Deubiquitinase USP10 promotes osteosarcoma autophagy and progression through regulating GSK3ß-ULK1 axis.

BACKGROUND: Deubiquitinating enzymes (DUBs) are pivotal in maintaining cell homeostasis by regulating substrate protein ubiquitination in both healthy and cancer cells. Ubiquitin-specific protease 10 (USP10) belongs to the DUB family. In this study, we investigated the clinical and pathological significance of USP10 and Unc-51-like autophagy activating kinase 1 (ULK1) in osteosarcoma (OS), as well as the mechanism of USP10 action in ULK1-mediated autophagy and disease progression. RESULTS: The analysis of OS and adjacent normal tissues demonstrated that USP10 and ULK1 were significantly overexpressed in OS, and a positive association between their expression and malignant properties was observed. USP10 knockdown in OS cells reduced ULK1 mRNA and protein expression, whereas USP10 overexpression increased ULK1 mRNA and protein expression. In vitro experiments showed that USP10 induced autophagy, cell proliferation, and invasion by enhancing ULK1 expression in OS cell lines. Furthermore, we found that the regulation of ULK1-mediated autophagy, cell proliferation, and invasion in OS by USP10 was dependent on glycogen synthase kinase 3ß (GSK3ß) activity. Mechanistically, USP10 promoted ULK1 transcription by interacting with and stabilising GSK3ß through deubiquitination, which, in turn, increased the activity of the ULK1 promoter, thereby accelerating OS progression. Using a xenograft mouse model, we showed that Spautin-1, a small-molecule inhibitor targeting USP10, significantly reduced OS development, with its anti-tumour activity significantly enhanced when combined with the chemotherapeutic agent cisplatin. CONCLUSION: Collectively, we demonstrated that the USP10-GSK3ß-ULK1 axis promoted autophagy, cell proliferation, and invasion in OS. The findings imply that targeting USP10 may offer a promising therapeutic avenue for treating OS.

Knee arthroscopy has limited effects on relieving local symptoms of knee osteoarthritis: an analysis of data from the Osteoarthritis Initiative.

BACKGROUND: Knee arthroscopy's efficacy in symptom improvement for knee osteoarthritis remains debated. In this study, we analyzed a multicenter database to investigate local symptom improvement. METHODS: We extracted and analyzed the data of 163 patients from the Osteoarthritis Initiative cohort who underwent unilateral knee arthroscopy (UKA) and were followed up for at least 24 months. UKA patients were matched to non-UKA patients (n = 163) according to sex, age, abdominal circumference, and Kellgren-Lawrence grade. The verified KOOS questionnaires (knee catching, locking, grinding, or clicking) and common local symptoms (frequent knee pain, aching, or stiffness) were set as outcomes. Furthermore, we built a binary logistic regression model to examine the relationship between UKA and local symptom improvement and new-onset symptoms, adjusting for conservative therapeutic covariables (injection of steroids or transparent acid into the knee joint, oral chondroitin sulfate, amino glucose, or analgesics). RESULT: Analysis showed that the UKA and non-UKA groups showed no obvious difference in the three knee symptoms, but the probability of new-onset grinding or clicking, and frequent knee pain, aching, or stiffness symptoms in the UKA group were respectively 5.82 and 5.65-fold higher than that in the non-UKA group. After analyzing conservative treatment data using a multiple imputation method, the results were consistent with previous regression analyses. CONCLUSION: Compared to the non-UKA group, the UKA group showed no noticeable differences in the improvement of the three knee symptoms and showed an increased the probability of new-onset grinding or clicking and frequent knee pain, aching, or stiffness symptoms. Key Points • Knee arthroscopy may increase the probability of new-onset grinding or clicking and frequent knee pain, aching, or stiffness symptoms. • We found no difference in the improvement of local knee symptoms (knee catching, locking, grinding, clicking or frequent pain, aching, or stiffness) improvement between the two groups with or without knee arthroscopy.

Anterior knee pain as a potential risk factor for falls in older adults: insights from the osteoarthritis initiative data.

