Yizhou Hu

Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants

Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions.

Netrin-1-induced activation of Notch signaling mediates glioblastoma cell invasion.

Summary Glioblastoma multiforme is an aggressively invasive human brain cancer, which lacks effective treatment. The axonal guidance protein, netrin-1, is overexpressed in glioblastoma tumor biopsies. In Matrigel invasion assays we observed that experimental overexpression of netrin-1 increased cell invasiveness and its downregulation decreased invasiveness. Using tandem affinity purification and mass spectrometry protein identification we found that netrin-1 forms a complex with both Notch2 and Jagged1. Recombinant netrin-1 colocalized with Jagged1 and Notch2 at the cell surface and was also present in the intracellular vesicles with Jagged1, but not with Notch2. Netrin-1 activated Notch signaling and subsequent glioblastoma cell invasion. Interestingly, the recombinant central domain of netrin-1 counteracted the effects of the full-length netrin-1: it inhibited glioblastoma cell invasion and Notch activation by retaining the Notch signaling complex at the cell surface. This finding may give rise to therapeutic applications. These results reveal a new mechanism leading to glioblastoma cell invasion, in which netrin-1 activates Notch signaling.

Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

BackgroundGlioblastoma is an untreatable brain cancer. The tumors contain a population of stem-like cells which are highly invasive and resistant to therapies. These cells are the main reason for the lethality of glioblastoma. Extracellular guidance molecule netrin-1 promotes the invasiveness and survival of various cancer cell types. We have previously found that netrin-1 activates Notch signaling, and Notch signaling associates with cell stemness. Therefore, we have here investigated the effects of netrin-1 on glioblastoma pathogenesis and glioblastoma cell stemness.MethodsGlioma tissue microarrays were stained with immunohistochemistry and the results were used to evaluate the association between netrin-1 and survival of glioma patients. The localization of netrin-1 was analyzed utilizing fresh frozen glioblastoma tissues. The glioma cell invasion was investigated using ex vivo glioma tissue cultures and newly established primary cell cultures in 3D in vitro invasion assays. Intracranial mouse xenograft models were utilized to investigate the effects of netrin-1 on glioblastoma growth and invasion in vivo.ResultsNetrin-1 expression associated with poor patient prognosis in grade II-III gliomas. In addition, its expression correlated with the stem-like cell marker nestin. Netrin-1 overexpression in cultured cells led to increased formation of stem-like cell spheroids. In glioblastoma tumor biopsies netrin-1 localized to hypoxic tumor areas known to be rich in the stem-like cells. In xenograft mouse models netrin-1 expression altered the phenotype of non-invasive glioblastoma cells into diffusively invading and increased the expression of glioma stem-like cell markers. Furthermore, a distinct invasion pattern where netrin-1 positive cells were following the invasive stem-like cells was detected both in mouse models and ex vivo human glioblastoma tissue cultures. Inhibition of netrin-1 signaling targeted especially the stem-like cells and inhibited their infiltrative growth.ConclusionsOur findings describe netrin-1 as an important regulator of glioblastoma cell stemness and motility. Netrin-1 activates Notch signaling in glioblastoma cells resulting in subsequent gain of stemness and enhanced invasiveness of these cells. Moreover, inhibition of netrin-1 signaling may offer a way to target stem-like cells.

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.

Fms‑like tyrosine kinase 3 ligand is required for thymic dendritic cell generation from bone marrow‑derived CD117+ hematopoietic progenitor cells

Thymic dendritic cells (TDCs) are a type of dendritic cell (DC) in the thymus, which can enhance the proliferation of thymic T lymphocytes, regulate negative selection and induce central tolerance through autoantigen presentation. However, further investigations using TDCs has been restricted due to insufficient numbers. Therefore, an effective expansion method for TDCs in vitro is urgently required to further examine their biological characteristics. In the present study, a novel system was established using fetal thymus organ culture (FTOC) and a hanging drop culture system in the presence of fms‑like tyrosine kinase 3 ligand (Flt3L), termed the Flt3L/FTOC system. TDCs were successfully generated and expanded from CD117+ bone marrow hematopoietic progenitor cells. Conventional DCs (cDCs; CD11c+B220‑ DCs) and plasmacytoid DCs (pDCs; CD11c+B220+ DCs) were found in the TDCs generated using the Flt3L/FTOC system. These cells exhibited the specific morphological features of DCs, which were confirmed using Giemsa staining. Furthermore, the cytokine and surface marker profiles were also analyzed. Higher expression levels of interferon‑α and interleukin‑12 were observed in the pDCs, compared with the cDCs, and higher expression levels of toll‑like receptor (TLR)7 and TLR9 were found in the pDCs than in the cDCs. In addition, the Flt3L/FTOC‑derived TDCs also exhibited the ability to stimulate the allogenic T cell response. In conclusion, a novel in vitro culture system of thymic cDCs and pDCs using Flt3L was established, and this may provide a methodological basis for understanding the properties of TDCs.

