The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs.

Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

Mitochondrial creatine kinase 1 regulates the cell cycle in non-small cell lung cancer via activation of cyclin-dependent kinase 4.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the main type of the most common malignant tumor in the world. Previous studies have shown that the expression level of mitochondrial creatine kinase 1 (CKMT1) is abnormal in NSCLC, but the mechanism of its effect remains unclear. Therefore, in this study, we intend to clarify the potential mechanism of CKMT1 in NSCLC and provide the theoretical basis for the clinical application of CKMT1. METHODS: The function of CKMT1 in NSCLC was identified by analyzing the GEO dataset and evaluating using in vitro and in vivo models. Protein mass spectrometry was used to find proteins interacting with CKMT1, and Co-immunoprecipitation (Co-IP) and GST-pull down experiments were used to verify the interaction between proteins. The immunofluorescence (IF) assay was used to explore the functional position of CKMT1 in cells. The effect of CKMT1 expression level on the efficacy of paclitaxel (TAX) in the treatment of NSCLC was analyzed by a combined TAX experiment in vivo and in vitro. RESULTS: CKMT1 expression was increased in NSCLC and CKMT1 promoted the proliferation of NSCLC cells in vitro and in vivo. CKMT1 knockdown resulted in a significantly increased G0/G1 fraction and decreased S phase cell fraction, indicating G1 phase arrest. Mechanically, the cyclin-dependent kinase 4 (CDK4) was identified to interact with CKMT1, and the crucial binding areas were focused on the DH domain of CKMT1 and the N- and C-terminal of CDK4. A fraction of the CDK4 proteins colocalize and interact with the CKMT1 at mitochondria, the level of phosphorylated CDK4 was regulated by CKMT1. Hence, the decrease in CKMT1 expression level could increase the antitumor effect of G2/M cell cycle antagonist-TAX in NSCLC in vitro and in vivo. CONCLUSIONS: CKMT1 could interact with CDK4 in mitochondria and regulate the phosphorylated level of CDK4, thus contributing to the proliferation and cell cycle transition of NSCLC cells. And CKMT1 could be a potential target to improve the sensitivity of chemotherapy based on TAX.

Human Papillomavirus Infection in Penile Cancer: Multidimensional Mechanisms and Vaccine Strategies.

Penile cancer (PC) is a rare male malignant tumor, with early lymph node metastasis and poor prognosis. Human papillomavirus (HPV) plays a key role in the carcinogenesis of PC. This review aims to summarize the association between HPV infection and PC in terms of virus-host genome integration patterns (the disrupted regions in the HPV and PC genome), genetic alterations, and epigenetic regulation (methylation and microRNA modification) occurring in HPV and PC DNA, as well as tumor immune microenvironment reprogramming. In addition, the potential of HPV vaccination strategies for PC prevention and treatment is discussed. Understanding of the HPV-related multidimensional mechanisms and the application of HPV vaccines will promote rational and novel management of PC.

Rice straw for energy and value-added products in China: a review.

The rise of global waste and the decline of fossil fuels are calling for recycling waste into energy and materials. For example, rice straw, a by-product of rice cultivation, can be converted into biogas and by-products with added value, e.g., biofertilizer, yet processing rice straw is limited by the low energy content, high ash and silica, low nitrogen, high moisture, and high-quality variability. Here, we review the recycling of rice straw with focus on the global and Chinese energy situations, conversion of rice straw into energy and gas, biogas digestate management, cogeneration, biogas upgrading, bioeconomy, and life cycle assessment. The quality of rice straw can be improved by pretreatments, such as baling, ensiling, and co-digestion of rice straw with other feedstocks. The biogas digestate can be used to fertilize soils. The average annual potential energy of collectable rice straw, with a lower heating value of 15.35 megajoule/kilogram, over the past ten years (2013-2022) could reach 2.41 × 109 megajoule.

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation.

It has recently been shown that CFIm25, a canonical mRNA 3' processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3' processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25's modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3' processing factor.

A comparative environmental life cycle assessment of rice straw-based bioenergy projects in China.

