KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Co-Expression of Chromatin Assembly Factor 1 Subunit A and Proliferating Cell Nuclear Antigen Is a Prognostic Biomarker of Esophageal Cancer.

(1) Background: Esophageal cancer (EC) is an important global health challenge. Due to the lack of necessary biomarkers and therapeutic targets, the survival of EC patients is poor. The EC proteomic data of 124 patients recently published by our group provides a database for research in this field. (2) Methods: Bioinformatics analysis was used to identify DNA replication and repair-related proteins in EC. Proximity ligation assay, colony formation assay, DNA fiber assay, and flow cytometry were used to study the effects of related proteins on EC cells. Kaplan-Meier survival analysis was used to evaluate the relationship between gene expression and the survival time of EC patients. (3) Results: Chromatin assembly factor 1 subunit A (CHAF1A) was highly correlated with proliferating cell nuclear antigen (PCNA) expression in EC. CHAF1A and PCNA colocalized in the nucleus of EC cells. Compared with the knockdown of CHAF1A or PCNA alone, the double knockdown of CHAF1A and PCNA could significantly inhibit EC cell proliferation. Mechanistically, CHAF1A and PCNA synergistically accelerated DNA replication and promoted S-phase progression. EC patients with high expression of both CHAF1A and PCNA had a worse survival rate. (4) Conclusion: we identify CHAF1A and PCNA as key cell cycle-related proteins leading to the malignant progression of EC, and these proteins could serve as important prognostic biomarkers and targets for EC.

The post-translational modification of Fascin: impact on cell biology and its associations with inhibiting tumor metastasis.

The post-translational modifications (PTMs), which are crucial in the regulation of protein functions, have great potential as biomarkers of cancer status. Fascin (Fascin actin-bundling protein 1, FSCN1), a key protein in the formation of filopodia that is structurally based on actin filaments (F-actin), is significantly associated with tumor invasion and metastasis. Studies have revealed various regulatory mechanisms of human Fascin, including PTMs. Although a number of Fascin PTM sites have been identified, their exact functions and clinical significance are much less explored. This review explores studies on the functions of Fascin and briefly discusses the regulatory mechanisms of Fascin. Next, to review the role of Fascin PTMs in cell biology and their associations with metastatic disease, we discuss the advances in the characterization of Fascin PTMs, including phosphorylation, ubiquitination, sumoylation, and acetylation, and the main regulatory mechanisms are discussed. Fascin PTMs may be potential targets for therapy for metastatic disease.

AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression.

Fascin is the main actin-bundling protein in filopodia and is highly expressed in metastatic tumor cells. The overexpression of Fascin has been associated with poor clinical prognosis and metastatic progression. Post-translational modifications of Fascin, such as phosphorylation, can affect the proliferation and invasion of tumor cells by regulating the actin-bundling activity of Fascin. However, the phosphorylation sites of Fascin and their corresponding kinases require further exploration. In the current study, we identified novel phosphorylation of Fascin Threonine 403 (Fascin-T403) mediated by AKT serine/threonine kinase 2 (AKT2), which was studied using mass spectrometry data from esophageal cancer tissues (iProX database: IPX0002501000). A molecular dynamics simulation revealed that Fascin-Threonine 403 phosphorylation (Fascin-T403D) had a distinct spatial structure and correlation of amino acid residues, which was different from that of the wild type (Fascin-WT). Low-speed centrifugation assay results showed that Fascin-T403D affected actin cross-linking. To investigate whether Fascin-T403D affected the function of esophageal cancer cells, either Fascin-WT or Fascin-T403D were rescued in Fascin-knockout or siRNA cell lines. We observed that Fascin-T403D could suppress the biological behavior of esophageal cancer cells, including filopodia formation, cell proliferation, and migration. Co-immunoprecipitation (Co-IP) and Duolink in situ proximity ligation assay (PLA) were performed to measure the interaction between Fascin and AKT2. Using in vitro and in vivo kinase assays, we confirmed that AKT2, but not AKT1 or AKT3, is an upstream kinase of Fascin Threonine 403. Taken together, the AKT2-catalyzed phosphorylation of Fascin Threonine 403 suppressed esophageal cancer cell behavior, actin-bundling activity, and filopodia formation.

Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting.

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes-S1 and S2-based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.

Metabolic and transcriptional changes in seminal plasma of asthenozoospermia patients.

