Comparison of genomic alterations in Epstein-Barr virus-positive and Epstein-Barr virus-negative diffuse large B-cell lymphoma.

BACKGROUND: Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV-posDLBCL) is an aggressive B-cell lymphoma that often presents similar morphological and immune phenotype features to that of EBV-negative DLBCL (EBV-negDLBCL). AIMS AND METHODS: To better understand their difference in genomic landscape, we performed whole-exome sequencing (WES) of EBV-posDLBCL and EBV-negDLBCL. RESULTS: This analysis revealed a new mutational signature 17 (unknown) and signature 29 (smoking) in EBV-posDLBCL as well as a specific mutational signature 24 (associated with aflatoxin) in EBV-negDLBCL. Compared with EBV-negDLBCL, more somatic copy number alterations (CNAs) and deletions were detected in EBV-posDLBCL (p = 0.01). The most frequent CNAs specifically detected in EBV-posDLBCL were gains at 9p24.1 (PDL1 and JAK2), 8q22.2-q24.23 (DEPTOR and MYC), and 7q31.31-q32.2 (MET), which were validated in additional EBV-posDLBCL cases. Overall, 53.7% (22/41) and 62.9% (22/35) of the cases expressed PD-L1 and c-MET, respectively, in neoplastic cells, whereas only 15.4% (4/26) expressed c-MYC. Neoplastic c-MET expression was positively correlated with PD-L1 (p < 0.001) and MYC expression (p = 0.016). However, EBV-posDLBCL cases did not show any differences in overall survival between PD-L1-, c-MET-, or c-MYC-positive and -negative cases or between age-related groups. Analysis of the association between somatic mutation load and EBV status showed no difference in the distribution of tumor mutant burden between the two lymphomas (p = 0.41). Recurrent mutations in EBV-posDLBCL implicated several genes, including DCAF8L1, KLF2, and NOL9, while in EBV-negDLBCL, ANK2, BPTF, and CNIH3 were more frequently mutated. Additionally, PIM1 is the most altered gene in all the WES-detected cases. CONCLUSIONS: Our results confirm that genomic alteration differs significantly between EBV-posDLBCL and EBV-negDLBCL, and reveal new genetic alterations in EBV-posDLBCL. The positive correlation of c-MET and PD-L1/c-Myc expression may be involved in the pathogenesis of EBV-posDLBCL, which is should be explored prospectively in trials involving MET-directed therapies.

Nuclear transport maintenance of USP22-AR by Importin-7 promotes breast cancer progression.

The translocation of biological macromolecules between cytoplasm and nucleus is of great significance to maintain various life processes in both normal and cancer cells. Disturbance of transport function likely leads to an unbalanced state between tumor suppressors and tumor-promoting factors. In this study, based on the unbiased analysis of protein expression differences with a mass spectrometer between human breast malignant tumors and benign hyperplastic tissues, we identified that Importin-7, a nuclear transport factor, is highly expressed in breast cancer (BC) and predicts poor outcomes. Further studies showed that Importin-7 promotes cell cycle progression and proliferation. Mechanistically, through co-immunoprecipitation, immunofluorescence, and nuclear-cytoplasmic protein separation experiments, we discovered that AR and USP22 can bind to Importin-7 as cargoes to promote BC progression. In addition, this study provides a rationale for a therapeutic strategy to restream the malignant progression of AR-positive BC by inhibiting the high expression state of Importin-7. Moreover, the knockdown of Importin-7 increased the responsiveness of BC cells to the AR signaling inhibitor, enzalutamide, suggesting that targeting Importin-7 may be a potential therapeutic strategy.

Functional L-Arginine Derivative as an Efficient Vector for Intracellular Protein Delivery for Potential Cancer Therapy.

The utilization of cytosolic protein delivery is a promising approach for treating various diseases by replacing dysfunctional proteins. Despite the development of various nanoparticle-based intracellular protein delivery methods, the complicated chemical synthesis of the vector, loading efficiency and endosomal escape efficiency of proteins remain a great challenge. Recently, 9-fluorenylmethyloxycarbonyl (Fmoc)-modified amino acid derivatives have been used to self-assemble into supramolecular nanomaterials for drug delivery. However, the instability of the Fmoc group in aqueous medium restricts its application. To address this issue, the Fmoc ligand neighboring arginine was substituted for dibenzocyclooctyne (DBCO) with a similar structure to Fmoc to obtain stable DBCO-functionalized L-arginine derivative (DR). Azide-modified triethylamine (crosslinker C) was combined with DR to construct self-assembled DRC via a click chemical reaction for delivering various proteins, such as BSA and saporin (SA), into the cytosol of cells. The hyaluronic-acid-coated DRC/SA was able to not only shield the cationic toxicity, but also enhance the intracellular delivery efficiency of proteins by targeting CD44 overexpression on the cell membrane. The DRC/SA/HA exhibited higher growth inhibition efficiency and lower IC50 compared to DRC/SA toward various cancer cell lines. In conclusion, DBCO-functionalized L-arginine derivative represents an excellent potential vector for protein-based cancer therapy.

