Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages.

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

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.

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

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.

CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization.

Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin⁻/CD271⁺/CD45⁻/CD146⁺ stem-cell fraction, but also in lin⁻/CD271⁺/CD45⁻/CD146(⁻/low) cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34⁺ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment.

Inactivation of mTORC2 in macrophages is a signature of colorectal cancer that promotes tumorigenesis.

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.

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

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.Implications: High SR-BI expression in melanoma is linked with increased cellular glycosylation and hence is essential for a metastasis-specific expression signature. Mol Cancer Res; 16(1); 135-46. ©2017 AACR.

The unfolded protein response impacts melanoma progression by enhancing FGF expression and can be antagonized by a chemical chaperone.

The mechanisms hallmarking melanoma progression are insufficiently understood. Here we studied the impact of the unfolded protein response (UPR) - a signalling cascade playing ambiguous roles in carcinogenesis - in melanoma malignancy. We identified isogenic patient-derived melanoma cell lines harboring BRAFV600E-mutations as a model system to study the role of intrinsic UPR in melanoma progression. We show that the activity of the three effector pathways of the UPR (ATF6, PERK and IRE1) was increased in metastatic compared to non-metastatic cells. Increased UPR-activity was associated with increased flexibility to cope with ER stress. The activity of the ATF6- and the PERK-, but not the IRE-pathway, correlated with poor survival in melanoma patients. Using whole-genome expression analysis, we show that the UPR is an inducer of FGF1 and FGF2 expression and cell migration. Antagonization of the UPR using the chemical chaperone 4-phenylbutyric acid (4-PBA) reduced FGF expression and inhibited cell migration and viability. Consistently, FGF expression positively correlated with the activity of ATF6 and PERK in human melanomas. We conclude that chronic UPR stimulates the FGF/FGF-receptor signalling axis and promotes melanoma progression. Hence, the development of potent chemical chaperones to antagonize the UPR might be a therapeutic approach to target melanoma.

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

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.

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.

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.