mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway

The role of scavenger receptor class B type I in melanoma and its contribution to the metastatic process

Da die Häufigkeit von Melanomerkrankungen stetig steigt, besteht eine hohe Nachfrage nach heilenden Behandlungsmöglichkeiten. Eines der vielen Kennzeichen für Krebs, ist ein abweichender metabolischer Stoffwechsel. Diese Veränderung beinhaltet unter anderem die Lipidsynthese und eine erhöhte Glukoseaufnahme. Erstgenanntes ist mittlerweile anerkannt ein Teil der Krebsbildung und begleitender Metastasen zu sein. Jedoch wurde die Rolle der cholesterintransportierenden Rezeptoren in der humanen Melanomprogression noch nicht abgeklärt. Hier wollen wir den Beitrag von SR-BI, auch bekannt als HDL-Rezeptor, im invasiven Prozess vom humanen Melanom studieren. Dafür haben wir sechs humane Melanomzelllinien untersucht und einen Mausversuch durchgeführt. Zusätzlich haben wir klinische mRNA Daten evaluiert um einen allgemeinen Überblick von SR-BI im Melanom zu erhalten. Um den molekularen Pfad zu enträtseln, haben wir eine Expressionsanalyse des gesamten Genoms von drei stark metastasierenden Melanomzelllinien durchführen lassen. Die Zelllinien wurden entweder mit SR-BI siRNA inkubiert oder mit einem SR-BI Inhibitor behandelt, der die funktionelle Aufnahme vom Cholesterin hemmt. Wir wollen hervorheben, dass SR-BI als Marker der Melanomentwicklung angewendet werden kann, da metastasierende Melanomzelllinien stark erhöhte SR-BI Level aufgewiesen haben verglichen zu primären Tumorlinien. Wir haben ähnliche Ergebnisse bei den klinischen Daten beobachtet, wo Melanompatienten mit hohem SR-BI Level eine schlechtere Überlebensrate aufgewiesen haben als Patienten mit einem niedrigerem. Die Expressionsanalyse hat gezeigt, dass die onkogene Signatur der metastasierenden Melanomzellen verschwunden war. Gleiches haben wir bei funktionellen Experimenten gesehen, wo die Migration, Invasion und das Tumorwachstum stark abgenommen haben nach dem SR-BI knockdown. Ein bekannter Mitwirkender der EMT ist STAT5. Das Entfernen von SR-BI hat eine Abnahme der STAT5 Glykosylierung und eine Schwächung der STAT5 DNA Bindung verursacht. Außerdem wurden die untersuchten STAT5 Zielgene weniger exprimiert. Letztendlich haben wir ein Genprofil entdeckt, welches zwischen nicht-metastasierenden und metastasierenden Tumoren unterscheiden kann. Abschließend weisen wir darauf hin, dass SR-BI ein bestimmtes Genexpressionsmuster über STAT5 antreibt, welches das Wachstum von Metastasen fördert.The incidence of melanoma rises continually hence there is great demand for curative treatments. One of the hallmarks of cancer is an aberrant metabolic pathway. This alteration comprises among others, lipid synthesis and up-regulated glucose flux. This concept is now part of the accepted hallmarks of cancer formation and accompanying metastatic lesions. However, the role of cholesterol transporting receptors has not been examined in the context of human melanoma progression to metastasis. Here we aimed to evaluate the contribution of the scavenger receptor class B type I receptor (SR-BI), also known as HDL receptor, to the invasive process in human melanoma. Therefore, we investigated six human-derived melanoma cell lines and performed an in vivo experiment. Additionally, we evaluated clinical mRNA data sets to obtain a general overview of SR-BI in melanoma. To unravel molecular pathways after SR-BI siRNA or functional SR-BI inhibition we performed whole genome expression analysis on a set of three highly metastatic melanoma cell lines. We propose that SR-BI can be applied as a marker for melanoma progression since metastatic cell lines showed strong up-regulated SR-BI levels compared to primary tumor cells in vitro. We observed similar results in the clinical samples where melanoma patients with high SR-BI levels had poorer survival outcomes compared to patients with low SR-BI levels. Whole genome expression analysis revealed the loss of an oncogenic signature, reassembling epithelial to mesenchymal transition, upon SR-BI deletion. Parallel, functional assays showed decreased migratory and invasive capabilities of metastatic cell lines after SR-BI knockdown as well as reduced xenograft tumor growth. A well-known EMT mediator is STAT5 and SR-BI depletion resulted in attenuated glycosylation and diminished DNA binding of STAT5. Furthermore, specific STAT5 targets showed a reduced expression pattern. Finally, a novel gene profile, regulated by SR-BI, was found to discriminate non-metastatic from metastatic tumors. In conclusion this indicates that SR-BI drives a distinct gene expression pattern via STAT5, enhancing growth at metastatic sites.submitted by Katharina KinslechnerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)273881

