Zirong Chen

Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion.

LKB1 is a tumor susceptibility gene for the Peutz–Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1–CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1–CRTC signaling might represent a crucial mechanism for esophageal cancer progression.

DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia

Notch signaling is a highly conserved cell–cell communication pathway regulating normal development and tissue homeostasis. Aberrant Notch signaling represents an important oncogenic mechanism for T cell acute lymphoblastic leukemia (T-ALL), an aggressive subset of the most common malignant childhood cancer ALL. Therefore, understanding the molecular regulation of Notch signaling is critical to identify new approaches to block aberrant Notch oncogenic activity. The family of three MAML transcriptional coactivators is crucial for Notch signaling activation. The prototypic member MAML1 is the major coactivator that regulates Notch oncogenic activities in leukemic cells. However, the molecular basis underlying MAML1 coactivator function that contributes to Notch signaling remains unclear. In this study, we performed proteomic studies and identified DDX5, an ATP-dependent DEAD-box RNA helicase, as a component of the MAML1 protein complex. DDX5 interacts with MAML1 in vitro and in vivo, and is associated with the endogenous NOTCH1 transcription activation complex in human T-ALL leukemic cells. Lentivirus-mediated short-hairpin RNA knock-down of DDX5 resulted in decreased expression of Notch target genes, reduced cell proliferation and increased apoptosis in cultured human leukemic cells with constitutive activation of Notch signaling. Also, DDX5 depletion inhibited the growth of human leukemia xenograft in nude mice. Moreover, DDX5 is highly expressed in primary human T-ALL leukemic cells based on the analyses of Oncomine and GEO databases, and Immunohistochemical staining. Our overall findings revealed a critical role of DDX5 in promoting efficient Notch-mediated transcription in leukemic cells, suggesting that DDX5 might be critical for NOTCH1-mediated T-ALL pathogenesis and thus is a potential new target for modulating the Notch signaling in leukemia.

cAMP/CREB-regulated LINC00473 marks LKB1- inactivated lung cancer and mediates tumor growth

The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.

The Mastermind-like 1 (MAML1) Co-activator Regulates Constitutive NF-κB Signaling and Cell Survival

Nuclear factor-κB (NF-κB)-based signaling regulates diverse biological processes, and its deregulation is associated with various disorders including autoimmune diseases and cancer. Identification of novel factors that modulate NF-κB function is therefore of significant importance. The Mastermind-like 1 (MAML1) transcriptional co-activator regulates transcriptional activity in the Notch pathway and is emerging as a co-activator of other pathways. In this study, we found that MAML1 regulates NF-κB signaling via two mechanisms. First, MAML1 co-activates the NF-κB subunit RelA (p65) in NF-κB-dependent transcription. Second, MAML1 causes degradation of the inhibitor of NF-κB (IκBα). Maml1-deficient mouse embryonic fibroblasts showed impaired tumor necrosis factor-α (TNFα)-induced NF-κB responses. Moreover, MAML1 expression level directly influences cellular sensitivity to TNFα-induced cytotoxicity. In vivo, mice deficient in the Maml1 gene exhibited spontaneous cell death in the liver, with a large increase in the number of apoptotic hepatic cells. These findings indicate that MAML1 is a novel modulator for NF-κB signaling and regulates cellular survival.

Brief Report: Blockade of Notch Signaling in Muscle Stem Cells Causes Muscular Dystrophic Phenotype and Impaired Muscle Regeneration

Muscular dystrophies are a group of devastating diseases characterized by progressive muscle weakness and degeneration, with etiologies including muscle gene mutations and regenerative defects of muscle stem cells. Notch signaling is critical for skeletal myogenesis and has important roles in maintaining the muscle stem cell pool and preventing premature muscle differentiation. To investigate the functional impact of Notch signaling blockade in muscle stem cells, we developed a conditional knock‐in mouse model in which endogenous Notch signaling is specifically blocked in muscle stem cell compartment. Mice with Notch signaling inhibition in muscle stem cells showed several muscular dystrophic features and impaired muscle regeneration. Analyses of satellite cells and isolated primary myoblasts revealed that Notch signaling blockade in muscle stem cells caused reduced activation and proliferation of satellite cells but enhanced differentiation of myoblasts. Our data thus indicate that Notch signaling controls processes that are critical to regeneration in muscular dystrophy, suggesting that Notch inhibitor therapies could have potential side effects on muscle functions. STEM CELLS 2013;31:823–828

Proteomic and Functional Analyses Reveal the Role of Chromatin Reader SFMBT1 in Regulating Epigenetic Silencing and the Myogenic Gene Program

Background: SFMBT1 is a poorly characterized chromatin reader. Results: SFMBT1 is associated with multiple transcriptional corepressor complexes and required for MyoD-mediated transcriptional silencing. Conclusion: Novel mechanisms of SFMBT1-mediated transcription repression and its regulation of myogenesis are identified. Significance: Our findings contribute to the understanding of how histone modification language is interpreted to regulate gene expression and biological processes. SFMBT1 belongs to the malignant brain tumor domain-containing chromatin reader family that recognizes repressive histone marks and represses transcription. The biological functions and molecular basis underlying SFMBT1-mediated transcriptional repression are poorly elucidated. Here, our proteomic analysis revealed that SFMBT1 is associated with multiple transcriptional corepressor complexes, including CtBP/LSD1/HDAC complexes, polycomb repressive complexes, and malignant brain tumor family proteins, that collectively contribute to SFMBT1 repressor activity. During myogenesis, Sfmbt1 represses myogenic differentiation of cultured and primary myoblasts. Mechanistically, Sfmbt1 interacts with MyoD and mediates epigenetic silencing of MyoD target genes via recruitment of its associated corepressors and subsequent induction of epigenetic modifications and chromatin compaction. Therefore, our study identified novel mechanisms accounting for SFMBT1-mediated transcription repression and revealed an essential role of Sfmbt1 in regulating MyoD-mediated transcriptional silencing that is required for the maintenance of undifferentiated states of myogenic progenitor cells.

