The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser 180 , within the FGF23 R 176 XXR 179 /S 180 AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.phosphate homeostasis | rickets | Fam20 | familial tumoral calcinosis | chronic kidney disease P rotein kinases are evolutionarily conserved enzymes that regulate numerous cellular processes by transferring a molecule of phosphate from ATP to target substrates (1, 2). The vast majority of theses enzymes function within the nucleus and cytosol. In contrast, there are several examples of phosphorylated proteins that are secreted from the cell, which raises the question: What are the kinases that phosphorylate these secreted phosphoproteins? We recently identified a small family of secretory pathway kinases that phosphorylate secreted proteins and proteoglycans (3). These enzymes have N-terminal signal sequences that direct them to the lumen of the endoplasmic reticulum, where they encounter the proteins or proteoglycans that they phosphorylate. One member of this atypical kinase family is the family with sequence similarity 20, member C (Fam20C), which phosphorylates secreted proteins on Ser(S)-xGlu(E)/pSer(pS) (S-x-E/pS) motifs (3, 4). Because Fam20C localizes within the secretory pathway and the vast majority of secreted phosphoproteins are phosphorylated on S-x-E/pS motifs, Fam20C has been proposed to play a major role in the generation of the secreted phosphoproteome (5-7). For example, ∼75% of human serum and cerebrospinal fluid phosphoproteins are phosphorylated on S-x-E/pS motifs (8, 9). This includes proteins important for tooth and bone formation, as well as numerous hormones. In most cases, the functional importance of these phosphorylation events is unknown.Loss-of-function mutations in the human FAM20C gene cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia (10, 11)....

BackgroundBreast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.MethodsMCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.ResultsTwo thousand thirty five genes were differentially expressed (p < 0.001, fold change ≥ 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFβ, NF-κB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-κB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-κB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.ConclusionsEMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.

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IntroductionSerum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.MethodsSera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease.ResultsU6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization.ConclusionsElevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.

Estrogen regulates several biological processes through estrogen receptor ␣ (ER␣) and ER␤. ER␣-estrogenEstrogen-mediated transcriptional regulation has been studied quite extensively because of its relevance to breast cancer (1, 26). It also serves as a model system to study integration of several signaling events to transcription (26). The transcriptional effects of estrogen are largely mediated by two receptors belonging to nuclear receptor superfamily: estrogen receptor ␣ (ER␣) and ER␤ (26). ERs bind DNA either directly via estrogen response elements (ERE) or tethered through other transcription factors (8,30,47). Genome-wide analyses of ER␣ binding to DNA by chromatin immunoprecipitation combined with microarrays (ChIP-on-chip) have revealed the presence of complete (GGTCAnnnTGACC) or half-ERE (GGTCA)-like sequences in ϳ95% of ER␣ binding regions (13,36). These studies also revealed enrichment of binding sites for C/EBP, Oct, and Forkhead transcription factors adjoining ERE sequences in the ER␣ binding regions (13). Importantly, only ϳ5% of ER␣ binding sites are within 5-kb promoter-proximal region, with a great majority being found in the intronic or distal locations of the transcription start site of a gene (13,36).The transcriptional activity of ER␣ is regulated by distinct posttranslational modifications, including phosphorylation. Several extracellular signal-activated kinases phosphorylate ER␣ and regulate DNA binding, transactivation, coregulator interaction, stability, and/or subcellular distribution. Kinases known to phosphorylate ER␣ include mitogen-activated protein (MAPK), IKK␣, RSK, AKT/PKB, p38 kinase, PKA, Src, cyclin A/cdk2, and cdk7 (1,11,27,31,45,48). It has been suggested that changes in the phosphorylation status of the receptor contributes to ER␣ dysfunction in various pathological conditions, including breast cancer (1).The effects of phosphorylation on ER␣ activity have been studied by many methods, including in vitro DNA-binding assays, ChIP analysis of few endogenous target genes, and transient-transfection experiments with synthetic ERE-containing reporters or with the promoter regions of endogenous target genes (2,11,27,45,48). Kinase-induced changes in ER␣ DNA-binding activity in a genome-wide scale have not been analyzed. Here, we focus on the serine/threonine kinase AKT/ PKB because it phosphorylates ER␣, confers anti-estrogen resistance, and is aberrantly activated in ϳ50% of human malignancies (17,28). In addition, transgenic mice overexpressing AKT1 in the mammary gland predominantly develop ER␣-positive mammary tumors when exposed to chemical carcinogen (6). Several other studies also intimately link AKT to ER␣-positive breast cancers (5, 40).