Increasing Sufu gene dosage reveals its unorthodox role in promoting polydactyly and medulloblastoma tumorigenesis.

Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.

Hedgehog signal activates AMPK via Smoothened to promote autophagy and lipid degradation in hepatocytes.

Studies in the past decade have shown that lipid droplets stored in liver cells under starvation are encapsulated by autophagosomes and fused to lysosomes via the endocytic system. Autophagy responds to a variety of environmental factors inside and outside the cell, so it has a complex signal regulation network. To this end, we first explored the role of Hedgehog (Hh) in autophagy and lipid metabolism. Treatment of normal mouse liver cells with SAG and GDC-0449 revealed elevated phosphorylation of AMP-activated protein kinase (AMPK) and increased lipidation of LC3. SAG, and GDC-0449 were agonist and antagonist of Smoothened (Smo) in canonical Hh pathway, respectively, but they played a consistent role in the regulation of autophagy in hepatocytes. Moreover, SAG and GDC-0449 did not affect the expression of glioma-associated oncogene (Gli1) and patched 1, suggesting the absence of canonical Hh signaling in hepatocytes. We further knocked down the Smo and found that the effects of SAG and GDC-0449 disappeared, indicating that the non-canonical Smo pathway was involved in the regulation of autophagy in hepatocytes. In addition, SAG and GDC-0449 promoted lipid degradation and inhibited lipid production signals. Knockdown of Smo slowed down the rate of lipid degradation rather than Sufu or Gli1, indicating that Hh signaling regulated the lipid metabolism via Smo. In summary, activates AMPK via Smo to promote autophagy and lipid degradation.

AMPK attenuates SHH subgroup medulloblastoma growth and metastasis by inhibiting NF-κB activation.

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant pediatric brain tumors. Metastasis and relapse are the leading causes of death in MB patients. The initiation of the SHH subgroup of MB (SHH-MB) is due to the aberrant activation of Sonic Hedgehog (Shh) signaling. However, the mechanisms for its metastasis are still unknown. RESULTS: AMP-dependent protein kinase (AMPK) restrains the activation of Shh signaling pathway, thereby impeding the proliferation of SHH-MB cells. More importantly, AMPK also hinders the growth and metastasis of SHH-MB cells by regulating NF-κB signaling pathway. Furthermore, Vismodegib and TPCA-1, which block the Shh and NF-κB pathways, respectively, synergistically restrained the growth, migration, and invasion of SHH-MB cells. CONCLUSIONS: This work demonstrates that AMPK functions through two signaling pathways, SHH-GLI1 and NF-κB. AMPK-NF-κB axis is a potential target for molecular therapy of SHH-MB, and the combinational blockade of NF-κB and Shh pathways confers synergy for SHH-MB therapy.

Macroautophagy supports Sonic Hedgehog signaling by promoting Patched1 degradation.

Autophagy is a highly conservative self-digestion process to maintain intracellular homeostasis and to ensure the survival of cells under stress. Activation of Sonic Hedgehog (Shh) signaling depends on the normal endocytic degradation of pathway receptor Patched1 (Ptch1). It is unclear whether autophagy participates in the receptor endocytosis and modulates Shh signaling transduction. Here we found that blocking macroautophagy attenuates Shh signaling due to the failed transport of Smoothened (Smo) into primary cilia. At the upstream of Smo, Ptch1 was poly-ubiquitinated through K63-conjugated ubiquitin chains. Macroautophagy participates Shh-induced degradation of poly-ubiquitinated Ptch1, contributing to the activation of Shh signaling.

Sufu negatively regulates both initiations of centrosome duplication and DNA replication.

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.

SPOP-PTEN-SUFU axis promotes progression of clear cell renal cell carcinoma via activating SHH and WNT pathway.