BACKGROUND: Knee joint pain has been demonstrated to be a separate risk factor for falling. A common pain site in the knee, anterior knee pain(AKP), is believed to be associated with early knee osteoarthritis (KOA).This study investigated the relationship between falls and AKP in people with or at risk for KOA. METHODS: Four years of follow-up data from the Osteoarthritis Initiative cohort trial, a large-scale, multicenter observational investigation, were analyzed in this study. A patellar quadriceps tenderness/tendinitis knee exam was performed to evaluate AKP. Falls were self-reported. The associations between falls (recurrent falls: ≥2 falls/year; any falls: ≥1 fall(s)/year) and AKP were analyzed using the generalized estimation equation of repeated logistic regression and adjusted for confounding variables. RESULTS: The study analyzed data from 3,318 participants, split into two groups: those with AKP (720 participants) and those without AKP (2,598 participants). The primary outcome of the study, which focused on repeated falls, revealed that participants with AKP were 1.27 times more likely to experience repeated falls compared to those without AKP (95% CI: 1.07-1.52, P = 0.007). However, when considering any falls experienced by an individual as an additional outcome, it is important to note that our findings did not indicate a significant predictive effect of AKP on any falls investigated. Sensitivity analyses, which excluded knee arthroplasty cases, yielded consistent results with the aforementioned findings. CONCLUSIONS: Older adults with AKP experience a higher frequency of falls compared to those without AKP in individuals diagnosed with KOA or at a high risk of developing KOA.

Anserine bursa palpation tenderness is a risk factor for knee osteoarthritis progression and arthroplasty: data from the Osteoarthritis Initiative.

OBJECTIVE: Anserine bursa pain (ABP) is defined as the presence of palpation tenderness medially below the joint line, which is 2 cm from the tibial tuberosity. This study aimed to determine a link between ABP and three knee outcomes: frequent pain, joint space narrowing (JSN) progression, and total knee arthroplasty (TKA). METHODS: Participants from the Osteoarthritis Initiative cohort were included in this study. Frequent ABP was defined as presenting thrice at four-time points. The Chi-square test and binary logistic regression analyses examined the associations between ABP and the three knee outcomes. Furthermore, Cox Proportional Hazards Model explored the association between ABP and TKA. RESULTS: Baseline ABP was linked to a higher risk of frequent pain (odds ratio (OR): 2.28, 95% confidence interval (CI): 1.76-2.97, P < 0 .001) and TKA (OR: 1.54, 95% CI 1.01-2.36, P = 0 .044) after adjusting for gender, baseline age, body mass index (BMI), and Kellgren-Lawrence (KL) grade. In the frequent ABP group from baseline to the 4-year follow-up (≥ 3 of four-time points), frequent pain (OR: 3.14, 95% CI: 2.34-4.22, P < 0 .001) and TKA (OR: 1.79, 95% CI: 1.11-2.90, P = 0 .017) had a high association with ABP after adjusting for gender, baseline age, BMI, and KL grade. CONCLUSION: This study highlights the association between ABP and knee outcomes; therefore, clinicians should pay closer attention during the physical examination, especially in middle-aged and older female patients. Moreover, understanding ABP cause aids in better diagnosis and treatment. Key Points • This is the first study to identify an association between anserine bursa palpation tenderness and symptomatic knee osteoarthritis. • As opposed to most studies, which focus on intra-articular symptoms and signs, this study focused on extra-articular symptoms and signs. • Clinically, anserine bursa palpation tenderness can be utilized to determine patients at risk for the progression of knee osteoarthritis, thereby aiding in providing early therapeutic intervention.

[Research progress of arthroscopic long head of biceps tendon transposition in treatment of irreparable massive rotator cuff tears].

