ALBERT

Description: Background The absence of dystrophin has gave a massive impact on myotube development in Muscular Dystrophy pathogenesis. One of the conserved signaling pathways involved in skeletal muscle differentiation is the PI3K/Akt/mTOR pathway that plays a vital role in autophagy regulation. To further understand and establish targeted therapy in dystrophin-deficient myoblasts, protein expression profiling has been determined which provides information on perturbed autophagy modulation and activation. Methods In this study, a dystrophin-deficient myoblast cell line established from the skeletal muscle of a dystrophic (mdx) mouse was used as a model. The dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation. The cells were subjected to total protein extraction prior to Western blotting assay technique. Protein sub-fractionation has been conducted to determine protein localization. The live-cell analysis of autophagy assay was done using a flow cytometer. Results In our culture system, the dfd13 myoblasts did not achieve terminal differentiation. PTEN expression was profoundly increased in dfd13 myoblasts throughout the differentiation day subsequently indicates perturbation of PI3K/Akt/mTOR regulation. In addition, rictor-mTORC2 was also found inactivated in this event. This occurrence has caused FoxO3 misregulation leads to higher activation of autophagy-related genes in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, the ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features. Conclusion Perturbation of the PTEN-PI3K/Akt pathway triggers excessive autophagosome formation and subsequently reduced autophagic flux within dystrophin-deficient myoblasts where these findings are of importance to understand Duchenne Muscular Dystrophy (DMD) patients. We believe that some manipulation within its regulatory signaling reported in this study could help restore muscle homeostasis and attenuate disease progression.

Description: Intercellular communication is a critical process that ensures cooperation between distinct cell types and maintains homeostasis. EVs, which were initially described as cellular debris and devoid of biological function, are now recognized as key components in cell–cell communication. EVs are known to carry multiple factors derived from their cell of origin, including cytokines and chemokines, active enzymes, metabolites, nucleic acids, and surface molecules, that can alter the behavior of recipient cells. Since the cargo of EVs reflects their parental cells, EVs from damaged and dysfunctional tissue environments offer an abundance of information toward elucidating the molecular mechanisms of various diseases and pathological conditions. In this review, we discuss the most recent findings regarding the role of EVs in the progression of cancer, metabolic disorders, and inflammatory lung diseases given the high prevalence of these conditions worldwide and the important role that intercellular communication between immune, parenchymal, and stromal cells plays in the development of these pathological states. We also consider the clinical applications of EVs, including the possibilities for their use as novel therapeutics.

Description: Background Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. Methods A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. Results Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Conclusions CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.

Description: Glioma is the most common primary brain tumor and its prognosis is poor. Despite surgical removal, glioma is still prone to recurrence because it grows rapidly in the brain, is resistant to chemotherapy, and is highly aggressive. Therefore, there is an urgent need for a platform to study the cell dynamics of gliomas in order to discover the characteristics of the disease and develop more effective treatments. Although 2D cell models and animal models in previous studies have provided great help for our research, they also have many defects. Recently, scientific researchers have constructed a 3D structure called Organoids, which is similar to the structure of human tissues and organs. Organoids can perfectly compensate for the shortcomings of previous glioma models and are currently the most suitable research platform for glioma research. Therefore, we review the three methods currently used to establish glioma organoids. And introduced how they play a role in the diagnosis and treatment of glioma. Finally, we also summarized the current bottlenecks and difficulties encountered by glioma organoids, and the current efforts to solve these difficulties.

Description: Background The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells. Methods Cellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB. Results CAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics. Conclusion This study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures.

Description: Background Recent studies have shown that bone morphogenetic protein receptor 2 (BMPR2) regulates cell survival signaling events in cancer cells independent of the BMP type 1 receptor (BMPR1) or the Smad-1/5 transcription factor. Mutations in BMPR2 trafficking proteins leads to overactive BMP signaling, which leads to neurological diseases caused by BMPR2 stabilization of the microtubules. It is not known whether BMPR2 regulates the microtubules in cancer cells and what effect this has on cell survival. It is also not known whether alterations in BMPR2 trafficking effects activity and response to BMPR2 inhibitors. Methods We utilized BMPR2 siRNA and the BMP receptor inhibitors JL5 and Ym155, which decrease BMPR2 signaling and cause its mislocalization to the cytoplasm. Using the JL5 resistant MDA-MD-468 cell line and sensitive lung cancer cell lines, we examined the effects of BMPR2 inhibition on BMPR2 mislocalization to the cytoplasm, microtubule destabilization, lysosome activation and cell survival. Results We show that the inhibition of BMPR2 destabilizes the microtubules. Destabilization of the microtubules leads to the activation of the lysosomes. Activated lysosomes further decreases BMPR2 signaling by causing it to mislocalizated to the cytoplasm and/or lysosome for degradation. Inhibition of the lysosomes with chloroquine attenuates BMPR2 trafficking to the lysosome and cell death induced by BMPR2 inhibitors. Furthermore, in MDA-MD-468 cells that are resistant to JL5 induced cell death, BMPR2 was predominately located in the cytoplasm. BMPR2 failed to localize to the cytoplasm and/or lysosome following treatment with JL5 and did not destabilize the microtubules or activate the lysosomes. Conclusions These studies reveal that the inhibition of BMPR2 destabilizes the microtubules promoting cell death of cancer cells that involves the activation of the lysosomes. Resistance to small molecules targeting BMPR2 may occur if the BMPR2 is localized predominantly to the cytoplasm and/or fails to localize to the lysosome for degradation.

