Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an aciddependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.Genetic variation in proprotein convertase subtilisin/kexin type 9 (PCSK9) 4 contributes to differences in plasma levels of low density lipoprotein (LDL) cholesterol (1, 2), the primary cholesterol-carrying lipoprotein in humans. Selected missense mutations in PCSK9 cause dominant hypercholesterolemia and premature atherosclerosis (1, 3, 4), whereas loss-of-function mutations in PCSK9 reduce plasma LDL levels and protect against coronary heart disease (5-7). Studies in mice suggest that the major metabolic effect of PCSK9 is to reduce the amount of hepatic LDL receptor (LDLR), the primary conduit for the clearance of LDL from the circulation (8). Expression of recombinant PCSK9 in the livers of mice causes a reduction in hepatic LDLR protein (but not mRNA) and produces severe hypercholesterolemia (9 -11). Conversely, mice lacking PCSK9 manifest increased levels of LDLR protein in the liver and accelerated clearance of circulating LDL (12). PCSK9 is expressed predominantly in the liver, small intestine, kidney, and brain (13) and is present in human plasma (14,15). Introduction of PCSK9 into the circulation of mice through parabiosis reduces hepatic LDLR levels, which is consistent with PCSK9 interacting with the LDLR on the cell surface (14). In cultured cells, the addition of recombinant PCSK9 to the medium results in LDLR degradation, providing further evidence that PCSK9 can promote the degradation of the LDLR by acting at the cell surface (14, 16). Autocatalytic cleavage is required for PCSK9 maturation and secretion (10), but whether the catalytic activity of PCSK9 is required for LDLR d...

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy.

SUMMARY Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused 13C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.

Summary Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We further elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human datasets analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases. Significance In advanced stages, breast cancer bone metastases are driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts, which constitute a vicious osteolytic cycle. Current therapies targeting this process limit tumor progression, but do not improve patient survival. On the other hand, bone micrometastases may remain indolent for years before activating the vicious cycle, providing a therapeutic opportunity to prevent macrometastases. Here, we show that bone colonization is initiated in a microenvironment niche exhibiting active osteogenesis. Cancer and osteogenic cells form heterotypic adherens junctions, which enhance mTOR activity and drive early-stage bone colonization prior to osteolysis. These results reveal a strong connection between osteogenesis and micrometastasis and suggest potential therapeutic targets to prevent bone macrometastases.

CSPGs (chondroitin sulfate proteoglycans) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane LAR (the leukocyte common antigen-related phosphatase). CSPGs bind LAR with high affinity in transfected COS-7 cells and co-immunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knockout mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.

Background Several somatic mutation hotspots were recently identified in the TERT promoter region in human cancers. Large scale studies of these mutations in multiple tumor types are limited, in particular in Asian populations. This study aimed to: analyze TERT promoter mutations in multiple tumor types in a large Chinese patient cohort, investigate novel tumor types and assess the functional significance of the mutations. Methods TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumor types and 799 tumor tissues from Chinese cancer patients. Thymic epithelial tumors, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by RT-qPCR, telomerase activity by the TRAP assay, and promoter activity by the luciferase reporter assay. Results TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%), and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in GIST, thymic epithelial tumors, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. Conclusions TERT promoter mutations are frequent in multiple tumor types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumorigenesis, making them potential therapeutic targets.

Alterations in p53 or p16/p14ARF are found in a fourth of ES cases and define a subset with highly aggressive behavior and poor chemoresponse.

The gene hypermethylated in cancer 1 (HIC1) is epigenetically inactivated, but not mutated, in cancer. Here we show that cooperative loss of Hic1 with p53, but not INK4a, yields distinct tumor phenotypes in mice. Germline deletion of one allele of each gene on the opposite chromosome yields breast and ovarian carcinomas and metastatic osteosarcomas with epigenetic inactivation of the wild-type Hic1 allele. Germline deletion of the two genes on the same chromosome results in earlier appearance and increased prevalence and aggressiveness of osteosarcomas with genetic deletion of both wild-type genes. In human osteosarcomas, hypermethylation of HIC1 is frequent only in tumors with p53 mutations. Our results indicate the importance of genes altered only through epigenetic mechanisms in cancer progression in conjunction with genetically modified tumor suppressor genes.