DNA mismatch repair-proficient colon cancer is the most common type of colon cancer, but its initiation and progression are still unknown. Our previous study has revealed that a long noncoding RNA (lncRNA) ENST00000455974 was significantly associated with TNM stage and distant metastasis in patients with DNA mismatch repair-proficient (pMMR) colon cancer (CC). Here, firstly, we observed that ENST00000455974 was gradual increased across colon normal-adenoma-carcinoma-metastasis sequence by quantitative real-time PCR. Secondly, ENST00000455974 showed a better sensitivity and specificity than CEA and CA19-9 in the diagnosis of pMMR CC by drawing the receiver operating characteristic (ROC) curve. Thirdly, a higher level of ENST00000455974 was associated with a poorer patient survival. Furthermore, Knockdown of ENST00000455974 led to reduced proliferation and migration of colon cancer cells. Mechanistically, ENST00000455974 was mainly located in the nucleus of colon cancer cells and it promoted the growth and metastasis of pMMR CC cells through up-regulating JAG2.

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5.

miR-206 plays an important role in regulating the growth of multiple cancer cells. Cyclin-dependent kinase 9 (CDK9) stimulates the production of abundant prosurvival proteins, leading to impaired apoptosis of cancer cells. However, it is unknown whether CDK9 is involved in the miR-206-mediated growth suppression of hepatocellular carcinoma (HCC) cells. In this study, we found that the expression level of miR-206 was significantly lower in HCC cell lines than that in normal hepatic cell line (L02). Meanwhile, CDK9 was upregulated in HCC cell lines. Moreover, miR-206 downregulated CDK9 in HCC cells via directly binding to its mRNA 3' UTR, which resulted in a decrease of RNA PolII Ser2 phosphorylation and Mcl-1 level. Additionally, miR-206 suppressed the cell proliferation, and induced cell cycle arrest and apoptosis. Similarly, silence or inhibition of CDK9 also repressed the cell proliferation, and induced cell cycle arrest and apoptosis. Taken together, the results demonstrated that miR-206 inhibited the growth of HCC cells through targeting CDK9, suggesting that the miR-206-CDK9 pathway may be a novel target for the treatment of HCC.

Several studies have shown that tRNAs can be enzymatically cleaved to generate distinct classes of tRNA-derived fragments (tRF). Here, we report that tRF/miR-1280, a 17-bp fragment derived from tRNA

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche.We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease.This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche.This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

Notch signaling dysregulation plays an important role in altering cancer cell behaviors; however, its role in oral squamous cell carcinoma (OSCC) remains controversial. This study aimed to investigate the role of Notch signaling related genes in human OSCC using a meta-analysis of Gene Expression Omnibus database (GEO-publicly available gene expression microarray data) and to examine the role of Notch signaling in OSCC behaviors. The meta-analysis included 13 GEO datasets and was performed by combining effect sizes in a random effect model. The results demonstrated that in OSCC dysregulated genes participated in the metabolic process and protein binding as determined by gene ontology analysis. Enriched pathway analysis demonstrated the majority of the dysregulated genes were involved in pathway categories as follow; pathway in cancers, small cell lung cancer, extracellular matrix-receptor interaction, focal adhesion, and cell cycle progression. Interestingly, the enriched pathway analysis also demonstrated that OSCC samples exhibited an upregulation of genes in Notch signaling pathway, namely JAG1, JAG2, ADAM17, NCSTN, PSEN1, NCOR2, NUMB, DVL3, HDAC1, and HDAC2. Furthermore, Notch signaling inhibition by a γ-secretase inhibitor significantly decreased OSCC cell proliferation in vitro, corresponding with a decrease in C-FOS mRNA expression. The study demonstrated that Notch signaling is dysregulated in human OSCC and plays a role in cell proliferation. (J Oral Sci 58, 283-294, 2016).

Sphere formation in conditioned serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem-like cells, also known as tumor-initiating cells. We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem-like cells (MCSLCs). The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588). Immunofluorescence staining showed tumorosphere cells highly expressed Notch4. Notch4(high) B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial-mesenchymal transition (EMT)-associated proteins. Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE-cadherin and the overexpression of E-cadherin was observed in human melanoma A375 and MUM-2B cells. In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ-secretase inhibitor, DAPT. Mechanistically, the re-overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE-cadherin expression and a decrease in E-cadherin expression. Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma. Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.