OBJECTIVE: To review the research progress of arthroscopic long head of biceps tendon (LHBT) transposition in treatment of irreparable massive rotator cuff tears. METHODS: The domestic and foreign related literature in recent years on the treatment of irreparable massive rotator cuff tears with different LHBT transposition methods under arthroscopy was reviewed and analyzed. RESULTS: Arthroscopic LHBT transposition is an effective method for irreparable massive rotator cuff tears, which mainly includes "proximal cut", "both two cuts", "distal cut", and "no cut". Different methods of LHBT transposition can achieve good effectiveness, but its long-term effectiveness needs further follow-up. CONCLUSION: Arthroscopic LHBT transposition in treatment of irreparable massive rotator cuff tears is simple and effective. The patients can recover quickly after operation with less injury. But the technique has higher requirements for surgeons, and the indications must be strictly controlled.

Downregulation of NEAT1 sensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine through modulation of the miR-506-3p/ZEB2/EMT axis.

Chemoresistance is a major cause of treatment failure in pancreatic cancer (PC). It has been demonstrated that epithelial-to-mesenchymal transition (EMT) is closely related to drug resistance in PC; however, the underlying mechanisms are not yet fully understood. Recently found evidence has suggested that nuclear-enriched abundant transcript 1 (NEAT1) is involved in the development of chemoresistance. However, the role and mechanism of NEAT1 in PC gemcitabine resistance remain unknown. In the present study, we first established two independent gemcitabine-resistant (GR) PC cell lines, PANC-1/GR and SW1990/GR. We found that GR cells displayed markedly enhanced migration and invasion abilities, decreased expression of E-cadherin, and upregulation of N-cadherin, Vimentin, Snail, ZEB1, and ZEB2. Our findings suggested that downregulation of NEAT1 enhanced the sensitivity of GR cells to gemcitabine by reversing the EMT process. Mechanistically, NEAT1 mediates ZEB2 mRNA expression through sponging miR-506-3p. Downregulation of NEAT1 can reverse the EMT process of GR PC cells by reducing the expression of ZEB2, thus enhancing the sensitivity of GR PC cells to gemcitabine. These findings were further confirmed in a nude mouse xenograft model. Taken together, downregulation of NEAT1 sensitized the GR PC cells to gemcitabine through modulation of the miR-506-3p/ZEB2/EMT axis. These results provide the novel evidence for understanding the function and molecular mechanism of NEAT1, and a new direction for improving the chemotherapeutic effects in PC.

E3 ubiquitin ligase TRIM29 promotes pancreatic cancer growth and progression via stabilizing Yes-associated protein 1.

BACKGROUND: Pancreatic cancer (PC) is one of the most fatal digestive system cancers. tripartite motif-29 (TRIM29) has been reported as oncogene in several human cancers. However, the precise role and underlying signal cascade of TRIM29 in PC progression remain unclear. METHODS: Western blot, qRT-PCR and immunohistochemistry were used to analyze TRIM29 and Yes-associated protein 1 (YAP1) levels. CCK8 assays, EdU assays and flow cytometry were designed to explore the function and potential mechanism of TRIM29 and YAP1 in the proliferation of PC. Next, a nude mouse model of PC was established for validating the roles of TRIM29 and YAP1 in vivo. The relationship among TRIM29 and YAP1 was explored by co-immunoprecipitation and in vitro ubiquitination assay. RESULTS: TRIM29 and YAP1 was significantly upregulated in PC patient samples, and TRIM29 expression was closely related to a malignant phenotype and poorer overall survival (OS) of PC patients. Functional assays revealed that TRIM29 knockdown suppresses cell growth, arrests cell cycle progression and promotes cell apoptosis of PC cells in vivo and in vitro. Furthermore, the rescue experiments demonstrated that TRIM29-induced proliferation is dependent on YAP1 in PC cells. Mechanistically, TRIM29 regulates YAP1 expression by directly binding to YAP1, and reduced its ubiquitination and degradation. CONCLUSION: Taken together, these results identify a novel mechanism used by PC growth, and provide insight regarding the role of TRIM29 in PC.

E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating FBP1 via destabilization of C/EBPα.

