👤 Christian Dombrowski

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%; Show less

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 ( Show less

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. Show less

In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma. Show less

Metabolic reprogramming is a hallmark of many cancer types, including hepatocellular carcinoma (HCC). Identifying the critical players in this process might be crucial for the generation of novel and effective anti-neoplastic therapies. In the present investigation, we determined the importance of carbohydrate responsive element binding protein (ChREBP), a central player in the regulation of lipid and glucose metabolism in the liver, on the development of HCC in in vitro and in vivo models. We found that genetic deletion of ChREBP (that will be referred to as ChREBPKO mice) strongly delays or impairs hepatocarcinogenesis driven by AKT or AKT/c-Met overexpression in mice, respectively. In contrast, HCC development was found to be completely unaffected by ChREBP depletion in mice co-expressing AKT and N-Ras protooncogenes. In mouse and human HCC cell lines, suppression of ChREBP via specific small interfering RNAs (siRNAs) resulted in decreased proliferation and induction of apoptosis. Of note, these cellular events were strongly augmented by concomitant inhibition of the mitogen-activated protein kinase (MAPK) pathway. The present data indicate that ChREBP activity might be required or dispensable for HCC growth, depending on the oncogenes involved. In particular, the activation of Ras/MAPK signaling might represent a possible mechanism of resistance to ChREBP depletion in this tumor type. Additional studies are needed to unravel the molecular mechanisms rendering HCC cells insensitive to ChREBP suppression. Show less

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program. Show less

After low-number transplantation of islets of Langerhans into the liver of streptozotocin-diabetic rats, the hepatocytes in the acini, draining the blood from the islets, are exposed to a local hyperinsulinemia, whereas the remaining tissue is affected by hypoinsulinemia. In this model, insulin induces alterations that resemble preneoplastic foci of altered hepatocytes (FAH) and develop into hepatocellular tumors in later stages of carcinogenesis. In rodents, apolipoprotein A-IV (A-IV) is synthesized in the small intestine and the liver. Whereas intestinal production is mainly influenced by lipid intake and chylomicrone formation, little is known about mechanisms regulating hepatic A-IV synthesis. As it is known that insulin modulates lipoprotein metabolism in different ways, we investigated the effect of insulin on hepatocytic A-IV mRNA expression in this model. After Laser microdissection of FAH and quantitative RT-PCR (LightCycler), we found a 3.2 to 7.4-fold increase of A-IV mRNA in the FAH. To the best of our knowledge, these results represent the first data of insulin-stimulated A-IV mRNA overexpression in rat hepatocytes in vivo, and are in line with previously reported results of experiments with cultured hepatocytes. It remains to be elucidated whether A-IV mRNA overexpression is only an epiphenomenon of insulin action or is relevant for hepatocarcinogenesis in this model. Show less