Colon cancer is among the most common cancer types worldwide. Signaling pathways that control cell proliferation and migration play a crucial role in its progression. The G-protein-coupled protease-activated receptors (PARs) are associated mediators in this process. Both activated coagulation factors thrombin and FXa are capable of activating PARs. While thrombin, beyond its intrinsic role in hemostasis, primarily activates PAR1, FXa mediates its cellular effects independently via PAR2. Although the role of thrombin and PAR1 activation in cancer development has been established for some time, the impact of FXa-PAR2 on tumor progression represents a relatively novel area of investigation. Therefore, the current study was conducted to examine the role of FXa and PAR2 signaling in colon cancer progression using the murine colon cancer cell line MC38. Proliferation and migration assays were performed in vitro and signaling pathways analyzed by Western blot. In vivo, tumor growth and health status were investigated in WT and PAR2-KO mice. The findings demonstrate that FXa considerably augments the proliferation and migration of colon cancer (CC) cells in vitro. A molecular mechanism of action has been identified in the activation of PAR2 by FXa. The coagulation factor significantly induces MAPK- and AKT-signaling with EGFR transactivation in the murine MC38 cells utilized. Although oral treatment with a direct FXa inhibitor (Apixaban) at a dosage of up to 50 mg/kg did not significantly affect tumor growth in vivo, PAR2 deficiency resulted in significantly reduced tumor growth and enhanced health condition status, indicating a key role of PAR2 in the progression of colon cancer.

Amyloidoses are a group of diseases characterized by the pathological deposition of non-degradable misfolded protein fibrils. These include plasma cell neoplasms, chronic inflammatory conditions, and age-related disorders, among others. Precise identification of the fibril-forming, and thereby amyloidosis-type defining protein is crucial for prognosis and correct therapeutic intervention. While immunohistochemistry (IHC) is widely used for amyloid typing, it requires extensive interpretation expertise and can be limited by inconclusive staining results. Thus, mass spectrometry (MS), if available, has been proposed as the preferred method for amyloid typing by international specialized centers (United States and United Kingdom) using primarily spectral counts for quantification. Here, we introduce an alternative method of relative quantification to further enhance the accuracy and reliability of proteomic amyloid typing. We analyzed 62 formalin-fixed, paraffin-embedded (FFPE) tissue samples, primarily endomyocardial biopsies, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employed internal normalization of iBAQ values of amyloid-related proteins relative to serum amyloid P component (APCS) for amyloidosis typing. The APCS method demonstrated robust performance across multiple LC-MS/MS platforms and achieved complete concordance with clear cut IHC typed amyloidosis cases. More importantly, it resolved unclear amyloid cases with inconclusive staining results. Additionally, for samples without a distinct fibril-forming protein identified in the standard procedure, de novo sequencing uncovered immunoglobulin light chain components, enabling the diagnosis of rare AL-amyloidosis subtypes. Finally, we established machine learning approach (XGBoost) achieving 94% accuracy by using ∼160 amyloid-related proteins as input variables. In summary, the iBAQ APCS normalization method extended by de novo sequencing allows robust, accurate, and reliable diagnostic amyloid typing, and can be complemented by an AI-based classification. Careful reviewing of each histological sample and the clinical context, nevertheless, remains indispensable for accurate interpretation.

Die S3-Leitlinie "Diagnostik und Therapie des Hepatozellulären Karzinoms und der biliären Karzinome" umfasst zwei Tumorentitäten. Die letzte Version der Leitlinie wurde im August 2023 veröffentlicht.

