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.

Late stage melanoma is hallmarked by low NLGN4X expression leading to HIF1A accumulation.

BACKGROUND: Despite ongoing research and recent advances in therapy, metastatic melanoma remains one of the cancers with the worst prognosis. Here we studied the postsynaptic cell adhesion molecule Neuroligin 4X (NLGN4X) and investigated its role in melanoma progression. METHODS: We analysed histologic samples to assess the expression and predictive value of NLGN4X in human melanoma. The oncogenic role of NLGN4X was determined by loss or gain-of-function experiments in vitro as well as by analysis of tumorspheres, which were grafted to human skin organoids derived from pluripotent stem cells. Whole genome expression analysis and validation experiments were performed to clarify the molecular mechanism. RESULTS: We identified that suppression of NLGN4X down regulated the prefoldin member Von Hippel-Lindau binding protein 1 (VBP1). Moreover, loss of VBP1 was sufficient for accumulation of HIF1A and HIF1A signalling was further shown to be essential for the acquisition of migratory properties in melanoma. We re-established NLGN4X expression in late stage melanoma lines and observed decreased tumour growth after transplantation to human skin organoids generated from pluripotent stem cells. In line, we showed that high amounts of NLGN4X and its target VBP1 in human patient samples had a beneficial prognostic effect on patient survival. CONCLUSION: In view of these findings, we propose that decreased amounts of NLGN4X are indicative of a metastatic melanoma phenotype and that loss of NLGN4X provides a novel mechanism for HIF induction.

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.

Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α) and interleukin (IL)-1ß are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC)1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1ß was added to the media (=OA-model). The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG) release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH) assay and quantitative real time polymerase chain reaction (PCR). Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study highlights that blocking mTORC1 is a new and promising approach for treating OA. Low side effects make rapamycin an attractive implementation to existing therapeutic strategies. We showed that rapamycin's chondroprotective property might be due to an interference with IL-1ß triggered inflammatory processes.

Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

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.

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

MET expression in melanoma correlates with a lymphangiogenic phenotype.

Melanomas contain high frequencies of tumorigenic cells and their tumorigenic capacity resides in several distinct subpopulations within melanoma. Since their metastatic potential is linked to their ability to recruit lymphatic vessels, we aimed at identifying lymphangiogenic subpopulations by comparative in vitro analysis of single cell clones derived from a melanoma of a single patient. Selected lymphangiogenic clones were then grafted into severe combined immunodeficient mice, where they induced lymphangiogenesis and metastasized into sentinel nodes, whereas non-lymphangiogenic clones from the same patient did not metastasize. Transcriptome analysis revealed high expression of vascular endothelial growth factor C (VEGF-C) and platelet derived growth factor C (PDGF-C) as well as of the met proto-oncogene (MET) and its targets to be associated with this lymphangiogenic phenotype. Screening of a set of independently isolated melanoma cell lines from other patients confirmed this association between expression of high levels of MET and of VEGF-C and PDGF-C. Hence, we provide a model to screen for the lymphangiogenic potential of tumor cells. We show that the lymphangiogenic potential is heterogeneously distributed among melanoma cells within one given tumor and is associated with activation of MET signaling.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues.

BACKGROUND & AIMS: The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), a multifunctional protein, plays a central role in intracellular targeting of lysosomal enzymes and control of insulin-like growth factor II (IGF-II) bioactivity. Importantly, the gene encoding this receptor is frequently inactivated in a wide range of malignant tumors including hepatocellular carcinomas. Thus, M6P/IGF2R is considered a putative liver tumor suppressor. The aim of this study was to establish the impact of the receptor on the invasive properties of liver cells. METHODS: Reconstitution experiments were performed by expression of wild type and mutant M6P/IGF2R in receptor-deficient FRL14 fetal rat liver cells. RNA interference was used to induce M6P/IGF2R downregulation in receptor-positive MIM-1-4 mouse hepatocytes. RESULTS: We show that the M6P/IGF2R status exerts a strong impact on the invasiveness of tumorigenic rodent liver cells. M6P/IGF2R-deficient fetal rat liver cells hypersecrete lysosomal cathepsins and penetrate extracellular matrix barriers in a cathepsin-dependent manner. Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells. Conversely, M6P/IGF2R knock-down in receptor-positive mouse hepatocytes causes increased cathepsin secretion as well as enhanced cell motility and invasiveness. We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells. CONCLUSIONS: M6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.

Identification of genetic disparity between primary and metastatic melanoma in human patients.

