HSF1 promotes CD69+ Treg differentiation to inhibit colitis progression.

Regulatory T cells (Tregs) are critical for generating and maintaining peripheral tolerance. Treg-based immunotherapy is valuable for the clinical management of diseases resulting from dysregulation of immune tolerance. However, the lack of potency is a potential limitation of Treg therapy. In addition, CD69 positive-Treg (CD69+ Treg) represent a newly identified subset of Tregs with potent immune suppressive capability. Methods: Foxp3 YFP-Cre CD69 fl/fl and CD4 Cre CD69 fl/fl mice were generated to determine the relevance of CD69 to Treg. Chromatin Immunoprecipitation Assay (ChIP) and luciferase Assay were performed to detect the regulation of CD69 transcription by heat shock transcription factor 1(HSF1). Gene expression was measured by western blotting and qRT-PCR. The differentiation of naive T cells to CD69+Foxp3+ iTregs was determined by flow cytometry. The immunosuppressive ability of Tregs was analyzed by ELISA and flow cytometry. Colon inflammation in mice was reflected by changes in body weight and colon length, the disease activity index (DAI), and H&E staining of colon tissues. Results: Induced Tregs (iTregs) from CD4 Cre CD69 fl/fl mice failed to alleviate colitis. The transcription factor HSF1 interacted with the promoter of the CD69 gene to prompt its transcription during Treg differentiation. Genetic and chemical inhibition of HSF1 impaired CD69+ Treg differentiation and promoted the pathogenesis of colitis in mice. In contrast, HSF1 protein stabilized by inhibiting its proteasomal degradation promoted CD69+ Treg differentiation and alleviated colitis in mice. Moreover, adoptive transfer of iTregs with HSF1 stabilization by proteasome inhibitor (PSI) dramatically prevented the development of colitis in mice and was accompanied by decreased production of pro-inflammatory cytokines and reduced accumulation of pro-inflammatory lymphocytes in colitis tissue, whereas Tregs induced in the absence of PSI were less stable and ineffective in suppressing colitis. Conclusions: HSF1 promotes CD69+ Tregs differentiation by activating the CD69 transcription, which is critical for the immunosuppressive function of Tregs. Stabilization of HSF1 by PSIs results in the efficient generation of Tregs with high potency to treat colitis and probably other autoimmune diseases involving Tregs deficiency.

Development and validation of a machine learning model for survival risk stratification after esophageal cancer surgery.

Background: Prediction of prognosis for patients with esophageal cancer(EC) is beneficial for their postoperative clinical decision-making. This study's goal was to create a dependable machine learning (ML) model for predicting the prognosis of patients with EC after surgery. Methods: The files of patients with esophageal squamous cell carcinoma (ESCC) of the thoracic segment from China who received radical surgery for EC were analyzed. The data were separated into training and test sets, and prognostic risk variables were identified in the training set using univariate and multifactor COX regression. Based on the screened features, training and validation of five ML models were carried out through nested cross-validation (nCV). The performance of each model was evaluated using Area under the curve (AUC), accuracy(ACC), and F1-Score, and the optimum model was chosen as the final model for risk stratification and survival analysis in order to build a valid model for predicting the prognosis of patients with EC after surgery. Results: This study enrolled 810 patients with thoracic ESCC. 6 variables were ultimately included for modeling. Five ML models were trained and validated. The XGBoost model was selected as the optimum for final modeling. The XGBoost model was trained, optimized, and tested (AUC = 0.855; 95% CI, 0.808-0.902). Patients were separated into three risk groups. Statistically significant differences (p < 0.001) were found among all three groups for both the training and test sets. Conclusions: A ML model that was highly practical and reliable for predicting the prognosis of patients with EC after surgery was established, and an application to facilitate clinical utility was developed.

McKeown esophagectomy: robot-assisted versus conventional minimally invasive technique-systematic review and meta-analysis.

