VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Fructose-1,6-bisphosphatase 1 dephosphorylates and inhibits TERT for tumor suppression.

Telomere dysfunction is intricately linked to the aging process and stands out as a prominent cancer hallmark. Here we demonstrate that telomerase activity is differentially regulated in cancer and normal cells depending on the expression status of fructose-1,6-bisphosphatase 1 (FBP1). In FBP1-expressing cells, FBP1 directly interacts with and dephosphorylates telomerase reverse transcriptase (TERT) at Ser227. Dephosphorylated TERT fails to translocate into the nucleus, leading to the inhibition of telomerase activity, reduction in telomere lengths, enhanced senescence and suppressed tumor cell proliferation and growth in mice. Lipid nanoparticle-mediated delivery of FBP1 mRNA inhibits liver tumor growth. Additionally, FBP1 expression levels inversely correlate with TERT pSer227 levels in renal and hepatocellular carcinoma specimens and with poor prognosis of the patients. These findings demonstrate that FBP1 governs cell immortality through its protein phosphatase activity and uncover a unique telomerase regulation in tumor cells attributed to the downregulation or deficiency of FBP1 expression.

Nuclear PGK1 Alleviates ADP-Dependent Inhibition of CDC7 to Promote DNA Replication.

DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development.

The moonlighting function of glycolytic enzyme enolase-1 promotes choline phospholipid metabolism and tumor cell proliferation.

Aberrantly upregulated choline phospholipid metabolism is a novel emerging hallmark of cancer, and choline kinase α (CHKα), a key enzyme for phosphatidylcholine production, is overexpressed in many types of human cancer through undefined mechanisms. Here, we demonstrate that the expression levels of the glycolytic enzyme enolase-1 (ENO1) are positively correlated with CHKα expression levels in human glioblastoma specimens and that ENO1 tightly governs CHKα expression via posttranslational regulation. Mechanistically, we reveal that both ENO1 and the ubiquitin E3 ligase TRIM25 are associated with CHKα. Highly expressed ENO1 in tumor cells binds to I199/F200 of CHKα, thereby abrogating the interaction between CHKα and TRIM25. This abrogation leads to the inhibition of TRIM25-mediated polyubiquitylation of CHKα at K195, increased stability of CHKα, enhanced choline metabolism in glioblastoma cells, and accelerated brain tumor growth. In addition, the expression levels of both ENO1 and CHKα are associated with poor prognosis in glioblastoma patients. These findings highlight a critical moonlighting function of ENO1 in choline phospholipid metabolism and provide unprecedented insight into the integrated regulation of cancer metabolism by crosstalk between glycolytic and lipidic enzymes.

Base editing of the mutated TERT promoter inhibits liver tumor growth.

BACKGROUND AND AIMS: Base editing has shown great potential for treating human diseases with mutated genes. However, its potential for treating HCC has not yet been explored. APPROACH AND RESULTS: We employed adenine base editors (ABEs) to correct a telomerase reverse transcriptase ( TERT ) promoter mutation, which frequently occurs in various human cancers, including HCC. The mutated TERT promoter -124 C>T is corrected to -124 C by a single guide (sg) RNA-guided and deactivated Campylobacter jejuni Cas9 (CjCas9)-fused adenine base editor (CjABE). This edit impairs the binding of the E-twenty six/ternary complex factor transcription factor family, including E-twenty six-1 and GABPA, to the TERT promoter, leading to suppressed TERT promoter and telomerase activity, decreased TERT expression and cell proliferation, and increased cell senescence. Importantly, injection of adeno-associated viruses expressing sgRNA-guided CjABE or employment of lipid nanoparticle-mediated delivery of CjABE mRNA and sgRNA inhibits the growth of liver tumors harboring TERT promoter mutations. CONCLUSIONS: These findings demonstrate that a sgRNA-guided CjABE efficiently converts the mutated TERT promoter -124 C>T to -124 C in HCC cells and underscore the potential to treat HCC by the base editing-mediated correction of TERT promoter mutations.

Safety and Feasibility of Regional Citrate Anticoagulation for Continuous Renal Replacement Therapy With Calcium-Containing Solutions: A Randomized Controlled Trial.