Surface expression of the disialoganglioside subtype GD2 has been observed on Ewing sarcoma (ES) cells, making it a suitable target for immunotherapy with the anti-GD2 antibody dinutuximab beta (DB). Here we report our experience of using DB in a cohort of 13 patients with GD2-positive, metastatic ES, in both the frontline (n=9) and relapsed/refractory (n=4) settings, when added to standard chemotherapeutic regimens. Outcomes were compared with 24 patients, primarily with localized ES, who were also treated at our center with standard therapy alone (without DB). Patients treated with DB had a median overall survival (OS) of 1877 days in the frontline setting and 810 days in the relapsed/refractory setting. Median time to progression was 1811 days and 782 days, respectively. In contrast, those treated with standard therapy alone in our center demonstrated a median OS of 1547 days and 210 days in the frontline and relapsed/refractory setting, respectively, with median times of progression of 1261 days and 113 days. DB treatment was well tolerated, with no new or unexpected adverse events reported. Anti-GD2 immunotherapy with DB represents a promising therapeutic option to improve outcomes in patients with metastatic ES, in both the frontline and relapsed/refractory settings.

The transcription factor carbohydrate response element binding protein (ChREBP) has emerged as a crucial regulator of hepatic glucose and lipid metabolism. The increased ChREBP activity involves the pro-oncogenic PI3K/AKT/mTOR signaling pathway that induces aberrant lipogenesis, thereby promoting hepatocellular carcinomas (HCC). However, the molecular pathogenesis of ChREBP-related hepatocarcinogenesis remains unexplored in the high-fat diet (HFD)-induced mouse model. Male C57BL/6J (WT) and liver-specific (L)-ChREBP-KO mice were maintained on either a HFD or a control diet for 12, 24, and 48 weeks, starting at the age of 4 weeks. At the end of the feeding period, mice were perfused, and liver tissues were formalin-fixed, paraffin-embedded, sectioned, and stained for histological and immunohistochemical analysis. Biochemical and gene expression analysis were conducted using serum and frozen liver tissue. Mice fed with HFD showed a significant increase (p < 0.05) in body weight from 8 weeks onwards compared to the control. WT and L-ChREBP-KO mice also demonstrated a significant increase (p < 0.05) in liver-to-body weight ratio in the 48-week HFD group. HFD mice exhibited a gradual rise in hepatic lipid accumulation over time, with 24-week mice showing a 20-30% increase in fat content, which further advanced to 80-100% fat accumulation at 48 weeks. Both dietary source and the increased expression of lipogenic pathways at transcriptional and protein levels induced steatosis and steatohepatitis in the HFD group. Moreover, WT mice on a HFD exhibited markedly higher inflammation compared to the L-ChREBP-KO mice. The enhanced lipogenesis, glycolysis, persistent inflammation, and activation of the AKT/mTOR pathway collectively resulted in significant metabolic disturbances, thereby promoting HCC development and progression in WT mice. In contrast, hepatic loss of ChREBP resulted in reduced hepatocyte proliferation in the HFD group, which significantly contributed to the impaired hepatocarcinogenesis and a reduced HCC occurrence in the L-ChREBP-KO mice. Our present study implicates that prolonged HFD feeding contributes to NAFLD/NASH, which in turn progresses to HCC development in WT mice. Collectively, hepatic ChREBP deletion ameliorates hepatic inflammation and metabolic alterations, thereby impairing NASH-driven hepatocarcinogenesis.

The Carbohydrate-Responsive Element-Binding Protein (ChREBP) is a glucose-sensitive transcription factor that regulates the carbohydrate and lipid metabolism. We investigated its cell-type-specific role in hepatocarcinogenesis using a chemically induced mouse model. Additionally, we examined the functions of its isoforms, ChREBPα and ChREBPβ. After the diethylnitrosamine (DEN) administration, we analyzed hepatocellular adenomas and carcinomas in systemic ChREBP-knockout (KO), hepatocyte-specific ChREBP-KO (L-KO), and wildtype (WT) mice at 4, 12, and 36 weeks using histology, morphometry, proliferation measurements, immunohistochemistry, a Western blot, and a quantitative PCR. Tumors developed 36 weeks after the DEN administration in 27% of WT mice but less frequently in KO (18%) and L-KO (9%) mice. However, preneoplastic foci were less common in KO mice but not in L-KO mice (39% vs. 9%; p < 0.05). L-KO hepatocytes exhibited lower proliferation, while KO tumors showed the downregulation of AKT/mTOR signaling, glycolysis, and lipogenesis compared to WT tumors. Our results showed that the liver-specific loss of ChREBPα, while ChREBPβ remained active, significantly reduced the tumor progression, suggesting an oncogenic role for ChREBPα. In contrast, the systemic knockout of both ChREBPα and ChREBPβ reduced the tumor initiation but did slightly prevent tumor progression, indicating that ChREBPβ may exert tumor-suppressive functions.