Although E3 ligase Speckle type BTB/POZ protein (SPOP) promotes tumorigenesis by acting as a key regulatory hub in clear cell renal cell carcinoma (ccRCC), the detailed molecular mechanism remains unclear. Here, we demonstrate that a well-known tumor suppressor, Suppressor of Fused (SUFU), is downregulated by SPOP. Interestingly, this downregulation depends on cullin-3(Cul3)-SPOP E3 ligase, but SUFU is not a direct substrate of SPOP. Phosphatase and tensin homolog (PTEN), a ubiquitinated substrate of SPOP, is involved in SPOP-mediated SUFU reduction. Importantly, inhibition of SUFU leads to elevated SHH and WNT signaling, consequently rescuing the reduced proliferation, migration, and invasion abilities of ccRCC cells caused by SPOP-knockdown. Moreover, combinatorial treatment with SHH and WNT inhibitors shows more effective for suppressing ccRCC cell proliferation and aggressiveness. These findings demonstrate that a novel SPOP-PTEN-SUFU axis promotes ccRCC carcinogenesis by activating SHH and WNT pathway, providing a new treatment strategy for ccRCC.

Identification of a potent antagonist of smoothened in hedgehog signaling.

BACKGROUND: Hedgehog signaling is essential to the regulation of embryonic development, tissue homeostasis, and stem cell self-renewal, making it a prime target for developing cancer therapeutics. Given the close link between aberrant Hedgehog signaling and cancers, many small molecular compounds have been developed to inhibit Smoothened, a key signal transducer of this pathway, for treating cancer and several such compounds have been approved by the United States Food and Drug Administration (GDC-0449 and LDE-225). However, acquired drug resistance has emerged as an important obstacle to the effective use of these first generation Hedgehog pathway blockers. Thus, new Smoothened inhibitors that can overcome such resistance is an urgent need going forward. RESULTS: We established the Smoothened/ßarrestin2-GFP high-throughput screening platform based on the mechanistic discovery of Hedgehog signaling pathway, and discovered several active small molecules targeting Smoothened including 0025A. Here we show that 0025A can block the translocation of ßarrestin2-GFP to Smoothened, displace Bodipy-cyclopamine binding to wild-type Smoothened or mutant Smoothened-D473H, reduce the accumulation of Smo on primary cilia and the expression of Gli upon Hedgehog stimulation. In addition, we show that 0025A can effectively suppress hair follicle morphogenesis and hair growth in mice. CONCLUSIONS: Our results demonstrate that 0025A is a potent antagonist targeting Smoothened wild-type and mutant receptors in the Hedgehog signaling pathway and may provide a new therapy for refractory cancers.

Deletion at an 1q24 locus reveals a critical role of long noncoding RNA DNM3OS in skeletal development.

BACKGROUND: Skeletal development and maintenance are complex processes known to be coordinated by multiple genetic and epigenetic signaling pathways. However, the role of long non-coding RNAs (lncRNAs), a class of crucial epigenetic regulatory molecules, has been under explored in skeletal biology. RESULTS: Here we report a young patient with short stature, hypothalamic dysfunction and mild macrocephaly, who carries a maternally inherited 690 kb deletion at Chr.1q24.2 encompassing a noncoding RNA gene, DNM3OS, embedded on the opposite strand in an intron of the DYNAMIN 3 (DNM3) gene. We show that lncRNA DNM3OS sustains the proliferation of chondrocytes independent of two co-cistronic microRNAs miR-199a and miR-214. We further show that nerve growth factor (NGF), a known factor of chondrocyte growth, is a key target of DNM3OS-mediated control of chondrocyte proliferation. CONCLUSIONS: This work demonstrates that DNM3OS is essential for preventing premature differentiation of chondrocytes required for bone growth through endochondral ossification.

Identification of four novel prognosis biomarkers and potential therapeutic drugs for human colorectal cancer by bioinformatics analysis.

Colorectal cancer (CRC) is one of the most deadly cancers in the world with few reliable biomarkers that have been selected into clinical guidelines for prognosis of CRC patients. In this study, mRNA microarray datasets GSE113513, GSE21510, GSE44076, and GSE32323 were obtained from the Gene Expression Omnibus (GEO) and analyzed with bioinformatics to identify hub genes in CRC development. Differentially expressed genes (DEGs) were analyzed using the GEO2R tool. Gene ontology (GO) and KEGG analyses were performed through the DAVID database. STRING database and Cytoscape software were used to construct a protein-protein interaction (PPI) network and identify key modules and hub genes. Survival analyses of the DEGs were performed on GEPIA database. The Connectivity Map database was used to screen potential drugs. A total of 865 DEGs were identified, including 374 upregulated and 491 downregulated genes. These DEGs were mainly associated with metabolic pathways, pathways in cancer, cell cycle and so on. The PPI network was identified with 863 nodes and 5817 edges. Survival analysis revealed that HMMR, PAICS, ETFDH, and SCG2 were significantly associated with overall survival of CRC patients. And blebbistatin and sulconazole were identified as candidate drugs. In conclusion, our study found four hub genes involved in CRC, which may provide novel potential biomarkers for CRC prognosis, and two potential candidate drugs for CRC.

