Furin processing dictates ectodomain shedding of human FAT1 cadherin.

Fat1 is a single pass transmembrane protein and the largest member of the cadherin superfamily. Mouse knockout models and in vitro studies have suggested that Fat1 influences cell polarity and motility. Fat1 is also an upstream regulator of the Hippo pathway, at least in lower vertebrates, and hence may play a role in growth control. In previous work we have established that FAT1 cadherin is initially cleaved by proprotein convertases to form a noncovalently linked heterodimer prior to expression on the cell surface. Such processing was not a requirement for cell surface expression, since melanoma cells expressed both unprocessed FAT1 and the heterodimer on the cell surface. Here we further establish that the site 1 (S1) cleavage step to promote FAT1 heterodimerisation is catalysed by furin and we identify the cleavage site utilised. For a number of other transmembrane receptors that undergo heterodimerisation the S1 processing step is thought to occur constitutively but the functional significance of heterodimerisation has been controversial. It has also been generally unclear as to the significance of receptor heterodimerisation with respect to subsequent post-translational proteolysis that often occurs in transmembrane proteins. Exploiting the partial deficiency of FAT1 processing in melanoma cells together with furin-deficient LoVo cells, we manipulated furin expression to demonstrate that only the heterodimer form of FAT1 is subject to cleavage and subsequent release of the extracellular domain. This work establishes S1-processing as a clear functional prerequisite for ectodomain shedding of FAT1 with general implications for the shedding of other transmembrane receptors.

Phytochemical properties and anti-proliferative activity of Olea europaea L. leaf extracts against pancreatic cancer cells.

Olea europaea L. leaves are an agricultural waste product with a high concentration of phenolic compounds; especially oleuropein. Oleuropein has been shown to exhibit anti-proliferative activity against a number of cancer types. However, they have not been tested against pancreatic cancer, the fifth leading cause of cancer related death in Western countries. Therefore, water, 50% ethanol and 50% methanol extracts of Corregiola and Frantoio variety Olea europaea L. leaves were investigated for their total phenolic compounds, total flavonoids and oleuropein content, antioxidant capacity and anti-proliferative activity against MiaPaCa-2 pancreatic cancer cells. The extracts only had slight differences in their phytochemical properties, and at 100 and 200 µg/mL, all decreased the viability of the pancreatic cancer cells relative to controls. At 50 µg/mL, the water extract from the Corregiola leaves exhibited the highest anti-proliferative activity with the effect possibly due to early eluting HPLC peaks. For this reason, olive leaf extracts warrant further investigation into their potential anti-pancreatic cancer benefits.

Sleeping giants: emerging roles for the fat cadherins in health and disease.

The vertebrate Fat cadherins comprise a small gene family of four members, Fat1-Fat4, all closely related in structure to Drosophila ft and ft2. Over the past decade, knock-out mouse studies, genetic manipulation, and large sequencing projects has aided our understanding of the function of vertebrate Fat cadherins in tissue development and disease. The majority of studies of this family have focused on Fat1, with evidence now showing it can bind enable (ENA)/Vasodilator-stimulated phosphoprotein (VASP), ß-catenin and Atrophin proteins to influence cell polarity and motility; HOMER-1 and HOMER-3 proteins to regulate actin accumulation in neuronal synapses; and scribble to influence the Hippo signaling pathway. Fat2 and Fat3 can regulate cell migration in a tissue specific manner and Fat4 appears to influence both planar cell polarity and Hippo signaling recapitulating the activity of Drosophila ft. Knowledge about the exact downstream signaling pathways activated by each family member remains in its infancy, but it is becoming clearer that they have tissue specific and redundant roles in development and may be lost or gained in cancer. In this review, we summarize the recent progress on understanding the role of the Fat cadherin family, integrating the current knowledge of molecular interactions and tissue distributions, together with the accumulating evidence of their changed expression in human disease. The latter is now beginning to promote interest in these molecules as both biomarkers and new targets for therapeutic intervention.