BACKGROUND: Calcium-free (Ca-free) solutions are theoretically the most ideal for regional citrate anticoagulation (RCA) in continuous renal replacement therapy (CRRT). However, the majority of medical centers in China had to make a compromise of using commercially available calcium-containing (Ca-containing) solutions instead of Ca-free ones due to their scarcity. This study was designed to probe into the potential of Ca-containing solution as a secure and efficient substitution for Ca-free solutions. METHODS: In this prospective, randomized single-center trial, 99 patients scheduled for CRRT were randomly assigned in a 1:1:1 ratio to one of three treatment groups: continuous veno-venous hemodialysis Ca-free dialysate (CVVHD Ca-free) group, continuous veno-venous hemodiafiltration calcium-free dialysate (CVVHDF Ca-free) group, and continuous veno-venous hemodiafiltration Ca-containing dialysate (CVVHDF Ca-containing) group at cardiac intensive care unit (CICU). The primary endpoint was the incidence of metabolic complications. The secondary endpoints included premature termination of treatment, thrombus of filter, and bubble trap after the process. RESULTS: The incidence of citrate accumulation (18.2% vs. 12.1% vs. 21.2%) and metabolic alkalosis (12.1% vs. 0% vs. 9.1%) did not significantly differ among three groups (p > 0.05 for both). The incidence of premature termination was comparable among the groups (18.2% vs. 9.1% vs. 9.1%, p = 0.582). The thrombus level of the filter and bubble trap was similar in the three groups (p > 0.05 for all). CONCLUSIONS: In RCA-CRRT for CICU population, RCA-CVVHDF with Ca-containing solutions and traditional RCA with Ca-free solutions had a comparable safety and feasibility. TRIAL REGISTRATION: ChiCTR2100048238 in the Chinese Clinical Trial Registry.

3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines inhibit tubulin polymerisation and act as anticancer agents.

A series of cis-restricted 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines as novel tubulin polymerisation inhibitors was designed based on molecular docking. Compound 9p, exhibited potent antiproliferative activity against HeLa, MCF-7, and A549 cell lines. Mechanism studies indicated that 9p potently inhibited tubulin polymerisation and disrupted the microtubule dynamics of tubulin in HeLa cells. Moreover, 9p could cause G2/M phase cell cycle arrest and apoptosis in HeLa cells. In addition, the prediction of physicochemical properties disclosed that 9p conformed well to the Lipinski's rule of five. The initial results suggest that the 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines could serve as a promising scaffold for the development of novel anticancer drugs.

Novel application potential of cinaciguat in the treatment of mixed hyperlipidemia through targeting PTL/NPC1L1 and alleviating intestinal microbiota dysbiosis and metabolic disorders.

Mixed hyperlipidemia, characterized by high levels of triglycerides and cholesterol, is a key risk factor leading to atherosclerosis and other cardiovascular diseases. Existing clinical drugs usually only work on a single indicator, decreasing either triglyceride or cholesterol levels. Developing dual-acting agents that reduce both triglycerides and cholesterol remains a great challenge. Pancreatic triglyceride lipase (PTL) and Niemann-Pick C1-like 1 (NPC1L1) have been identified as crucial proteins in the transport of triglycerides and cholesterol. Here, cinaciguat, a known agent used in the treatment of acute decompensated heart failure, was identified as a potent dual inhibitor targeting PTL and NPC1L1. We presented in vitro evidence from surface plasmon resonance analysis that cinaciguat interacted with PTL and NPC1L1. Furthermore, cinaciguat exhibited potent PTL-inhibition activity. Fluorescence-labeled cholesterol uptake analysis and confocal imaging showed that cinaciguat effectively inhibited cholesterol uptake. In vivo evaluation showed that cinaciguat significantly reduced the plasma levels of triglycerides and cholesterol, and effectively alleviated high-fat diet-induced intestinal microbiota dysbiosis and metabolic disorders. These results collectively suggest that cinaciguat has the potential to be further developed for the therapy of mixed hyperlipidemia.

Photosensitizer-based small molecule theranostic agents for tumor-targeted monitoring and phototherapy.