Protein phosphatase 4 promotes Hedgehog signaling through dephosphorylation of Suppressor of fused.

Reversible phosphorylation of Suppressor of fused (Sufu) is essential for Sonic Hedgehog (Shh) signal transduction. Sufu is stabilized under dual phosphorylation of protein kinase A (PKA) and glycogen synthase kinase 3ß (GSK3ß). Its phosphorylation is reduced with the activation of Shh signaling. However, the phosphatase in this reversible phosphorylation has not been found. Taking advantage of a proteomic approach, we identified Protein phosphatase 4 regulatory subunit 2 (Ppp4r2), an interacting protein of Sufu. Shh signaling promotes the interaction of these two proteins in the nucleus, and Ppp4 also promotes dephosphorylation of Sufu, leading to its degradation and enhancing the Gli1 transcriptional activity. Finally, Ppp4-mediated dephosphorylation of Sufu promotes proliferation of medulloblastoma tumor cells, and expression of Ppp4 is positively correlated with up-regulation of Shh pathway target genes in the Shh-subtype medulloblastoma, underscoring the important role of this regulation in Shh signaling.

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation. Simultaneous loss of Spop, an E3 ubiquitin ligase targeting Gli2, restores robust Gli2 activation and induces rapid MB formation in Sufu knockout background. We also demonstrated a tumor-promoting role of Sufu in Smo-activated MB (∼60% of human SHH MB) by maintaining robust Gli activity. Having established Gli2 activation as a key driver of SHH MB, we report a comprehensive analysis of its targetome. Furthermore, we identified Atoh1 as a target and molecular accomplice of Gli2 that activates core SHH MB signature genes in a synergistic manner. Overall, our work establishes the dual role of SUFU in SHH MB and provides mechanistic insights into transcriptional regulation underlying Gli2-mediated SHH MB tumorigenesis.

Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor.

Direct interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three anti-apoptotic BCL-2 proteins (MCL-1, BCL-2 and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Anti-apoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in SUFU (suppressor of fused), a tumour suppressor and antagonist of the GLI DNA-binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU-GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1 and BCL-XL. Anti-apoptotic BCL-2 protein/SUFU feedforward signalling promotes cancer cell survival and growth, and can be disabled with BH3 mimetics-small molecules that target anti-apoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumours and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.

DGKδ triggers endoplasmic reticulum release of IFT88-containing vesicles destined for the assembly of primary cilia.

The morphogenic factor Sonic hedgehog (Shh) signals through the primary cilium, which relies on intraflagellar transport to maintain its structural integrity and function. However, the process by which protein and lipid cargos are delivered to the primary cilium from their sites of synthesis still remains poorly characterized. Here, we report that diacylglycerol kinase δ (DGKδ), a residential lipid kinase in the endoplasmic reticulum, triggers the release of IFT88-containing vesicles from the ER exit sites (ERES), thereby setting forth their movement to the primary cilium. Encoded by the gene whose mutations originally implicated the primary cilium as the venue of Shh signaling, IFT88 is known to be part of the complex B that drives the anterograde transport within cilia. We show that IFT88 interacts with DGKδ, and is associated with COPII-coated vesicles at the ERES. Using a combination of RNAi silencing and gene knockout strategies, we further show that DGKδ is required for supporting Shh signaling both in vitro and in vivo, demonstrating the physiological significance of this regulation.

DeSUMOylation of Gli1 by SENP1 Attenuates Sonic Hedgehog Signaling.

