Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis --relapse of precursor B-cell (preB) ALL pediatric samples (n ¼ 32 and n ¼ 27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.Leukemia ( INTRODUCTIONChildren with acute lymphoblastic leukemia (ALL) continue to suffer a 20% incidence of relapse after treatment with the best available therapy. High-resolution genomic profiling, including analysis of single-nucleotide polymorphisms and copy number abnormalities, has greatly aided an understanding of the molecular mechanisms underlying treatment outcome, therapy response and the biology of relapse. 1,2 For precursor B-cell (preB) ALL, genomic studies have shown that copy number abnormalities in genes involved in lymphoid differentiation and cell cycle control are common, with deletions, or part thereof, found in PAX5, EBF1, IKZF1, TCF-4, CDKN2A and RB1. 2 --5 Recent reports also indicate that deletions and nonsense mutations of the IKZF1 gene are significantly associated with poor relapse-free and overall survival rates in preB-ALL. 6 However, in light of these studies, questions remain on the biology of relapse, with marker analysis complicated by the fact that phenotypic shifts in preB-ALL blasts can occur between diagnostic and post-chemotherapy or relapse samples. 7 Those cells that give rise to relapse in some cases appear to be selected during treatment, with clonal evolution occurring of a minor subclone present at diagnosis rather than simply being the development of chemotherapeutic resistance of the original leukemic clone. 8,9 The inherent genetic heterogeneity

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins ␣ 3 ␤ 1 and ␣ 6 ␤ 1 and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of ␣ 3 and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous ␤ 1 integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.Integrins comprise a large family of ␣␤ heterodimeric transmembrane proteins that function as key receptors for cellular attachment to the extracellular matrix and to cellular ligands (1). Integrin function extends beyond cell adhesion per se, and integrin-mediated signaling influences a range of biological processes including cellular division and migration, differentiation, and apoptosis (reviewed in Refs.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum- and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells. Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors.

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Objective:Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36+MPs in persons with T2DM.Design:An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI)=39.9±6.4 kg m−2; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI=23.6±1.8 kg m−2 and 33.5±5.9 kg m−2, respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235a, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations.Results:CD36+MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235a+=35.8±14.6%); although this did not correlate with haemoglobin A1c. By contrast, the main source of CD36+MPs in non-T2DM individuals was endothelial cells (CD105+=40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 μg ml−1 and was positively correlated with CD36+MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36+MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively).Conclusions:Both the levels and cellular profile of CD36+MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis. In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1. By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress. Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1. Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress. This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown. Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1. Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1. These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

To cite this article: Alkhatatbeh MJ, Mhaidat NM, Enjeti AK, Lincz LF, Thorne RF. The putative diabetic plasma marker, soluble CD36, is noncleaved, non-soluble and entirely associated with microparticles. J Thromb Haemost 2011; 9: 844-51.Summary. Background: CD36 is a widely expressed cell surface receptor that binds lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell-free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. Objective: To determine the nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). Methods: Lipoproteins were fractionated by density gradient centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight-488 anti-CD36 conjugate in combination with antibodies against different cellular markers. Results: Cell-free plasma CD36 was not observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. Conclusions: sCD36 is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.