Bioenergy is a promising solution for greenhouse gas (GHG) emissions mitigation. However, the emissions resulting from the different production stages must be quantified and evaluated. The life cycle assessment (LCA) method was used to compare and quantify the environmental burdens of three rice straw (RS) utilization scenarios for producing biogas, briquette fuel, and syngas. To our knowledge, this is the first study that applies the LCA approach to assess these three bioenergy scenarios in a single study where the main goal was to determine the most sustainable option. A total of 10 mid-point impact categories were investigated. The results indicated that the three scenarios achieved net positive energy and net negative GHG balances. The briquette fuel scenarios had the highest net energy balance (11,115 MJ/tonne dry RS), while the syngas scenario had the highest net GHG (-2,315 kg CO2-eq./tonne dry RS). Moreover, the syngas scenario was the most beneficial to the environment, achieving negative results in 9 out of the 10 impact categories; the largest was marine ecotoxicity (-853,897 kg 1,4-DB-eq./tonne dry RS). The biogas scenario achieved emission savings in 3 out of the 10 categories. Although the briquette fuel scenario had no negative values in the 10 categories, its overall contribution to environmental burdens was relatively low. Overall, the order of the three scenarios in terms of the most sustainable option is syngas > briquette fuel > biogas.

Agro-environmental sustainability of using digestate fertilizer for solanaceous and leafy vegetables cultivation: Insights on fertilizer efficiency and risk assessment.

Digestate generated from anaerobic digestion (AD) has been widely used as digestate fertilizer (DF) for plant growth, but its application should be comprehensively investigated. This study evaluates the effects of different amounts of DF on crop growth, nutrient use efficiency (NUE), soil properties, and potential negative impacts of DF application (salinity and heavy metals (HMs)) with two different crops (Eggplant and Shanghai cabbage). In eggplant cultivation, the yield increased with the increase of DF amount, and the yield of the DF-680 group was the highest (65.4 t/ha) under the highest fertilizer amount. However, due to high ammonia volatilization loss and excessive application, the NUE of DF was only about half of that of chemical fertilizer (CF). Significantly different from eggplant, the high application amount of DF resulted in yield reduction in Shanghai cabbage cultivation. The yield and NUE of the DF-170 group were the highest, the yield was 46.5 t/ha, and the NUE was more than twice compared to CF. Moreover, DF can raise soil nitrogen storage and alleviate soil acidification caused by fertilization in both batches of cultivation. Nevertheless, the electrical conductivity (EC) value of the soil was increased by 2-3 times, and the long-term application may lead to soil salinization. On the other hand, the increase of DF application elevated the content of copper (Cu), zinc (Zn), and cadmium (Cd) in soil significantly but did not cause HMs contamination in crops and tillage soil. In summary, reasonable application amounts and methods should be considered when applying DF.

U1 snRNP telescripting: molecular mechanisms and beyond.

U1 snRNP is one of the most abundant ribonucleoprotein (RNP) complexes in eukaryotic cells and is estimated to be approximately 1 million copies per cell. Apart from its canonical role in mRNA splicing, this complex has emerged as a key regulator of eukaryotic mRNA length via inhibition of mRNA 3'-end processing at numerous intronic polyadenylation sites, in a process that is also termed 'U1 snRNP telescripting'. Several reviews have extensively described the concept of U1 telescripting and subsequently highlighted its potential impacts in mRNA metabolism. Here, we review what is currently known regarding the underlying mechanisms of this important phenomenon and discuss open questions and future challenges.

A potential mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 3' processing.

It is well established that U1 snRNP inhibits the cleavage of cryptic polyadenylation site (PAS) within introns, thereby facilitating full-length mRNA transcription for numerous genes in vertebrate cells, yet the underlying mechanism remains poorly understood. Here, by using a model PAS of wdr26 mRNA, we show that U1 snRNP predominantly interferes with the association of PAS with a core 3' processing factor CstF64, which can promote the cleavage step of mRNA 3' processing. Furthermore, we provide evidence that U1A, a component of U1 snRNP, might directly interfere with CstF64 binding on PAS through its RNA binding capacity. Consistently, U1A could potentially associate with U1-suppressed intronic PASs at the transcriptome level in human cells, showing a binding peak ∼50 nt downstream of the cleavage site, as revealed by U1A iCLIP-seq (individual-nucleotide resolution UV crosslinking and immunoprecipitation coupled with RNA sequencing) analysis. Together, our data suggest a molecular mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 3' processing. More generally, we argue that U1 snRNP might inhibit the usage of cryptic PASs through disturbing the recruitment of core 3' processing factors.

Exome sequencing reveals the genetic landscape and frequent inactivation of PCDHB3 in Chinese rectal cancers.