SNS-023 sensitizes hepatocellular carcinoma to sorafenib by inducing degradation of cancer drivers SIX1 and RPS16.

Hepatocellular carcinoma (HCC) remains challenging due to the lack of efficient therapy. Promoting degradation of certain cancer drivers has become an innovative therapy. The nuclear transcription factor sine oculis homeobox 1 (SIX1) is a key driver for the progression of HCC. Here, we explored the molecular mechanisms of ubiquitination of SIX1 and whether targeting SIX1 degradation might represent a potential strategy for HCC therapy. Through detecting the ubiquitination level of SIX1 in clinical HCC tissues and analyzing TCGA and GEPIA databases, we found that ubiquitin specific peptidase 1 (USP1), a deubiquitinating enzyme, contributed to the lower ubiquitination and high protein level of SIX1 in HCC tissues. In HepG2 and Hep3B cells, activation of EGFR-AKT signaling pathway promoted the expression of USP1 and the stability of its substrates, including SIX1 and ribosomal protein S16 (RPS16). In contrast, suppression of EGFR with gefitinib or knockdown of USP1 restrained EGF-elevated levels of SIX1 and RPS16. We further revealed that SNS-023 (formerly known as BMS-387032) induced degradation of SIX1 and RPS16, whereas this process was reversed by reactivation of EGFR-AKT pathway or overexpression of USP1. Consequently, inactivation of the EGFR-AKT-USP1 axis with SNS-032 led to cell cycle arrest, apoptosis, and suppression of cell proliferation and migration in HCC. Moreover, we showed that sorafenib combined with SNS-032 or gefitinib synergistically inhibited the growth of Hep3B xenografts in vivo. Overall, we identify that both SIX1 and RPS16 are crucial substrates for the EGFR-AKT-USP1 axis-driven growth of HCC, suggesting a potential anti-HCC strategy from a novel perspective.

Development and Validation of a Predictive Nomogram for Possible REM Sleep Behavior Disorders.

Objectives: To develop and validate a predictive nomogram for idiopathic rapid eye movement (REM) sleep behavior disorder (RBD) in a community population in Beijing, China. Methods: Based on the validated RBD questionnaire-Hong Kong (RBDQ-HK), we identified 78 individuals with possible RBD (pRBD) in 1,030 community residents from two communities in Beijing. The least absolute shrinkage and selection operator (LASSO) regression was applied to identify candidate features and develop the nomogram. Internal validation was performed using bootstrap resampling. The discrimination of the nomogram was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, and the predictive accuracy was assessed via a calibration curve. Decision curve analysis (DCA) was performed to evaluate the clinical value of the model. Results: From 31 potential predictors, 7 variables were identified as the independent predictive factors and assembled into the nomogram: family history of Parkinson's disease (PD) or dementia [odds ratio (OR), 4.59; 95% confidence interval (CI), 1.35-14.45; p = 0.011], smoking (OR, 3.24; 95% CI, 1.84-5.81; p < 0.001), physical activity (≥4 times/week) (OR, 0.23; 95% CI, 0.12-0.42; p < 0.001), exposure to pesticides (OR, 3.73; 95%CI, 2.08-6.65; p < 0.001), constipation (OR, 6.25; 95% CI, 3.58-11.07; p < 0.001), depression (OR, 3.66; 95% CI, 1.96-6.75; p < 0.001), and daytime somnolence (OR, 3.28; 95% CI, 1.65-6.38; p = 0.001). The nomogram displayed good discrimination, with original AUC of 0.885 (95% CI, 0.845-0.925), while the bias-corrected concordance index (C-index) with 1,000 bootstraps was 0.876. The calibration curve and DCA indicated the high accuracy and clinical usefulness of the nomogram. Conclusions: This study proposed an effective nomogram with potential application in the individualized prediction for pRBD.

