Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

Cell-permeable PI3 kinase competitive peptide inhibits KIT mutant mediated tumorigenesis of gastrointestinal stromal tumor (GIST).

BACKGROUND: Mutations in the receptor tyrosine kinase KIT are the main cause of gastrointestinal stromal tumor (GIST), and the KIT mutants mediated PI3 kinase activation plays a key role in the tumorigenesis of GIST. In this study, we aimed to block PI3 kinase activation by cell-permeable peptide and investigate its possible application in the treatment of GIST. METHODS AND RESULTS: We designed cell-permeable peptides based on the binding domain of PI3 kinase subunit p85 to KIT or PI3 kinase subunit p110, respectively, in order to compete for the binding between p85 and KIT or p110 and therefore inhibit the activation of PI3 kinases mediated by KIT. The results showed that the peptide can penetrate the cells, and inhibit the activation of PI3 kinases, leading to reduced cell survival and cell proliferation mediated by KIT mutants in vitro. Treatment of mice carrying germline KIT/V558A mutation, which can develop GIST, with the peptide that can compete for the binding between p85 and p110, led to reduced tumorigenesis of GIST. The peptide can further enhance the inhibition of the tumor growth by imatinib which is used as the first line targeted therapy of GIST. CONCLUSIONS: Our results showed that cell-permeable PI3 kinase competitive peptide can inhibit KIT-mediated PI3 kinase activation and tumorigenesis of GIST, providing a rationale to further test the peptide in the treatment of GIST and even other tumors with over-activation of PI3 kinases.

SPRY4 inhibits and sensitizes the primary KIT mutants in gastrointestinal stromal tumors (GISTs) to imatinib.

BACKGROUND: KIT is frequently mutated in gastrointestinal stromal tumors (GISTs), and the treatment of GISTs largely relies on targeting KIT currently. In this study, we aimed to investigate the role of sprouty RTK signaling antagonist 4 (SPRY4) in GISTs and related mechanisms. METHODS: Ba/F3 cells and GIST-T1 cell were used as cell models, and mice carrying germline KIT/V558A mutation were used as animal model. Gene expression was examined by qRT-PCR and western blot. Protein association was examined by immunoprecipitation. RESULTS: Our study revealed that KIT increased the expression of SPRY4 in GISTs. SPRY4 was found to bind to both wild-type KIT and primary KIT mutants in GISTs, and inhibited KIT expression and activation, leading to decreased cell survival and proliferation mediated by KIT. We also observed that inhibition of SPRY4 expression in KITV558A/WT mice led to increased tumorigenesis of GISTs in vivo. Moreover, our results demonstrated that SPRY4 enhanced the inhibitory effect of imatinib on the activation of primary KIT mutants, as well as on cell proliferation and survival mediated by the primary KIT mutants. However, in contrast to this, SPRY4 did not affect the expression and activation of drug-resistant secondary KIT mutants, nor did it affect the sensitivity of secondary KIT mutants to imatinib. These findings suggested that secondary KIT mutants regulate a different downstream signaling cascade than primary KIT mutants. CONCLUSIONS: Our results suggested that SPRY4 acts as negative feedback of primary KIT mutants in GISTs by inhibiting KIT expression and activation. It can increase the sensitivity of primary KIT mutants to imatinib. In contrast, secondary KIT mutants are resistant to the inhibition of SPRY4.

High diversity of potential nitrate-reducing Fe(II)-oxidizing bacteria enriched from activated sludge.