BACKGROUND AND PURPOSE: This meta-analysis assesses the surgical outcomes between robot-assisted minimally-invasive McKeown esophagectomy and conventional one. METHOD: This meta-analysis searched the Web of Science, PUBMED, and EMBASE from the database's inception to January 2022. Altogether, 1073 records were identified in the literature search. Studies that evaluated the outcomes between robot-assisted minimally-invasive McKeown esophagectomy and conventional one among postoperative patients with oesophageal neoplasms were included. The assessed outcomes involved complications and clinical outcomes. In addition, heterogeneity was analyzed, and evidence quality was evaluated. RESULT: Evidence indicated that RAMIE (minimally-invasive esophagectomy assisted with robot) decreased incidences of lung complications and hospital stay as well as increased harvested lymph nodes. CONCLUSIONS: There was currently little evidence from randomized studies depicting that robot surgery manifested a clear overall advantage, but there was growing evidence regarding the clinical benefits of robot-assisted minimally invasive McKeown esophagectomy over conventional one.

Chronic stress induced depressive-like behaviors in a classical murine model of Parkinson's disease.

Depression occurs in around 40 % of patients with Parkinson's disease (PD) and contributes to severe disability and a poor quality of life. The underlying mechanisms and pathophysiology of depression in PD (PDD) remain obscure, due to a lack of stable animal models of PDD. In this study, we established a PDD model by inducing exposure to chronic mild (CMS) and strong stress (CSS) using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PD mice. We detected changes in motor and non-motor symptoms, brain structure, neurotransmitters, levels of 5-HT related genes and inflammation. CMS exposed PD (PDMS) mice exhibited obviously decreased levels of neuromuscular strength and enhanced levels of inflammation, compared with that of control mice. CSS exposed MPTP (PDSS) mice exhibited the highest level of motor impairment and depression states along with the highest levels of inflammation enhancement and a decrease in the expression levels of 5-hydroxytryptamine (5-HT) related genes in all groups. Our results suggested that CSS can successfully induce stable depression like symptoms in sub-chronic MPTP PD mice and appears to be a valuable tool for investigating PDD. Furthermore, it was found that 5-HT system dysfunction may contribute to depression like symptoms in PD.

Metabolic enzyme PDK3 forms a positive feedback loop with transcription factor HSF1 to drive chemoresistance.

Background & Aims: Dysregulation of metabolism plays an important role in the development and progression of cancers, while the underlying mechanisms remain largely unknown. This study aims to explore the regulation and relevance of glycolysis in chemoresistance of gastric cancer. Methods: Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by metabolism profiling, microarray gene expression, PCR or western blotting. Cancer cell growth in vitro or in vivo were analyzed by viability, apoptosis and nude mice assay. Immunoprecipation was used to explore the interaction of proteins with other proteins or DNAs. Results: By metabolic and gene expression profiling, we found that pyruvate dehydrogenase kinase 3 (PDK3) was highly expressed to promote glycolysis in chemoresistant cancer cells. Its genetic or chemical inhibition reverted chemoresistance in vitro and in vivo. It was transcriptionally regulated by transcription factor HSF1 (Heat shock factor 1). Interestingly, PDK3 can localize in the nucleus and interact with HSF1 to disrupt its phosphorylation by GSK3ß. Since HSF1 was subjected to FBXW7-catalyzed polyubiquitination in a phosphorylation-dependent manner, PDK3 prevented HSF1 from proteasomal degradation. Thus, metabolic enzyme PDK3 and transcription factor HSF1 forms a positive feedback loop to promote glycolysis. As a result, inhibition of HSF1 impaired enhanced glycolysis and reverted chemoresistance both in vitro and in vivo. Conclusions: PDK3 forms a positive feedback loop with HSF1 to drive glycolysis in chemoresistance. Targeting this mitonuclear communication may represent a novel approach to overcome chemoresistance.

SIRT1 deacetylated and stabilized XRCC1 to promote chemoresistance in lung cancer.

Chemoresistance is one of the most important challenges in the clinical management of lung cancer. SIRT1 is a NAD dependent protein deacetylase and implicated in diverse cellular processes such as DNA damage repair, and cancer progression. SIRT1 is upregulated in chemoresistant lung cancer cells, genetic knockdown or chemical inhibition of SIRT1 reversed chemoresistance by enhancing DNA damage and apoptosis activation, accompanied with XRCC1 degradation. E3 ligase ß-TrCP catalyzed the poly-ubiquitination of XRCC1 to promote its proteasome-dependent degradation. SIRT1 bound and deacetylated XRCC1 at lysine K260, K298 and K431, preventing it from ß-TrCP-dependent ubiquitination. Mutations of these three lysine sites in XRCC1 abrogated the interaction with ß-TrCP and prolonged the half-life of XRCC1 protein. Here, we describes SIRT1 confers chemoresistance to lung cancer cells by deacetylating and stabilizing XRCC1. Therefore, targeting SIRT1 might be a new strategy to manage the chemoresistance of lung cancer, and probably other cancers.

