Vps37a regulates hepatic glucose production by controlling glucagon receptor localization to endosomes.

During mammalian energy homeostasis, the glucagon receptor (Gcgr) plays a key role in regulating both glucose and lipid metabolisms. However, the mechanisms by which these distinct signaling arms are differentially regulated remain poorly understood. Using a Cy5-glucagon agonist, we show that the endosomal protein Vps37a uncouples glucose production from lipid usage downstream of Gcgr signaling by altering intracellular receptor localization. Hepatocyte-specific knockdown of Vps37a causes an accumulation of Gcgr in endosomes, resulting in overactivation of the cAMP/PKA/p-Creb signaling pathway to gluconeogenesis without affecting ß-oxidation. Shifting the receptor back to the plasma membrane rescues the differential signaling and highlights the importance of the spatiotemporal localization of Gcgr for its metabolic effects. Importantly, since Vps37a knockdown in animals fed with a high-fat diet leads to hyperglycemia, although its overexpression reduces blood glucose levels, these data reveal a contribution of endosomal signaling to metabolic diseases that could be exploited for treatments of type 2 diabetes.

Estimating HLA haplotype frequencies from homozygous individuals - A Technical Report.

We estimated HLA haplotype frequencies based on individuals homozygous for 4, 5 or 6 loci. Validation of our approach using a sample of over 3.4 million German individuals was successful. Compared to an expectation-maximization algorithm, the errors were larger. However, our approach allows the unequivocal detection of rare haplotypes.

Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism.

By accentuating drug efficacy and impeding resistance mechanisms, combinatorial, multi-agent therapies have emerged as key approaches in the treatment of complex diseases, most notably cancer. Using high-throughput drug screens, we uncovered distinct metabolic vulnerabilities and thereby identified drug combinations synergistically causing a starvation-like lethal catabolic response in tumor cells from different cancer entities. Domperidone, a dopamine receptor antagonist, as well as several tricyclic antidepressants (TCAs), including imipramine, induced cancer cell death in combination with the mitochondrial uncoupler niclosamide ethanolamine (NEN) through activation of the integrated stress response pathway and the catabolic CLEAR network. Using transcriptome and metabolome analyses, we characterized a combinatorial response, mainly driven by the transcription factors CHOP and TFE3, which resulted in cell death through enhanced pyrimidine catabolism as well as reduced pyrimidine synthesis. Remarkably, the drug combinations sensitized human organoid cultures to the standard-of-care chemotherapy paclitaxel. Thus, our combinatorial approach could be clinically implemented into established treatment regimen, which would be further facilitated by the advantages of drug repurposing.

Stem cell donor registry activities during the COVID-19 pandemic: a field report by DKMS.

The COVID-19 pandemic has serious implications also for patients with other diseases. Here, we describe the effects of the pandemic on unrelated hematopoietic stem cell donation and transplantation from the perspective of DKMS, a large international donor registry. Especially, we cover the development of PBSC and bone marrow collection figures, donor management including Health and Availability Check (HAC), transport and cryopreservation of stem cell products, donor recruitment and business continuity measures. The total number of stem cell products provided declined by around 15% during the crisis with a particularly strong decrease in bone marrow products. We modified donor management processes to ensure donor and product safety. HAC instead of confirmatory typing was helpful especially in countries with strict lockdowns. New transport modes were developed so that stem cell products could be safely delivered despite COVID-19-related travel restrictions. Cryopreservation of stem cell products became the new temporary standard during the pandemic to minimize risks related to transport logistics and donor availability. However, many products from unrelated donors will never be transfused. DKMS discontinued public offline donor recruitment, leading to a 40% decline in new donors during the crisis. Most DKMS employees worked from home to ensure business continuity during the crisis.

Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates.

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.

Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity.

Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.

Organellar Proteomics and Phospho-Proteomics Reveal Subcellular Reorganization in Diet-Induced Hepatic Steatosis.