Colorectal cancer (CRC) is the third most common cancer worldwide, with more than 1.3 million new cases and 690 000 deaths each year. In China, the incidence of CRC has increased dramatically due to dietary and lifestyle changes, to become the fifth leading cause of cancer-related death. Here, we performed whole-exome sequencing in 50 rectal cancer cases among the Chinese population as part of the International Cancer Genome Consortium research project. Frequently mutated genes and enriched pathways were identified. Moreover, a previously unreported gene, PCDHB3, was found frequently mutated in 5.19% cases. Additionally, PCDHB3 expression was found decreased in 81.6% of CRC tissues and all eight CRC cell lines tested. Low expression and cytoplasmic localization of PCDHB3 predict poor prognosis in advanced CRC. Copy number decrease and/or CpG island hypermethylation contributes to the pervasive decreased expression of PCDHB3. PCDHB3 inhibits CRC cell proliferation, migration, and epithelial-mesenchymal transition. The tumor-suppressive effects of PCDHB3 are partially due to inhibition of NF-κB transcriptional activity through K63 deubiquitination of p50 at lysine 244/252, which increases the binding affinity of inactive p50 homodimer to κB DNA, resulting in competitive inhibition of the transcription of NF-κB target genes by p65 dimers. Our study identified PCDHB3 as a novel tumor suppressor in CRC via inhibition of the NF-κB pathway, and its expression and localization may serve as prognostic markers for advanced CRC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recovery of unstable digestion of vegetable waste by adding trace elements using the bicarbonate alkalinity to total alkalinity ratio as an early warning indicator.

Vegetable waste (VW) is highly perishable and susceptible to acidification during anaerobic digestion, which inhibits biogas production. Effective process monitoring, diagnosis and control are necessary to maintain stable anaerobic digestion at a high organic loading rate (OLR). Continuous mesophilic digestion was conducted at OLRs of 0.5, 1.0, 1.5, 2.0, 3.0, 3.5 and 4.0 g volatile solids (VS)/(L d) with effluent recirculation (ER) in a reactor with total volume of 70 L. The effectiveness of three early warning indicators was validated. The ability of trace elements (TEs) (Fe, Co and Ni) to recover unstable VW digestion systems was evaluated. The results showed that the ratio of bicarbonate alkalinity (BA) to total alkalinity (TA) was a more effective warning indicator than the ratios of methane (CH4) to carbon dioxide and volatile fatty acids (VFAs) to TA. When the ratio of BA/TA was lower than 0.9, the digestion system tended to be unstable. ER maintained a stable OLR of 1.5 g VS/(L d). The addition of TEs achieved a maximum stable OLR of 3.5 g VS/(L d) with an average volumetric biogas production rate of 1.91 L/(L d). Severe VFAs accumulation and unrecoverable instability occurred at an OLR of 4.0 g VS/(L d). The supplementation of ammonium bicarbonate was not useful for the recovery of the unstable system when the OLR was greater than 3.5 g VS/(L d) for the digestion of VW. The specific methane production was approximately 340 L/kg VS during the stable period with a digestion efficiency of 85%.

Laparoscopic common bile duct exploration with primary closure is safe for management of choledocholithiasis in elderly patients.

BACKGROUND: Laparoscopic common bile duct exploration (LCBDE) is one of the minimally invasive options for choledocholithiasis. Primary closure of the common bile duct (CBD) upon completion of laparoscopic choledochotomy is safe in selected patients. The present study aimed to evaluate the feasibility and safety of primary closure of CBD after LCBDE in patients aged 70 years or older. METHODS: A total of 116 patients (51 males and 65 females) who suffered from choledocholithiasis and underwent primary closure of the CBD (without T-tube drainage) after LCBDE from January 2003 to December 2017 were recruited. They were classified into two groups according to age: group A (≥70 years, n = 56), and group B (<70 years, n = 60). The preoperative characteristics, intraoperative details, and postoperative outcomes of the two groups were evaluated. RESULTS: The mean operative time was 172.02 min for group A and 169.92 min for group B (P = 0.853). The mean hospital stay was 7.40 days for group A and 5.38 days for group B (P < 0.001). Bile leakage occurred in two patients in group A and one in group B (3.57% vs 1.67%, P = 0.952). There were no significant differences in the rates of postoperative complications and mortality between the two groups. At median follow-up time of 60 months, stone recurrence was detected in one patient in group A and two in group B (1.79% vs 3.33%, P = 1.000). Stenosis of CBD was not observed in group A and slight stenosis in one patient in group B (0 vs 1.67%, P = 1.000). CONCLUSION: Primary closure of the CBD upon completion of laparoscopic choledochotomy is safe and feasible in elderly patients ≥70 years old.

ARHGEF19 interacts with BRAF to activate MAPK signaling during the tumorigenesis of non-small cell lung cancer.