Non-small-cell lung cancer (NSCLC) is a lethal and aggressive malignancy. Currently, the identities of prognostic and predictive makers of NSCLC have not been fully established. Dysregulated Notch signaling has been implicated in many human malignancies, including NSCLC. However, the prognostic value of measuring Notch signaling and the utility of developing Notch-targeted therapies in NSCLC remain inconclusive. The present study investigated the association of individual Notch receptor and ligand levels with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) prognosis using the Kaplan-Meier plotte database. This online database encompasses 2437 lung cancer samples. Hazard ratios with 95% confidence intervals were calculated. The results showed that higher Notch1, Notch2, JAG1, and DLL1 mRNA expression predicted better overall survival (OS) in lung ADC, but showed no significance in SCC patients. Elevated Notch3, JAG2, and DLL3 mRNA expression was associated with poor OS of ADC patients, but not in SCC patients. There was no association between Notch4 and OS in either lung ADC or SCC patients. In conclusion, the set of Notch1, Notch2, JAG1, DLL1 and that of Notch3, JAG2, DLL3 played opposing prognostic roles in lung ADC patients. Neither set of Notch receptors and ligands was indicative of lung SCC prognosis. Notch signaling could serve as promising marker to predict outcomes in lung ADC patients. The distinct features of lung cancer subtypes and Notch components should be considered when developing future Notch-targeted therapies.

Accurate markers and molecular mechanisms of stem cell dormancy and activation are poorly understood. In this study, the anti-cancer drug, 5-fluorouracil, was used to selectively kill proliferating cells of human bronchial epithelial (HBE) cell line. This method can enrich and purify stem cell population. The dormant versus active status of stem cells was determined by phosphorylation of RNAp II Ser2. The surviving stem cells were cultured to form stem cell spheres expressing stem cell markers and transplanted into nude mice to form a teratoma. The results demonstrated the properties of stem cells and potential for multi-directional differentiation. Bisulfite sequencing polymerase chain reaction showed that demethylation of the Sox2 promoter by 5-FU resulted in Sox2 expression in the dormant stem cells. This study shows that the dormancy and activation of HBE stem cells is closely related to epigenetic modification.

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

In cervical squamous cell carcinomas, high-risk human papillomavirus (HRHPV) DNA is usually integrated into host chromosomes. Multiple integration events are thought to be present within the cells of a polyclonal premalignant lesion and the features that underpin clonal selection of one particular integrant remain poorly understood. We previously used the W12 model system to generate a panel of cervical keratinocyte clones, derived from cells of a low-grade premalignant lesion naturally infected with the major HRHPV type, HPV16. The cells were isolated regardless of their selective advantage and differed only by the site of HPV16 integration into the host genome. We used this resource to test the hypothesis that levels of HPV16 E6/E7 oncogene expression in premalignant cells are regulated epigenetically. We performed a comprehensive analysis of the epigenetic landscape of the integrated HPV16 DNA in selected clones, in which levels of virus oncogene expression per DNA template varied ~6.6-fold. Across the cells examined, higher levels of virus expression per template were associated with more open chromatin at the HPV16 long control region, together with greater loading of chromatin remodelling enzymes and lower nucleosome occupancy. There were higher levels of histone post-translational modification hallmarks of transcriptionally active chromatin and lower levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor b complex responsible for Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyltransferases and CDK9 and less sensitivity to histone deacetylase inhibition. We conclude that virus gene expression per template following HPV16 integration is determined through multiple layers of epigenetic regulation, which are likely to contribute to selection of individual cells during cervical carcinogenesis.

Glioblastoma is the most common malignant brain tumor accounting for more than 54 % of all gliomas. Despite aggressive treatments, median survival remains less than 1 year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2(-ΔΔCt) method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85 % of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis.

BACKGROUND: MicroRNA-200 (miR-200) suppresses the epithelial-mesenchymal transition of various cancer cells, including lung adenocarcinoma cells. We found that bone morphogenetic protein 4 (BMP4) was decreased in miR-200-overexpressing cells and epithelial-like lung cancer cells. In this study, we investigated the mechanism and role of BMP4 depletion by miR-200 in murine lung adenocarcinoma cells.
METHODS: BMP4 expression levels in murine lung cancer cells were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Promoter and 3'-untranslated region (UTR) luciferase reporter assays were performed to discover the mechanism of regulation of BMP4 by miR-200. Murine lung cancer cells were transfected with Bmp4 shRNAs, which were then injected into syngeneic mice to measure their tumorigenic and metastatic potential and cultured on Matrigel to study the influence of BMP4 on 3-D acinus formation.
RESULTS: miR-200 down-regulated BMP4 via direct targeting of the GATA4 and GATA6 transcription factors that stimulate Bmp4 transcription. BMP4 up-regulated JAG2, an upstream factor of miR-200; therefore, JAG2, miR-200, and BMP4 form a regulatory loop. Bmp4 knockdown suppressed cancer cell growth, migration, and invasion and inhibited tumorigenesis and metastasis of lung cancer cells when injected into syngeneic mice. In addition, BMP4 was required for normal acinus formation in Matrigel 3-D culture of murine lung cancer cells, which may be mediated by MYH10, a downstream target of BMP4.
CONCLUSION: BMP4 functions as a pro-tumorigenic factor in a murine lung cancer model, and its transcription is regulated by miR-200 and GATA4/6. Thus, we propose that BMP4 and its antagonists may be suitable therapeutic targets for the treatment of lung cancer.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