Lipid metabolism is highly compartmentalized between cellular organelles that dynamically adapt their compositions and interactions in response to metabolic challenges. Here, we investigate how diet-induced hepatic lipid accumulation, observed in non-alcoholic fatty liver disease (NAFLD), affects protein localization, organelle organization, and protein phosphorylation in vivo. We develop a mass spectrometric workflow for protein and phosphopeptide correlation profiling to monitor levels and cellular distributions of ∼6,000 liver proteins and ∼16,000 phosphopeptides during development of steatosis. Several organelle contact site proteins are targeted to lipid droplets (LDs) in steatotic liver, tethering organelles orchestrating lipid metabolism. Proteins of the secretory pathway dramatically redistribute, including the mis-localization of the COPI complex and sequestration of the Golgi apparatus at LDs. This correlates with reduced hepatic protein secretion. Our systematic in vivo analysis of subcellular rearrangements and organelle-specific phosphorylation reveals how nutrient overload leads to organellar reorganization and cellular dysfunction.

A Hepatic GAbp-AMPK Axis Links Inflammatory Signaling to Systemic Vascular Damage.

Increased pro-inflammatory signaling is a hallmark of metabolic dysfunction in obesity and diabetes. Although both inflammatory and energy substrate handling processes represent critical layers of metabolic control, their molecular integration sites remain largely unknown. Here, we identify the heterodimerization interface between the α and ß subunits of transcription factor GA-binding protein (GAbp) as a negative target of tumor necrosis factor alpha (TNF-α) signaling. TNF-α prevented GAbpα and ß complex formation via reactive oxygen species (ROS), leading to the non-energy-dependent transcriptional inactivation of AMP-activated kinase (AMPK) ß1, which was identified as a direct hepatic GAbp target. Impairment of AMPKß1, in turn, elevated downstream cellular cholesterol biosynthesis, and hepatocyte-specific ablation of GAbpα induced systemic hypercholesterolemia and early macro-vascular lesion formation in mice. As GAbpα and AMPKß1 levels were also found to correlate in obese human patients, the ROS-GAbp-AMPK pathway may represent a key component of a hepato-vascular axis in diabetic long-term complications.

Decreased expression of angiogenesis antagonist EFEMP1 in sporadic breast cancer is caused by aberrant promoter methylation and points to an impact of EFEMP1 as molecular biomarker.

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real-time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down-regulation in 59% and 61% of the analyzed tissues, respectively. This down-regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57-62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down-regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease-free (p = 0.037) and overall survival (p = 0.032), particularly in those node-positive patients who received adjuvant anthracycline-based chemotherapy, but not in those treated by either cyclophosphamide-methotrexate-5-fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.

Identification of brain- and bone-specific breast cancer metastasis genes.

In breast cancer, metastases are relatively widely distributed, with the most common sites being bone, regional lymph nodes, lung, liver, and brain. The detailed mechanism of organ-specific metastasis is poorly understood. In this study, we initiated a search for genes that are implicated in brain or bone metastasis of primary human breast cancer. We generated gene expression profiles of 18 brain and eight bone metastases derived from primary breast tumors. We identified 73 genes differentially expressed between brain and bone metastases. Visualization of the differential gene expression profiles by correspondence and cluster analyses shows that the metastases clearly separate into two distinct groups as an exact reflection of their site of metastasis. Moreover, the analysis of this gene set in primary breast tumors relapsing to either bone or brain allowed accurate categorization of the tumors according to their metastatic site. The identified genes may prove to be excellent markers to predict the site of metastasis in breast cancer patients and could lead to tailor-made therapy to an individual patient.

SV40 large T antigen-transformed human primary normal and cancerous mammary epithelial cells are phenotypically similar but can be distinguished in 3D culture with selection medium.

Human normal mammary epithelial cells (NMECs) have 2 major in vitro growth restrictions, senescence and crisis. Cellular immortalization is considered a hallmark of malignancy. However, cancerous mammary epithelial cells (CMECs) that are thought to have passed growth barriers in vivo usually cannot be established long-term in vitro. Here we show that CMECs deprived of their natural environment and grown in conventional complete medium behave similar to NMECs, e.g., they stop producing telomerase and become senescent. Like NMECs, CMECs are rescued by SV40 large T (LT) from senescence but not from crisis. The telomere length of both LT-transformed NMEC (N-LT) and CMEC (C-LT) cells first shortens but later partially recovers after telomerase activation. Both cell types upregulate ErbB2 expression, acquire genetic changes, remain long-term dependent on LT and ErbB2 and are nontumorigenic. Despite these similarities, N-LT and C-LT cells cultured in selection medium show different growth characteristics in 3D culture and in vivo tumorigenesis. Thus, CMECs are under a comparable in vitro selective pressure in conventional monolayer culture as NMECs despite their in vivo malignancy. This data demonstrate that most primary breast cancer cells are still unable to overcome the in vitro growth restrictions and suggest that the relationship of in vitro immortalization and in vivo carcinogenesis should be re-evaluated.