Small molecule theranostic agents for tumor treatment exhibited triadic properties in tumor targeting, imaging, and therapy, which have attracted increasing attention as a potential complement for, or improved to, classical small molecule antitumor drugs. Photosensitizer have dual functions of imaging and phototherapy, and have been widely used in the construction of small molecule theranostic agents over the last decade. In this review, we summarized representative agents that have been studied in the field of small molecule theranostic agents based on photosensitizer in the last decade, and highlighted their characteristics and application in tumor-targeted monitoring and phototherapy. The challenges and future perspectives of photosensitizers in building small molecule theranostic agents for diagnosis and therapy of tumors were also discussed.

Intelligent delivery system targeting PD-1/PD-L1 pathway for cancer immunotherapy.

The drugs targeting the PD-1/PD-L1 pathway have gained abundant clinical applications for cancer immunotherapy. However, only a part of patients benefit from such immunotherapy. Thus, brilliant novel tactic to increase the response rate of patients is on the agenda. Nanocarriers, particularly the rationally designed intelligent delivery systems with controllable therapeutic agent release ability and improved tumor targeting capacity, are firmly recommended. In light of this, state-of-the-art nanocarriers that are responsive to tumor-specific microenvironments (internal stimuli, including tumor acidic microenvironment, high level of GSH and ROS, specifically upregulated enzymes) or external stimuli (e.g., light, ultrasound, radiation) and release the target immunomodulators at tumor sites feature the advantages of increased anti-tumor potency but decreased off-target toxicity. Given the fantastic past achievements and the rapid developments in this field, the future is promising. In this review, intelligent delivery platforms targeting the PD-1/PD-L1 axis are attentively appraised. Specifically, mechanisms of the action of these stimuli-responsive drug release platforms are summarized to raise some guidelines for prior PD-1/PD-L1-based nanocarrier designs. Finally, the conclusion and outlook in intelligent delivery system targeting PD-1/PD-L1 pathway for cancer immunotherapy are outlined.

S100P is Overexpressed in Squamous Cell and Adenosquamous Carcinoma Subtypes of Endometrial Cancer and Promotes Cancer Cell Proliferation and Invasion.

S100P is known to affect tumor development and metastasis of various cancers, but its role in endometrial cancer is unclear. We reported that S100P expression was dramatically elevated in both endometrial squamous cell carcinoma and adenosquamous carcinoma, but not in adenocarcinoma and normal endometrial samples. Moreover, we revealed an oncogenic role of S100P promoting cell proliferation, invasion, and migration while reducing apoptosis, possibly via its upregulation and/or activation of receptors of advanced glycation end products and consequently the oncogenic PI3K-AKT and MAPK pathways. Therefore, S100P might be a specific biomarker and a potential drug target for squamous cell and adenosquamous carcinoma subtypes of endometrial cancer.

Treatment of Calcified Insertional Achilles Tendinopathy by the Posterior Midline Approach.

The present study investigated the clinical outcomes of the posterior midline approach in the treatment of 34 patients with significantly calcified insertional Achilles tendinopathy. The posterior midline approach was applied for the surgical treatment of 34 patients with chronic significantly calcified insertional Achilles tendinopathy after failed conservative treatment. Gastrocnemius recession was performed simultaneously for patients with gastrocnemius contracture. The Fowler-Philip angle and parallel pitch lines were measured before surgery, and the visual analog scale, Tegner score, and Victorian Institute of Sport tendon study group score were recorded before and after surgery. The mean follow-up period was 45.2 ± 17.7 (range 24 to 84) months. After surgery, the visual analog scale score had decreased notably, and the Tegner score and Victorian Institute of Sport tendon study group score had increased significantly. The posterior midline approach can achieve satisfactory outcomes in the treatment of significantly calcified insertional Achilles tendinopathy, and gastrocnemius recession (Strayer procedure) should be performed for patients with gastrocnemius contracture to improve the surgical outcome.

AGEs and chronic subclinical inflammation in diabetes: disorders of immune system.