ARV-771 Acts as an Inducer of Cell Cycle Arrest and Apoptosis to Suppress Hepatocellular Carcinoma Progression.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed liver cancer with limited treatment options and extremely poor prognosis worldwide. Recently, the proteolysis targeting chimeras (PROTACs), which aim to induce proteasome-mediated degradation of interesting proteins via recruiting E3 ligases, have become the advanced tools and attractive molecules for cancer treatment. However, the anticancer effects of PROTACs in HCC remain to be clarified. Here, we evaluate the anticancer activity of ARV-771, a previously reported PROTAC compound designed for bromodomain and extra-terminal domain (BET) proteins, in HCC. We show that ARV-771 suppresses the cell viability and colony formation of HCC cells via arresting cell cycle progression and triggering apoptosis. Further investigations reveal that ARV-771 notably downregulates multiple non-proteasomal deubiquitinases which are critical to the development of cancers. Additionally, HCC cells can decrease their sensitivity to ARV-771 via activating the MEK/ERK and p38 MAPKs. ARV-771 also inhibits HCC progression in vivo. Moreover, we show that ARV-771 and sorafenib, a Raf inhibitor that clinically used for targeted therapy of liver cancer, can synergistically inhibit the growth of HCC cells. Overall, this study not only explores the anticancer activity of ARV-771 and its underlying mechanisms in HCC, but also deepens our understanding of deubiquitinases, MAPKs, cell cycle, and apoptosis induction in cancer therapy.

A SIX1 degradation inducer blocks excessive proliferation of prostate cancer.

Prostate cancer (PC) remains a great medical challenge due to its high incidence and the development of castration resistance in patients treated with androgen deprivation therapy. Deubiquitinases, the enzymes that specifically hydrolyze ubiquitin chains on their substrates, were recently proposed as a serious of critical therapeutic targets for cancer treatment. Our previous study has been reported that the ubiquitin specific peptidase 1 (USP1) functionally acts as a deubiquitinase of sine oculis homeobox homolog 1 (SIX1) and contributes to the proliferation and castration resistance of PC. The stabilization of SIX1 by USP1 partially depends on the status of glucose-regulated protein 75 (GRP75). In this study, we aimed to identify a SIX1 degradation inducer via inhibiting the USP1-SIX1 axis. we screened a range of kinase inhibitors and showed that SNS-032 is the best candidate to trigger the ubiquitinated degradation of SIX1. SNS-032 not only restrains activity of the USP1-SIX1 axis and cell cycle progression, but also results in apoptosis of PC cells. Moreover, the combination of SNS-032 and enzalutamide synergistically induces apoptosis and downregulates expression of USP1, SIX1, and AR/AR-V7 in AR-V7 highly expressed 22Rv1 cells. Overall, our findings may develop a novel and effective strategy to overcome castration resistance in PC for the identification of a SIX1 degradation inducer via targeting the USP1-SIX1 axis.

Selective degradation of AR-V7 to overcome castration resistance of prostate cancer.

Androgen receptor splice variant 7 (AR-V7), a form of ligand-independent and constitutively activating variant of androgen receptor (AR), is considered as the key driver to initiate castration-resistant prostate cancer (CRPC). Because AR-V7 lacks ligand-binding domain, the AR-targeted therapies that aim to inactivate AR signaling through disrupting the interaction between AR and androgen are limited in CRPC. Thus, the emergence of AR-V7 has become the greatest challenge for treating CRPC. Targeting protein degradation is a recently proposed novel avenue for cancer treatment. Our previous studies have been shown that the oncoprotein AR-V7 is a substrate of the proteasome. Identifying novel drugs that can trigger the degradation of AR-V7 is therefore critical to cure CRPC. Here we show that nobiletin, a polymethoxylated flavonoid derived from the peel of Citrus fruits, exerts a potent anticancer activity via inducing G0/G1 phase arrest and enhancing the sensitivity of cells to enzalutamide in AR-V7 positive PC cells. Mechanically, we unravel that nobiletin selectively induces proteasomal degradation of AR-V7 (but not AR). This effect relies on its selective inhibition of the interactions between AR-V7 and two deubiquitinases USP14 and USP22. These findings not only enrich our understanding on the mechanism of AR-V7 degradation, but also provide an efficient and druggable target for overcoming CRPC through interfering the stability of AR-V7 mediated by the interaction between AR-V7 and deubiquitinase.