Over-expression of PDGF receptors (PDGFRs) has been previously implicated in high-risk medulloblastoma (MB) pathogenesis. However, the exact biological functions of PDGFRα and PDGFRβ signaling in MB biology remain poorly understood. Here, we report the subgroup specific expression of PDGFRα and PDGFRβ and their associated biological pathways in MB tumors. c-MYC, a downstream target of PDGFRβ but not PDGFRα, is involved in PDGFRβ signaling associated with cell proliferation, cell death, and invasion. Concurrent inhibition of PDGFRβ and c-MYC blocks MB cell proliferation and migration synergistically. Integrated analysis of miRNA and miRNA targets regulated by both PDGFRβ and c-MYC reveals that increased expression of JAG2, a target of miR-1280, is associated with high metastatic dissemination at diagnosis and a poor outcome in MB patients. Our study may resolve the controversy on the role of PDGFRs in MB and unveils JAG2 as a key downstream effector of a PDGFRβ-driven signaling cascade and a potential therapeutic target.

CYLD, an ubiquitin hydrolase, has an expanding repertoire of regulatory roles in cell signalling and is dysregulated in a number of cancers. To dissect CYLD function we used a proteomics approach to identify CYLD interacting proteins and identified MIB2, an ubiquitin ligase enzyme involved in Notch signalling, as a protein which interacts with CYLD. Coexpression of CYLD and MIB2 resulted in stabilisation of MIB2 protein levels and was associated with reduced levels of JAG2, a ligand implicated in Notch signalling. Conversely, gene silencing of CYLD using siRNA, resulted in increased JAG2 expression and upregulation of Notch signalling. We investigated Notch pathway activity in skin tumours from patients with germline mutations in CYLD and found that JAG2 protein levels and Notch target genes were upregulated. In particular, RUNX1 was overexpressed in CYLD defective tumour cells. Finally, primary cell cultures of CYLD defective tumours demonstrated reduced viability when exposed to γ-secretase inhibitors that pharmacologically target Notch signalling. Taken together these data indicate an oncogenic dependency on Notch signalling and suggest potential novel therapeutic approaches for patients with CYLD defective tumours.

A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

Medulloblastoma (MB), the most common pediatric malignant brain cancer, typically arises as pathological result of deregulated developmental pathways, including the NOTCH signaling cascade. Unlike the evidence supporting a role for NOTCH receptors in MB development, the pathological functions of NOTCH ligands remain largely unexplored. By examining the expression in large cohorts of MB primary tumors, and in established in vitro MB models, this research study demonstrates that MB cells bear abnormal levels of distinct NOTCH ligands. We explored the potential association between NOTCH ligands and the clinical outcome of MB patients, and investigated the rational of inhibiting NOTCH signaling by targeting specific ligands to ultimately provide therapeutic benefits in MB. The research revealed a significant over-expression of ligand JAG1 in the vast majority of MBs, and proved that JAG1 mediates pro-proliferative signals via activation of NOTCH2 receptor and induction of HES1 expression, thus representing an attractive therapeutic target. Furthermore, we could identify a clinically relevant association between ligand JAG2 and the oncogene MYC, specific for MYC-driven Group 3 MB cases. We describe for the first time a mechanistic link between the oncogene MYC and NOTCH pathway in MB, by identifying JAG2 as MYC target, and by showing that MB cells acquire induced expression of JAG2 through MYC-induced transcriptional activation. Finally, the positive correlation of MYC and JAG2 also with aggressive anaplastic tumors and highly metastatic MB stages suggested that high JAG2 expression may be useful as additional marker to identify aggressive MBs.

NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

Genomic, transcriptional, and proteomic analyses of brain tumors reveal subtypes that differ in pathway activity, progression, and response to therapy. However, a number of small molecule inhibitors under development vary in strength of subset and pathway-specificity, with molecularly targeted experimental agents tending toward stronger specificity. The Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes. We investigated the effects of Notch pathway inhibition in glioma tumor-initiating cell (GIC, hereafter GIC) populations using γ secretase inhibitors. Drug cytotoxicity testing of 16 GICs showed differential growth responses to the inhibitors, stratifying GICs into responders and nonresponders. Responder GICs had an enriched proneural gene signature in comparison to nonresponders. Also gene set enrichment analysis revealed 17 genes set representing active Notch signaling components NOTCH1, NOTCH3, HES1, MAML1, DLL-3, JAG2, and so on, enriched in responder group. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation suggesting γ secretase inhibitors might be of potential value to treat that particular group of proneural glioblastoma (GBM). Inhibition of Notch pathway by γ secretase inhibitor treatment attenuated proliferation and self-renewal of responder GICs and induces both neuronal and astrocytic differentiation. In vivo evaluation demonstrated prolongation of median survival in an intracranial mouse model. Our results suggest that proneural GBM characterized by high Notch pathway activation may exhibit greater sensitivity to γ secretase inhibitor treatment, holding a promise to improve the efficiency of current glioma therapy.

Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

The key molecular events required for the formation of ductal carcinoma in situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here, we show that the nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal three-dimensional mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both before DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ breast cancer-initiating cell (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. The loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, human epidermal growth factor receptor 2 (HER2) and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 has a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through the maintenance of BCIC, facilitates formation of DCIS, a necessary step before development of invasive disease.

Notch signaling pathway is a highly conserved developmental pathway, which plays an important role in the regulation of cellular proliferation, differentiation and apoptosis. Deregulation of Notch pathway has been connected with the carcinogenesis in a variety of cancers. In this study, we investigated the expression of Notch receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4), ligands (JAG1, JAG2 and DLL1) and target gene HES1. Fifty paired samples of endometrial cancer and adjacent nontumor endometrial tissue from endometrial cancer patients were analyzed by quantitative PCR. The mRNA levels of all investigated molecules were lower in endometrial cancer compared to adjacent nontumor tissue. The expression of NOTCH1, NOTCH4 and DLL1 in IB stage adenocarcinoma was significantly lower (P < 0.05) than the expression in IA stage adenocarcinoma. Significant correlations were found between mRNA expression levels of Notch target gene HES1 and several Notch signaling molecules: NOTCH1, NOTCH3, DLL1 (P < 0.001) and NOTCH2, JAG2 (P < 0.05). This supports the notion that Notch pathway can function as tumor suppressor in human endometrial cancer.

AIMS: Notch signalling plays diverse roles in malignant tumours as well as in normal tissue development. In this study we investigated the expression of Notch signalling pathway genes and their clinicopathological significance in gastric carcinomas.
METHODS AND RESULTS: Notch1, Notch3, Jagged1, Jagged2 and Hes1 expression were analysed by quantitative real-time polymerase chain reaction (qRT-PCR) (n = 81) and immunohistochemistry (n = 103) in gastric carcinomas. MUC2 and MUC5AC expression were also assessed, using immunohistochemistry only. With qRT-PCR, Notch1, Notch3, Jagged1 and Jagged2 expression were increased significantly in tumour compared to normal tissue (P < 0.001, P = 0.002, P = 0.008 and P < 0.001, respectively). Overexpression of Notch3 and Jagged2 was associated with intestinal-type carcinomas (P = 0.024) and better histological differentiation (P = 0.047), respectively. Immunohistochemistry showed a reverse correlation between MUC2 and Notch3 or Jagged1 (P = 0.033 and P = 0.005, respectively) and between MUC5AC and Jagged1 or Hes1 (P = 0.004 and P = 0.002, respectively). Notch3 and Jagged2 gene overexpression related to a favourable outcome on univariate (P = 0.046 and P = 0.042, respectively) and multivariate (P = 0.045, Notch3) analysis.
CONCLUSION: The expression of Notch3 and Jagged2 is associated not only with gastric cancer development but also with the intestinal/glandular differentiation of gastric carcinoma cells, suggesting a role as a possible favourable prognostic indicator.

PURPOSE: The "gate-keeper" mutations T674I platelet-derived growth factor receptor α (PDGFRα) in hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to efficiently kill resistant malignant hematologic cells. The purpose of this study was to evaluate the strategy of shutting down the transcription and expression of FIP1-like-1 (FIP1L1)-PDGFRα and Bcr-Abl with SNS-032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials.
EXPERIMENTAL DESIGN: The effects of SNS-032 on PDGFRα and Bcr-Abl signaling pathways, apoptosis, and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRα and KBM5-T315I Bcr-Abl cells in nude mouse models.
RESULTS: SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032 decreased both the mRNA and protein levels of FIP1L1-PDGFRα and Bcr-Abl and inhibited the proliferation of malignant cells expressing FIP1L1-PDGFRα or Bcr-Abl. It also decreased the phosphorylation of downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death receptor pathway.
CONCLUSIONS: This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRα-positive HES cells and Bcr-Abl-positive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating hematologic malignancy by abrogating oncogene addiction.

