Splicing factor arginine/serine-rich 8 promotes multiple myeloma malignancy and bone lesion through alternative splicing of CACYBP and exosome-based cellular communication.

BACKGROUND: Multiple myeloma (MM) is a distinctive malignancy of plasma cell within the bone marrow (BM), of which alternative splicing factors play vital roles in the progression. Splicing factor arginine/serine-rich 8 (SFRS8) is the exclusive factor associated with MM prognosis, however its role in MM remains undefined. METHODS: The analyses of 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, immunohistochemistry, flow cytometry and xenograft model were performed to examine cell proliferation, cell cycle and apoptosis in SFRS8 overexpression or knockdown MM cells in vitro and in vivo. The SFRS8-regulated alternative splicing events were identified by RNA immunoprecipitation sequencing (RIP-seq) and validated by RIP-qPCR and Co-IP methods. Exosomes were extracted from the supernatant of myeloma cells by ultracentrifugation. Bone lesion was evaluated by TRAP staining in vitro and SCID/NOD-TIBIA mouse model. A neon electroporation system was utilised to deliver siRNA through exosomes. The effect of siRNA-loaded exosomes in vivo was evaluated by using a patient-derived tumor xenograft (PDX) model and SCID/NOD-TIBIA mouse model. RESULTS: SFRS8 was significantly upregulated in MM samples and positively associated with poor overall survival (OS) in MM patients. SFRS8 promoted MM cell proliferation in vitro and in vivo. Furthermore, calcyclin binding protein (CACYBP) was identified as the downstream target of SFRS8. Particularly, SFRS8 could reduce CACYBP isoform1 (NM_014412.3) and increase CACYBP isoform2 (NM_001007214.1) by mediating the alternative splicing of CACYBP, thereby altering the ubiquitination degradation of ß-catenin to promote MM progression. In addition, SFRS8 promoted osteoclast differentiation through exosomes in vitro and in vivo. More importantly, exosomal siRNA targeting CACYBP isoform2 inhibited tumour growth in PDX and SCID/NOD-TIBIA mouse models. CONCLUSION: Our findings demonstrate that targeting the SFRS8/CACYBP/ß-catenin axis may be a promising strategy for MM diagnosis and treatment.

Enhanced photocatalytic disinfection of Escherichia coli K-12 by porous g-C3N4 nanosheets: Combined effect of photo-generated and intracellular ROSs.

The porous graphitic carbon nitride nanosheets (PCNSs) with high yields were synthesized by using one-step chemical exfoliation method. PCNSs accelerated separation efficiency of photo-generated electron-hole pairs in comparison to bulk graphitic carbon nitride. The PCNS5 (exfoliation for 5 h) exhibited optimal photocatalytic disinfection capability towards Escherichia coli K-12 under simulated solar light irradiation with complete disinfection of 6.5 log10 cfu/mL of E. coil K-12 within 2 h. The enhanced photocatalytic activity of PCNS5 originated from mesoporous nanosheet structure. The possible mechanism of photocatalytic disinfection has proposed that intracellular reactive oxygen species levels and the activities of antioxidant enzymes (e.g., catalase and superoxide dismutase) were enhanced. Transmission electron microscope images observed during photocatalytic disinfection process suggested that the cell membrane was regarded as the first target for oxidation, resulting in a faster leakage of cytoplasmic content and finally degradation of DNA leading to bacterial death. Furthermore, the trapping experiment showed that superoxide radical (•O2-) and holes (h+) were responsible for E. coli K-12 disinfection by PCNS5.

Trop2 is a potential biomarker for the promotion of EMT in human breast cancer.

Trop2 is considered to have an important function in tumor metastasis and the promotion of epithelial­mesenchymal transition (EMT). E­cadherin is a crucial factor in intercellular adhesion and EMT transformation. In the present study, we detected the expression of Trop2 and E­cadherin in breast cancer (BC) to better define their prognostic value. The mRNA expression levels of these two genes in 20 cases of fresh BC tissues were detected by quantitative real­time polymerase chain reaction (qRT­PCR). We also detected the expression levels of these two genes by immunohistochemistry (IHC) in 312 BC tissues, and the correlations between the expression of these two genes and the clinicopathological characteristics in BC patients were analyzed. The mRNA and protein expression levels of the two genes in BC cell lines were studied by qRT­PCR and western blotting. The results indicated that Trop2+/E­cadherin­ was expressed in BC tissues more than that in the matched adjacent tissues. The protein expression results obtained via IHC were similar to the mRNA expression results. Trop2+/E­cadherin­ that was expressed in BC was associated with lymph node status, metastasis, tumor­node­metastasis (TNM) stage, and ER­/PR­/HER2­ expression. BC patients that expressed Trop2+/E­cadherin­ had poor overall survival rates. The results of Trop2 and E­cadherin expression levels obtained in the BC cell lines were the same as those obtained in the BC tissues. Overall, Trop2 has a potential role in the promotion of EMT in BC and it could be considered as a therapeutic target in the future.

Chromosomal instability and acquired drug resistance in multiple myeloma.

Chromosomal instability (CIN) is an important hallmark of human cancer. CIN not only contributes to all stages of tumor development (initiation, promotion and progression) but also drives, in large measure, the acquisition of drug resistance by cancer cells. Although CIN is a cornerstone of the complex mutational architecture that underlies neoplastic cell development and tumor heterogeneity and has been tightly associated with treatment responses and survival of cancer patients, it may be one of the least understood features of the malignant phenotype in terms of genetic pathways and molecular mechanisms. Here we review new insights into the type of CIN seen in multiple myeloma (MM), a blood cancer of terminally differentiated, immunoglobulin-producing B-lymphocytes called plasma cells that remains incurable in the great majority of cases. We will consider bona fide myeloma CIN genes, methods for measuring CIN in myeloma cells, and novel approaches to CIN-targeted treatments of patients with myeloma. The new findings generate optimism that enhanced understanding of CIN will lead to the design and testing of new therapeutic strategies to overcome drug resistance in MM in the not-so-distant future.

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling.

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using ß-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro. Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo. Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM.