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Article ; Online: Generation of metastatic melanoma specific antibodies by affinity purification.

Schütz, Birgit / Koppensteiner, Anita / Schörghofer, David / Kinslechner, Katharina / Timelthaler, Gerald / Eferl, Robert / Hengstschläger, Markus / Missbichler, Albert / Hundsberger, Harald / Mikula, Mario

Scientific reports

2016 Volume 6, Page(s) 37253

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Neoplasm/chemistry ; Antibodies, Neoplasm/immunology ; Antibodies, Neoplasm/isolation & purification ; Antigens, Neoplasm/chemistry ; Antigens, Neoplasm/immunology ; Biomarkers, Tumor/chemistry ; Biomarkers, Tumor/immunology ; Chromatography, Affinity ; Female ; Humans ; Melanoma/immunology ; Melanoma/mortality ; Melanoma/pathology ; Mice ; Mice, SCID ; Neoplasm Metastasis ; Rabbits
Chemical Substances	Antibodies, Neoplasm ; Antigens, Neoplasm ; Biomarkers, Tumor
Language	English
Publishing date	2016-11-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep37253
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Article ; Online: The HDL receptor SR-BI is associated with human prostate cancer progression and plays a possible role in establishing androgen independence.

Schörghofer, David / Kinslechner, Katharina / Preitschopf, Andrea / Schütz, Birgit / Röhrl, Clemens / Hengstschläger, Markus / Stangl, Herbert / Mikula, Mario

Reproductive biology and endocrinology : RB&E

2015 Volume 13, Page(s) 88

Abstract: Background: Human prostate cancer represents one of the most frequently diagnosed cancers in men worldwide. Currently, diagnostic methods are insufficient to identify patients at risk for aggressive prostate cancer, which is essential for early ... ...

Abstract	Background: Human prostate cancer represents one of the most frequently diagnosed cancers in men worldwide. Currently, diagnostic methods are insufficient to identify patients at risk for aggressive prostate cancer, which is essential for early treatment. Recent data indicate that elevated cholesterol levels in the plasma are a prerequisite for the progression of prostate cancer. Here, we analyzed clinical prostate cancer samples for the expression of receptors involved in cellular cholesterol uptake. Methods: We screened mRNA microarray files of prostate cancer samples for alterations in the expression levels of cholesterol transporters. Furthermore, we performed immunohistochemistry analysis on human primary prostate cancer tissue sections derived from patients to investigate the correlation of SR-BI with clinicopathological parameters and the mTOR target pS6. Results: In contrast to LDLR, we identified SR-BI mRNA and protein expression to be induced in high Gleason grade primary prostate cancers. Histologic analysis of prostate biopsies revealed that 53.6 % of all cancer samples and none of the non-cancer samples showed high SR-BI staining intensity. The disease-free survival time was reduced (P = 0.02) in patients expressing high intra-tumor levels of SR-BI. SR-BI mRNA correlated with HSD17B1 and HSD3B1 and SR-BI protein staining showed correlation with active ribosomal protein S6 (RS = 0.828, P < 0.00001). Conclusions: We identified SR-BI to indicate human prostate cancer formation, suggesting that increased levels of SR-BI may be involved in the generation of a castration-resistant phenotype.
MeSH term(s)	Adenocarcinoma/genetics ; Adenocarcinoma/metabolism ; Adenocarcinoma/pathology ; CD36 Antigens/genetics ; CD36 Antigens/metabolism ; Disease Progression ; Disease-Free Survival ; Humans ; Male ; Neoplasm Grading ; Prostate/metabolism ; Prostate/pathology ; Prostatic Neoplasms/genetics ; Prostatic Neoplasms/metabolism ; Prostatic Neoplasms/pathology
Chemical Substances	CD36 Antigens
Language	English
Publishing date	2015-08-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2119215-7
ISSN	1477-7827 ; 1477-7827
ISSN (online)	1477-7827
ISSN	1477-7827
DOI	10.1186/s12958-015-0087-z
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Article ; Online: Inactivation of mTORC2 in macrophages is a signature of colorectal cancer that promotes tumorigenesis.

JCI insight

2019 Volume 4, Issue 20

Abstract: The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 ...