The purpose of this study was to identify the pathways associated with the ability of CD138(+) human myeloma cells to form colonies in a serum-free semi-solid human collagen-based assay. Only 26% (7 of 27) of human myeloma cell lines were able to spontaneously form colonies. This spontaneous clonogenic growth correlated with the expression of the NOTCH ligand JAG2 (p<0.001). Blocking JAG-NOTCH interactions with NOTCH-Fc chimeric molecules impaired self-colony formation, indicating a role for JAG-NOTCH pathway in colony formation. In two cell lines, silencing of JAG2 blocked both colony formation and in vivo tumor formation in immunocompromised mice. RT-PCR and flow cytometry analysis revealed that JAG2 is often expressed by CD138(+) primary cells. Our results indicate that spontaneous clonogenic growth of myeloma cells requires the expression of JAG2.

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation are frequently associated with transcriptional repression. Here, we show that gene silencing at selected genes with signs of DNA hypermethylation in breast cancer cells involves Pol II stalling. We studied several repressed genes with DNA hypermethylation within a region 1-kb upstream of the transcriptional start site that were upregulated after treatment with DNA demethylating agents, such as Azacytidine and several natural products. All those selected genes had stalled Pol II at their transcriptional start site and showed enhanced ser2 phosphorylated Pol II and elevated transcripts after drug treatment indicating successful elongation. In addition, a decrease of the epigenetic regulator LSH in a breast cancer cell line by siRNA treatment reduced DNA methylation and overcame Pol II stalling, whereas overexpression of LSH in a normal breast epithelial cell line increased DNA methylation and resulted in repression. Decrease of LSH was associated with reduced DNMT3b binding to promoter sequences, and depletion of DNMT3b by siRNA could release Pol II suggesting that DNMT3b is functionally involved. The release of paused Pol II was accompanied by a dynamic switch from repressive to active chromatin marks. Thus release of Pol II stalling can act as a mechanism for gene reactivation at specific target genes after DNA demethylating treatment in cancer cells.

BACKGROUND: Glioblastoma is a complex multifactorial disorder that has swift and devastating consequences. Few genes have been consistently identified as prognostic biomarkers of glioblastoma survival. The goal of this study was to identify general and clinical-dependent biomarker genes and biological processes of three complementary events: lifetime, overall and progression-free glioblastoma survival.
METHODS: A novel analytical strategy was developed to identify general associations between the biomarkers and glioblastoma, and associations that depend on cohort groups, such as race, gender, and therapy. Gene network inference, cross-validation and functional analyses further supported the identified biomarkers.
RESULTS: A total of 61, 47 and 60 gene expression profiles were significantly associated with lifetime, overall, and progression-free survival, respectively. The vast majority of these genes have been previously reported to be associated with glioblastoma (35, 24, and 35 genes, respectively) or with other cancers (10, 19, and 15 genes, respectively) and the rest (16, 4, and 10 genes, respectively) are novel associations. Pik3r1, E2f3, Akr1c3, Csf1, Jag2, Plcg1, Rpl37a, Sod2, Topors, Hras, Mdm2, Camk2g, Fstl1, Il13ra1, Mtap and Tp53 were associated with multiple survival events.Most genes (from 90 to 96%) were associated with survival in a general or cohort-independent manner and thus the same trend is observed across all clinical levels studied. The most extreme associations between profiles and survival were observed for Syne1, Pdcd4, Ighg1, Tgfa, Pla2g7, and Paics. Several genes were found to have a cohort-dependent association with survival and these associations are the basis for individualized prognostic and gene-based therapies. C2, Egfr, Prkcb, Igf2bp3, and Gdf10 had gender-dependent associations; Sox10, Rps20, Rab31, and Vav3 had race-dependent associations; Chi3l1, Prkcb, Polr2d, and Apool had therapy-dependent associations. Biological processes associated glioblastoma survival included morphogenesis, cell cycle, aging, response to stimuli, and programmed cell death.
CONCLUSIONS: Known biomarkers of glioblastoma survival were confirmed, and new general and clinical-dependent gene profiles were uncovered. The comparison of biomarkers across glioblastoma phases and functional analyses offered insights into the role of genes. These findings support the development of more accurate and personalized prognostic tools and gene-based therapies that improve the survival and quality of life of individuals afflicted by glioblastoma multiforme.