It is commonly accepted that cancer cell progression is accompanied by accumulation of genetic changes. Here we searched for copy number variations in melanoma and asked whether homozygous losses always cumulate during tumor cell progression. Therefore we investigated either melanoma cell lines or tissue derived from the primary lesion and from the lymph node metastasis of the same individual patient. In vitro studies of melanoma cell lines revealed high migratory and anchorage independent growth of metastasis-derived cells. Surprisingly, whole genome DNA analysis of a primum-derived cell line revealed a total of 10 homozygous losses, whereas the matched metastasis-derived cell line only shared five of those losses. We further tested these cells in a mouse model for intradermal melanoma growth and detected fast growth of the metastasis-derived cell line and no growth of primum-derived cells. Additionally, we screened matched pairs of patient-derived melanoma primum and metastasis samples and we could also identify a case with homozygous deletions exclusively present in the primary lesion. Therefore, we suggest that tumor cell progression at the metastatic niche can occur parallel and independently from the primary tumor. We propose that for mutation-targeted therapy genotyping should be performed not only from primary, but also from metastatic melanoma.

Proteome analysis of NRF2 inhibition in melanoma reveals CD44 up-regulation and increased apoptosis resistance upon vemurafenib treatment.

Malignant melanoma is the deadliest form of skin cancer and NRF2 has been proposed as a main regulator of tumor cell malignancy. Still the mechanisms how NRF2 is contributing to melanoma progression are incompletely understood. Here we analyzed the effects of either NRF2 induction or depletion, and we also quantified changes on the whole cell proteome level. Our results showed that inhibition of NRF2 leads to a loss of reactive oxygen species protection, but at the same time to an induction of an epithelial mesenchymal transition (EMT) phenotype and an up-regulation of the stem cell marker CD44. Additionally, cells devoid of NRF2 showed increased cell viability after treatment with a MYC and a BRAF inhibitor. Importantly, survival upon vemurafenib treatment was dependent on CD44 expression. Finally, analysis of archival melanoma patient samples confirmed a vice versa relationship of NRF2 and CD44 expression. In summary, we recorded changes in the proteome after NRF2 modulation in melanoma cells. Surprisingly, we identified that NRF2 inhibition lead to induction of an EMT phenotype and an increase in survival of cells after apoptosis induction. Therefore, we propose that it is important for future therapies targeting NRF2 to consider blocking EMT promoting pathways in order to achieve efficient tumor therapy.

Nuclear beta-catenin induces an early liver progenitor phenotype in hepatocellular carcinoma and promotes tumor recurrence.

Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.

Plasma Membrane Lipids: An Important Binding Site for All Lipoprotein Classes.

Cholesterol is one of the main constituents of plasma membranes; thus, its supply is of utmost importance. This review covers the known mechanisms of cholesterol transfer from circulating lipoprotein particles to the plasma membrane, and vice versa. To achieve homeostasis, the human body utilizes cellular de novo synthesis and extracellular transport particles for supply of cholesterol and other lipids via the blood stream. These lipoprotein particles can be classified according to their density: chylomicrons, very low, low, and high-density lipoprotein (VLDL, LDL, and HDL, respectively). They deliver and receive their lipid loads, most importantly cholesterol, to and from cells by several redundant routes. Defects in one of these pathways (e.g., due to mutations in receptors) usually are not immediately fatal. Several redundant pathways, at least temporarily, compensate for the loss of one or more of them, but the defects trigger systemic diseases, such as atherosclerosis later on. Recently, intracellular membrane-membrane contact sites were shown to be involved in intracellular cholesterol transfer and the plasma membrane itself has been proposed to act as a binding site for lipoprotein-mediated cargo unloading.

STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

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 NRASQ61K in an Ink4a-deficient background, two frequent driver mutations in human melanoma. Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain, and liver metastases. Whole-genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in the upregulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3-induced CAAT Box Enhancer Binding Protein (CEBP) expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus. Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression. We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.

Insights into Differentiation of Melanocytes from Human Stem Cells and Their Relevance for Melanoma Treatment.

Malignant melanoma represents a highly aggressive form of skin cancer. The metastatic process itself is mostly governed by the so-called epithelial mesenchymal transition (EMT), which confers cancer cells migrative, invasive and resistance abilities. Since EMT represents a conserved developmental process, it is worthwhile further examining the nature of early developmental steps fundamental for melanocyte differentiation. This can be done either in vivo by analyzing the physiologic embryo development in different species or by in vitro studies of melanocytic differentiation originating from embryonic human stem cells. Most importantly, external cues drive progenitor cell differentiation, which can be divided in stages favoring neural crest specification or melanocytic differentiation and proliferation. In this review, we describe ectopic factors which drive human pluripotent stem cell differentiation to melanocytes in 2D, as well as in organoid models. Furthermore, we compare developmental mechanisms with processes described to occur during melanoma development. Finally, we suggest differentiation factors as potential co-treatment options for metastatic melanoma patients.

The mannose 6-phosphate/insulin-like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R-deficient SCC-VII murine squamous cell carcinoma cells secrete large amounts of pro-invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC-VII cells. To assess the link between M6P/IGF2R status and tumour invasion, we have now generated SCC-VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC-VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC-VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC-VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas.

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.