Rho guanine nucleotide exchange factors (RhoGEFs) are proteins that activate Rho GTPases in response to extracellular stimuli and regulate various biologic processes. ARHGEF19, one of RhoGEFs, was reported to activate RhoA in the Wnt-PCP pathway controlling convergent extension in Xenopus gastrulation. The goal of our study was to identify the role and molecular mechanisms of ARHGEF19 in the tumorigenesis of non-small cell lung cancer (NSCLC). ARHGEF19 expression was significantly elevated in NSCLC tissues, and ARHGEF19 levels were significantly associated with lymph node status, distant metastasis and TNM stage; Patients with high ARHGEF19 levels had poor overall survival (OS) and progression-free survival (PFS). Our investigations revealed that ARHGEF19 overexpression promoted the cell proliferation, invasion and metastasis of lung cancer cells, whereas knockdown of this gene inhibited these processes. Mechanistically, ARHGEF19 activated the mitogen-activated protein kinase (MAPK) pathway in a RhoA-independent manner: ARHGEF19 interacted with BRAF and facilitated the phosphorylation of its downstream kinase MEK1/2; both the Dbl homology (DH) and Pleckstrin homology (PH) domains of ARHGEF19 were indispensable for the phosphorylation of MEK1/2. Furthermore, downregulation of miR-29b was likely responsible for the increased expression of ARHGEF19 in lung cancer tissues and, consequently, the abnormal activation of MAPK signaling. These findings suggest that ARHGEF19 upregulation, due to the low expression of miR-29 in NSCLC tissues, may play a crucial role in NSCLC tumorigenesis by activating MAPK signaling. ARHGEF19 could serve as a negative prognostic marker as well as a therapeutic target for NSCLC patients.

Interaction of NS2 with AIMP2 facilitates the switch from ubiquitination to SUMOylation of M1 in influenza A virus-infected cells.

UNLABELLED: Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication. IMPORTANCE: Although the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.

OTUB1 promotes metastasis and serves as a marker of poor prognosis in colorectal cancer.

BACKGROUND: OTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) is a deubiquitinating enzyme (DUB) that belongs to the OTU (ovarian tumor) superfamily. The aim of this study was to clarify the role of OTUB1 in colorectal cancer (CRC) and to identify the mechanism underlying its function. METHODS: Two hundred and sixty CRC samples were subjected to association analysis of OTUB1 expression and clinicopathological variables using immunohistochemical (IHC) staining. Overexpression of OTUB1 was achieved in SW480 and DLD-1 cells, and downregulation of OTUB1 was employed in SW620 cells. Then, migration and invasion assays were performed, and markers of the epithelial-mesenchymal transition (EMT) were analyzed. In addition, hepatic metastasis models in mice were used to validate the function of OTUB1 in vivo. RESULTS: OTUB1 was overexpressed in CRC tissues, and the expression level of OTUB1 was associated with metastasis. A high expression level of OTUB1 was also associated with poor survival, and OTUB1 served as an independent prognostic factor in multivariate analysis. OTUB1 also promoted the metastasis of CRC cell lines in vitro and in vivo by regulating EMT. CONCLUSIONS: OTUB1 promotes CRC metastasis by facilitating EMT and acts as a potential distant metastasis marker and prognostic factor in CRC. Targeting OTUB1 may be helpful for the treatment of CRC.

Genetic and epigenetic down-regulation of microRNA-212 promotes colorectal tumor metastasis via dysregulation of MnSOD.

BACKGROUND & AIMS: Altered functions of microRNAs (miRNAs) have been associated with colorectal cancer (CRC). miR-212 is transcribed from a stable intron of a non-protein coding gene, and is reportedly down-regulated in different tumor types. We investigated the role of miR-212 in colorectal carcinogenesis and progression. METHODS: We analyzed the expression of miR-212 by real-time polymerase chain reaction (PCR) analysis of colorectal cell lines and 180 paired tumor samples and surrounding healthy tissue. We overexpressed and knocked down miR-212 in CRC cell lines and assessed the in vitro effects. We also studied the effects of miR-212 overexpression on metastasis of tumors grown from HCT116 cells in nude mice. RESULTS: Overexpression of miR-212 inhibited CRC cell migration and invasion in vitro and formation of intrahepatic and pulmonary metastasis in vivo. We identified manganese superoxide dismutase (MnSOD) messenger RNA as a direct target of miR-212, and observed an inverse correlation between the level of miR-212 and MnSOD protein in colorectal tumor samples. MnSOD was required for down-regulation of epithelial markers and up-regulation of mesenchymal markers in CRC cells, indicating that it promoted the epithelial-mesenchymal transition. Overexpression of miR-212 reduced the levels of MnSOD to block the epithelial-mesenchymal transition process. Loss of heterozygosity and promoter hypermethylation each contributed to the down-regulation of miR-212. Reduced levels of miR-212 were associated with a more aggressive tumor phenotype and short disease-free survival times of patients (P = .0045; overall survival, P = .0015). CONCLUSIONS: miR-212 is down-regulated in human CRC tissues via genetic and epigenetic mechanisms. miR-212 might prevent tumor progression by targeting MnSOD messenger RNA; reduction of miR-212 could be a prognostic marker for patients with CRC. miR-212 and MnSOD might also be therapeutic targets for cancer.

