LIN28 expression and function in medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA-binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration-dependently reduced LIN28B expression, as well as the growth of D283 MB cells. While LIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cells and the sphere formation of CHLA-01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and open the possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.     

p53、survivin在食管癌中的作用

目的:检测p53和survivin蛋白在食管癌组织中的表达,探讨其意义。方法:应用免疫组织化学法检测食管癌及正常食管粘膜组织中p53、survivin蛋白的表达情况。结果:p53、survivin在食管癌组织中的表达量均高于正常食管粘膜组织(P<0.05),p53、survivin高表达与食管癌淋巴结转移状况差异有关(P<0.05),与性别、年龄、肿瘤长度、浸润深度、分化程度无关(P>0.05)。p53和survivin蛋白在食管癌中表达呈明显正相关性(R=0.218,P<0.05)。结论:p53、survivin在食管癌中高表达(82.1%&71.9%),二者在食管癌的淋巴结转移中发挥重要作用,并具有协同作用。     

On predicting medulloblastoma metastasis by gene expression profiling

Accurately predicting clinical outcome or metastatic status from gene expression profiles remains one of the biggest hurdles facing the adoption of predictive medicine. Recently, MacDonald et al. (Nat. Genet. 2001, 29, 143-152) used gene expression profiles, from samples taken at diagnosis, to distinguish between clinically designated metastatic and nonmetastatic primary medulloblastomas, helping to elucidate the genetic mechanisms underlying metastasis and suggesting novel therapeutic targets. The obtained accuracy of predicting metastatic status does not, however, reach statistical significance on Fisher's exact test, although 22 training samples were used to make each prediction via leave-one-out testing. This paper introduces readily implemented nonlinear filters to transform sequences of gene expression levels into output signals that are significantly easier to classify and predict metastasis. It is shown that when only 3 exemplars each from the metastatic and nonmetastatic classes were assumed known, a predictor was constructed whose accuracy is statistically significant over the remaining profiles set aside as a test set. The predictor was as effective in recognizing metastatic as nonmetastatic medulloblastomas, and may be helpful in deciding which patients require more aggressive therapy. The same predictor was similarly effective on an independent set of 5 nonmetastatic tumors and 3 metastatic cell lines also used by MacDonald et al.     

Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.     

Gene expression monitoring accurately predicts medulloblastoma positive and negative clinical outcomes

Prediction of medulloblastoma clinical outcome is crucial to personalizing treatment, both to identify high-risk patients for aggressive or alternative therapy and to spare those at low risk from excessive treatment. The best predictors [Pomeroy et al. (2002) Nature 415, 436–442], based on gene expression monitoring at diagnosis, have shown much less accuracy in recognizing patients with eventual failed outcomes – <50% for the predictor making fewest total errors – than those who would survive, while a single gene predictor exhibited reverse asymmetry. Such inaccuracy in recognizing one of the outcomes is a problem for clinical use. We hypothesized that a non-linear model could be built to significantly improve prediction of medulloblastoma outcome, thereby promoting use of gene-expression-based predictors in a clinical setting. In fact, this approach resulted in fewer errors and much less asymmetry in prediction, and bidirectional accuracy of about 80% could be obtained via its combination with other methods. Indeed, three combinations of methods were identified that yielded significantly better predictions of clinical outcome than previously attained, making feasible predictors of medulloblastoma treatment response with greatly improved bidirectional accuracy essential for clinical use.     

Angiotensin II-induced changes in G-protein expression and resistance of renal microvessels in young genetically hypertensive rats

Altered regulation of cAMP may contribute to enhanced renal reactivity to angiotensin II (Ang II) in spontaneously hypertensive rats (SHR). Such a phenomenon may occur in renal preglomerular arterioles and may involve changes in expression of GTP-binding regulatory proteins. We have examined the effects of Ang II on steady state levels of G_i-1,2, G_i-3 G_s and G_q in preglomerular arterioles from young marginally hypertensive SHR and on mean arterial pressure (MAP), renal vascular resistance (RVR) and renal cAMP excretion (UcAMP.V). Young (5-6 week old) SHR and Wistar Kyoto (WKY) rats received Ang II (35 ng/kg/min, s.c.) or vehicle for 7 days via osmotic minipumps. Urine was collected over the last 24 h. On day seven, MAP and renal blood flow were measured in anesthetized rats and RVR was determined. Preglomerular arterioles were isolated by perfusing the kidneys with iron oxide and using a series of mechanical steps coupled with the use of a magnet to retain iron-laden vessels. Membranes were prepared and the expressions of G_i-1,2, G_i-3, G_s and G_q were evaluated by Western immunoblotting. Baseline MAP (124 ± 6 mmHg) was only marginally (p > 0.05) higher in SHR when compared with WKY rats (110 ± 4 mmHg). RBF (3.04 ± 0.16 mL/min) was significantly lower and RVR (41.10 ± 1.37 mmHg.min/mL) was significantly higher in SHR when compared to age-matched WKY rats (4.36 ± 0.30 mL/min and 25.79 ± 1.58 mmHg.min/mL, respectively). Ang II significantly increased MAP in SHR (17 mmHg) but not in WKY rats. These increases in MAP were accompanied by significant increases in RVR in SHR (48% over control) but not in WKY rats. Compared to WKY rats, preglomerular arterioles from SHR exhibited significantly higher basal expression of G_i-1,2 (11- fold), G_i-3 (13-fold) and G_s (3-fold). Chronic infusion of Ang II, however, downregulated the expression of G_s (by 53%; p < 0.05), G_i-1,2 ( by 72%; p < 0.05) and G_i-3 (by 35%; p > 0.05) in SHR preglomerular arterioles but significantly upregulated the expression of these proteins in WKY by 3-, 8- and 15-fold, respectively. Basal levels of G_q were not different in preglomerular arterioles from the two strains but were downregulated by Ang II in both WKY (74% of basal) and SHR (52% of control). Baseline UcAMP.V was significantly lower in SHR (31.22 ± 6.51 nmol/24 h) compared with WKY rats (65.33 ± 3.60 nmol/24 h). Chronic Ang II infusion significantly increased UcAMP.V in SHR as well as WKY rats. These data clearly demonstrate that expressions of G_i isoforms as well as G_s in renal microvessels are elevated during early stages of hypertension and suggest that the elevated levels of G_i proteins may be directly associated with a blunted adenylyl cyclase-cAMP cascade in the renal microvasculature. Furthermore, Ang II appears to directly downregulate the expression of G_s in young SHR but not in young WKY renal microvessels. Such diversity in its effect on G-protein expression may be important for enhanced renal sensitivity to Ang II in SHR.     

