Identification of prognostic values defined by copy number variation, mRNA and protein expression of LANCL2 and EGFR in glioblastoma patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) and lanthionine synthetase C-like 2 (LanCL2) genes locate in the same amplicon, and co-amplification of EGFR and LANCL2 is frequent in glioblastoma. However, the prognostic value of LANCL2 and EGFR co-amplification, and their mRNA and protein expression in glioblastoma remain unclear yet. METHODS: This study analyzed the prognostic values of the copy number variations (CNVs), mRNA and protein expression of LANCL2 and EGFR in 575 glioblastoma patients in TCGA database and 100 glioblastoma patients in tumor banks of the Shenzhen Second People's Hospital and the Sun Yat-sen University Cancer Center. RESULTS: The amplification of LANCL2 or EGFR, and their co-amplification were frequent in glioblastoma of TCGA database and our tumor banks. A significant correlation was found between the CNVs of LANCL2 and EGFR (p < 0.001). CNVs of LANCL2 or EGFR were significantly correlated with IDH1/2 mutation but not MGMT promoter methylation. Multivariate analysis showed that LANCL2 amplification was significantly correlated with reduced overall survival (OS) in younger (< 60 years) glioblastoma patients of TCGA database (p = 0.043, HR = 1.657) and our tumor banks (p = 0.018, HR = 2.199). However, LANCL2 or EGFR amplification, and their co-amplification had no significant impact on OS in older (≥ 60 years) or IDH1/2-wild-type glioblastoma patients. mRNA and protein overexpression of LANCL2 and EGFR was also frequently found in glioblastoma. The mRNA expression rather than the protein expression of LANCL2 and EGFR was positively correlated (p < 0.001). However, mRNA or protein expression of EGFR and LANCL2 was not significantly correlated with OS of glioblastoma patients. The protein expression level of LANCL2, rather than EGFR, was elevated in relapsing glioblastoma, compared with newly diagnosed glioblastoma. In addition, the intracellular localization of LanCL2, not EGFR, was associated with the grade of gliomas. CONCLUSIONS: Taken together, amplification and mRNA overexpression of LANCL2 and EGFR, and their co-amplification and co-expression were frequent in glioblastoma patients. Our findings suggest that amplification of LANCL2 and EGFR were the independent diagnostic biomarkers for glioblastoma patients, and LANCL2 amplification was a significant prognostic factor for OS in younger glioblastoma patients.

Synergism between the phosphatidylinositol 3-kinase p110ß isoform inhibitor AZD6482 and the mixed lineage kinase 3 inhibitor URMC-099 on the blockade of glioblastoma cell motility and focal adhesion formation.

BACKGROUND: Glioblastoma multiforme, the most aggressive and malignant primary brain tumor, is characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Our previous studies delineated a crosstalk between PI3K/Akt and JNK signaling pathways, and a moderate anti-glioblastoma synergism caused by the combined inhibition of PI3K p110ß (PI3Kß) isoform and JNK. However, this combination strategy is not potent enough. MLK3, an upstream regulator of ERK and JNK, may replace JNK to exert stronger synergism with PI3Kß. METHODS: To develop a new combination strategy with stronger synergism, the expression pattern and roles of MLK3 in glioblastoma patient's specimens and cell lines were firstly investigated. Then glioblastoma cells and xenografts in nude mice were treated with the PI3Kß inhibitor AZD6482 and the MLK3 inhibitor URMC-099 alone or in combination to evaluate their combination effects on tumor cell growth and motility. The combination effects on cytoskeletal structures such as lamellipodia and focal adhesions were also evaluated. RESULTS: MLK3 protein was overexpressed in both newly diagnosed and relapsing glioblastoma patients' specimens. Silencing of MLK3 using siRNA duplexes significantly suppressed migration and invasion, but promoted attachment of glioblastoma cells. Combined inhibition of PI3Kß and MLK3 exhibited synergistic inhibitory effects on glioblastoma cell proliferation, migration and invasion, as well as the formation of lamellipodia and focal adhesions. Furthermore, combination of AZD6482 and URMC-099 effectively decreased glioblastoma xenograft growth in nude mice. Glioblastoma cells treated with this drug combination showed reduced phosphorylation of Akt and ERK, and decreased protein expression of ROCK2 and Zyxin. CONCLUSION: Taken together, combination of AZD6482 and URMC-099 showed strong synergistic anti-tumor effects on glioblastoma in vitro and in vivo. Our findings suggest that combined inhibition of PI3Kß and MLK3 may serve as an attractive therapeutic approach for glioblastoma multiforme.