Macrophage migration inhibitory factor engages PI3K/Akt signalling and is a prognostic factor in metastatic melanoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a widely expressed cytokine involved in a variety of cellular processes including cell cycle regulation and the control of proliferation. Overexpression of MIF has been reported in a number of cancer types and it has previously been shown that MIF is upregulated in melanocytic tumours with the highest expression levels occurring in malignant melanoma. However, the clinical significance of high MIF expression in melanoma has not been reported. METHODS: MIF expression was depleted in human melanoma cell lines using siRNA-mediated gene knockdown and effects monitored using in vitro assays of proliferation, cell cycle, apoptosis, clonogenicity and Akt signalling. In silico analyses of expression microarray data were used to correlate MIF expression levels in melanoma tumours with overall patient survival using a univariate Cox regression model. RESULTS: Knockdown of MIF significantly decreased proliferation, increased apoptosis and decreased anchorage-independent growth. Effects were associated with reduced numbers of cells entering S phase concomitant with decreased cyclin D1 and CDK4 expression, increased p27 expression and decreased Akt phosphorylation. Analysis of clinical outcome data showed that MIF expression levels in primary melanoma were not associated with outcome (HR = 1.091, p = 0.892) whereas higher levels of MIF in metastatic lesions were significantly associated with faster disease progression (HR = 2.946, p = 0.003 and HR = 4.600, p = 0.004, respectively in two independent studies). CONCLUSIONS: Our in vitro analyses show that MIF functions upstream of the PI3K/Akt pathway in human melanoma cell lines. Moreover, depletion of MIF inhibited melanoma proliferation, viability and clonogenic capacity. Clinically, high MIF levels in metastatic melanoma were found to be associated with faster disease recurrence. These findings support the clinical significance of MIF signalling in melanoma and provide a strong rationale for both targeting and monitoring MIF expression in clinical melanoma.

Dual processing of FAT1 cadherin protein by human melanoma cells generates distinct protein products.

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.

Zinc status of Iranian preschool children.

BACKGROUND: Zinc deficiency is one of the most prevalent micronutrient deficiencies in developing countries, including Iran. The main direct causes of zinc deficiency are insufficient zinc intake, absorption or metabolic disorder, and increase in need during acute growth periods. OBJECTIVE: To determine the prevalence of zinc deficiency in preschool boys and girls in urban and rural populations in order to assist policy makers. Children of preschool age (i.e., 6 years old in Iran) were studied because interventions in this age group are believed to result in greater improvement in learning skills once these children enter school. METHODS: A national cross-sectional study was carried out on 4,374 randomly selected healthy preschool children from Iranian families in 2001. Serum zinc concentration was measured by atomic absorption spectrometry. The cutoff point for zinc deficiency was set at a serum level of 10 micromol/L (65 microg/dL). RESULTS: The prevalence of zinc deficiency was estimated at approximately 19.3%. The highest prevalence was seen in the region that includes Sistan and Baluchistan, South Khorasan, and the southeast area of Kerman and the lowest in the region of Boushehr, Hormozgan, and South Khoozestan. The prevalence of zinc deficiency was significantly higher in rural areas than in urban areas. No significant difference in prevalence was seen between boys and girls. CONCLUSIONS: In the long run, nutritional security and increased access to and intake of foods with high levels of zinc are the most sustainable strategies to overcome zinc deficiency. Fortification of staple foods, improved quality of traditional bread, and supplementation for at-risk population groups are considered short- and mid-term interventions. Nutrition education and behavioral change may be long-term strategies.

Protein interaction screening identifies SH3RF1 as a new regulator of FAT1 protein levels.

Mutations and ectopic FAT1 cadherin expression are implicated in a broad spectrum of diseases ranging from developmental disorders to cancer. The regulation of FAT1 and its downstream signalling pathways remain incompletely understood. We hypothesized that identification of additional proteins interacting with the FAT1 cytoplasmic tail would further delineate its regulation and function. A yeast two-hybrid library screen carried out against the juxtamembrane region of the cytoplasmic tail of FAT1 identified the E3 ubiquitin-protein ligase SH3RF1 as the most frequently recovered protein-binding partner. Ablating SH3RF1 using siRNA increased cellular FAT1 protein levels and stabilized expression at the cell surface, while overexpression of SH3RF1 reduced FAT1 levels. We conclude that SH3RF1 acts as a negative post-translational regulator of FAT1 levels.