Abstract 3348: Netrin-4/Integrin beta-4 interaction promotes glioblastoma cell proliferation and protects from temozolomide induced cellular senescence via activating PI3K/AKT pathway

Netrin-4 (NTN4) is a secreted laminin-related protein, which was originally observed to guide neuronal axons during neuronal development. Recently, it has been found to be expressed in many other tissues and tumor types, and to contribute to the regulation of cell adhesion, migration, proliferation, and apoptosis. Glioblastoma multiforme (GBM) is the most common primary tumor of central nervous system (CNS). Although there is no remedy for this fatal disease, the efficacy of temozolomide (TMZ) -an orally taken alkylating agent- has been demonstrated in the treatment of glioblastoma. However, the sensitivity of GBM cells to TMZ is interfered by many factors such as MGMT expression, AKT activation, etc. Here we have explored the functions and molecular mechanisms of NTN4 in GBM. The suppression of NTN4 expression in GBM cell lines significantly reduced cell proliferation and motility and increased serum deprivation-induced apoptosis. By using tandem affinity purification and mass spectrometric analysis, we identified the physical interaction between integrin beta-4 (ITGB4) and NTN4. This interaction mediates AKT phosphorylation and concomitant mitogenic effects. Furthermore, silencing either NTN4 or ITGB4 in GBM cell induced cellular senescence. Temozolomide induces cellular senescence in MGMT devoid glioma cell lines. Addition of exogenous recombinant NTN4 protein rescued TMZ induced senescence and AKT dephosphorylation in GBM cells in an ITGB4 dependent manner. Current data suggest that the NTN4-ITGB4 interaction promotes GBM cell proliferation and protects GBM cell from TMZ triggered cellular senescence via activating PI3K/AKT pathway. Citation Format: Yizhou Hu, Irene Ylivinkka, Li Li, Ping Chen, Sampsa Hautaniemi, Tuula A. Nyman, Jorma Keski-Oja Keski-Oja, Marko Hyytiainen. Netrin-4/Integrin beta-4 interaction promotes glioblastoma cell proliferation and protects from temozolomide induced cellular senescence via activating PI3K/AKT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3348. doi:10.1158/1538-7445.AM2014-3348

Abstract 4104: Netrin-1 promotes the invasiveness and survival of human glioblastoma cells.

Glioblastoma multiforme (GBM) is the most severe human brain cancer, which has no efficient treatment. The main reason for the lethality of GBM is its resistance to all current therapies and highly invasive. According to microarray data on Oncomine database netrin-1 is upregulated in glioblastoma tumors. Netrin-1 is a secreted laminin related extracellular matrix protein. Its main function is to regulate the axon guidance during embryogenesis. Recently it has been observed to act as a survival factor for different forms of cancer such as aggressive neuroblastoma, metastatic breast cancer and non small cell lung cancer. It also promotes tumorigenesis of colorectal cancer. Based on these observations we explored the possibility whether netrin-1 plays a role in human GBM. We analyzed the effects of netrin-1 expression on human GBM cells. According to Matrigel invasion assays overexpression of netrin-1 increased the invasiveness whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness of human GBM cells. On the other hand, complete knock-down of netrin-1 led to apoptotic death of those cells. To further analyze netrin-1 signaling in human GBM we performed a mass spectrometry screen to identify binding partners of netrin-1. We identified Notch as a novel receptor for netrin-1. In addition, we observed that netrin-1 overexpression increased and knockdown decreased Notch activation. The role of Notch signaling regarding the aggressiveness of various cancers has been controversial. Our results confirm that increased Notch signaling increases glioblastoma cell invasiveness, and that netrin-1 increases Notch signaling. Further, we identified here a recombinant netrin-1 fragment that inhibits Notch signaling by blocking the Notch signaling complex to the cell surface. Current studies provide new mechanistic information on the tumorigenesis of GBM. Activation of Notch signaling pathway has been linked to the initiation of EMT which is essential for cancer cell invasion. Our results suggest that netrin-1 regulates GBM invasion by activating Notch signaling. The inhibition of netrin-1 signaling may prove out to be a treatment option for human glioblastoma. Citation Format: Jorma K. Keski-Oja, Irene Ylivinkka, Yizhou Hu, Tuula Nyman, Marko Hyytiainen. Netrin-1 promotes the invasiveness and survival of human glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4104. doi:10.1158/1538-7445.AM2013-4104