Abstract	The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in distinct cells within malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity, which was downregulated in stromal macrophages residing within human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a proinflammatory reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worsened clinical prognosis. Treatment of mice with a second-generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in CRC and limits tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colitis-associated CRC.
MeSH term(s)	Animals ; Carcinogenesis/drug effects ; Carcinogenesis/immunology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cells, Cultured ; Colitis, Ulcerative/blood ; Colitis, Ulcerative/chemically induced ; Colitis, Ulcerative/immunology ; Colitis, Ulcerative/pathology ; Colon/cytology ; Colon/drug effects ; Colon/immunology ; Colon/pathology ; Colorectal Neoplasms/immunology ; Colorectal Neoplasms/mortality ; Colorectal Neoplasms/pathology ; Colorectal Neoplasms/prevention & control ; Dextran Sulfate/toxicity ; Disease Models, Animal ; Female ; Humans ; Intestinal Mucosa/cytology ; Intestinal Mucosa/drug effects ; Intestinal Mucosa/immunology ; Intestinal Mucosa/pathology ; Kaplan-Meier Estimate ; Macrophages/immunology ; Macrophages/metabolism ; Male ; Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors ; Mechanistic Target of Rapamycin Complex 2/metabolism ; Mice ; Mice, Transgenic ; Morpholines/pharmacology ; Osteopontin/blood ; Osteopontin/metabolism ; Primary Cell Culture ; Prognosis ; Survival Rate
Chemical Substances	Morpholines ; SPP1 protein, human ; Spp1 protein, mouse ; Osteopontin (106441-73-0) ; Dextran Sulfate (9042-14-2) ; (5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol (970JJ37FPW) ; Mechanistic Target of Rapamycin Complex 2 (EC 2.7.11.1)
Language	English
Publishing date	2019-10-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2379-3708
ISSN (online)	2379-3708
DOI	10.1172/jci.insight.124164
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Article ; Online: STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

Swoboda, Alexander / Soukup, Robert / Eckel, Oliver / Kinslechner, Katharina / Wingelhofer, Bettina / Schörghofer, David / Sternberg, Christina / Pham, Ha T T / Vallianou, Maria / Horvath, Jaqueline / Stoiber, Dagmar / Kenner, Lukas / Larue, Lionel / Poli, Valeria / Beermann, Friedrich / Yokota, Takashi / Kubicek, Stefan / Krausgruber, Thomas / Rendeiro, André F /

Bock, Christoph / Zenz, Rainer / Kovacic, Boris / Aberger, Fritz / Hengstschläger, Markus / Petzelbauer, Peter / Mikula, Mario / Moriggl, Richard

Oncogene

2020 Volume 40, Issue 6, Page(s) 1091–1105

Abstract: Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in ... ...

Abstract	Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels. We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRAS
MeSH term(s)	Animals ; CCAAT-Enhancer-Binding Protein-beta/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Melanocytes/drug effects ; Melanoma/genetics ; Melanoma/pathology ; Mice ; Microphthalmia-Associated Transcription Factor/genetics ; Neoplasm Metastasis ; STAT3 Transcription Factor/genetics ; Signal Transduction/drug effects
Chemical Substances	CCAAT-Enhancer-Binding Protein-beta ; Cebpb protein, mouse ; Microphthalmia-Associated Transcription Factor ; Mitf protein, mouse ; STAT3 Transcription Factor ; Stat3 protein, mouse
Language	English
Publishing date	2020-12-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639046-8
ISSN	1476-5594 ; 0950-9232
ISSN (online)	1476-5594
ISSN	0950-9232
DOI	10.1038/s41388-020-01584-6
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Zs.A 2218: Show issues

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Article ; Online: Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation.

Kinslechner, Katharina / Schörghofer, David / Schütz, Birgit / Vallianou, Maria / Wingelhofer, Bettina / Mikulits, Wolfgang / Röhrl, Clemens / Hengstschläger, Markus / Moriggl, Richard / Stangl, Herbert / Mikula, Mario

Molecular cancer research : MCR

2017 Volume 16, Issue 1, Page(s) 135–146

Abstract: Metastatic melanoma is hallmarked by elevated glycolytic flux and alterations in cholesterol homeostasis. The contribution of cholesterol transporting receptors for the maintenance of a migratory and invasive phenotype is not well defined. Here, the ... ...

