👤 Alex Odermatt

Hepatocellular carcinoma (HCC) has been observed in neonatal mice following the integration of recombinant Adeno-Associated Viruses (rAAV) into the Rian locus. rAAV-related oncogenic risk for patients remains unclear, and the lack of relevant in vitro methods hinders its proper assessment. The soft agar colony-forming (SACF) assay and the growth in low attachment assay (GILA) monitor anchorage-independent growth, a hallmark of transformed adherent cells, and have been previously proposed to assess the tumorigenicity of CRISPR/Cas9-edited human MCF10A cells. Here, we introduce murine versions of SACF and GILA as surrogate in vitro systems to evaluate the risk of HCC development following genome editing or rAAV induced insertional mutagenesis. Selected tumor suppressors linked to HCC onset in vivo were edited through CRISPR/Cas9 in the hepatic murine cell line AML12. The knockout of neurofibromin (Nf2) and the dual inactivation of tumor protein p53 (Tp53) and phosphatase and tensin homolog (Pten) induced anchorage-independence, while the editing of Axin1, Ctnnb1 (coding for β-catenin), and tuberous sclerosis complex 1 (Tsc1) did not promote growth in anchorage-free conditions. Additionally, we generated stable AML12 and MCF10A clones with the rAAV genome respectively integrated into Rian and MEG8, the human homolog of Rian; however, these clones did not show anchorage independence when seeded in SACF and GILA. Overall, the murine SACF and GILA exhibit low predictive value for HCC development, failing to detect rAAV- and tumor-suppressors-associated oncogenicity. While further optimization may improve assays performance, these results highlight the need for more appropriate in vitro methodologies to accurately evaluate rAAV genotoxicity. Show less

17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) converts Δ4-androstene-3,17-dione (androstenedione) to testosterone. It is expressed almost exclusively in the testes and is essential for appropriate male sexual development. More than 70 mutations in the HSD17B3 gene that cause 17β-HSD3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD) have been reported. This study describes three novel Tunisian cases with mutations in HSD17B3. The first patient is homozygous for the previously reported mutation p.C206X. The inheritance of this mutation seemed to be independent of consanguineous marriage, which can be explained by its high frequency in the Tunisian population. The second patient has a novel splice site mutation in intron 6 at position c.490 -6 T > C. A splicing assay revealed a complete omission of exon 7 in the resulting HSD17B3 mRNA transcript. Skipping of exon 7 in HSD17B3 is predicted to cause a frame shift in exon 8 that affects the catalytic site and results in a truncation in exon 9, leading to an inactive enzyme. The third patient is homozygous for the novel missense mutation p.K202M, representing the first mutation identified in the catalytic tetrad of 17β-HSD3. Site-directed mutagenesis and enzyme activity measurements revealed a completely abolished 17β-HSD3 activity of the p.K202M mutant, despite unaffected protein expression, compared to the wild-type enzyme. Furthermore, the present study emphasizes the importance of genetic counselling, detabooization of 46,XY DSD, and a sensitization of the Tunisian population for the risks of consanguineous marriage. Show less

Metabolic reprogramming of tumor cells involves upregulation of fatty acid (FA) synthesis to support high bioenergetic demands and membrane synthesis. This has been shown for cytosolic synthesis of FAs with up to 16 carbon atoms. Synthesis of long-chain fatty acids (LCFAs), including ω-6 and ω-3 polyunsaturated FAs, takes place at the endoplasmic reticulum. Despite increasing evidence for an important role of LCFAs in cancer, the impact of their synthesis in cancer cell growth has scarcely been studied. Here, we demonstrated that silencing of 17β-hydroxysteroid dehydrogenase type 12 (17β-HSD12), essentially catalyzing the 3-ketoacyl-CoA reduction step in LCFA production, modulates proliferation and migration of breast cancer cells in a cell line-dependent manner. Increased proliferation and migration after 17β-HSD12 knockdown were partly mediated by metabolism of arachidonic acid towards COX2 and CYP1B1-derived eicosanoids. Decreased proliferation was rescued by increased glucose concentration and was preceded by reduced ATP production through oxidative phosphorylation and spare respiratory capacity. In addition, 17β-HSD12 silencing was accompanied by alterations in unfolded protein response, including a decrease in CHOP expression and increase in eIF2α activation and the folding chaperone ERp44. Our study highlights the significance of LCFA biosynthesis for tumor cell physiology and unveils unknown aspects of breast cancer cell heterogeneity. Show less

Mutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. Expression plasmids for wild-type 17β-hydroxysteroid hydrogenase type 3 (17β-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ Testosterone formation by wild-type and mutant 17β-HSD3 enzymes was compared. Mutations T54A and L194P, despite normal protein expression, completely abolished 17β-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17β-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17β-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Biochemical analysis of mutant 17β-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. The 17β-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients. Engeli RT, Tsachaki M, Hassan HA, et al. Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients. J Sex Med 2017;14:1165-1174. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17β-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17β-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17β-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17β-HSD3 deficiency pedigrees in Tunisia. Show less

Mutations in the HSD17B3 gene resulting in 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency cause 46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; only few mutant enzymes have been mechanistically investigated. Here, we report novel compound heterozygous mutations in HSD17B3, composed of the nonsense mutation C206X and the missense mutation G133R, in three Tunisian patients from two non-consanguineous families. Mutants C206X and G133R were constructed by site-directed mutagenesis and expressed in HEK-293 cells. The truncated C206X enzyme, lacking part of the substrate binding pocket, was moderately expressed and completely lost its enzymatic activity. Wild-type 17β-HSD3 and mutant G133R showed comparable expression levels and intracellular localization. The conversion of Δ4-androstene-3,17-dione (androstenedione) to testosterone was almost completely abolished for mutant G133R compared with wild-type 17β-HSD3. To obtain further mechanistic insight, G133 was mutated to alanine, phenylalanine and glutamine. G133Q and G133F were almost completely inactive, whereas G133A displayed about 70% of wild-type activity. Sequence analysis revealed that G133 on 17β-HSD3 is located in a motif highly conserved in 17β-HSDs and other short-chain dehydrogenase/reductase (SDR) enzymes. A homology model of 17β-HSD3 predicted that arginine or any other bulky residue at position 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected. The results indicate an essential role of G133 in the arrangement of the cofactor binding pocket, thus explaining the loss-of-function of 17β-HSD3 mutant G133R in the patients investigated. Show less

Here, we analyzed the transcriptional effects of the antiprogestin mifepristone (MIF, RU486) and progesterone (P4) in zebrafish as well as their in vitro activities in yeast-based reporter gene assays. This study is associated with the reproduction study in adult zebrafish and embryos exposed for 21 days to 5, 39, 77 ng/L MIF, and 25 ng/L P4 (Blüthgen et al., 2013a). The in vitro activities of MIF and P4 were investigated using a series of recombinant yeast-based assays (YES, YAS, YPS) and compared to transcriptional alterations obtained in fish tissues and embryos from the exposure study. MIF elicited antiestrogenic, androgenic and progestogenic activities in recombinant yeast, similar to P4, and no antiprogestogenic activity in vitro. The transcriptional alterations of steroid hormone receptors were similar in adult males and females, and more pronounced in embryos. MIF tended to transcriptionally down-regulate the androgen (ar), progesterone (pgr) and glucocorticoid (gr) receptors in adult fish and embryos. Transcripts of the estrogen receptor (esr1) and vitellogenin (vtg1) were not significantly altered. A trend for down-regulation was observed for transcripts of genes belonging to steroidogenic enzymes including 17β-hydroxysteroid dehydrogenase type 3 (hsd17b3), 3 β-hydroxysteroid dehydrogenase (hsd3b), P450 aromatase A (cyp19a) and 11β-hydroxylase (cyp11b). P4 resulted in similar transcriptional alterations as MIF. The data indicate that gene expression changes (here and later gene expression is taken as synonym to gene transcription) and in vitro activities match only in part including the lack of antiprogestogenic activity of MIF. Additionally, effects on reproduction and gonad histology described in the associated report (Blüthgen et al., 2013a) can only partly be explained by gene expression data presented here. Show less

Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism. Show less

17β-Hydroxysteroid dehydrogenase type 3 and 5 (17β-HSD3 and 17β-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17βHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17β-HSD3 and 17β-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors. Show less

The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17beta-HSD3 inhibitors. Biological analyses revealed (2,4-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17beta-HSD3 (IC(50) 1.05microM). BP-1 also efficiently blocked conversion of androstenedione to testosterone by mouse and rat 17beta-HSD3 in whole-organ enzyme assays. Moreover, BP-1 antagonized the testosterone-dependent activation of androgen receptors (IC(50) 5.7microM), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17beta-HSD enzymes revealed 3-benzylidene camphor (3-BC) and 4-methylbenzylidene camphor (4-MBC) as low micromolar 17beta-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17beta-HSD enzyme function by the UV-filters BP-1, 3-BC and 4-MBC requires further investigation and should be considered for safety assessment of these chemicals. Show less