Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

The methionine salvage pathway-involving ADI1 inhibits hepatoma growth by epigenetically altering genes expression via elevating S-adenosylmethionine.

The 5'-methylthioadenosine (MTA) cycle-participating human acireductone dioxygenase 1 (ADI1) has been implicated as a tumor suppressor in prostate cancer, yet its role remains unclear in hepatocellular carcinoma (HCC). Here, we demonstrated a significant reduction of ADI1, either in protein or mRNA level, in HCC tissues. Additionally, higher ADI1 levels were associated with favorable postoperative recurrence-free survival in HCC patients. By altering ADI1 expression in HCC cells, a negative correlation between ADI1 and cell proliferation was observed. Cell-based and xenograft experiments were performed by using cells overexpressing ADI1 mutants carrying mutations at the metal-binding sites (E94A and H133A, respectively), which selectively disrupted differential catalytic steps, resulting in staying or leaving the MTA cycle. The results showed that the growth suppression effect was mediated by accelerating the MTA cycle. A cDNA microarray analysis followed by verification experiments identified that caveolin-1 (CAV1), a growth-promoting protein in HCC, was markedly decreased upon ADI1 overexpression. Suppression of CAV1 expression was mediated by an increase of S-adenosylmethionine (SAMe) level. The methylation status of CAV1 promoter was significantly altered upon ADI1 overexpression. Finally, a genome-wide methylation analysis revealed that ADI1 overexpression altered promoter methylation profiles in a set of cancer-related genes, including CAV1 and genes encoding antisense non-coding RNAs, long non-coding RNAs, and microRNAs, resulting in significant changes of their expression levels. In conclusion, ADI1 expression promoted MTA cycle to increase SAMe levels, which altered genome-wide promoter methylation profiles, resulting in altered gene expression and HCC growth suppression.

Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase.

CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.

LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration.

Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber-associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA-resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.

Regulation of CTP Synthase Filament Formation During DNA Endoreplication in Drosophila.

CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. Recent studies in bacteria, yeast, and Drosophila all reveal a polymeric CTPsyn structure, which dynamically regulates its enzymatic activity. However, the molecular mechanism underlying the formation of CTPsyn polymers is not completely understood. In this study, we found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. We further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme and reveals a role for Cbl in endocycles during Drosophila development.

Cytoophidium assembly reflects upregulation of IMPDH activity.

Cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) (both of which have two isoforms) can form fiber-like subcellular structures termed 'cytoophidia' under certain circumstances in mammalian cells. Although it has been shown that filamentation of CTPS downregulates its activity by disturbing conformational changes, the activity of IMPDH within cytoophidia is still unclear. Most previous IMPDH cytoophidium studies were performed under conditions involving inhibitors that impair GTP synthesis. Here, we show that IMPDH forms cytoophidia without inhibition of GTP synthesis. First, we find that an elevated intracellular CTP concentration or treatment with 3'-deazauridine, a CTPS inhibitor, promotes IMPDH cytoophidium formation and increases the intracellular GTP pool size. Moreover, restriction of cell growth triggers the disassembly of IMPDH cytoophidia, implying that their presence is correlated with active cell metabolism. Finally, we show that the presence of IMPDH cytoophidia in mouse pancreatic islet cells might correlate with nutrient uptake in the animal. Collectively, our findings reveal that formation of IMPDH cytoophidia reflects upregulation of purine nucleotide synthesis, suggesting that the IMPDH cytoophidium plays a role distinct from that of the CTPS cytoophidium in controlling intracellular nucleotide homeostasis.

Effects of RING-SH2Grb², a chimeric protein containing the E3 ligase domain of Cbl, on the EGFR pathway.

The E3 ubiquitin-protein ligase Casitas B-lineage lymphoma protein (Cbl) negatively regulates epidermal growth factor receptor (EGFR) signaling pathway in many organisms, and has crucial roles in cell growth, development and human pathologies, including lung cancers. RING-SH2Grb² a chimeric protein of 215 amino acids containing the RING domain of Cbl that provides E3 ligase activity, and the SH2 domain of Grb2 that serves as an adaptor for EGFR. In this study, we demonstrated that RING-SH2Grb² could promote the ubiquitinylation and degradation of EGFR in a human non-small cell lung carcinoma cell line H1299. Moreover, we discovered that the RING-SH2Grb² chimera promoted the internalization of ligand-bound EGFR, inhibited the growth of H1299 cells, and significantly suppressed tumor growth in a xenograft mouse model. In summary, our results revealed a potential new cancer therapeutic approach for non-small cell lung cancer.