Abstract	Metastatic melanoma is hallmarked by elevated glycolytic flux and alterations in cholesterol homeostasis. The contribution of cholesterol transporting receptors for the maintenance of a migratory and invasive phenotype is not well defined. Here, the scavenger receptor class B type I (SCARB1/SR-BI), a high-density lipoprotein (HDL) receptor, was identified as an estimator of melanoma progression in patients. We further aimed to identify the SR-BI-controlled gene expression signature and its related cellular phenotypes. On the basis of whole transcriptome analysis, it was found that SR-BI knockdown, but not functional inhibition of its cholesterol-transporting capacity, perturbed the metastasis-associated epithelial-to-mesenchymal transition (EMT) phenotype. Furthermore, SR-BI knockdown was accompanied by decreased migration and invasion of melanoma cells and reduced xenograft tumor growth. STAT5 is an important mediator of the EMT process and loss of SR-BI resulted in decreased glycosylation, reduced DNA binding, and target gene expression of STAT5. When human metastatic melanoma clinical specimens were analyzed for the abundance of SR-BI and STAT5 protein, a positive correlation was found. Finally, a novel SR-BI-regulated gene profile was determined, which discriminates metastatic from nonmetastatic melanoma specimens indicating that SR-BI drives gene expression contributing to growth at metastatic sites. Overall, these results demonstrate that SR-BI is a highly expressed receptor in human metastatic melanoma and is crucial for the maintenance of the metastatic phenotype.
MeSH term(s)	Animals ; Cell Line, Tumor ; Cell Movement/physiology ; Female ; Glycosylation ; Heterografts ; Humans ; Melanoma/genetics ; Melanoma/metabolism ; Melanoma/pathology ; Mice ; Mice, SCID ; Phenotype ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Receptors, LDL/biosynthesis ; STAT5 Transcription Factor/metabolism ; Scavenger Receptors, Class B/biosynthesis ; Scavenger Receptors, Class B/genetics ; Scavenger Receptors, Class B/metabolism ; Transfection
Chemical Substances	LDLR protein, human ; RNA, Messenger ; Receptors, LDL ; SCARB1 protein, human ; STAT5 Transcription Factor ; Scavenger Receptors, Class B
Language	English
Publishing date	2017-10-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2098788-2
ISSN	1557-3125 ; 1541-7786
ISSN (online)	1557-3125
ISSN	1541-7786
DOI	10.1158/1541-7786.MCR-17-0292
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Article: Rapamycin-Induced Hypoxia Inducible Factor 2A Is Essential for Chondrogenic Differentiation of Amniotic Fluid Stem Cells.

Preitschopf, Andrea / Schörghofer, David / Kinslechner, Katharina / Schütz, Birgit / Zwickl, Hannes / Rosner, Margit / Joó, József Gabor / Nehrer, Stefan / Hengstschläger, Markus / Mikula, Mario

Stem cells translational medicine

2016 Volume 5, Issue 5, Page(s) 580–590

Abstract: Unlabelled: Amniotic fluid stem (AFS) cells represent a major source of donor cells for cartilage repair. Recently, it became clear that mammalian target of rapamycin (mTOR) inhibition has beneficial effects on cartilage homeostasis, but the effect of ... ...