FAT1 cadherin is multiply phosphorylated on its ectodomain but phosphorylation is not catalysed by the four-jointed homologue.

The interaction between the Drosophila cadherins fat and dachsous is regulated by phosphorylation of their respective ectodomains, a process catalysed by the atypical kinase four-jointed. Given that many signalling functions are conserved between Drosophila and vertebrate Fat cadherins, we sought to determine whether ectodomain phosphorylation is conserved in FAT1 cadherin, and also whether FJX1, the vertebrate orthologue of four-jointed, was involved in such phosphorylation events. Potential Fj consensus phosphorylation motifs were identified in FAT1 and biochemical experiments revealed the presence of phosphoserine and phosphothreonine residues in its extracellular domain. However, silencing FJX1 did not influence the levels of FAT1 ectodomain phosphorylation, indicating that other mechanisms are likely responsible.

Optimisation of Ultrasound-Assisted Extraction Conditions for Phenolic Content and Antioxidant Capacity from Euphorbia tirucalli Using Response Surface Methodology.

Euphorbia tirucalli (E. tirucalli) is now widely distributed around the world and is well known as a source of traditional medicine in many countries. This study aimed to utilise response surface methodology (RSM) to optimise ultrasonic-assisted extraction (UAE) conditions for total phenolic compounds (TPC) and antioxidant capacity from E. tirucalli leaf. The results showed that ultrasonic temperature, time and power effected TPC and antioxidant capacity; however, the effects varied. Ultrasonic power had the strongest influence on TPC; whereas ultrasonic temperature had the greatest impact on antioxidant capacity. Ultrasonic time had the least impact on both TPC and antioxidant capacity. The optimum UAE conditions were determined to be 50 °C, 90 min. and 200 W. Under these conditions, the E. tirucalli leaf extract yielded 2.93 mg GAE/g FW of TPC and exhibited potent antioxidant capacity. These conditions can be utilised for further isolation and purification of phenolic compounds from E. tirucalli leaf.

A soluble form of the giant cadherin Fat1 is released from pancreatic cancer cells by ADAM10 mediated ectodomain shedding.

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.

Combined MUC1-specific nanobody-tagged PEG-polyethylenimine polyplex targeting and transcriptional targeting of tBid transgene for directed killing of MUC1 over-expressing tumour cells.

We provide evidence for combining a single domain antibody (nanobody)-based targeting approach with transcriptional targeting as a safe way to deliver lethal transgenes to MUC1 over-expressing cancer cells. From a nanobody immune library, we have isolated an anti-DF3/Mucin1 (MUC1) nanobody with high specificity for the MUC1 antigen, which is an aberrantly glycosylated glycoprotein over-expressed in tumours of epithelial origin. The anti-MUC1 nanobody was covalently linked to the distal end of poly(ethylene glycol)(3500) (PEG(3500)) in PEG(3500)-25kDa polyethylenimine (PEI) conjugates and the resultant macromolecular entity successfully condensed plasmids coding a transcriptionally targeted truncated-Bid (tBid) killer gene under the control of the cancer-specific MUC1 promoter. The engineered polyplexes exhibited favourable physicochemical characteristics for transfection and dramatically elevated the level of Bid/tBid expression in both MUC1 over-expressing caspase 3-deficient (MCF7 cells) and caspase 3-positive (T47D and SKBR3) tumour cell lines and, concomitantly, induced considerable cell death. Neither transgene expression nor cell death occurred when the MUC1 promoter was replaced with the CNS-specific synapsin I promoter. Since PEGylated PEI was only responsible for DNA compaction and played no significant role in direct transfection and cell killing, our attempts overcome previously reported PEI-mediated apoptotic and necrotic cell death, which is advantageous for future in vivo transcriptional targeting as this will minimize (or eliminate) non-targeted cell damage.