USP1-dependent RPS16 protein stability drives growth and metastasis of human hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a medical challenge due to its high proliferation and metastasis. Although deubiquitinating enzymes (DUBs) play a key role in regulating protein degradation, their pathological roles in HCC have not been fully elucidated. METHODS: By using biomass spectrometry, co-immunoprecipitation, western blotting and immunofluorescence assays, we identify ribosomal protein S16 (RPS16) as a key substrate of ubiquitin-specific peptidase 1 (USP1). The role of USP1-RPS16 axis in the progression of HCC was evaluated in cell cultures, in xenograft mouse models, and in clinical observations. RESULTS: We show that USP1 interacts with RPS16. The depletion of USP1 increases the level of K48-linked ubiquitinated-RPS16, leading to proteasome-dependent RPS16 degradation. In contrast, overexpression of USP1-WT instead of USP1-C90A (DUB inactivation mutant) reduces the level of K48-linked ubiquitinated RPS16, thereby stabilizing RPS16. Consequently, USP1 depletion mimics RPS16 deficiency with respect to the inhibition of growth and metastasis, whereas transfection-enforced re-expression of RPS16 restores oncogenic-like activity in USP1-deficient HCC cells. Importantly, the high expression of USP1 and RPS16 in liver tissue is a prognostic factor for poor survival of HCC patients. CONCLUSIONS: These findings reveal a previously unrecognized role for the activation of USP1-RPS16 pathway in driving HCC, which may be further developed as a novel strategy for cancer treatment.

A new role of GRP75-USP1-SIX1 protein complex in driving prostate cancer progression and castration resistance.

Prostate cancer (PC) is the second most common cancer with limited treatment option in males. Although the reactivation of embryonic signals in adult cells is one of the characteristics of cancer, the underlying protein degradation mechanism remains elusive. Here, we show that the molecular chaperone GRP75 is a key player in PC cells by maintaining the protein stability of SIX1, a transcription factor for embryonic development. Mechanistically, GRP75 provides a platform to recruit the deubiquitinating enzyme USP1 to inhibit K48-linked polyubiquitination of SIX1. Structurally, the C-terminus of GRP75 (433-679 aa) contains a peptide binding domain, which is required for the formation of GRP75-USP1-SIX1 protein complex. Functionally, pharmacological or genetic inhibition of the GRP75-USP1-SIX1 protein complex suppresses tumor growth and overcomes the castration resistance of PC cells in vitro and in xenograft mouse models. Clinically, the protein expression of SIX1 in PC tumor tissues is positively correlated with the expression of GRP75 and USP1. These new findings not only enhance our understanding of the protein degradation mechanism, but also may provide a potential way to enhance the anti-cancer activity of androgen suppression therapy.

TSPAN31 suppresses cell proliferation in human cervical cancer through down-regulation of its antisense pairing with CDK4.

Natural antisense transcripts (NAT) are prevalent phenomena in the mammalian genome and play significant regulatory roles in gene expression. While new insights into NAT continue to be revealed, their exact function and their underlying mechanisms in human cancer remain largely unclear. We identified a NAT of CDK4, referred to TSPAN31, which inhibits CDK4 mRNA and protein expression in human cervical cancer by targeting the 3'-untranslated region (3'-UTR) of the CDK4 mRNA. Furthermore, silencing the expression of the TSPAN31 mRNA rescued the TSPAN31 3'-UTR- or the TSPAN31 full-length-induced decrease in CDK4 expression. Noteworthy, we discovered that TSPAN31, as a member of the tetraspanin family, suppressed cell proliferation by down-regulating its antisense pairing with CDK4 and decreasing retinoblastoma protein phosphorylation in human cervical cancer. Therefore, the results of the present study suggest that TSPAN31 may serve as a potential molecular target for the development of novel anti-cancer agents. SIGNIFICANCE OF THE STUDY: Natural antisense transcripts are widely found in the genome and play an important role in the growth and development of cells. TSPAN31 is natural antisense transcript, and CDK4 is an important gene in the regulation of the cell cycle. Therefore, TSPAN31 and CDK4 have great significance in the study of tumour therapeutic targets.

LXW7 attenuates inflammation via suppressing Akt/nuclear factor kappa B and mitogen-activated protein kinases signaling pathways in lipopolysaccharide-stimulated BV2 microglial cells.