Nitrate-dependent Fe(II) oxidation (NDFO) has been discovered in various environments including activated sludge and can potentially be used to remove nitrate from wastewater. In this study, NDFO sludge was successfully enriched from activated sludge under high Fe(II) concentrations over 100 days and the denitrification rate achieved 1.37 mmol N/(gVSS day). High-throughput sequencing of the bacterial 16S rRNA gene was used to investigate the microbial community structure dynamics during the enrichment process. The results showed that the microbial community changed significantly and high diversity of potential Fe(II)-oxidizing bacteria (FeOB) was observed in the enriched sludge. Thermomonas and Gallionella were the dominant bacterial genera in the enriched sludge and their relative abundances accounted for 9.49 and 4.08%, respectively. Furthermore, it was found that potential FeOB were also abundantly present in activated sludge samples of common municipal wastewater treatment plants. Collectively, this study demonstrated that NDFO could be successfully performed by enriched activated sludge and high diversity of bacteria is involved in this process, and the results also provide baseline information for future research and engineering application of NDFO process.

RAF1 facilitates KIT signaling and serves as a potential treatment target for gastrointestinal stromal tumor.

The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.

Entry of ZSWIM4 to the nucleus is crucial for its inhibition of KIT and BMAL1 in gastrointestinal stromal tumors.

BACKGROUND: Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs). RESULTS: We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4-/- mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice. CONCLUSIONS: Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment.

[D816V mutation of KIT specifically induces phosphorylation of HNRNPL and HNRNPK in COS-1 cells].

Objective To study the regulation of D816V mutation of III tyrosine kinase receptor KIT on RNA binding proteins HNRNPL and HNRNPK. Methods In COS-1 cells, wild-type KIT or KIT D816V mutation were expressed alone or together with HNRNPL or HNRNPK. Activation of KIT and phosphorylation of HNRNPL and HNRNPK were detected by immunoprecipitation and Western blot analysis. The localization of KIT, HNRNPL and HNRNPK in COS-1 cells were examined by confocal microscopy. Results Wild-type KIT needs to bind its ligand stem cell factor (SCF) for phosphorylation, while KIT D816V could auto-phosphorylation without SCF stimulation. In addition, KIT D816V can induce phosphorylation of HNRNPL and HNRNPK, which is not possible in wild-type KIT. HNRNPL and HNRNPK are expressed in the nucleus, and wild-type KIT is expressed in cytosol and cell membrane, while KIT D816V is mainly found in cytosol. Conclusion Wild-type KIT needs SCF binding for activation, while KIT D816V can autoactivate without SCF stimulation, and induces phosphorylation of HNRNPL and HNRNPK specifically.

Farnesyltransferase (FTase) Inhibitors Increase Inhibition of KIT Mutants by Imatinib.

Background: Mutations in the receptor tyrosine kinase KIT are the major cause of gastrointestinal stromal tumors. KIT-mediated activation of the RAS/RAF/MEK/ERK and PI3 kinase/AKT pathways plays an important role in KIT mutant-mediated cell transformation. Methods: The frequently seen primary KIT mutations W557K558del and V560D, and the secondary KIT mutations V654A and N822K, in gastrointestinal stromal tumors were stably transfected into Ba/F3 cells. Cell proliferation was examined with a CCK kit, and cell survival and cell cycle were examined by flow cytometry. Cell signaling was examined by western blot. Results: We found that farnesyltransferase inhibitors tipifarnib and lonafarnib, which inhibit RAS activity, inhibited ERK activation mediated by both wild-type and KIT mutants, which often occur in gastrointestinal stromal tumors. Correspondingly, both wild-type and KIT mutant-mediated cell survival and proliferation were inhibited by both inhibitors. Imatinib is used as the first-line targeted therapy for gastrointestinal stromal tumors in the clinic. In our study, both inhibitors increased imatinib-mediated inhibition of cell survival and proliferation induced by both wild-type and KIT mutants. Similar to the primary KIT mutations, secondary mutations of KIT-induced ERK activation and cell response were inhibited by both inhibitors. Conclusions: Our results suggested the potential benefit of farnesyltransferase inhibitors either alone or combined with imatinib in the treatment of gastrointestinal stromal tumors carrying KIT mutations.

A JAK tyrosine kinase and pseudokinase Co-inhibition strategy combines enhanced potency and on-demand activation.

