Cadherin‐3 is a novel oncogenic biomarker with prognostic value in glioblastoma

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin‐3 (also known as P‐cadherin), a cell–cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P‐cadherin in this highly malignant brain tumor. CDH3/P‐cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P‐cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3‐associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P‐cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high‐grade gliomas. P‐cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P‐cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P‐cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor.     

Whole-exome sequencing,EGFR amplification and infiltration patterns in human glioblastoma

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. This cancer shows rapid, highly infiltrative growth, that invades individually or in small groups the surrounding tissue. The aggressive tumor biology of GBM has devastating consequences with a median survival of 15 months. GBM often has Epidermal Growth Factor Receptor (EGFR) abnormalities. Despite recent advances in the study of GBM tumor biology, it is unclear whether mutations in GBM are related to EGFR amplification and relevant phenotypes like tumor infiltration. This study aimed to perform whole-exome sequencing analysis in 30 human GBM samples for identifying mutational portraits associated with EGFR amplification and infiltrative patterns. Our results show that EGFR-amplified tumors have overall higher mutation rates than EGFR-no-amplified. Six genes out of 2029 candidate genes show mutations associated with EGFR amplification status. Mutations in these genes for GBM are novel, not previously reported in GBM, and with little presence in the TCGA database. GPR179, USP48, and BLK show mutation only in EGFR-amplified cases, and all the affected cases exhibit diffuse infiltrative patterns. On the other hand, mutations in ADGB, EHHADH, and PTPN13, were present only in the EGFR-no-amplified group with a more diverse infiltrative phenotype. Overall, our work identified different mutational portraits of GBM related to well-established features like EGFR amplification and tumor infiltration.     

Anti-malarial drug dihydroartemisinin downregulates the expression levels of CDK1 and CCNB1 in liver cancer

Liver cancer is the third leading cause of cancer-associated mortality worldwide. By the time liver cancer is diagnosed, it is already in the advanced stage. Therefore, novel therapeutic strategies need to be identified to improve the prognosis of patients with liver cancer. In the present study, the profiles of {"type":"entrez-geo","attrs":{"text":"GSE84402","term_id":"84402"}}GSE84402, {"type":"entrez-geo","attrs":{"text":"GSE19665","term_id":"19665"}}GSE19665 and {"type":"entrez-geo","attrs":{"text":"GSE121248","term_id":"121248"}}GSE121248 were used to screen differentially expressed genes (DEGs). Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses for DEGs were conducted using the Database for Annotation, Visualization and Integrated Discovery. The protein-protein interaction network was established to screen the hub genes associated with liver cancer. Additionally, the expression levels of hub genes were validated using the Gene Expression Profiling Interactive Analysis and Oncomine databases. In addition, the prognostic value of hub genes in patients with liver cancer was analyzed using Kaplan-Meier Plotter. It was demonstrated that 132 and 246 genes were upregulated and downregulated, respectively, in patients with liver cancer. Among these DEGs, 10 hub genes with high connected node values were identified, which were AURKA, BIRC5, BUB1B, CCNA2, CCNB1, CCNB2, CDC20, CDK1, DLGAP5 and MAD2L1. CDK1 and CCNB1 had the most connection nodes and the highest score and were therefore, the most significantly expressed. In addition, it was demonstrated that high expression levels of CDK1 and CCNB1 were associated with poor overall survival time of patients with liver cancer. Dihydroartemisinin (DHA) is a Food and Drug Administration-approved drug, which is derived from the traditional Chinese medicine Artemisia annua Linn. DHA inhibits cell proliferation in numerous cancer types, including liver cancer. In our previous study, it was revealed that DHA inhibited the proliferation of HepG2215 cells. In the present study, it was further demonstrated that DHA reduced the expression levels of CDK1 and CCNB1 in liver cancer. Overall, CDK1 and CCNB1 were the potential therapeutic targets of liver cancer, and DHA reduced the expression levels of CDK1 and CCNB1, and inhibited the proliferation of liver cancer cells.     

Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer

The high morbidity and mortality of colorectal cancer pose a significant public health problem worldwide. Here we assessed the pro-cancer efficacy and mechanism of action of CCNB1 in different colorectal cancer cells. We provided evidence that CCNB1 mRNA and protein level were upregulated in a subset of human colorectal tumors, and positively correlated with Chk1 expression. Repression of Chk1 caused a significant decrease in cell proliferation and CCNB1 protein expression in colorectal cancer cells. Furthermore, downregulation of CCNB1 impaired colorectal cancer proliferation in vitro and tumor growth in vivo. Specifically, suppression of CCNB1 caused a strong G₂/M phase arrest in both HCT116 and SW480 cells, interfering with the expression of cdc25c and CDK1. Additionally, CCNB1 inhibition induced apoptotic death in certain colorectal cancer cells. Together, these results suggest that CCNB1 is activated by Chk1, exerts its oncogenic role in colorectal cancer cells, and may play a key role in the development of a novel therapeutic approach against colorectal cancer.     

Pineal region glioblastomas display features of diffuse midline and non-midline gliomas

Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor, which is virtually incurable due to its therapeutic resistance to radiation and chemotherapy. To develop novel therapeutic approaches for treatment of GBM, we examined the role of miR-378 on tumor growth, angiogenesis, and radiation response in ectopic and orthotopic U87 glioblastoma models. Cell and tumor growth rates, in vitro and in vivo radiation sensitivities, and tumor vascular density were evaluated in U87-GFP and U87-miR-378 tumor lines. Ectopic tumor response to radiation was evaluated under normal blood flow and clamp hypoxic conditions. Results show that in vitro, miR-378 expression moderately increased cell growth rate and plating efficiency, but did not alter radiation sensitivity. U87-miR-378 tumors exhibited a higher transplantation take rate than U87-GFP tumors. In vivo, under oxygenated condition, subcutaneous U87-miR-378 tumors receiving 25 Gy showed a tendency for longer tumor growth delay (TGD) than control U87-GFP tumors. In contrast, under hypoxic condition, U87-miR-378 xenografts exhibited substantially shorter TGD than U87-GFP tumors, indicating that under normal blood flow conditions, U87-miR-378 tumors were substantially more oxygenated than U87-GFP tumors. Intracranial multi-photon laser-scanning microscopy demonstrated increased vascular density of U87-miR-378 versus control U87-GFP tumors. Finally, miR-378 increased TGD following 12 Gy irradiation in U87 intracranial xenografts, and significantly prolonged survival of U87-miR-378 tumor-bearing mice (P?=?0.04). In conclusion, higher miR-378 expression in U87-miR-378 cells promotes tumor growth, angiogenesis, radiation-induced TGD, and prolongs survival of orthotopic tumor-bearing hosts. Regulation of VEGFR2 by miR-378 significantly increased vascular density and oxygenation in U87 xenografts.     

Genomic and molecular aberrations in malignant peripheral nerve sheath tumor and their roles in personalized target therapy

Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors with a high rate of local recurrence and a significant tendency to metastasize. Its dismal outcome points to the urgent need to establish better therapeutic strategies for patients harboring MPNSTs. The investigations of genomic and molecular aberrations in MPNSTs which detect many chromosomal aberrations, pathway abnormalities, and specific molecular aberrant events would supply multiple potential therapy targets and contribute to achievement of personalized medicine. The involved genes in the significant gains aberrations include BIRC5, CCNE2, DAB2, DDX15, EGFR, DAB2, MSH2, CDK6, HGF, ITGB4, KCNK12, LAMA3, LOXL2, MET, and PDGFRA. The involved genes in the significant deletion aberrations include CDH1, GLTSCR2, EGR1, CTSB, GATA3, SULT2A1, GLTSCR2, HMMR/RHAMM, LICAM2, MMP13, p16/INK4a, RASSF2, NM-23H1, and TP53. These genetic aberrations involve in several important signaling pathways such as TFF, EGFR, ARF, IGF1R signaling pathways. The genomic and molecular aberrations of EGFR, IGF1R, SOX9, EYA4, TOP2A, ETV4, and BIRC5 exhibit great promise as personalized therapeutic targets for MPNST patients.     