Changes in the gene expression of C-myc and CD38 in HL-60 cells during differentiation induced by nicotinic acid-related compounds

Changes in gene expression levels of c-myc and CD38 were examined during the differentiation of HL-60 cells to granulocytes due to three nicotinic acid-related compounds. CD38 expression was increased by isonicotinic acid and all-trans-retinoic acid (ATRA). Nicotinamide and nicotinamide N-oxide drastically decreased c-myc expression, but isonicotinic acid had no effect, suggesting that these compounds differentiate HL-60 to granulocytes through different pathways. These results should provide useful information as to the mechanisms of cell differentiation.     

Signaling pathways in medulloblastoma

Medulloblastoma is the most common brain tumor of childhood. Multiple signaling pathways have been associated with medulloblastoma formation and growth. These include the developmental pathways Hedgehog, (Hh) Notch, and Wnt as well as the receptor tyrosine kinases (RTK) c-Met, erbB2, IGF-R and TrkC, and the oncoprotein Myc. Here we review the involvement of these pathways in medulloblastoma malignancy with a focus on their mode of deregulation, prognostic value, functional effects, cellular and molecular mechanisms of action, and implications for therapy.     

Immunogold study of altered expression of some interendothelial junctional molecules in the brain blood microvessels of diabetic scrapie-infected mice

Quantitative immunogold procedure was used to study the distribution of molecular components of interendothelial junctions in blood–brain barrier (BBB) microvessels of scrapie infected SJL/J hyperglycemic mice showing obesity and reduced glucose tolerance. Samples of brain (fronto-parietal cerebral cortex and thalamo-hypothalamic region) obtained from hyperglycemic (diabetic) mice and from non- infected, normoglycemic (non-diabetic) SJL/J mice, were processed for immunocytochemical examination. The localization of the following tight junction (TJ)-associated proteins was studied: occludin as an integral membrane (transmembrane) protein, and zonula occludens one (ZO-1) as a peripheral protein. The localization of β-catenin as a representative of the cadherin/catenin complex that is typical for adherens junctions (AJs) also was studied. Morphometric analysis revealed that the density of immunosignals for occludin, represented by colloidal gold particles (GPs), was significantly lower in the brain microvessels of diabetic than in non-diabetic mice. No significant differences in the density of immunosignals for ZO-1 and β-catenin between both experimental mouse groups were observed. It indicates that abnormal glucose metabolism affects mostly occludin which is believed to play a fundamental role in the maintenance of the tightness of endothelial lining in brain microvascular network and thereby in the preservation of its barrier function. These results also support the previously expressed opinion that occludin, detected with the applied morphological method, can be considered a sensitive indicator of altered molecular architecture of the interendothelial junctions due to the action of some metabolic or pathological insults.     

Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels

Introduction  

Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.     

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.     

Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.     

Malignant potential of dysplastic endocervical epithelium assessed by ploidy status,S-phase fraction and C-myc expression

The aim of this study was to determine if dysplastic endocervical cells (EC) posses a neoplastic potential as precursor lesions to adenocarcinoma in situ (AIS). The malignant potential was determined by assessing the ploidy status and proliferative activity by flow cytometry and c-myc expression by immunohistochemistry. The studied parameters were assessed separately in morphologically normal, dysplastic and malignant EC. The chi 2 test showed significant association of malignant EC with aneuploidy (p = 0.008) and high proliferative activity (p = 0.042). Since one third of the dysplastic EC are also aneuploid and show high mitotic activity, they probably have malignant potential as well. The dysplastic EC showed a significant association with c-myc oncogene expression (p = 0.028). Our results indicate the existence of pre-malignant glandular lesions, while the immunohistochemical detection of c-myc protooncogene could be helpful in detection of EC with malignant potential, even without any dysplastic morphological changes.