Integrated genomic analysis identifies clinically relevant subtypes of renal clear cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) account for over 80% of renal malignancies. The most common type of RCC can be classified into three subtypes including clear cell, papillary and chromophobe. ccRCC (the Clear Cell Renal Cell Carcinoma) is the most frequent form and shows variations in genetics and behavior. To improve accuracy and personalized care and increase the cure rate of cancer, molecular typing for individuals is necessary. METHODS: We adopted the genome, transcriptome and methylation HMK450 data of ccRCC in The Cancer Genome Atlas Network in this research. Consensus Clustering algorithm was used to cluster the expression data and three subtypes were found. To further validate our results, we analyzed an independent data set and arrived at a consistent conclusion. Next, we characterized the subtype by unifying genomic and clinical dimensions of ccRCC molecular stratification. We also implemented GSEA between the malignant subtype and the other subtypes to explore latent pathway varieties and WGCNA to discover intratumoral gene interaction network. Moreover, the epigenetic state changes between subgroups on methylation data are discovered and Kaplan-Meier survival analysis was performed to delve the relation between specific genes and prognosis. RESULTS: We found a subtype of poor prognosis in clear cell renal cell carcinoma, which is abnormally upregulated in focal adhesions and cytoskeleton related pathways, and the expression of core genes in the pathways are negatively correlated with patient outcomes. CONCLUSIONS: Our work of classification schema could provide an applicable framework of molecular typing to ccRCC patients which has implications to influence treatment decisions, judge biological mechanisms involved in ccRCC tumor progression, and potential future drug discovery.

Recent advances in the use of PI3K inhibitors for glioblastoma multiforme: current preclinical and clinical development.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in the central nervous system. One of the most widely used chemotherapeutic drugs for GBM is temozolomide, which is a DNA-alkylating agent and its efficacy is dependent on MGMT methylation status. Little progress in improving the prognosis of GBM patients has been made in the past ten years, urging the development of more effective molecular targeted therapies. Hyper-activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is frequently found in a variety of cancers including GBM, and it plays a central role in the regulation of tumor cell survival, growth, motility, angiogenesis and metabolism. Numerous PI3K inhibitors including pan-PI3K, isoform-selective and dual PI3K/mammalian target of rapamycin (mTOR) inhibitors have exhibited favorable preclinical results and entered clinical trials in a range of hematologic malignancies and solid tumors. Furthermore, combination of inhibitors targeting PI3K and other related pathways may exert synergism on suppressing tumor growth and improving patients' prognosis. Currently, only a handful of PI3K inhibitors are in phase I/II clinical trials for GBM treatment. In this review, we focus on the importance of PI3K/Akt pathway in GBM, and summarize the current development of PI3K inhibitors alone or in combination with other inhibitors for GBM treatment from preclinical to clinical studies.

Nuclear transport of phosphorylated LanCL2 promotes invadopodia formation and tumor progression of glioblastoma by activating STAT3/Cortactin signaling.

INTRODUCTION: Our previous study showed that the abscisic acid receptor lanthionine synthetase C-like 2 (LanCL2) is a significant prognostic factor for overall survival in young glioblastoma patients. However, the role of LanCL2 in glioblastoma remains unclear yet. OBJECTIVES: This study aims to investigate the role of LanCL2 in regulating in-vitro cell invasion and in-vivo tumor progression of glioblastoma and its underlying mechanism. METHODS: Tyrosine 198 or 295 residue of LanCL2 was mutated using site-directed mutagenesis to block its phosphorylation. The role of LanCL2 in glioblastoma was investigated using transwell or 3D invasion assay, matrix degradation assay and intracranial xenograft model. RESULTS: This study showed that nuclear transport of LanCL2 was enhanced by overexpression of LanCL2 or its ligand abscisic acid in glioblastoma cells. Knockdown of LanCL2 suppressed migration, invasion and invadopodia formation of glioblastoma cells, whereas overexpression of wild-type LanCL2 enhanced them. Blocking of Tyr295 residue phosphorylation of LanCL2 impeded its nuclear transport, retarded glioblastoma cell motility and invadopodia formation, and deceased the phosphorylation of Cortactin and STAT3. c-Met was identified as the upstream tyrosine kinase of Tyr295 residue of LanCL2, and inhibition of c-Met markedly suppressed the nuclear transport of LanCL2. Moreover, overexpression of wild-type LanCL2 significantly promoted orthotopic tumor growth of glioblastoma in vivo and led to poor survival of mice with median survival time of 33.5 days, whereas Tyr295 mutation rescued it with median survival time of 49 days. CONCLUSION: Our findings suggested that Tyr295 phosphorylation is crucial to the activation and nuclear transport of LanCL2, as well as invadopodia formation and tumor progression of glioblastoma, providing the evidence of a novel signaling axis c-Met/LanCL2/STAT3/Cortactin and the first observation of the importance of Tyr295 phosphorylation to LanCL2.