This study was designed to investigate the metabolic and transcriptional alterations in seminal fluid caused by asthenozoospermia (AS). To address these issues, a method of metabonomics based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and real-time quantitative PCR (RT-qPCR) was performed to identify some crucial biomarkers and transcription levels of the enzymes in seminal fluid. Seminal fluid samples were collected from 87 AS patients and 73 healthy males with normozoospermia. The quantitative analysis by UPLC-MS/MS showed that 19 metabolites in seminal plasma were associated with AS, and they were involved in several metabolic pathways, such as energy metabolism, purine metabolism, methionine cycle, and branched chain amino acid metabolism. Among these metabolites, the levels of citric acid, malic acid, succinic acid, and pyruvic acid, which are related to energy metabolism, were collectively reduced in the AS group, whereas the lactic acid level was enhanced. These results indicated that lesser energy source (adenosine triphosphate) was produced through the anaerobic glycolysis pathway rather than via aerobic catabolism of suger and tricarboxylic acid cycle, resulting in reduced power of sperms. Meanwhile, partial least squares discriminant analysis showed significant differences in metabolic profiles between the AS and control groups. In addition, RT-qPCR results revealed that the expression levels of four genes encoding fructokinase citrate synthase, succinate dehydrogenase, and spermine synthase, which were related to energy metabolism, were decreased in the AS group. The 23 descriptors with differential expression in AS may be valuable for the diagnosis and sequential study on AS. These results will help highlight the role of sperm inactivity in AS pathogenesis.

Let-7f-5p regulates TGFBR1 in glucocorticoid-inhibited osteoblast differentiation and ameliorates glucocorticoid-induced bone loss.

Previous studies indicated that let-7 enhances osteogenesis and bone formation of human adipose-derived mesenchymal stem cells (MSCs). We also have confirmed that let-7f-5p expression was upregulated during osteoblast differentiation in rat bone marrow-derived MSCs (BMSCs) and was downregulated in the vertebrae of patients with glucocorticoid (GC)-induced osteoporosis (GIOP). The study was performed to determine the role of let-7f-5p in GC-inhibited osteogenic differentiation of murine BMSCs in vitro and in GIOP in vivo. Here, we report that dexamethasone (Dex) inhibited osteogenic differentiation of BMSCs and let-7f-5p expression, while increasing the expression of transforming growth factor beta receptor 1 (TGFBR1), a direct target of let-7f-5p during osteoblast differentiation under Dex conditions. In addition, let-7f-5p promoted osteogenic differentiation of BMSCs, as indicated by the promotion of alkaline phosphatase (ALP) staining and activity, Von Kossa staining, and osteogenic marker expression (Runx2,Osx, Alp, and Ocn), but decreased TGFBR1 expression in the presence of Dex. However, overexpression of TGFBR1 reversed the upregulation of let-7f-5p during Dex-treated osteoblast differentiation. Knockdown of TGFBR1 reversed the effect of let-7f-5p downregulation during Dex-treated osteogenic differentiation of BMSCs. We also found that glucocorticoid receptor (GR) mediated transcriptional silencing of let-7f-5p and its knockdown enhanced Dex-inhibited osteogenic differentiation. Further, when injected in vivo, agomiR-let-7f-5p significantly reversed bone loss induced by Dex, as well as increased osteogenic marker expression (Runx2, Osx, Alp, and Ocn) and decreased TGFBR1 expression in bone extracts. These findings indicated that the regulatory axis of GR/let-7f-5p/TGFBR1 may be important for Dex-inhibited osteoblast differentiation and that let-7f-5p may be a useful therapeutic target for GIOP.

IGF-1R/ß-catenin signaling axis is involved in type 2 diabetic osteoporosis.

Insulin-like growth factor-1 receptor (IGF-1R) is involved in both glucose and bone metabolism. IGF-1R signaling regulates the canonical Wnt/ß-catenin signaling pathway. In this study, we investigated whether the IGF-1R/ ß-catenin signaling axis plays a role in the pathogenesis of diabetic osteoporosis (DOP). Serum from patients with or without DOP was collected to measure the IGF-1R level using enzyme-linked immunosorbent assay (ELISA). Rats were given streptozotocin following a four-week high-fat diet induction (DOP group), or received vehicle after the same period of a normal diet (control group). Dual energy X-ray absorption, a biomechanics test, and hematoxylin-eosin (HE) staining were performed to evaluate bone mass, bone strength, and histomorphology, respectively, in vertebrae. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to measure the total and phosphorylation levels of IGF-1R, glycogen synthase kinase-3ß (GSK-3ß), and ß-catenin. The serum IGF-1R level was much higher in patients with DOP than in controls. DOP rats exhibited strikingly reduced bone mass and attenuated compression strength of the vertebrae compared with the control group. HE staining showed that the histomorphology of DOP vertebrae was seriously impaired, which manifested as decreased and thinned trabeculae and increased lipid droplets within trabeculae. PCR analysis demonstrated that IGF-1R mRNA expression was significantly up-regulated, and western blotting detection showed that phosphorylation levels of IGF-1R, GSK-3ß, and ß-catenin were enhanced in DOP rat vertebrae. Our results suggest that the IGF-1R/ß-catenin signaling axis plays a role in the pathogenesis of DOP. This may contribute to development of the underlying therapeutic target for DOP.

