SummaryA functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

Notch signaling is a central mechanism for controlling embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and specifically, in bone homeostasis remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis due to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by up-regulating Cyclin D, Cyclin E, and Osterix. Notch also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all physiologic Notch signaling in osteoblasts, generated by deletion of Presenilin 1 and 2 in bone, is associated with late onset, age-related osteoporosis resulting from increased osteoblast-dependent osteoclastic activity due to decreased production of Osteoprotegerin. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.Evolutionarily conserved Notch signaling plays a critical role in cell fate determination, and various developmental processes by translating cell-cell interactions into specific transcriptional programs 1, 2 . Temporal and spatial modulation of this pathway can significantly affect proliferation, differentiation and apoptotic events 3 . Moreover, the timing of Notch signaling can lead to diverse effects within the same cell lineage 4, 5 . In mammals, activation of up to four Notch receptors by membrane-bound ligands initiates a process leading to presenilin-mediated cleavage and release of the Notch intracellular domain (NICD) from the membrane that then traffics to the nucleus. NICD subsequently regulates the expression of genes in cooperation with the transcription factor RBP-Jκ and Mastermind-like proteins.The observation that mutations in the Notch ligand Delta homologue-3 (Dll-3) and γ-secretase Presenilin1 both cause axial skeletal phenotypes originally linked Notch signaling with skeletal development 6, 7 . Recently, several in vitro studies with conflicting results implicated the Notch pathway in the regulation of osteoblast differentiation, but the in vivo role of Notch signaling in bone homeostasis still remains unknown 8-12 .Corresponding Author: Brendan Lee, M.D., Ph.D., One Baylor Plaza, Rm 635E, Houston, Tx 77030,, Email E-mail: blee@bcm.tmc.edu. In this study, we investigate the tissue, cellular, and molecular consequences of both gain and loss of function of Notch signaling in committed osteoblasts. NIH Public Access RESULTS Gain of function of Notch signaling results in severe osteosclerosisTo determine the pathological consequences of in vivo gain of Notch function during bone formation and homeostasis, we generated transgenic mice expressing the Notch1 intracellular domain (N1ICD) under the control of the type I collagen (Col1a1) promoter (Suppl. Fig. 1a,b). Here, gain of Notch function would occur in committed osteoblastic ce...

Osteogenesis Imperfecta (OI) is a heritable disorder of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations1. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms2. Here, we show that excessive transforming growth factor-beta (TGFβ) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGFβ target genes, ratio of pSmad2/Smad2 protein, and in vivo Smad2 reporter activity. Anti-TGFβ treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI, and improves the lung abnormalities in Crtap−/− mice. Moreover, type I collagen of Crtap−/− mice shows reduced binding to the small leucine rich proteoglycan decorin, a known regulator of TGFβ activity3–4. Hence, altered TGFβ matrix-cell signaling is a primary mechanism in the pathogenesis of OI, and could be a promising target for the treatment of OI.

Members of the Wnt family are secreted glycoproteins that trigger cellular signals essential for proper development of organisms. Cellular signaling induced by Wnt proteins is involved in diverse developmental processes and human diseases. Previous studies have generated an enormous wealth of knowledge on the events in signal-receiving cells. However, relatively little is known about the making of Wnt in signal-producing cells. Here, we describe that Gpr177, the mouse orthologue of Drosophila Wls, is expressed during formation of embryonic axes. Embryos with deficient Gpr177 exhibit defects in establishment of the body axis, a phenotype highly reminiscent to the loss of Wnt3. Although many different mammalian Wnt proteins are required for a wide range of developmental processes, the Wnt3 ablation exhibits the earliest developmental abnormality. This suggests that the Gpr177-mediated Wnt production cannot be substituted. As a direct target of Wnt, Gpr177 is activated by ␤-catenin and LEF/TCF-dependent transcription. This activation alters the cellular distributions of Gpr177 which binds to Wnt proteins and assists their sorting and secretion in a feedback regulatory mechanism. Our findings demonstrate that the loss of Gpr177 affects Wnt production in the signal-producing cells, leading to alterations of Wnt signaling in the signal-receiving cells. A reciprocal regulation of Wnt and Gpr177 is essential for the patterning of the anterior-posterior axis during mammalian development.A-P axis ͉ ␤-catenin ͉ developmental deformities ͉ primitive streak ͉ Wnt production

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

BackgroundEnhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets.ResultsUsing functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival.ConclusionsOur data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-016-0146-8) contains supplementary material, which is available to authorized users.

Summary Sox2 regulates the self-renewal of multiple types of stem cells. Recent studies suggest it also plays oncogenic roles in the formation of squamous carcinoma in several organs, including the esophagus where Sox2 is predominantly expressed in the basal progenitor cells of the stratified epithelium. Here, we use mouse genetic models to reveal a novel mechanism by which Sox2 cooperates with microenvironmental signals to malignantly transform epithelial progenitor cells. Conditional overexpression of Sox2 in basal cells expands the progenitor population in both the esophagus and forestomach. Significantly, carcinoma only develops in the forestomach where pathological progression correlates with inflammation and nuclear localization of Stat3 in progenitor cells. Importantly, co-overexpression of Sox2 and activated Stat3 (Stat3C) also transforms esophageal basal cells but not the differentiated suprabasal cells. These findings indicate basal stem/progenitor cells are the cells-of-origin of squamous carcinoma and that cooperation between Sox2 and microenvironment-activated Stat3 is required for Sox2-driven tumorigenesis.

In several organ systems the transitional zone between different types of epithelia is a hotspot for pre-neoplastic metaplasia and malignancy1–3. However, the cell-of-origin for the metaplastic epithelium and subsequent malignancy, remains obscure1–3. In the case of Barrett’s oesophagus (BE), intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells4. Based on different experimental models, several alternative cell types have been proposed as the source of the metaplasia, but in all cases the evidence is inconclusive and no model completely mimics BE with the presence of intestinal goblet cells5–8. Here, we describe a novel transitional columnar epithelium with distinct basal progenitor cells (p63+ KRT5+ KRT7+) in the squamous-columnar junction (SCJ) in the upper gastrointestinal tract of the mouse. We use multiple models and lineage tracing strategies to show that this unique SCJ basal cell population serves as a source of progenitors for the transitional epithelium. Moreover, upon ectopic expression of CDX2 these transitional basal progenitors differentiate into intestinal-like epithelium including goblet cells, thus reproducing Barrett’s metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues, including the anorectal junction, and, importantly, at the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (MLE) believed to be a precursor of BE are both characterized by the expansion of the transitional basal progenitor cells. Taken together our findings reveal the presence of a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+ KRT7+ basal cells in this zone are the cell-of-origin for MLE and BE.