Incorporation of copper ion promoted adsorption of anionic dye (Acid Yellow 36) by acrolein-crosslinked polyethyleneimine/chitosan hydrogel: Adsorption, dynamics, and mechanisms.

In this study, a novel adsorbent, A-PEI/CS-Cu2+, was developed by crosslinking polyethyleneimine/chitosan hydrogel with acrolein and loading it with copper ions. The adsorption process of A-PEI/CS-Cu2+ on the anionic dye acid yellow 36 (AY36) was investigated by kinetic, isothermal and thermodynamic modeling. It was noteworthy that A-PEI/CS-Cu2+ exhibited rapid adsorption with a 90 % removal rate achieved within just 5 min, which was much faster than the adsorption rate of A-PEI/CS without load of copper ions and showed its potential for rapid adsorption applications. The maximum adsorption capacity for AY36 could reach up to 3114 mg g-1. In addition, the high concentration of saline wastewater was found to have almost no effect on the adsorption reaction in the salt effect test experiment. In five desorption-regeneration cycle experiments, the sample exhibited good recyclability and regeneration performance. The driving force of the adsorption process mainly originated from the electrostatic interaction, hydrogen bonding, and intermolecular interaction, in which the addition of copper ions led to the enhancement of the electrostatic interaction and chelation between A-PEI/CS-Cu2+ and AY36. Overall, the findings suggest the excellent potential of A-PEI/CS-Cu2+ for rapid and efficient adsorption, as well as its suitability for practical applications in wastewater treatment.

Construction of imine-hydrazone dual linkage covalent organic frameworks.

The development of dual linkage covalent organic frameworks (DL-COFs) is fundamentally important for creating diversified structures and functions. Herein, imine-hydrazone DL-COFs were synthesized for the first time. Three novel imine-hydrazone DL-COFs have been constructed with bifunctional molecule 4-amino-2-methoxybenzohydrazide as the key building block via imine and hydrazone condensations simultaneously.

Electrochemical stereoselective synthesis of polysubstituted 1,4-dicarbonyl Z-alkenes via three-component coupling of sulfoxonium ylides and alkynes with water.

The first straightforward strategy for the synthesis of 1,4-dicarbonyl Z-alkenes has been developed via an electrochemical cross-coupling reaction of sulfoxonium ylides and alkynes with water. The metal-free protocol showed an easy-to-handle nature, good functional group tolerance, and high Z-stereoselectivity, which is rare in previous cases. The proposed reaction mechanism was convincingly established by carrying out a series of control experiments, cyclic voltammetry experiments, and density functional theory (DFT) studies.

Nilotinib in combination with sunitinib renders MCL-1 for degradation and activates autophagy that overcomes sunitinib resistance in renal cell carcinoma.

PURPOSE: Sunitinib is a recommended drug for metastatic renal cell carcinoma (RCC). However, the therapeutic potential of sunitinib is impaired by toxicity and resistance. Therefore, we seek to explore a combinatorial strategy to improve sunitinib efficacy of low-toxicity dose for better clinical application. METHODS: We screen synergistic reagents of sunitinib from a compound library containing 1374 FDA-approved drugs by in vitro cell viability evaluation. The synergistically antiproliferative and proapoptotic effects were demonstrated on in vitro and in vivo models. The molecular mechanism was investigated by phosphoproteomics, co-immunoprecipitation, immunofluorescence and western-blot assays, etc. RESULTS: From the four-step screening, nilotinib stood out as a potential synergistic killer combined with sunitinib. Subsequent functional evaluation demonstrated that nilotinib and sunitinib synergistically inhibit RCC cell proliferation and promote apoptosis in vitro and in vivo. Mechanistically, nilotinib activates E3-ligase HUWE1 and in combination with sunitinib renders MCL-1 for degradation via proteasome pathway, resulting in the release of Beclin-1 from MCL-1/Beclin-1 complex. Subsequently, Beclin-1 induces complete autophagy flux to promote antitumor effect. CONCLUSION: Our findings revealed that a novel mechanism that nilotinib in combination with sunitinib overcomes sunitinib resistance in RCC. Therefore, this novel rational combination regimen provides a promising therapeutic avenue for metastatic RCC and rationale for evaluating this combination clinically.