Schlüsselwörter biliäre Karzinome - hepatobiliäre Karzinome - CCA - Gallenblasenkarzinom - Gallengangskarzinom - cholangiozelluläres Karzinom - Diagnostik - Therapie - Risikofaktoren - Cholangiokarzinom

Backgroud Carbohydrate-response-element-binding-protein (ChREBP) is a glucose-mediated transcription-factor that is mainly expressed in liver & strongly involved in glycolytic & lipogenic-pathways [1, 2]. ChREBP has emerged as a pioneer-factor for regulation of de-novo lipogenesis (DNL), an important source of fatty-acids in the development of NAFLD (non-alcoholic-fatty-liver-disease) [3]. Previous studies from our group have suggested an associative role of ChREBP in development of hepatocellular-carcinoma (HCC) [4,5].

Glycogen metabolism and autophagy are two processes that are influenced by the protein known as Starch Binding Domain 1 (STBD1). The C-terminal CBM20 domain is important for stability and protein interaction with GABARAPL1, PGYL, GYS1, HSPs, and AMPK. Our laboratory has shown a decrease in STBD1 expression in hepatocellular carcinoma (HCC), whereas other studies have shown a similar decrease in other types of cancers. Furthermore, STBD1 is localized to the endoplasmic reticulum (ER) and is potentially involved in cellular stress responses. This suggests that STBD1 may have different target proteins that have not yet been identified. Therefore, we study protein-protein interactions by exogenous expression of STBD1 with a V5-TurboID tag for proximity labeling with biotin and pull-down to identify the STBD1 interactome using mass spectrometry. To accomplish this, we cloned STBD1 into a V5-TurboID containing plasmid and a control plasmid lacking STBD1 and confirmed its expression in HepG2 cells. Next, we generated several stable monoclonal cells showing different expression levels, validated by qPCR and western blotting, to avoid experimental discrepancies. To date, each stable clone has shown consistent exogenous STBD1-V5-TurboID and V5-TurboID expression between cell passages. Second, STBD1-V5-TurboID localized to the nucleus, similar to calnexin, an endoplasmic reticulum marker, and TOM20, a mitochondrial marker, in our immunofluorescence studies. Thus, utilizing STBD1-V5-TurboID expressing stable cells for cellular and biochemical analyses enabled us to perform mass spectrometric studies. In summary, our investigations with STBD1-V5-TurboID expressing cells will identify the missing link between proteins that connect STBD1 to glycogen metabolism, autophagy, and HCC.

AimEndometrial cancer (EC) is heterogeneous with respect to epidemiology, clinical course, histopathology and tumor biology. Recently, The Cancer Genome Atlas (TCGA) network has identified four molecular subtypes with distinct clinical courses by an integrated multi-omics approach. These subtypes are of critical importance in the clinical management of EC. However, determination of TCGA molecular subtypes requires a complex methodological approach that is resource intensive and difficult to implement in diagnostic routine procedures. In this context, Talhouk et al. reported the precise determination of modified subtypes based on molecular surrogates obtained by a two-method approach comprising immunohistochemistry and DNA-sequence analysis (Proactive Molecular Risk Classifier for Endometrial Cancer; ProMisE). In this study, we aimed to identify EC molecular subtypes in analogy to TCGA and ProMisE applying an innovative whole exome-sequencing (WES) based single-method approach.MethodsWES was performed in a cohort comprising N = 114 EC patients. WES data were analyzed using the oncology treatment decision support software MH Guide (Molecular Health, Heidelberg, Germany) and EC molecular subtypes in analogy to TCGA and ProMisE were determined. Results from both classifications were compared regarding their prognostic values using overall survival and progression-free survival analyses.ResultsApplying a single-method WES-approach, EC molecular subtypes analogue to TCGA and ProMisE were identified in the study cohort. The surrogate marker-analogue classification precisely identified high-risk and low-risk EC, whereas the TCGA-analogue classification failed to obtain significant prognostic values in this regard.ConclusionOur data demonstrate that determination of EC molecular subtypes analogue to TCGA and ProMisE is feasible by using a single-method WES approach. Within our EC cohort, prognostic implications were only reliably provided by applying the surrogate marker-analogue approach. Designation of molecular subtypes in EC will be increasingly important in routine clinical practice. Thus, the single-method WES approach provides an important simple tool to tailor therapeutic decisions in EC.