A toxicological, metabonomic and transcriptional analysis to investigate the property of mulberry 1-deoxynojirimycin against the growth of Samia cynthia ricini.

1-Deoxynojirimycin (DNJ) is a natural d-glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1-5, and mass deaths happened in DNJ groups on day 3-5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.

Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway.

BACKGROUND/AIMS: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. METHODS: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, ß-catenin, Gsk3ß, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-ß-CATENIN, and p-GSK3ß were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. RESULTS: The optimum concentration for PTE was 30 µg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, ß-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3ß mRNA expression. PTE inhibited TNFR2, TRAF2, and p-ß-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3ß protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. CONCLUSIONS: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

Effect of osteoporosis induced by ovariectomy on vertebral bone defect/fracture in rat.

Osteoporotic vertebral fracture (OVF) is a worldwide health concern and lacks sufficient basic studies. Suitable animal models should be the foundation for basic study and treatment of OVF. There have been few studies on the development of animal models of osteoporotic vertebral bone defects. OVF models using various animal species should be developed to evaluate the therapeutic strategy in preclinical testing. We developed an OVF model in rats. Rat osteoporosis was induced by ovariectomy (OVX), and 3 months after OVX, a 3 mm diameter hemispheric vertebral bone defect was developed in lumbar vertebra 6 (L6). Sagittal plain X-rays of the rats, their bone quantity, bone microarchitecture, and histomorphology were analyzed: 3 months after OVX, rats showed significantly lower bone quantity, relative bone volume, and total volume bone mineral density. After the vertebral bone defect had developed for 16 weeks, no significant indication of self-healing could be observed from the sagittal plain X-rays, three-dimensional images, and histomorphology. These results indicate that the rat model of osteoporotic vertebral bone defect, induced by OVX and a 3 mm diameter hemispheric vertebral bone defect, can sufficiently mimic OVF patients in clinic and provide a sound basis for subsequent studies.

[Risk factors for multiple level osteoporosis vertebral compression fractures].

OBJECTIVE: To identify risk factors associated with patients suffered multiple level osteoporosis vertebral compression fractures(OVCFs). METHODS: From March 2011 to March 2015, 199 patients suffered osteoporotic were classified into multiple level OVCFs group and single level OVCF group. Multivariate logistic regression analysis were performed to identify risks factors associated with multiple level OVCFs. RESULTS: All the patients underwent OVCF, including 71 multiple level OVCFs and 128 single level OVCF. There were no differences in the age, gender, BMI, hypertension and diabetes between two groups. While multiple level OVCFs were associated with spinal deformity index SDI[(2<=SDI<4, OR=2.587, 95% CI(1.148, 5.828);SDI>=-4, OR=7.775, 95% CI(3.272, 18.478)], BMD[(T<-4.5SD, OR=2.608, 95% CI(1.038, 6.551)]. CONCLUSIONS: SDI and BMD might be the risk factors for multiple level OVCFs.

[Case-control study on minimally invasive percutaneous locking compression plate internal fixation for the treatment of type II and III pilon fractures].

OBJECTIVE: To explore the clinical efficacy of delayed open reduction and internal fixation with minimally invasive percutaneous locking compression plate for the treatment of type II and III Pilon fractures. METHODS: From January 2007 to September 2009, 32 patients with type II and III Pilon fractures were treated with open reduction and anatomic plate fixation (AP group) and minimally invasive percutaneous locking compression plate osteosynthesis (LCP group). There were 11 males and 6 females in AP group, with an average age of (37.4 +/- 13.3) years (ranged, 19 to 55 years). And there were 10 males and 5 females in LCP group, with an average age of (34.6 +/- 11.3) years(ranged, 21 to 56 years). The operating time, fracture healing time, aligned angulation and ankle function were compared between the two groups. RESULTS: All the patients were followed up, and the during ranged from 12 to 25 months, with a mean of (15.0 +/- 1.7) months. The average operation time was (76.5 +/- 8.3) min for AP group and (58.3 +/- 3.4) min for LCP group; the average time of fracture healing was (20.5 +/- 0.4) weeks for AP group and (15.7 +/- 0.2) weeks for LCP group; the total angulation between anterior posterior film and lateral film was averaged (6.6 +/- 0.5) degrees for AP group and (3.6 +/- 0.2) degrees for LCP group. As to above index, the results of LCP group were better than those of AP group (P < 0.05). According to Kofoed criteria for ankle joint, the results of LCP group were better than those of AP group in ankle joint pain, wakling and ankle joint function (P < 0.05). CONCLUSION: The method of minimally invasive percutaneous locking compression plate internal fixation is effective in the treatment of Pilon fracture with less invasion, faster bone union, more stabilized fixation, quicker recovery of ankle function and fewer complications, which is more advantaged for type II and III Pilon fractures.