Journal of Orthopaedic Research / Galectins1 and 3 in Human Intervertebral Disc Degeneration: NonUniform Distribution Profiles and Activation of Disease Markers Involving NFB by Galectin1

Degeneration of the human intervertebral disc (IVD) is assumed to underlie severe clinical symptoms, in particular chronic back pain. Since adhesion/growthregulatory galectins are linked to arthritis/osteoarthritis pathogenesis by activating a prodegradative/inflammatory gene expression signature, we hypothesized a similar functional involvement of galectins in IVD degeneration. Immunohistochemical evidence for the presence of galectins1 and 3 in IVD is provided comparatively for specimens of spondylochondrosis, spondylolisthesis, and spinal deformity. Immunopositivity was detected in sections of fixed IVD specimens in each cellular compartment with age, disease, and galectintyperelated differences. Of note, presence of both galectins correlated with IVD degeneration, whereas correlation with age was seen only for galectin3. In addition, staining profiles for these two galectins showed different distribution patterns in serial sections, an indication for nonredundant functionalities. In vitro, both galectins bound to IVD cells in a glycandependent manner. However, exclusively galectin1 binding triggered a significant induction of functional disease markers (i.e., IL6, CXCL8, and MMP1/3/13) with involvement of the nuclear factorkB pathway. This study thus gives direction to further network analyses and functional studies on galectins in IVD degeneration. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:22042216, 2019(VLID)489515

Generation of metastatic melanoma specific antibodies by affinity purification

Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis.(VLID)468875

International Journal of Molecular Sciences / Loss of SR-BI Down-Regulates MITF and Suppresses Extracellular Vesicle Release in Human Melanoma

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide. Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release. We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway. Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells. Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET. In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex. Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI. In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype. Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.(VLID)491292

Scheme for the applied differentiation protocol.

<p>In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.</p

Rapamycin decreases Schwann cell markers, whereas statin induces Schwann cell markers.

<p>(A) During the last 72 hrs of differentiation, AFS cells were treated with 5 µM and 10 µM statin. After 15 days cDNA was generated and used for quantitative PCR of respective genes. The results are expressed as means ± SEM of three independent experiments. P<0,05 for * vs control treated cells. (B) AFS cells were differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. Fixed cells were stained with indicated antibodies (labeled in red, nuclei in green). Scale bar represents 10 µm. (C) Western blotting of cells differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. GFAP was detected at about 50 kDa, LDLR at 160 kDa and HMGCR as a double band at 90 kDa.</p

mTOR signaling is active in differentiated AFS cells and important for the differentiation process.

<p>(A) AKT phosphorylation at Ser 473 and ribosomal protein S6 phosphorylation at Ser 240/244 were quantified at the indicated time points during differentiation with and without rapamycin treatment. (B) NGFR, a marker for early differentiated AFS cells (labeled in green), was co-stained with phosphorylated S6 at Ser 240/244 protein (labeled in red) with and without rapamycin treatment (nuclei labeled in blue). Scale bar represents 10 µm. (C) Accumulation of free cholesterol was monitored by filipin III staining. Scale bar represents 10 µm.</p

Rapamycin resistant S6K1 induces S100b, but not LDLR or HMGCR expression.

<p>(A) AFS cells were differentiated without or (B) in the presence of rapamycin and at day 15 cells were transfected with an HA-fused S6K1 rapamycin-resistant mutant (HA-S6K1-RR). After 72 hours in differentiation media containing rapamycin, cells were fixed and stained with anti-HA antibody (shown in green) combined with antibodies detecting S100b, LDLR, HMGCR and phosphorylated S6 (shown in red). Scale bar represents 25 µm.</p