Correlation of somatic mutation and expression identifies genes important in human glioblastoma progression and survival

Cooperative dysregulation of gene sequence and expression may contribute to cancer formation and progression. The Cancer Genome Atlas (TCGA) Network recently catalogued gene sequence and expression data for a collection of glioblastoma multiforme (GBM) tumors. We developed an automated, model-free method to rapidly and exhaustively examine the correlation among somatic mutation and gene expression and interrogated 149 GBM tumor samples from the TCGA. The method identified 41 genes whose mutation status is highly correlated with drastic changes in the expression (z-score ± 2.0), across tumor samples, of other genes. Some of the 41 genes have been previously implicated in GBM pathogenesis (e.g., NF1, TP53, RB1, and IDH1) and others, while implicated in cancer, had not previously been highlighted in studies using TCGA data (e.g., SYNE1, KLF6, FGFR4, and EPHB4). The method also predicted that known oncogenes and tumor suppressors participate in GBM via drastic over- and underexpression, respectively. In addition, the method identified a known synthetic lethal interaction between TP53 and PLK1, other potential synthetic lethal interactions with TP53, and correlations between IDH1 mutation status and the overexpression of known GBM survival genes.     

Correlation between Rho-kinase pathway gene expressions and development and progression of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and the most aggressive primary malignant tumor of the brain. Prognostic factors in GBM can be sorted as age, tumor localization, tumor diameter, symptom period and type, the extent of surgery, postoperative tumor volume, and adjuvant radiotherapy and/or chemotherapy status. Besides the interactions between actin microfilaments, microtubules, and intermediate filaments, environmental factors and intracellular signals which regulate them affect the cell invasion. Rho proteins and therefore Rho-kinase activation play important role at these changes. The aim of this study is to evaluate the relationship between the Rho-kinase pathway gene expressions and prognosis in GBM. Ninety-eight patients diagnosed as GBM between 2001 and 2010 were enrolled into the study. RNA was obtained from the paraffinized tumor tissue of the patients with formalin-fixed, paraffin-embedded RNA isolation kit and the mRNA expressions of 26 genes were investigated. There was a statistically significant negative correlation between the ages at the diagnosis and survival. There was a significant relationship between the overexpression of Rho-kinase pathway-related genes LIMK1, CFL1, CFL2, and BCL2 and low expression of MAPK1 gene and the survival of the patients. These results demonstrate for the first time that there is a marked contribution of Rho-kinase pathway-related genes to the progression and survival of the GBM. The expression of these genes may be related to response of multimodal therapy or these parameters could be used to determine possible unresponsive patients before treatment.     

Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of glioblastoma

BackgroundGlioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo.MethodsWe utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections.ResultsGBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model.Conclusions SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.     

High-risk Stage III colon cancer patients identified by a novel five-gene mutational signature are characterized by upregulation of IL-23A and gut bacterial translocation of the tumor microenvironment

The heterogeneities of colorectal cancer (CRC) lead to staging inadequately of patients’ prognosis. Here, we performed a prognostic analysis based on the tumor mutational profile and explored the characteristics of the high-risk tumors. We sequenced 338 colorectal carcinomas as the training dataset, constructed a novel five-gene (SMAD4, MUC16, COL6A3, FLG and LRP1B) prognostic signature, and validated it in an independent dataset from The Cancer Genome Atlas (TCGA). Kaplan–Meier and Cox regression analyses confirmed that the five-gene signature is an independent predictor of recurrence and prognosis in patients with Stage III colon cancer. The mutant signature translated to an increased risk of death (hazard ratio = 2.45, 95% confidence interval = 1.15–5.22, p = 0.016 in our dataset; hazard ratio = 4.78, 95% confidence interval = 1.33–17.16, p = 0.008 in TCGA dataset). RNA and bacterial 16S rRNA sequencing of high-risk tumors indicated that mutations of the five-gene signature may lead to intestinal barrier integrity, translocation of gut bacteria and deregulation of immune response and extracellular related genes. The high-risk tumors overexpressed IL23A and IL1RN genes and enriched with cancer-related bacteria (Bacteroides fragilis,Peptostreptococcus, Parvimonas, Alloprevotella and Gemella) compared to the low-risk tumors. The signature identified the high-risk group characterized by gut bacterial translocation and upregulation of interleukins of the tumor microenvironment, which was worth further researching.     