Pancreatic cancer is one of the most fatal cancers in humans. While it thrives in a state of malnutrition, the mechanism by which pancreatic cancer cells adapt to metabolic stress through metabolic reprogramming remains unclear. Here, we showed that UBR5, an E3 ubiquitin ligase, was significantly upregulated in pancreatic cancer patient samples compared to the levels in adjacent normal tissues. Levels of UBR5 were closely related to a malignant phenotype and shorter survival among pancreatic cancer patients. Multivariate analyses also revealed that UBR5 overexpression was an independent predictor of poor outcomes among patients with pancreatic cancer. Functional assays revealed that UBR5 contributes to the growth of pancreatic cancer cells by inducing aerobic glycolysis. Furthermore, we demonstrated that UBR5 knockdown increased levels of fructose-1,6-bisphosphatase (FBP1), an important negative regulator in the process of aerobic glycolysis in many cancers. We found a significant negative correlation between levels of UBR5 and FBP1, further demonstrating that UBR5-induced aerobic glycolysis is dependent on FBP1 in pancreatic cancer cells. Mechanistically, UBR5 regulates FBP1 expression by modulating C/EBPα, directly binding to C/EBPα, and promoting its ubiquitination and degradation. Together, these results identify a mechanism used by pancreatic cancer cells to survive the nutrient-poor tumour microenvironment and also provide insight regarding the role of UBR5 in pancreatic cancer cell adaptation to metabolic stresses.

FAT10 promotes the progression of bladder cancer by upregulating HK2 through the EGFR/AKT pathway.

The ubiquitin-like protein FAT10 and the hexokinase protein HK2 play vital regulatory roles in several cellular processes. However, the relationship between these two proteins and their role in the pathogenesis of bladder cancer are not well understood. Here, we found that FAT10 and HK2 protein levels were markedly higher in bladder cancer tissues than in normal adjacent tissues. In addition, RNAi-mediated silencing of FAT10 led to reduced HK2 levels and suppressed bladder cancer progression in vivo and in vitro. The results of our in vivo and in vitro experiments revealed that HK2 is critical for FAT10-mediated progression of bladder cancer. The current study demonstrated that FAT10 enhanced the progression of bladder cancer by positively regulating HK2 via the EGFR/AKT pathway. Based on our findings, FAT10 is believed to stabilize EGFR expression by modulating its degradation and ubiquitination. The results of the current study indicate that there is a correlation between FAT10 and HK2 in the progression of bladder cancer. In addition, we identified a new pathway that may be involved in the regulation of HK2. These findings implicate dysfunction of the FAT10, EGFR/AKT, and HK2 regulatory circuit in the progression of bladder cancer.

Hsa_circularRNA_0079201 suppresses chondrocyte proliferation and endochondral ossification by regulating the microRNA­140­3p/SMAD2 signaling pathway in idiopathic short stature.

Circular (circ)RNAs are an important group of non­coding RNAs involved in different pathological and physiological functions, such as longitudinal bone growth. However, the effects of an increase or decrease in circRNA expression on idiopathic short stature (ISS) remain largely unknown. The present study compared the circRNA expression patterns of patients with ISS and healthy individuals to identify differentially expressed circRNAs involved in the regulation of ISS pathogenesis and their target microRNAs (miR). Microarray analysis revealed that 145 circRNAs were differentially expressed in patients with ISS, including 83 up­ and 62 downregulated circRNAs. Reverse transcription­quantitative PCR confirmed that hsa_circRNA_0079201 was increased in patients with ISS compared with that in the normal individuals, whilst hsa_circRNA_0079201 overexpression in human chondrocytes was shown to significantly suppress their proliferation, hypertrophy and endochondral ossification abilities. Luciferase reporter assays identified that circRNA_0079201 acted as an miR­140­3p sponge. In situ hybridization confirmed the co­localization of circRNA_0079201 and miR­140­3p in the human chondrocyte and neonatal femur growth plate of C57 mice, while rescue experiments demonstrated that miR­140­3p overexpression reversed the inhibition of human chondrocyte proliferation, hypertrophy and endochondral ossification, caused by circRNA_0079201 overexpression. Bioinformatics analysis and luciferase reporter assays revealed that SMAD2 was a potential target gene of miR­140­3p. Furthermore, overexpressing circRNA_0079201 in human chondrocytes suppressed miR­140­3p and increased SMAD2 protein expression level. Taken together, chondrocyte proliferation, hypertrophy and endochondral ossification in ISS was suppressed by a novel regulatory axis consisting of the hsa_circRNA_0079201/miR­140­3p/SMAD2 pathway. The present study provided evidence that hsa_circRNA_0079201 may be a potential target for ISS therapy.