Identification of 11-amino acid peptides that disrupt Notch-mediated processes in Drosophila.

BACKGROUND: The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification. RESULTS: From the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal) increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs.In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV) compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(spl)mß are target genes of the Notch pathway in DV boundary formation and vein specification, respectively. We also found that overexpression of Pri/Tal peptides abolishes Cut expression and co-expression of Pri/Tal peptides with phyl strongly reduces E(spl)mß expression. CONCLUSIONS: We show for the first time that the overexpression of Pri/Tal 11-amino acid peptides disrupts multiple Notch-mediated processes and reduces Notch target gene expression in Drosophila, suggesting that these peptides have novel antagonistic activity to the Notch pathway. Thus, our discovery might provide insights into designing new therapeutic reagents for Notch-related diseases.

D-Cbl binding to Drk leads to dose-dependent down-regulation of EGFR signaling and increases receptor-ligand endocytosis.

Proper control of Epidermal Growth Factor Receptor (EGFR) signaling is critical for normal development and regulated cell behaviors. Abnormal EGFR signaling is associated with tumorigenic process of various cancers. Complicated feedback networks control EGFR signaling through ligand production, and internalization-mediated destruction of ligand-receptor complexes. Previously, we found that two isoforms of D-Cbl, D-CblS and D-CblL, regulate EGFR signaling through distinct mechanisms. While D-CblL plays a crucial role in dose-dependent down-regulation of EGFR signaling, D-CblS acts in normal restriction of EGFR signaling and does not display dosage effect. Here, we determined the underlying molecular mechanism, and found that Drk facilitates the dose-dependent regulation of EGFR signaling through binding to the proline-rich motif of D-CblL, PR. Furthermore, the RING finger domain of D-CblL is essential for promoting endocytosis of the ligand-receptor complex. Interestingly, a fusion protein of the two essential domains of D-CblL, RING- PR, is sufficient to down-regulate EGFR signal in a dose-dependent manner by promoting internalization of the ligand, Gurken. Besides, RING-SH2(Drk), a fusion protein of the RING finger domain of D-Cbl and the SH2 domain of Drk, also effectively down-regulates EGFR signaling in Drosophila follicle cells, and suppresses the effects of constitutively activated EGFR. The RING-SH2(Drk) suppresses EGFR signaling by promoting the endosomal trafficking of ligand-receptor complexes, suggesting that Drk plays a negative role in EGFR signaling by enhancing receptor endocytosis through cooperating with the RING domain of D-Cbl. Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2(Drk) might further reduces EGFR signaling. The fusion proteins we developed may provide alternative strategies for therapy of cancers caused by hyper-activation of EGFR signaling.

Positive regulation of spondin 2 by thyroid hormone is associated with cell migration and invasion.

The thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T(3) treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T(3). However, the underlying mechanism and the physiological role of T(3) in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T(3) treatment in the HepG2-TR alpha 1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T(3) response element on the spondin 2 promoter was localized in the -1104/-1034 or -984/-925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T(3), has an important role in cell invasion, cell migration, and tumor progression.

Smiling Gurken gradient: An expansion of the Gurken gradient.

Morphogen gradients provide unique positional information within a tissue. Cells that are sensitive to the concentration of the morphogen integrate this signal and develop an appropriately distinct cell fate. A morphogen gradient is usually generated by a restricted source and shaped by the speed of diffusion and stability of the signaling molecule. In addition, the availability of receptor and Heparan Sulfate Proteoglycans (HSPGs) help to shape the gradient. We have shown that overexpression of Dally-like protein (Dlp) causes an expansion of Gurken distribution and a loss of cell fates which are specified by high levels of epidermal growth factor receptor (Egfr) signaling. In this article, we discuss how D-Cbl mediated Egfr endocytosis and the levels of Dlp affect the shape of the Gurken gradient.

The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis.

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.

Differential effects of Cbl isoforms on Egfr signaling in Drosophila.

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.