genes RASSF6, FXYD6 and PAX1. Conclusions: The major finding here is the so far unreported observation of different expression profiles of stem cells related genes in thymic and pulmonary SCCs and these findings suggest that thymic carcinomas suffer from overexpression of stem cell gene signature including PAX1, SPIC, PBX3 and FOXG1, which could be used as biomarker for the histopathology to distinguish the CD117 negative thymic and lung squamous cell carcinomas and for therapy application aiming to break treatment resistance in thymic cancers. The microRNA make-up in the current study defines decreased miR204, which might act as a tumor-suppressive gene in the tumorigenesis and progression in thymic SSC.