Ubiquitin-like protein FAT10 promotes osteosarcoma glycolysis and growth by upregulating PFKFB3 via stabilization of EGFR.

Osteosarcoma is a major cause of cancer-related deaths in adolescents. While it thrives in a state of malnutrition, the mechanism of metabolic stress adaptation via metabolic reprogramming is unclear. Here, we found that the level of FAT10, a ubiquitin-like protein, was significantly higher in tumors than in adjacent normal tissues. Moreover, high FAT10 levels were closely related to increased malignancy and shorter survival time in osteosarcoma patients. Multivariate analysis also showed that FAT10 overexpression was an independent predictor of poor prognosis. Functional assays indicated that FAT10 promoted osteosarcoma cell proliferation by inducing glycolysis. In addition, FAT10 knockdown reduced the level of PFKFB3, a positive regulator of glycolysis in many cancers. A positive correlation was found between FAT10 and PFKFB3 levels in osteosarcoma tissues, further indicating that FAT10 induced an increase in glycolysis and that cell growth depended on PFKFB3. Interestingly, FAT10 regulated PFKFB3 expression by directly binding to EGFR and inhibiting its ubiquitination and degradation. These results shed light on the mechanisms responsible for osteosarcoma cell survival in the malnourished tumor microenvironment. Further, the results provide insights into the role of FAT10 in the adaptation of osteosarcoma cells to metabolic stress.

Chondrocyte suppression is mediated by miR-129-5p via GDF11/SMAD3 signaling in developmental dysplasia of the hip.

Recent studies have shown that developmental dysplasia of the hip (DDH) during childhood and in animal models is associated with impaired endochondral ossification of the roof of the acetabulum, yet the molecular mechanism of this pathology remains unknown. To address this, an animal model of DDH was established in 4-week-old New Zealand white rabbits by cast immobilization of knee extension. Fifty-six rabbits of DDH were involved in this study, including 21 male rabbits and 25 female rabbits. High-throughput RNA sequencing identified 18 differentially expressed microRNAs; miR-129-5p downregulation was further confirmed by quantitative polymerase chain reaction. Bioinformatics and luciferase reporter assay identified growth differentiation factor 11 (GDF11) as the target gene of miR-129-5p in vitro. miR-129-5p downregulation increased GDF11 expression, which induced the phosphorylation of SMAD family member 3. As a result, the expression of runt-related transcription factor 2, Indian hedgehog homolog, and collagen type X was inhibited in vitro. Meanwhile, Alizarin Red S and Von Kossa staining revealed reduced formation of mineralized nodules by chondrocytes after miR-129-5P downregulation compared with the control. Additionally, proliferation assays and flow cytometry confirmed the suppression of chondrocyte proliferation and G1 cell cycle arrest following miR-129-5p downregulation. These findings indicate that miR-129-5p is able to suppress chondrocyte proliferation and hypertrophic differentiation and decrease mineralization via the miR-129-5p/GDF11/SMAD3 axis. This could present the underlying cause for the observed DDH-associated ossification impairment of the acetabular roof.

ROCK2 mediates osteosarcoma progression and TRAIL resistance by modulating O-GlcNAc transferase degradation.