Glycogen-storing so-called clear cell kidney tubules (CCTs), precursor lesions of renal cell carcinoma, have been described in diabetic rats and in humans. The lesions show upregulation of the Akt/mTOR-pathway and the related transcription factor carbohydrate responsive element binding protein (ChREBP), which is supposedly pro-oncogenic. We investigated the effect of ChREBP-knockout on nephrocarcinogenesis in streptozotocin-induced diabetic and normoglycemic mice. Diabetic, but not non-diabetic mice, showed CCTs at 3, 6 and 12 months of age. Glycogenosis was confirmed by periodic acid schiff reaction and transmission electron microscopy. CCTs in ChREBP-knockout mice consisted of larger cells and occurred more frequently compared to wildtype mice. Progression towards kidney tumors was observed in both diabetic groups but occurred earlier in ChREBP-knockout mice. Proliferative activity assessed by BrdU-labeling was lower in 1-week-old but higher in 12-month-old diabetic ChREBP-knockout mice. Surprisingly, renal neoplasms occurred spontaneously in non-diabetic ChREBP-knockout, but not non-diabetic wildtype mice, indicating an unexpected tumor-suppressive function of ChREBP. Immunohistochemistry showed upregulated glycolysis and lipogenesis, along with activated Akt/mTOR-signaling in tumors of ChREBP-knockout groups. Immunohistochemistry of human clear cell renal cell carcinomas revealed reduced ChREBP expression compared to normal kidney tissue. However, the molecular mechanisms by which loss of ChREBP might facilitate tumorigenesis require further investigation.

Background and Aims: Hepatocellular ballooning is a com-mon finding in chronic liver disease, mainly characterized by rarefied cytoplasm that often contains Mallory-Denk bodies (MDB). Ballooning has mostly been attributed to degenera-tion but its striking resemblance to glycogenotic/steatotic changes characterizing preneoplastic hepatocellular lesions in animal models and chronic human liver diseases prompts the question whether ballooned hepatocytes (BH) are dam-aged cells on the path to death or rather viable cells, possibly involved in neoplastic development. Methods: Using speci-mens from 96 cirrhotic human livers, BH characteristics were assessed for their glycogen/lipid stores, enzyme activities, and proto-oncogenic signaling cascades by enzyme-and immunohistochemical approaches with serial paraffin and cryostat sections. Results: BH were present in 43.8% of cirrhotic livers. Particularly pronounced excess glycogen stor-age of (glycogenosis) and/or lipids (steatosis) were charac-teristic, ground glass features and MDB were often observed. Decreased glucose-6-phosphatase, increased glucose-6-phosphate dehydrogenase activity and altered immunore-activity of enzymes involved in glycolysis, lipid metabolism, and cholesterol biosynthesis were discovered. Furthermore, components of the insulin signaling cascade were upregulat-ed along with insulin dependent glucose transporter glucose transporter 4 and the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin signaling pathway associated with de novo lipogenesis. Conclusions: BH are hallmarked by particularly pronounced glycogenosis with fac-ultative steatosis, many of their features being reminiscent of metabolic aberrations documented in preneoplastic hepa-tocellular lesions in experimental animals and chronic human liver diseases. Hence, BH are not damaged entities facing death but rather viable cells featuring metabolic reprogram-ming, indicative of a preneoplastic nature.