The transcriptional output of the Sonic Hedgehog morphogenic pathway is orchestrated by three Krüppel family transcription factors, Gli1 to -3, which undergo extensive posttranslational modifications, including ubiquitination and SUMOylation. Here, we report that the sentrin-specific peptidase SENP1 is the specific deSUMOylation enzyme for Gli1. We show that SUMOylation stabilizes Gli1 by competing with ubiquitination at conserved lysine residues and that SUMOylated Gli1 is enriched in the nucleus, suggesting that SUMOylation is a nuclear localization signal for Gli1. Finally, we show that small interfering RNA (siRNA)-mediated knockdown of SENP1 augments the ability of Shh to sustain the proliferation of cerebellar granule cell precursors, demonstrating the physiological significance of the negative regulation of Shh signaling by SENP1.

DAZ-interacting Protein 1 (Dzip1) Phosphorylation by Polo-like Kinase 1 (Plk1) Regulates the Centriolar Satellite Localization of the BBSome Protein during the Cell Cycle.

The function of the primary cilia, which is assembled in most vertebrate cells, is achieved by transport in and out of kinds of signaling receptors. The BBSome protein complex could recognize and target membrane proteins to the cilia, but how the BBSome itself is transported into the cilia is poorly understood. Here we demonstrate that the centrosome protein Dzip1 mediates the assembly of the BBSome-Dzip1-PCM1 complex in the centriolar satellites (CS) at the G0 phase for ciliary translocation of the BBSome. Phosphorylation of Dzip1 at Ser-210 by Plk1 (polo-like kinase 1) during the G2 phase promotes disassembly of this complex, resulting in removal of Dzip1 and the BBSome from the CS. Inhibiting the kinase activity of Plk1 maintains the CS localization of the BBSome and Dzip1 at the G2 phase. Collectively, our findings reveal the cell cycle-dependent regulation of BBSome transport to the CS and highlight a potential mechanism that the BBSome-mediated signaling pathways are accordingly regulated during the cell cycle.

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling.

Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1-/-, Sufu-/-, and Ptch1-/-; Sufu-/- double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function.

Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals.

Hedgehog signaling plays very important roles in development and cancers. Vertebrates have three transcriptional factors, Gli1, Gli2 and Gli3. Among them, Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus (Ci, in Drosophila) structurally and functionally as a 'double agent' for Shh target gene expression. Here we show that Gli3 full-length, but not the truncated form, can be methylated at K436 and K595. This methylation is specifically catalyzed by Set7, a lysine methyltransferase (KMT). Methylation at K436 and K595 respectively increases the stability and DNA binding ability of Gli3, resulting in an enhancement of Shh signaling activation. Furthermore, functional experiments indicate that the Gli3 methylation contributes to the tumor growth and metastasis in non-small cell lung cancer in vitro and in vivo. Therefore, we propose that Set7 mediated methylation is a novel PTM of Gli3, which positively regulates the transactivity of Gli3 and the activation of Shh signaling.

Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib.

BACKGROUND: The T790M mutation of epithelial growth factor receptor (EGFR) is a major cause of the acquired resistance to EGFR tyrosine kinase inhibitor (EGFR-TKIs) treatment for lung cancer patients. The Hippo pathway effector, TAZ, has emerged as a key player in organ growth and tumorigenesis, including lung cancer. RESULTS: In this study, we have discovered high TAZ expression in non-small cell lung cancer (NSCLC) cells harboring dual mutation and TAZ depletion sensitized their response to EGFR-TKIs. Mechanistically, knockdown of TAZ in T790M-induced resistant cells leaded to reduced anchorage-independent growth in vitro, tumor formation and resistance to gefitinib in vivo, correlated with epithelial-mesenchymal transition (EMT) and suppressed migration and invasion. Furthermore, we confirmed CTGF and AXL, novel EMT markers and potential therapeutic targets for overcoming EGFR inhibitor resistance, as directly transcriptional targets of TAZ. CONCLUSIONS: Taken together, this study suggests that expression of TAZ is an intrinsic mechanism of T790M-induced resistance in response to EGFR-TKIs. Combinational targeting on both EGFR and TAZ may enhance the efficacy of EGFR-TKIs in acquired resistance of NSCLC.