Expression of the protein phosphatase 1 inhibitor KEPI is downregulated in breast cancer cell lines and tissues and involved in the regulation of the tumor suppressor EGR1 via the MEK-ERK pathway.

KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.

Comparison of gene expression data from human and mouse breast cancers: identification of a conserved breast tumor gene set.

The aim of our work was to establish a database for breast cancer gene expression data in order to compare human and mouse breast cancer. We identified human and mouse homologues genes and compared the expression profile of 24 human breast tumors with 6 WAP-SVT/t breast tumors (WAP-SVT/t animals, line 8). Our studies confirmed the heterogeneity in gene expression of human as well as mouse breast cancer cells. However, 63 genes were found to be differentially expressed (upregulated: 40; downregulated: 23 genes) in at least 75% of the breast tumors of both species. To differentiate between early and late events in tumor formation, we compared the 63 differentially expressed genes with a mouse data set obtained from hyperplastic mammary glands. This revealed that the majority of the early deregulated genes are cell proliferation specific. These early changes seem to be necessary although not sufficient for breast cancer formation. Late alterations concern mainly genes belonging to the category of cell communication and metabolism. Interestingly, most of the 63 conserved genes are commonly associated with tumorigenesis.

Genetic background of different cancer cell lines influences the gene set involved in chromosome 8 mediated breast tumor suppression.

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.

Profile of differentially expressed genes after transfer of chromosome 17 into the breast cancer cell line CAL51.

Previous studies have shown that transfer of chromosome 17 suppresses the tumorigenic phenotype of the breast cancer cell line CAL51, suggesting the presence of putative tumor suppressor genes on this chromosome. Suppression subtractive hybridization and oligonucleotide microarray analyses were performed to identify differentially expressed genes in nontumorigenic microcell hybrids, CAL/17-1 and CAL/17-3, when compared with CAL51 cells. In total, 263 differentially expressed transcripts were associated with these phenotypes. Of these, a high percentage is involved in various biological processes associated with tumorigenesis, including DNA-dependent regulation of transcription, regulation of cell cycle, signal transduction, and cell proliferation. Microarray analysis of selected chromosome 17 genes in a series of 25 human primary breast tumors showed associations with clinicopathologic parameters of the tumors. Of these genes, TOB1 (transducer of ERBB2) was selected for further expression analysis. Using RT-PCR and immunohistochemical staining of tissue microarrays, we could reveal a differential mRNA and protein expression of TOB1 in the majority of breast tumors and lymph node metastases compared with normal breast tissues, indicating a potential role of this protein in breast tumorigenesis.

Characterization of {gamma}-aminobutyric acid type A receptor-associated protein, a novel tumor suppressor, showing reduced expression in breast cancer.

Frequent allelic loss of the chromosomal region 17p13 in breast cancer has suggested that more tumor suppressor genes, besides p53, are located in this region. By doing suppression subtractive hybridization to detect differentially expressed genes between the breast cancer cell line CAL51 and a nontumorigenic microcell hybrid CAL/17-1, we identified the gene for the gamma-aminobutyric acid type A (GABA(A)) receptor associated protein (GABARAP), located on 17p13.1. GABARAP displayed high expression levels in the microcell hybrid CAL/17-1 but only weak expression in CAL51 and other breast cancer cell lines tested. Furthermore, we observed large vesicles in CAL/17-1 by immunofluorescence staining, whereas no signal could be detected in the tumor cell line. GABARAP mRNA expression and protein expression were significantly down-regulated in invasive ductal and invasive lobular carcinomas compared with normal breast tissue measured by semiquantitative reverse transcription-PCR and immunohistochemistry, respectively. We assessed that neither mutations in the coding region of the gene nor hypermethylation of CpG islands in the promoter region are responsible for loss of gene expression in CAL51; however, 5-aza-2'-deoxycytidine treatment was effective in gene up-regulation, suggesting a methylation-dependent upstream effect. Stable transfection of GABARAP into CAL51 resulted in an increase of gene expression and remarkably influenced the ability of colony formation in soft agar and the growth rate in vitro and, moreover, suppressed the tumorigenicity of the cells in nude mice. In summary, our data suggest that GABARAP acts via a vesicle transport mechanism as a tumor suppressor in breast cancer.