Chronic subclinical inflammation represents a risk factor of type 2 diabetes and several diabetes complications, including neuropathy and atherosclerosis including macro-vasculopathy and micro-vasculopathy. However, the inflammatory response in the diabetic wound was shown to be remarkably hypocellular, unregulated and ineffective. Advanced glycation end products (AGEs) and one of its receptors, RAGE, were involved in inducing chronic immune imbalance in diabetic patients. Such interactions attracts immune cell into diffused glycated tissue and activates these cells to induce inflammatory damage, but disturbs the normal immune rhythm in diabetic wound. Traditional measurements of AGEs are high-performance liquid chromatography and immunohistochemistry staining, but their application faces the limitations including complexity, cost and lack of reproducibility. A new noninvasive method emerged in 2004, using skin autofluorescence as indicator for AGEs accumulation. It had been reported to be informative in evaluating the chronic risk of diabetic patients. Studies have indicated therapeutic potentials of anti-AGE recipes. These recipes can reduce AGE absorption/de novo formation, block AGE-RAGE interaction and arrest downstream signaling after RAGE activation.

Overexpression of zinc-finger protein 418 inhibits pathological cardiac remodelling after acute myocardial infarction.

AIMS: Zinc-finger protein 418 (ZNF418) has been confirmed to be expressed in myocardial tissue. However, the role and mechanism of ZNF418 in pathological myocardial remodelling after myocardial infarction (MI) have not been reported. This study was to elucidate the effect and mechanism of ZNF418 on ventricular remodelling after MI in mice. METHODS AND RESULTS: MI mice and H9c2 cardiomyocytes were used to conduct in vivo and in vitro experiments, respectively. ZNF418 expression was regulated by adeno-associated virus 9 and adenovirus vectors. Pathological analysis, echocardiography, and molecular analysis were performed. ZNF418 was down-regulated in the left ventricular tissues of post-MI mice. In contrast, ZNF418 overexpression decreased mortality and improved cardiac function in MI mice. The MI mice exhibited a significantly increased cross-sectional area of myocardial cells and elevated protein expression levels of myocardial hypertrophy markers ANP, BNP, and ß-MHC (all P < 0.05). Moreover, a significantly increased area of myocardial fibrosis and protein expression levels of myocardial fibrosis markers collagen I, collagen III, and CTGF were observed in MI mice (all P < 0.05) in MI mice. All of the above negative effects in MI mice were ameliorated in ZNF418 overexpressed mice (all P < 0.05). Mechanistically, ZNF418 overexpression inhibited the activation of the MAPK signalling pathway, as evidenced by the in vivo and in vitro experiments. CONCLUSIONS: Overexpression of ZNF418 could improve cardiac function and inhibit pathological cardiac remodelling by inhibiting the MAPK signalling pathway in post-MI mice.

The timing for embryo transfer after antibiotic therapy for chronic endometritis.

OBJECTIVE: To explore the optimal timing of embryo transfer after the first round treatment of chronic endometritis (CE) in vitro. MATERIALS AND METHODS: A total of 184 patients were recruited from a retrospective analysis of a large university-affiliated reproduction center in 2021. Some people chose to undergo embryo transfer in the same menstrual cycle with the first round of antibiotic treatment (Group 1, n = 29). Others received embryo transfer in the next cycle after the first round of treatment (Group 2, n = 69) or even one cycle later (Group 3，n = 96). RESULTS: Patients in Group 1 got significantly lower biochemical pregnancy rate and clinical pregnancy rate and live birth rate than Group 2 (p < 0.05) and also Group 3 (p < 0.05). Then after comparing the influence factors, we found embryo transfer in the next cycle after antibiotic treatment had a higher clinical pregnancy rate than group 1 (OR = 3.2 p < 0.05) and group 3(OR = 2.5, p < 0.05). The live birth rate in group 2 was higher than group 1(OR = 3.5, p < 0.05). CONCLUSION: These findings illustrate that embryo transfer in the next menstrual cycle is the optimal time. Embryo transfer in the same menstrual cycle with the first round of treatment reduces the pregnancy rate.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Clinical advances in TNC delivery vectors and their conjugate agents.