Microglia activation is closely linked to ischemia, various chronic neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis), and many other central nervous system diseases. Accumulating evidence suggests that depressing the microglial inflammatory response could be an effective treatment for inflammatory disorders. The integrin αvß3 inhibitor LXW7 has a neuroprotective effect; however, its anti-inflammatory effects and underlying mechanism remain unclear. Thus, we examined whether LXW7 would inhibit inflammatory cytokines and mediators, and we evaluated the potential mechanisms of its neuroprotective effects. Nitrite analysis revealed LXW7 reduced the nitric oxide (NO) level. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) suggested that LXW7 suppressed the expression of proinflammatory genes for tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and anti-inflammatory gene interleukin 10 (IL-10) at the messenger ribonucleic acid level. Enzyme-linked immunosorbent assay results demonstrated that LXW7 treatment reduced the expression of prostaglandin E2 (PGE2), TNF-α, IL-1ß and IL-10 at the protein level. Western blotting was conducted to confirm the upregulation of inflammatory factors, including iNOS and COX-2 at the protein level. LXW7 inhibited major genes in the Akt/NF-κB and c-Jun NH2-terminal kinase/ mitogen-activated protein kinases (JNK/MAPK) signaling pathways. Immunofluorescence revealed that LXW7 inhibited the nuclear translocation of nuclear factor kappa B (NF-κB). Thus, LXW7 effectively alleviated LPS-induced inflammatory damage and had neuroprotective effects. The anti-inflammatory effects of LXW7 may be associated with the inhibition of microglial activation via Akt/NF-κB and JNK/MAPK signaling pathways by blocking integrin αvß3 receptor. The present study's findings suggest that LXW7 has a substantial therapeutic potential for treating inflammatory and neurodegenerative diseases.

TSPAN31 is a critical regulator on transduction of survival and apoptotic signals in hepatocellular carcinoma cells.

Tetraspanins are commonly believed to act as 'molecular facilitators', not directly involved in signal transduction. Tetraspanin 31 (TSPAN31), recently discovered to be linked to cancer, has not yet been studied in hepatocellular carcinoma (HCC). Here, we show that TSPAN31 is the natural antisense transcript of cyclin dependent kinase 4 (CDK4), and regulates the expression of CDK4 mRNA and protein. Target analysis indicates that miR-135b can directly regulate TSPAN31 expression. miR-135b-induced TSPAN31 silencing increases CDK4 protein levels. Interestingly, p53 negatively regulates TSPAN31 expression. siRNA-induced TSPAN31 knockdown reduces the expression of Akt signaling pathway components phosphorylated Akt, p-GSK3ß and ß-catenin, and restrains ß-catenin migration to cell nucleus. TSPAN31 knockdown also significantly inhibits HCC cell invasion and migration. These findings thus point to TSPAN31 as a novel regulator in transduction of intracellular survival and apoptotic signals.

[Cannabinoid WIN55, 212-2 inhibits proliferation, invasion and migration of human SMMC-7721 hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects of WIN55, 212-2 (WIN) on the proliferation, invasion and migration of SMMC-7721 hepatocellular carcinoma cells and its underlying mechanisms. METHODS: SMMC-7721 cells were treated with (0, 1, 5, 10, 20) µmol/L WIN, and cell viability was determined by CCK-8 assay. The morphological changes of the cells were observed under a fluorescence microscope with Hoechst33258 staining. Cell apoptosis was measured by flow cytometry combined with annexin V-FITC/PI staining. The expression levels of apoptosis-related proteins P53, P21, Bcl-2 and Bax, and the phosphorylated AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were analyzed by Western blotting. Transwell(TM) invasion assay was used to detect cell invasion ability. Would healing assay was performed to test cell migration ability. The expression level of matrix metalloproteinase 14 (MMP-14) was evaluated by Western blotting. RESULTS: WIN inhibited the proliferation of SMMC-7721 cells and induced cell apoptosis in a dose-dependent manner. After treatment with WIN, the cell nucleus concentrated and broken, indicating obvious cell apoptosis. Western blotting exhibited an up-regulation in the protein expression of P53, P21 and Bax. And the anti-apoptotic protein Bcl-2 was repressed. The expression levels of AKT, p-AKT and p-ERK were down-regulated, whereas the expression of total ERK was not obviously changed. Compared with control group, there was a significant inhibition of cell invasion and migration abilities when SMMC-7721 cells were treated with WIN. The expression level of MMP-14 decreased as well. CONCLUSION: WIN can inhibit the proliferation of SMMC-7721 cells and induce cell apoptosis. The mechanism is associated with the activation of P53 and the inhibition of AKT, p-AKT and p-ERK. WIN can inhibit the invasion and migration of SMMC-7721 cells through down-regulating the protein expression of MMP-14.