Osteosarcoma is a common bone tumor, with a poor prognosis. New combinatorial therapies that sensitize anticancer drug-resistant osteosarcoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are, therefore, required. The GTPase RhoA effector, Rho-associated coiled-coil forming protein kinase 2 (ROCK2), is well known for its roles in various types of cancer; however, its involvement osteosarcoma has not yet been scrutinized. In this study, we analyzed ROCK2 expression, clinicopathological features, and prognosis in osteosarcoma patients. Apoptosis, colony formation, and cell proliferation were analyzed using flow cytometry, colony formation assays, and CCK8 assays, respectively. Proteomics analysis was used to evaluate osteosarcoma progression. We found that adjacent tissues had lower ROCK2 expression levels than osteosarcoma tissues and the level of expression was related to osteosarcoma tumor size and prognosis. Osteosarcoma prognosis was associated with ROCK2 expression level, which served as an independent marker in multivariate analysis. ROCK2 silencing inhibited proliferation in vivo and in vitro and triggered apoptotic osteosarcoma cell death. ROCK2 inhibited the TRAIL-mediated apoptotic pathway in osteosarcoma cells and promoted activation. Mechanistically, ROCK2 affected osteosarcoma progression and TRAIL resistance by modifying O-GlcNAcylation through O-GlcNAc transferase degradation. Taken together, our results demonstrated a unique mechanism whereby ROCK2 influences osteosarcoma progression and TRAIL resistance, hence improving osteosarcoma management.

Ubiquitin-like protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1.

Osteosarcoma is a common malignancy of the bone tissue. The rapid growth exhibited by this cancer is a primary challenge in its treatment. In many types of cancers, FAT10, a ubiquitin-like protein, is involved in several biological activities, especially cell proliferation. Herein, we demonstrate that FAT10 plays a vital role in tumorigenesis and is overexpressed in tumor tissues compared to its expression in adjacent normal tissues. Functional assays revealed that knockdown of FAT10 expression significantly repressed the proliferation of osteosarcoma in vitro and in vivo. Furthermore, our results indicate that FAT10 exhibits oncogenic functions by regulating the level of YAP1, a key protein of the Hippo/YAP signaling pathway, and a significant positive correlation exists between the levels of FAT10 and YAP1. Further analysis showed that FAT10-induced growth of osteosarcoma cells is dependent on YAP1. Mechanistically, FAT10 stabilizes YAP1 expression by regulating its ubiquitination and degradation. Taken together, our results link the two drivers of cell growth in osteosarcoma and reveal a novel pathway for FAT10 regulation. We provide new evidence for the biological and clinical significance of FAT10 as a potential biomarker for osteosarcoma.

ROCK2 promotes osteosarcoma growth and metastasis by modifying PFKFB3 ubiquitination and degradation.

Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) are widely involved in cell biological activities and play a key role in controlling various cell phenomena. However, the underlying mechanisms connecting ROCK2 and PFKFB3 in osteosarcoma growth and metastasis are poorly understood. In this study, we explored and analysed the role and molecular mechanism of ROCK2 and PFKFB3 in osteosarcoma. We analysed ROCK2 and PFKFB3 protein expression in 51 surgical specimens from osteosarcoma patients and determined the correlation between ROCK2 and PFKFB3. In addition, we used Transwell and wound-healing assays to detect cell invasion and migration and CCK8 and EdU assays to assess cell proliferation. Herein, we confirmed that ROCK2 and PFKFB3 proteins were significantly upregulated in osteosarcoma compared with adjacent normal tissues. Further studies revealed that knockdown of ROCK2 significantly decreased the expression levels of PFKFB3; moreover, growth and metastasis were decreased in shROCK2 osteosarcoma cells. Additionally, upregulation of PFKFB3 rescued the decreased proliferation and metastasis induced by ROCK2 knockdown, whereas knockdown of PFKFB3 decreased ROCK2-enhanced osteosarcoma proliferation and metastasis. These results suggest that PFKFB3 is essential for ROCK2-mediated proliferation and metastasis of osteosarcoma cells. Mechanistically, ROCK2 stabilizes PFKFB3 expression by modifying its ubiquitination and degradation. Taken together, our results link two drivers of proliferation and metastasis in osteosarcoma and identify a novel pathway for PFKFB3 regulation. Thus, we provide new evidence of the biological and clinical significance of PFKFB3 as a potential biomarker for osteosarcoma.