KDM5B demethylates H3K4 to recruit XRCC1 and promote chemoresistance.

Chemotherapy is the main treatment for human cancers including gastric cancer. However, in response to chemotherapeutic drugs, tumor cells can develop drug resistance by reprogramming intracellular metabolic and epigenetic networks to maintain their intrinsic homeostasis. Previously, we have established cisplatin-resistant gastric cancer cells as a drug resistant model, and elucidated the XRCC1 as the core DNA repair mechanism of drug resistance. This study investigated the regulation of XRCC1 by lysine demethylase 5B (KDM5B) in drug resistance. We found that the methylation level of H3K4 decreased significantly in drug-resistant cells. The chemical inhibitor of H3K4 demethylases, JIB-04, restored the methylation of H3K4 and blocked the co-localization of XRCC1 and γH2AX, eventually improved drug sensitivity. We further found that the expression level of KDM5B increased significantly in drug-resistant cells. Knockdown of KDM5B increased the methylation level of H3K4 and blocked the localization of XRCC1 to the DNA damage site, leads to increased drug sensitivity. In the sensitive cells, overexpression of KDM5B suppressed H3K4 methylation levels, which resulted to resistance to cisplatin. Moreover, we found that the posttranslational modification of KDM5B is responsible for its high expression in drug-resistant cells. Through mass spectrometry screening and co-immunoprecipitation validation, we found that the molecular chaperone HSP90 forms a complex with KDM5B in drug resistance cells. Interestingly, HSP90 inhibitor 17-AAG induced KDM5B degradation in a time-and-dose-dependent manner, indicating that HSP90 protected KDM5B from protein degradation. Targeting inhibition of HSP90 and KDM5B reversed drug resistance both in vitro and in vivo. Taken together, molecular chaperon HSP90 interacted with KDM5B to protect it from ubiquitin-dependent proteasomal degradation. Increased KDM5B demethylated H3K4 and facilitated the recruitment of XRCC1 to repair damaged DNA. Therefore, inhibition of HSP90 or KDM5B represented a novel approach to reverse chemoresistance in human cancers.

Rab5a suppresses autophagy to promote drug resistance in cancer cells.

Cancers are huge problems that need to be investigated thoroughly. Rab5a plays an important part in the regulation of intracellular membrane trafficking. However, its role in cancer and autophagy has not been fully determined. In this study, we analyzed the correlation between Rab5a expression and patients' prognosis and then explored the effect of Rab5a knockdown on different cell lines using western blotting and fluorescence. Our results showed that up-regulated Rab5a positively correlated with the prognosis of gastric cancer patients. After knocking down Rab5a, mTOR activity was inhibited and autophagy flux increased. We also found that in our cisplatin-resistant cells, knockdown of Rab5a activated autophagy via mTOR pathway and could reverse drug resistance while overexpression of Rab5a in drug sensitive cells increased drug tolerance. In conclusion, our study demonstrates that Rab5a can suppress autophagy through mTOR and promote drug resistance in gastric cancer cells.

Enolase 1 stimulates glycolysis to promote chemoresistance in gastric cancer.