Janus tyrosine kinase (JAK) inhibitors have been on the market for several years, but their use is limited by drug resistance and intolerable side effects. Herein, we propose a novel strategy of JAK tyrosine kinase (TK) and pseudokinase (PK) domain co-inhibition system to consolidate robust JAK inhibition and on-demand activation. A photoexcited prodrug PAT-SIL-TG-1&AT exhibits the synergy effects of TK-PK co-inhibition and enable the spatiotemporal control of JAK2 signaling. The hypoxia-activated prodrug HAT-SIL-TG-1&AT significantly inhibited HEL cells proliferation and downregulated phosphorylated STAT3/5 under hypoxic conditions. Importantly, HAT-SIL-TG-1&AT showed synergistic antitumor effects and selectively inhibited the JAK-STAT signaling in tumor tissues in vivo. This work demonstrates a viable solution to achieve superior JAK2 inhibition, and provides an inspiration for other kinases containing PK domain.

Protein tyrosine phosphatase receptor type E (PTPRE) regulates the activation of wild-type KIT and KIT mutants differently.

Activation of receptor tyrosine kinases needs tight control by tyrosine phosphatases to keep their normal function. In this study, we investigated the regulation of activation of the type III receptor tyrosine kinase KIT by protein tyrosine phosphatase receptor type E (PTPRE). We found that PTPRE can associate with wild-type KIT and inhibit KIT activation in a dose-dependent manner, although the activation of wild-type KIT is dramatically inhibited even when PTPRE is expressed at low level. The D816V mutation of KIT is the most frequently found oncogenic mutation in mastocytosis, and we found that PTPRE can associate and inhibit the activation of KIT/D816V in a dose dependent manner, but the inhibition is much weaker compared with wild-type KIT. Similar to mastocytosis, KIT mutations are the main oncogenic mutations in gastrointestinal stromal tumors (GISTs) although GISTs carry different types of KIT mutations. We further studied the regulation of the activation of GISTs-type KIT mutants and other mastocytosis-type KIT mutants by PTPRE. Indeed, PTPRE can almost block the activation of GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to the inhibition of PTPRE. Taken together, our results suggest that PTPRE can associate with KIT, and inhibit the activation of both wild-type KIT and GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to PTPRE.

[PI3K isoforms PI3Kß and PI3Kδ play different roles in KIT mutation-mediated cell transformation].

Objective To investigate the role of phosphatidylinositol 3-kinase (PI3K) isoforms in type III receptor tyrosine kinase KIT mutation-mediated signaling and cell proliferation. Methods The wild-type KIT and the common KIT mutations V560D and W557K558del in gastrointestinal stromal tumors (GIST) were stably expressed in BaF3 cells. The cells were treated with PI3K isoforms PI3Kα, PI3Kß and PI3Kδ specific inhibitors or pan PI3K inhibitor. The activation of KIT and its downstream signals was detected by immunoprecipitation and Western blot analysis. GIST-T1 cells were treated with the same drug, and the activation of KIT and its downstream signals was also detected by immunoprecipitation and Western blot analysis, and cell proliferation and apoptosis were detected by MTT assay and flow cytometry, respectively. Results Compared with the controls, in BaF3 cells expressing wild-type KIT and its mutants, the activation of KIT and its downstream signaling molecules AKT and ERK was inhibited the most by PI3Kδ specific inhibitor, followed by the specific inhibitors of PI3Kα and PI3Kß subtype. In GIST-T1 cells, the activation of KIT and its downstream signals was inhibited the most by PI3Kß specific inhibitor, followed by PI3Kδ and PI3Kα specific inhibitors. Conclusion In BaF3 cells, PI3Kδ subtype plays a major role in KIT activation and its downstream signal transduction, while in GIST-T1 cells, PI3Kß subtype plays a major role in KIT activation and its downstream signal transduction. These results indicate that PI3K isoforms play different roles in KIT mutation-mediated cell transformation depending on the host cells.