Collagen type VI regulates the CDK4/6-p-Rb signaling pathway and promotes ovarian cancer invasiveness,stemness, and metastasis

The expression of collagen VI in primary ovarian tumors may correlate with tumor grade and response to chemotherapy. We have sought to elucidate the role of collagen VI in promoting ovarian cancer tumor growth and metastasis. Here we examined the effects of collagen VI on ovarian carcinoma stromal progenitor cells (OCSPCs). Epithelial-like OCSPCs (epi-OCSPCs) and mesenchymal-like OCSPCs (msc-OCSPCs) were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Differentially expressed genes were integrated with survival-related genes using The Cancer Genome Atlas (TCGA) data and confirmed in our samples. The roles of candidate genes and signaling pathways were further explored. We found that SKOV3/msc-OCSPCs possessed greater migration, invasion, and spheroid formation than SKOV3/epi-OCSPCs (P < 0.001). Expression of collagen alpha-3 (VI; COL6A3), which encodes collagen VI, was 90-fold higher in msc-OCSPCs than in epi-OCSPCs. Analysis of TCGA data and our samples indicated that high expression of COL6A3 was correlated with advanced-stage carcinoma (P < 0.01) and shorter overall survival (P < 0.01). In vitro, adding collagen VI, msc-OCSPCs, or knockdown collagen VI in msc-OCSPCs to epithelial ovarian carcinoma (EOC) cells augmented or decreased invasion and spheroid formation. Tumor dissemination to the peritoneal cavity and lung in mice following intraperitoneal coinjection with msc-OCSPCs and SKOV3-Luc cells and intravenous injection with COL6A3 and ES2 cells derived spheroids was significantly greater compare to coinjection with SKOV3-Luc cells alone or in combination with msc-OCSPCs/shCOL6A3 cells and msc-OCSPCs and ES2 derived spheroids. Knockdown of COL6A3 abolished the expression of DNMT1, CDK4, CDK6, and p-Rb in msc-OCSPCs and EOC spheroids. In contrast, overexpression of COL6A3 enhanced the expression of CDK4, CDK6, and p-Rb in SKOV3 cells. EOC spheroid formation, invasion, tumor growth, and metastasis were inhibited when COL6A3 downstream signaling pathway was blocked using CDK4/6 inhibitor LEE011. Our results suggested that collagen VI regulates the CDK4/6-p-Rb signaling pathway and promotes EOC invasiveness, stemness, and metastasis.     

High-mobility group box 2 is associated with prognosis of glioblastoma by promoting cell viability,invasion, and chemotherapeutic resistance

Background

The expression profile of high-mobility group box 2 (HMGB2) in patients with glioblastoma multiforme (GBM) and its clinical signature with underlying mechanisms were not fully explored.

Methods

HMGB2 protein levels were measured in 51 GBM patients by immunohistochemical studies. To clarify the precise role of HMGB2 on cell invasion and viability of 3 GBM cell lines, we did in vitro and in vivo analyses with lentivirus vectors and small interfering RNA. Transwell invasion assays and wound-healing assays were used to analyze the invasion of GBM cells. Expression of p53 and matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot.

Results

HMGB2 protein expression was significantly higher in GBM than in controlled brain tissues (P < .0001). HMGB2 overexpression was significantly correlated with shorter overall survival time, which was the only independent prognostic factor for overall survival in a multivariate analysis (P = .017). HMGB2 knockdown by small interfering RNA decreased cell viability and invasion in vitro and significantly decreased tumor volume in vivo, which might be involved in the change of p53 expression and the balance of MMP2/TIMP2. Moreover, silencing of HMGB2 could significantly increase the sensitivity of GBM cells to temozolomide chemotherapy.