Chemotherapy is the major choice for the cancer treatment of early and advanced stages. However, intrinsic or acquired drug resistance significantly restricts the clinical efficacy of chemotherapy. It is critical to develop novel approaches to detect and overcome drug resistance. In this study, we demonstrated that accelerated glycolysis played a pivotal role in both intrinsic and acquired cisplatin-resistance of gastric cancer cells. The metabolic reprogramming of cisplatin-resistant cells was characterized by increased glycolysis dependence. Inhibition of glycolysis with glucose starvation or 2-Deoxy-D-glucose (2-DG) treatment significantly reversed drug resistance. By proteomic screening, we found the increased expression of the glycolytic enzyme Enolase 1 (ENO1) in cisplatin-resistant gastric cancer cells. Depletion of ENO1 by siRNA significantly reduced glycolysis and reversed drug resistance. Moreover, the increased expression of ENO1 was attributed to the down-regulation of ENO1-targeting miR-22, rather than activated gene transcriptional or prolonged protein stability. Finally, the elevated levels of ENO1 proteins were associated with the shorter overall survival of gastric cancer patients. In conclusion, ENO1 is a novel biomarker to predict drug resistance and overall prognosis in gastric cancer. Targeting ENO1 by chemical inhibitors or up-regulating miR-22 could be valuable to overcome drug resistance.

Loss of PIG3 increases HIF-1α level by promoting protein synthesis via mTOR pathway in renal cell carcinoma cells.

PIG3 is a target of the tumor suppressor p53 and is thought to be involved in p53-mediated cell apoptosis. Although PIG3 is similar to oxidoreductases involved in generating ROS, whether PIG3 would regulate HIF-1α was never characterized directly. Here we demonstrated that knockdown of PIG3 by transfecting with specific siRNA could increase the expression of HIF-1α in several human cancer cell lines, including CAKI, FTC-133 and A549. It indicates that PIG3 may be involved in the regulation of HIF-1α. Furthermore, we revealed that PIG3-siliencing increased HIF-1α protein level through promoting its protein biosynthesis via mTOR pathway. In addition, the effect of PIG3 on the production of HIF-1α was further related to VEGF secretion and cell migration. PIG3-downregulation increased the secretion of VEGF and promoted the migration of renal cancer cells obviously. Taken together, these data suggest that PIG3 was involved in HIF-1α regulation, and reveal a novel signaling pathway of PIG3/HIF-1α in the regulation of cell migration in renal cell carcinoma.

Expression of HIF-1α and HIF-2α correlates to biological and clinical significance in papillary thyroid carcinoma.

BACKGROUND: The aim of this study was to detect the expression of hypoxia-inducible factor (HIF)-1α and HIF-2α in papillary thyroid carcinoma (PTC) compared with normal thyroid tissues. METHODS: The mRNA levels and protein levels of HIF-1α and HIF-2α were detected by real-time PCR and Western blot separately in 30 pairs of PTCs and normal thyroid cases. The protein levels were also detected by immunohistochemistry (IHC) using 92 samples of PTC group and 46 normal samples as control group for analyzing the biological and clinical significance of the expression of HIF-1α/HIF-2α. RESULTS: Real-time PCR results showed the mRNA level of HIF-1α and HIF-2α were significantly higher in PTC than normal group (P<0.001). Also, significantly higher positive rates (73%/65%) of HIF-1α and HIF-2α were observed in PTC compared with the control group (27%/35%) by IHC (P<0.01); the consistent results were gotten with Western blot. Although we did not find a significant correlation between the expression of HIF-1α and HIF-2α with gender, age, calcification, or Hashimoto's disease in the present study (P>0.05), both of their expressions were correlated to lymph node metastasis (P<0.05), capsular invasion (P<0.05), and TNM stage (P<0.05). CONCLUSIONS: Overexpression of HIF-1α and HIF-2α are associated with the carcinogenesis of PTC, served as potential biomarkers of PTC.

PIG3 plays an oncogenic role in papillary thyroid cancer by activating the PI3K/AKT/PTEN pathway.

The p53-inducible gene 3 (PIG3 or TP53I3) is a downstream gene of p53, which can be involved in the process of apoptosis induced by p53 via the production of reactive oxygen species (ROS). However, the functional significance of PIG3 in cancer remains to be determined. This aim of this study was to examine the mRNA and protein expression of PIG3 in papillary thyroid carcinoma (PTC) and normal thyroid tissues, assess the relationship between PIG3 expression and clinicopathological parameters in PTC and examine its role in the proliferation of PTC cell lines. The results showed that PIG3 was aberrantly overexpressed in the majority of specimens of PTC while the expression of p53 was lower in PTC compared with normal thyroid tissues. Anti-PIG3 immuno-reactivity positively correlated with TNM grade. In the PTC cell lines, PIG3 silencing using small interfering RNA (siRNAs) impaired their ability of proliferation and decreased the activity of the PI3K/AKT/PTEN pathway. The results suggested that PIG3 plays an oncogenic role in PTC via the regulation of the PI3K/AKT/PTEN pathway and support the exploration of PIG3 as a novel biomarker for patients with PTC.