Hydrogen Peroxide Inducible JAK3 Covalent Inhibitor: Prodrug for the Treatment of RA with Enhanced Safety Profile.

Selective inhibition of Janus kinases (JAKs) is an arising strategy in drug discovery. Covalent inhibitors targeting a unique cysteine in JAK3 exhibit ultraselectivity among JAK family members. However, safety and tissue specific concerns still remain. A prodrug of a known JAK3 covalent inhibitor sensitive to H2O2 was designed and synthesized and its therapeutic effect was evaluated in the CIA (collagen-induced arthritis) mice model of RA (rheumatoid arthritis). The prodrug strategy relied on the introduction of a hydrogen peroxide-sensitive borate trigger group to avoid random covalent binding to thiol functionalities in biomacromolecules. The results show that the prodrug can be activated and released under pathophysiological concentration of H2O2. In addition, the prodrug demonstrated stability to the physiological environment. In comparison to the parent compound, the prodrug showed a similar therapeutic effect in the CIA model but notably exhibited lower toxicity and a larger therapeutic window.

Denitrification using excess activated sludge as carbon source: Performance and the microbial community dynamics.

In this study, the feasibility of using excess activated sludge as carbon source for denitrification to remove low concentrations of nitrate from wastewater was investigated. With an optimized upflow anaerobic reactor, high denitrification performance for both synthetic wastewater and effluent from municipal wastewater treatment plant was achieved. The results showed that the nitrate nitrogen can be reduced down to <1mgL-1 with a hydraulic retention time of 6h. The total nitrogen removal increased with the amount of sludge added, while, the nitrogen removal per unit of added sludge decreased with the amount of sludge added. 16S rRNA gene high-throughput sequencing showed that the bacterial community changed during the startup period and the subsequent denitrification process. And, it was found that some bacterial species in genus Pseudomonas and Thauera obviously decreased during the reactor operation, suggesting that they were likely digested and released organic matters for the denitrification.

Analysis of anthocyanins and flavonols in petals of 10 Rhododendron species from the Sygera Mountains in Southeast Tibet.

Flower color is one of the major ornamental characteristics of the genus Rhododendron, but few studies on flower color in alpine Rhododendron have been reported. In our study, the flower colors and the pigment constituents of petals from 10 Rhododendron species sampled in the Sygera Mountains of Southeast Tibet were analyzed using high-performance liquid chromatography-diode array detection and mass spectrometry (HPLC-DAD-ESI-MS(2)). The color analysis showed that the 10 Rhododendron species could be divided into five color groupings: yellow, red, red-purple, purple-violet, and purple. A total of 5 anthocyanin compounds and 23 flavonol compounds were tentatively identified and quantified. There were obvious differences in the composition of anthocyanin and flavonol among the petals of the 10 Rhododendron species. The color parameter L* decreased as the TA (total anthocyanin) content increased in the red-purple group. However, there was no obvious correlation between the L* value and the TA content in the other sampled Rhododendron species. In this study, the TA values of most of the Rhododendron species were quite low, but the TF (total flavonol) content was high. These results indicate the existence of copigmentation effects in these 10 Rhododendron species.

A novel fabrication of meso-porous silica film by sol-gol of TEOS.

A homogeneous crack-free nano- or meso-porous silica films on silicon was fabricated by colloidal silica sol derived by hydrolyzing tetraethyl orthosilicate (TEOS) catalyzing with (C4H9)4N+OH- in water medium. The solution with ratio of H2O/TEOS> or =15, R4N+ and glycerol as templates, combining with the hydrolyzed intermediate, controlled the silica aggregating; the templated silica film with heterostructure was developed into homogeneous nano-porous then meso-porous silica films after being annealed from 750 degrees C to 850 degrees C; the formation mechanism of the porous silica films was discussed; morphologies of the silica films were characterized. The refractive indexes of the porous silica films were 1.256-1.458, the thermal conductivity < 0.7 W/m/K. The fabricating procedure and the sequence had not been reported before.