Conclusions

Our present data suggest that HMGB2 expression is a significant prognostic factor and might play an important role in cell invasion and temozolomide-induced chemotherapeutic sensitivity of GBM. This study highlights the importance of HMGB2 as a novel prognostic marker and an attractive therapeutic target of GBM.     

Molecular profiling of patient-derived breast cancer xenografts

Introduction

Identification of new therapeutic agents for breast cancer (BC) requires preclinical models that reproduce the molecular characteristics of their respective clinical tumors. In this work, we analyzed the genomic and gene expression profiles of human BC xenografts and the corresponding patient tumors.

Methods

Eighteen BC xenografts were obtained by grafting tumor fragments from patients into Swiss nude mice. Molecular characterization of patient tumors and xenografts was performed by DNA copy number analysis and gene expression analysis using Affymetrix Microarrays.

Results

Comparison analysis showed that 14/18 pairs of tumors shared more than 56% of copy number alterations (CNA). Unsupervised hierarchical clustering analysis showed that 16/18 pairs segregated together, confirming the similarity between tumor pairs. Analysis of recurrent CNA changes between patient tumors and xenografts showed losses in 176 chromosomal regions and gains in 202 chromosomal regions. Gene expression profile analysis showed that less than 5% of genes had recurrent variations between patient tumors and their respective xenografts; these genes largely corresponded to human stromal compartment genes. Finally, analysis of different passages of the same tumor showed that sequential mouse-to-mouse tumor grafts did not affect genomic rearrangements or gene expression profiles, suggesting genetic stability of these models over time.

Conclusions

This panel of human BC xenografts maintains the overall genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations. The observed genomic profile and gene expression differences appear to be due to the loss of human stromal genes. These xenografts, therefore, represent a validated model for preclinical investigation of new therapeutic agents.     

Optical Tomography of MMP Activity Allows a Sensitive Noninvasive Characterization of the Invasiveness and Angiogenesis of SCC Xenografts

For improved tumor staging and therapy control, imaging biomarkers are of great interest allowing a noninvasive characterization of invasiveness. In squamous epithelial skin and cervix lesions, transition to invasive stages is associated with enhanced matrix metalloproteinase (MMP) activity, increased angiogenesis, and worsened prognosis. Thus, we investigated MMP activity as imaging biomarker of invasiveness and the potential of optical tomography in characterizing the angiogenic and invasive behavior of skin squamous cell carcinoma (SCC) xenografts. MMP activity was measured in vivo in HaCaT-ras A-5RT3 tumors at different angiogenic and invasive stages (onset of angiogenesis, intermediate and highly angiogenic, invasive stage) and after 1 week of sunitinib treatment by fluorescence molecular tomography–microcomputed tomography imaging using an activatable probe. Treatment response was additionally assessed morphologically by optical coherence tomography (OCT). In vivo MMP activity significantly differed between the groups, revealing highest levels in the highly angiogenic, invasive tumors that were confirmed by immunohistochemistry. At the onset of angiogenesis with lowest MMP activity, fibroblasts were detected in the MMP-positive areas, whereas macrophages were absent. Accumulation of both cell types occurred in both invasive groups, again to a significantly higher degree at the most invasive and angiogenic stage. Sunitinib treatment significantly reduced the MMP activity and accumulation of fibroblasts and macrophages and blocked tumor invasion that was additionally visualized by OCT. Human cervical SCCs also showed high MMP activity and a similar stromal composition as the HaCaT xenografts, whereas normal tissue was negative. This study strongly suggests MMP activity as imaging biomarker and demonstrates the high sensitivity of optical tomography in determining tumor invasiveness that can morphologically be supported by OCT.Abbreviations: SCC, squamous cell carcinoma; MMP, matrix metalloproteinase; ECM, extracellular matrix; FMT, fluorescence molecular tomography; μCT, micro-computed tomography; OCT, optical coherence tomography; i.d., intradermal; i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous; SMA, smooth muscle actin; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor number 2; PK, pan-keratin