Nanofibrous collagen nerve conduits for spinal cord repair.

Nerve regeneration in an injured spinal cord is often restricted, contributing to the devastating outcome of neurologic impairment below the site of injury. Although implantation of tissue-engineered scaffolds has evolved as a potential treatment method, the outcomes remain sub-optimal. One possible reason may be the lack of topographical signals from these constructs to provide contact guidance to invading cells or regrowing axons. Nanofibers mimic the natural extracellular matrix architecturally and may therefore promote physiologically relevant cellular phenotypes. In this study, the potential application of electrospun collagen nanofibers (diameter=208.2±90.4 nm) for spinal cord injury (SCI) treatment was evaluated in vitro and in vivo. Primary rat astrocytes and dorsal root ganglias (DRGs) were seeded on collagen-coated glass cover slips (two-dimensional [2D] substrate controls), and randomly oriented or aligned collagen fibers to evaluate scaffold topographical effects on astrocyte behavior and neurite outgrowth, respectively. When cultured on collagen nanofibers, astrocyte proliferation and expression of glial fibrillary acidic protein (GFAP) were suppressed as compared to cells on 2D controls at days 3 (p<0.05) and 7 (p<0.01). Aligned fibers resulted in elongated astrocytes (elongation factor >4, p<0.01) and directed the orientation of neurite outgrowth from DRGs along fiber axes. In the contrast, neurites emanated radially on randomly oriented collagen fibers. By forming collagen scaffolds into spiral tubular structures, we demonstrated the feasibility of using electrospun nanofibers for the treatment of acute SCI using a rat hemi-section model. At days 10 and 30 postimplantation, extensive cellular penetration into the constructs was observed regardless of fiber orientation. However, scaffolds with aligned fibers appeared more structurally intact at day 30. ED1 immunofluorescent staining revealed macrophage invasion by day 10, which decreased significantly by day 30. Neural fiber sprouting as evaluated by neurofilament staining was observed as early as day 10. In addition, GFAP immunostained astrocytes were found only at the boundary of the lesion site, and no astrocyte accumulation was observed in the implantation area at any time point. These findings indicate the feasibility of fabricating 3D spiral constructs using electrospun collagen fibers and demonstrated the potential of these scaffolds for SCI repair.

Sustained release of neurotrophin-3 and chondroitinase ABC from electrospun collagen nanofiber scaffold for spinal cord injury repair.

Nerve regeneration after spinal cord injuries (SCI) remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of its biomimicking architecture. In this study, we investigated the feasibility of incorporating neurotrophin-3 (NT-3) and chondroitinase ABC (ChABC) onto electrospun collagen nanofibers for SCI treatment. By using microbial transglutaminase (mTG) mediated crosslinking, proteins were loaded onto electrospun collagen nanofibers at an efficiency of ∼45-48%. By combining NT-3 with heparin during the protein incorporation process, a sustained release of NT-3 was obtained (∼96% by day 28). As indicated by dorsal root ganglion outgrowth assay, NT-3 incorporated collagen scaffolds supported neuronal culture and neurite outgrowth for a longer time period than bolus delivery of NT-3. The presence of heparin also protected ChABC from degradation. Specifically, as evaluated by dimethylmethylene blue assay, bioactive ChABC was detected from collagen scaffolds for at least 32 days in vitro in the presence of heparin (∼32% of bioactivity retained). In contrast, ChABC bioactivity was only ∼1.9% by day 22 in the absence of heparin. Taken together, these results clearly demonstrated the feasibility of incorporating NT-3 and ChABC via mTG immobilization to produce protein-incorporated collagen nanofibers. Such biofunctional nanofiber constructs may find useful applications in SCI treatment by providing topographical signals and multiple biochemical cues that can promote nerve regeneration while antagonizing axonal growth inhibition for CNS regeneration.