Recent insights into the biological activities and drug delivery systems of tanshinones

Tanshinones, the major lipid-soluble pharmacological constituents of the Chinese medicinal herb Tanshen (Salvia miltiorrhiza), have attracted growing scientific attention because of the prospective biomedical applications of these compounds. Numerous pharmacological activities, including anti-inflammatory, anticancer, and cardio-cerebrovascular protection activities, are exhibited by the three primary bioactive constituents among the tanshinones, ie, tanshinone I (TNI), tanshinone IIA (TNIIA), and cryptotanshinone (CPT). However, due to their poor solubility and low dissolution rate, the clinical applications of TNI, TNIIA, and CPT are limited. To solve these problems, many studies have focused on loading tanshinones into liposomes, nanoparticles, microemulsions, cyclodextrin inclusions, solid dispersions, and so on. In this review, we aim to offer an updated summary of the biological activities and drug delivery systems of tanshinones to provide a reference for these constituents in clinical applications.

Role of LKB1-CRTC1 on Glycosylated COX-2 and Response to COX-2 Inhibition in Lung Cancer

BACKGROUND Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins including PGE-2 linking inflammation with mitogenic signaling. COX-2 is also an anticancer target, however, treatment strategies have been limited by unreliable expression assays and by inconsistent tumor responses to COX-2 inhibition. METHODS We analyzed the TCGA and Directors Challenge lung cancer datasets (n = 188) and also generated an LKB1-null lung cancer gene signature (n = 53) to search the Broad Institute/Connectivity-MAP (C-MAP) dataset. We performed ChIP analyses, real-time polymerase chain reaction, immunoblotting, and drug testing of tumor cell lines (n = 8) and primary lung adenocarcinoma surgical resections (n = 13). RESULTS We show that COX-2 is a target of the cAMP/CREB coactivator CRTC1 signaling pathway. In addition, we detected a correlation between LKB1 status, CRTC1 activation, and presence of glycosylated, but not inactive hypoglycosylated COX-2 in primary lung adenocarcinoma. A search of the C-MAP drug database discovered that all high-ranking drugs positively associated with the LKB1-null signature are known CRTC1 activators, including forskolin and six different PGE-2 analogues. Somatic LKB1 mutations are present in 20.0% of lung adenocarcinomas, and we observed growth inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1 as compared with LKB1-wildtype cells (NS-398, P = .002 and Niflumic acid, P = .006; two-tailed t test). CONCLUSION CRTC1 activation is a key event that drives the LKB1-null mRNA signature in lung cancer. We also identified a positive feedback LKB1/CRTC1 signaling loop for COX-2/PGE2 regulation. These data suggest a role for LKB1 status and glycosylated COX-2 as specific biomarkers that provide a framework for selecting patients for COX-2 inhibition studies.

Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells

BackgroundMucoepidermoid carcinoma (MEC) arises from multiple organs and accounts for the most common types of salivary gland malignancies. Currently, patients with unresectable and metastatic MEC have poor long-term clinical outcomes and no targeted therapies are available. The majority of MEC tumors contain a t(11;19) chromosomal translocation that fuses two genes, CRTC1 and MAML2, to generate the chimeric protein CRTC1-MAML2. CRTC1-MAML2 displays transforming activity in vitro and is required for human MEC cell growth and survival, partially due to its ability to constitutively activate CREB-mediated transcription. Consequently, CRTC1-MAML2 is implicated as a major etiologic molecular event and a therapeutic target for MEC. However, the molecular mechanisms underlying CRTC1-MAML2 oncogenic action in MEC have not yet been systematically analyzed. Elucidation of the CRTC1-MAML2-regulated transcriptional program and its underlying mechanisms will provide important insights into MEC pathogenesis that are essential for the development of targeted therapeutics.MethodsTranscriptional profiling was performed on human MEC cells with the depletion of endogenous CRTC1-MAML2 fusion or its interacting partner CREB via shRNA-mediated gene knockdown. A subset of target genes was validated via real-time RT-PCR assays. CRTC1-MAML2-perturbed molecular pathways in MEC were identified through pathway analyses. Finally, comparative analysis of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles was carried out to assess the contribution of CREB in mediating CRTC1-MAML2-induced transcription.ResultsA total of 808 differentially expressed genes were identified in human MEC cells after CRTC1-MAML2 knockdown and a subset of known and novel fusion target genes was confirmed by real-time RT-PCR. Pathway Analysis revealed that CRTC1-MAML2-regulated genes were associated with network functions that are important for cell growth, proliferation, survival, migration, and metabolism. Comparison of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles revealed common and distinct genes regulated by CRTC1-MAML2 and CREB, respectively.ConclusionThis study identified a specific CRTC1-MAML2-induced transcriptional program in human MEC cells and demonstrated that CRTC1-MAML2 regulates gene expression in CREB-dependent and independent manners. Our data provide the molecular basis underlying CRTC1-MAML2 oncogenic functions and lay a foundation for further functional investigation of CRTC1-MAML2-induced signaling in MEC initiation and maintenance.

DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation.

CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.