A robust semi-supervised NMF model for single cell RNA-seq data.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) technology is a powerful tool to study organism from a single cell perspective and explore the heterogeneity between cells. Clustering is a fundamental step in scRNA-seq data analysis and it is the key to understand cell function and constitutes the basis of other advanced analysis. Nonnegative Matrix Factorization (NMF) has been widely used in clustering analysis of transcriptome data and achieved good performance. However, the existing NMF model is unsupervised and ignores known gene functions in the process of clustering. Knowledges of cell markers genes (genes that only express in specific cells) in human and model organisms have been accumulated a lot, such as the Molecular Signatures Database (MSigDB), which can be used as prior information in the clustering analysis of scRNA-seq data. Because the same kind of cells is likely to have similar biological functions and specific gene expression patterns, the marker genes of cells can be utilized as prior knowledge in the clustering analysis. METHODS: We propose a robust and semi-supervised NMF (rssNMF) model, which introduces a new variable to absorb noises of data and incorporates marker genes as prior information into a graph regularization term. We use rssNMF to solve the clustering problem of scRNA-seq data. RESULTS: Twelve scRNA-seq datasets with true labels are used to test the model performance and the results illustrate that our model outperforms original NMF and other common methods such as KMeans and Hierarchical Clustering. Biological significance analysis shows that rssNMF can identify key subclasses and latent biological processes. To our knowledge, this study is the first method that incorporates prior knowledge into the clustering analysis of scRNA-seq data.

Clinical Applications of and Challenges in Single-Cell Analysis of Circulating Tumor Cells.

Technological advancements in next-generation sequencing are continually changing the landscape of genomic, transcriptomic, and epigenetic research at the single-cell level. These technologies have been used to detect and analyze circulating tumor cells (CTCs) at the molecular level and provide a new approach for the management of cancer patients. A series of unanticipated discoveries, including the heterogeneity of cancer cell populations, new driver mutations responsible for the resistance of tumors to chemotherapy, and the mechanism of tumor metastasis, have been made using single CTC sequencing. CTC detection has been used in cancer diagnosis and monitoring and in determining the prognosis of cancer patients. Traditional treatment for cancer patients is universal and does not consider genetic variations among patients, but in the era of precision medicine, giving the right drug to the right patient at the right time is the core philosophy. In this study, we review the fundamental principles of CTC isolation and single-cell sequencing and discuss recent progress in their application in both basic research and clinical fields and describe the current challenges.

A Multi-targeted Natural Flavonoid Myricetin Suppresses Lamellipodia and Focal Adhesions Formation and Impedes Glioblastoma Cell Invasiveness and Abnormal Motility.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma, which contribute to tumor recurrence and poor prognosis. Myricetin is a natural flavonoid with potent anti-oxidant, anti-inflammatory and anti-cancer activities, which may serve as a potential and harmless agent for GBM treatment. METHODS: To investigate the anti-glioblastoma effects of myricetin, GBM cells were treated with myricetin alone or in combination with temozolomide. Its effects on GBM cell motility and cytoskeletal structures including lamellipodia, focal adhesions and membrane ruffles were also evaluated. RESULTS: We showed that myricetin alone inhibited glioblastoma U-87 MG cell proliferation, migration and invasion, whereas combination of myricetin and temozolomide did not exhibit any synergistic effect. The inhibitory effect on GBM cell proliferation is independent of PTEN status. Moreover, myricetin showed less cytotoxicity to normal astrocytes than GBM cells. Formation of lamellipodia, focal adhesions, membrane ruffles and vasculogenic mimicry were blocked by myricetin, and phosphorylation of ROCK2, paxillin and cortactin was suppressed. In addition, myricetin could inhibit PI3K/Akt and JNK signaling, and bind to a series of kinases and scaffold proteins including PI3K catalytic isoforms (p110α, p110ß and p110δ), PDK1, JNK, c-Jun, ROCK2, paxillin, vinculin and VEcadherin. CONCLUSIONS: In conclusion, myricetin is a multi-targeted drug that has potent anti-migratory and antiinvasive effects on GBM cells, and suppresses formation of lamellipodia and focal adhesions, suggesting that it may serve as an alternative option for GBM treatment.