Abstract	Unlabelled: Amniotic fluid stem (AFS) cells represent a major source of donor cells for cartilage repair. Recently, it became clear that mammalian target of rapamycin (mTOR) inhibition has beneficial effects on cartilage homeostasis, but the effect of mTOR on chondrogenic differentiation is still elusive. Therefore, the objectives of this study were to investigate the effects of mammalian target of rapamycin complex 1 (mTORC1) modulation on the expression of SOX9 and on its downstream targets during chondrogenic differentiation of AFS cells. We performed three-dimensional pellet culturing of AFS cells and of in vitro-expanded, human-derived chondrocytes in the presence of chondrogenic factors. Inhibition of mTORC1 by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR) led to increased AKT activation, upregulation of hypoxia inducible factor (HIF) 2A, and an increase in SOX9, COL2A1, and ACAN abundance. Here we show that HIF2A expression is essential for chondrogenic differentiation and that AKT activity regulates HIF2A amounts. Importantly, engraftment of AFS cells in cell pellets composed of human chondrocytes revealed an advantage of raptor knockdown cells compared with control cells in their ability to express SOX9. Our results demonstrate that mTORC1 inhibition leads to AKT activation and an increase in HIF2A expression. Therefore, we suggest that mTORC1 inhibition is a powerful tool for enhancing chondrogenic differentiation of AFS cells and also of in vitro-expanded adult chondrocytes before transplantation. Significance: Repair of cartilage defects is still an unresolved issue in regenerative medicine. Results of this study showed that inhibition of the mammalian target of rapamycin complex 1 (mTORC1) pathway, by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR), enhanced amniotic fluid stem cell differentiation toward a chondrocytic phenotype and increased their engrafting efficiency into cartilaginous structures. Moreover, freshly isolated and in vitro passaged human chondrocytes also showed redifferentiation upon mTORC1 inhibition during culturing. Therefore, this study revealed that rapamycin could enable a more efficient clinical use of cell-based therapy approaches to treat articular cartilage defects.
MeSH term(s)	Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Aggrecans/genetics ; Aggrecans/metabolism ; Amniotic Fluid/cytology ; Basic Helix-Loop-Helix Transcription Factors/genetics ; Basic Helix-Loop-Helix Transcription Factors/metabolism ; Cell Differentiation/drug effects ; Cells, Cultured ; Chondrocytes/drug effects ; Chondrocytes/metabolism ; Chondrogenesis/drug effects ; Collagen Type II/genetics ; Collagen Type II/metabolism ; Enzyme Activation ; Female ; Gene Expression Regulation, Developmental/drug effects ; Humans ; Mechanistic Target of Rapamycin Complex 1 ; Multipotent Stem Cells/drug effects ; Multipotent Stem Cells/metabolism ; Multiprotein Complexes/antagonists & inhibitors ; Multiprotein Complexes/metabolism ; Phenotype ; Pregnancy ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Regulatory-Associated Protein of mTOR ; SOX9 Transcription Factor/genetics ; SOX9 Transcription Factor/metabolism ; Signal Transduction/drug effects ; Sirolimus/pharmacology ; TOR Serine-Threonine Kinases/antagonists & inhibitors ; TOR Serine-Threonine Kinases/metabolism ; Transfection ; Up-Regulation
Chemical Substances	ACAN protein, human ; Adaptor Proteins, Signal Transducing ; Aggrecans ; Basic Helix-Loop-Helix Transcription Factors ; COL2A1 protein, human ; Collagen Type II ; Multiprotein Complexes ; RPTOR protein, human ; Regulatory-Associated Protein of mTOR ; SOX9 Transcription Factor ; SOX9 protein, human ; endothelial PAS domain-containing protein 1 (1B37H0967P) ; Mechanistic Target of Rapamycin Complex 1 (EC 2.7.11.1) ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; TOR Serine-Threonine Kinases (EC 2.7.11.1) ; Sirolimus (W36ZG6FT64)
Language	English
Publishing date	2016-03-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2642270-0
ISSN	2157-6580 ; 2157-6564
ISSN (online)	2157-6580
ISSN	2157-6564
DOI	10.5966/sctm.2015-0262
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Article ; Online: mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

Preitschopf, Andrea / Li, Kongzhao / Schörghofer, David / Kinslechner, Katharina / Schütz, Birgit / Thi Thanh Pham, Ha / Rosner, Margit / Joo, Gabor Jozsef / Röhrl, Clemens / Weichhart, Thomas / Stangl, Herbert / Lubec, Gert / Hengstschläger, Markus / Mikula, Mario

PloS one

2014 Volume 9, Issue 9, Page(s) e107004

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Amniotic Fluid/cytology ; Animals ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Lipogenesis/genetics ; Mechanistic Target of Rapamycin Complex 1 ; Mice, Inbred C57BL ; Multiprotein Complexes/genetics ; Multiprotein Complexes/metabolism ; Multiprotein Complexes/physiology ; Schwann Cells/cytology ; Schwann Cells/metabolism ; Signal Transduction ; Stem Cells/cytology ; Stem Cells/metabolism ; TOR Serine-Threonine Kinases/genetics ; TOR Serine-Threonine Kinases/metabolism ; TOR Serine-Threonine Kinases/physiology
Chemical Substances	Multiprotein Complexes ; MTOR protein, human (EC 2.7.1.1) ; Mechanistic Target of Rapamycin Complex 1 (EC 2.7.11.1) ; TOR Serine-Threonine Kinases (EC 2.7.11.1)
Language	English
Publishing date	2014-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0107004
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Generation of metastatic melanoma specific antibodies by affinity purification.

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Article ; Online: The HDL receptor SR-BI is associated with human prostate cancer progression and plays a possible role in establishing androgen independence.

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Article ; Online: Inactivation of mTORC2 in macrophages is a signature of colorectal cancer that promotes tumorigenesis.

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Article ; Online: STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

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Article ; Online: Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation.

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Article: Rapamycin-Induced Hypoxia Inducible Factor 2A Is Essential for Chondrogenic Differentiation of Amniotic Fluid Stem Cells.

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

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