Tenascin C (TNC), a glycoprotein that is abundant in the tumor extracellular matrix (ECM), is strongly overexpressed in tumor tissues but virtually undetectable in most normal tissues. Many TNC antibodies, peptides, aptamers, and nanobodies have been investigated as delivery vectors, including 20A1, α-A2, α-A3, α-IIIB, α-D, BC-2, BC-4 BC-8, 81C6, ch81C6, F16, FHK, Ft, Ft-NP, G11, G11-iRGD, GBI-10, 19H12, J1/TN1, J1/TN2, J1/TN3, J1/TN4, J1/TN5, NJT3, NJT4, NJT6, P12, PL1, PL3, R6N, SMART, ST2146, ST2485, TN11, TN12, TNFnA1A2-Fc, TNfnA1D-Fc, TNfnBD-Fc, TNFnCD-Fc, TNfnD6-Fc, TNfn78-Fc, TTA1, TTA1.1, and TTA1.2. In particular, BC-2, BC-4, 81C6, ch81C6, F16, FHK, G11, PL1, PL3, R6N, ST2146, TN11, and TN12 have been tested in human tissues. G11-iRGD and simultaneous multiple aptamers and arginine-glycine-aspartic acid (RGD) targeting (SMART) may be assessed in clinical trials because G11, iRGD and AS1411 (SMART components) are already in clinical trials. Many TNC-conjugate agents, including antibody-drug conjugates (ADCs), antibody fragment-drug conjugates (FDCs), immune-stimulating antibody conjugates (ISACs), and radionuclide-drug conjugates (RDCs), have been investigated in preclinical and clinical trials. RDCs investigated in clinical trials include 111In-DTPA-BC-2, 131I-BC-2, 131I-BC-4, 90Y-BC4, 131I81C6, 131I-ch81C6, 211At-ch81C6, F16124I, 131I-tenatumomab, ST2146biot, FDC 131I-F16S1PF(ab')2, and ISAC F16IL2. ADCs (including FHK-SSL-Nav, FHK-NB-DOX, Ft-NP-PTX, and F16*-MMAE) and ISACs (IL12-R6N and 125I-G11-IL2) may enter clinical trials because they contain components of marketed treatments or agents that were investigated in previous clinical studies. This comprehensive review presents historical perspectives on clinical advances in TNC-conjugate agents to provide timely information to facilitate tumor-targeting drug development using TNC.

Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance ß-oxidation and tumor cell proliferation.

Lipid droplet tethering with mitochondria for fatty acid oxidation is critical for tumor cells to counteract energy stress. However, the underlying mechanism remains unclear. Here, we demonstrate that glucose deprivation induces phosphorylation of the glycolytic enzyme phosphofructokinase, liver type (PFKL), reducing its activity and favoring its interaction with perilipin 2 (PLIN2). On lipid droplets, PFKL acts as a protein kinase and phosphorylates PLIN2 to promote the binding of PLIN2 to carnitine palmitoyltransferase 1A (CPT1A). This results in the tethering of lipid droplets and mitochondria and the recruitment of adipose triglyceride lipase to the lipid droplet-mitochondria tethering regions to engage lipid mobilization. Interfering with this cascade inhibits tumor cell proliferation, promotes apoptosis and blunts liver tumor growth in male mice. These results reveal that energy stress confers a moonlight function to PFKL as a protein kinase to tether lipid droplets with mitochondria and highlight the crucial role of PFKL in the integrated regulation of glycolysis, lipid metabolism and mitochondrial oxidation.

Doxorubicin prodrug-based nanomedicines for the treatment of cancer.

The chemotherapeutic drug of doxorubicin (DOX) has witnessed widespread applications for treating various cancers. DOX-treated dying cells bear cellular modifications which allow enhanced presentation of tumor antigen and neighboring dendritic cell activation. Furthermore, DOX also facilitate the immune-mediated clearance of tumor cells. However, disadvantages such as severe off-target toxicity, and prominent hydrophobicity have resulted in unsatisfactory clinical therapeutic outcomes. The effective delivery of DOX drug molecules is still challenging despite the rapid advances in nanotechnology and biomaterials. Huge progress has been witnessed in DOX nanoprodrugs owing to their brilliant benefits such as tumor stimuli-responsive drug release capacity, high drug loading efficiency and so on. This review summarized recent progresses of DOX prodrug-based nanomedicines to provide deep insights into future development and inspire researchers to explore DOX nanoprodrugs with real clinical applications.