Description: Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

Description: Background Exosomes mediated crosstalk between tumor cells and other stromal cells including tumor associated macrophages plays an essential role in reprogramming tumor microenvironment (TME) to facilitate tumor progression. However, the mechanism of tumor derived exosomes promotes bladder cancer progression have not been defined. Methods Exosomes were extracted from bladder cancer cells MB49 conditioned medium by ultracentrifugation. The effects of MB49-derived exosomes on macrophages polarization were analyzed by qPCR, flow cytometry, and Western blot. The immunosuppressive phenotype and function of MB49-derived exosomes stimulated macrophages were verified by tumor xenograft assays and T cell co-culture experiments. Exosomal miRNAs were analyzed by microarray to identify potential targets regulating macrophage polarization. Results MB49-derived exosomes could be ingested by macrophages, consequently promoting macrophages immunosuppressive polarization. Mechanically, the MB49-derived exosomes induced macrophage M2 polarization was mediated by down-regulation of PTEN and activation of AKT/STAT3/6 signaling. Moreover, hindrance of the generation or secretion of exosomes by GW4869 inhibited macrophages differentiation into immunosuppressive phenotype and function, thereby suppressed tumor growth in a mouse subcutaneous tumor model. Conclusion Our study confirmed the contribution of bladder cancer derived exosomes on the establishment of immunosuppressive TME and provided a potential therapeutic target for bladder cancer treatment.

Description: Ulcerative colitis (UC) has closely been associated with an increased risk of colorectal cancer. However, the exact mechanisms underlying colitis-associated cancer (CAC) development remain unclear. As a classic pattern-recognition receptor, Toll like receptor (TLR)4 is a canonical receptor for lipopolysaccharide of Gram-negative bacteria (including two CAC-associated pathogens Fusobacterium nucleatum and Salmonella), and functions as a key bridge molecule linking oncogenic infection to colonic inflammatory and malignant processes. Accumulating studies verified the overexpression of TLR4 in colitis and CAC, and the over-expressed TLR4 might promote colitis-associated tumorigenesis via facilitating cell proliferation, protecting malignant cells against apoptosis, accelerating invasion and metastasis, as well as contributing to the creation of tumor-favouring cellular microenvironment. In recent years, considerable attention has been focused on the regulation of TLR4 signaling in the context of colitis-associated tumorigenesis. MicroRNA (miR)-155 and TLR4 exhibited a similar dynamic expression change during CAC development and shared similar CAC-promoting properties. The available data demonstrated an interplay between TLR4 and miR-155 in the context of different disorders or cell lines. miR-155 could augment TLR4 signaling through targeting negative regulators SOCS1 and SHIP1; and TLR4 activation would induce miR-155 expression via transcriptional and post-transcriptional mechanisms. This possible TLR4-miR-155 positive feedback loop might result in the synergistic accelerating effect of TLR4 and miR-155 on CAC development.

Description: Background Metastasis represents the leading cause of death in patients with breast cancer. Traditional Chinese medicine is particularly appreciated for metastatic diseases in Asian countries due to its benefits for survival period prolongation and immune balance modulation. However, the underlying molecular mechanisms remain largely unknown. This study aimed to explore the antimetastatic effect and immunomodulatory function of a clinical formula Aiduqing (ADQ). Methods Naive CD4+ T cells, regulatory T cells (Tregs), and CD8+ T cells were sorted by flow cytometry. Then, breast cancer cells and these immune cells were co-cultured in vitro or co-injected into mice in vivo to simulate their coexistence. Flow cytometry, ELISA, qPCR, double luciferase reporter gene assay, and chromatin immunoprecipitation assay were conducted to investigate the immunomodulatory and antimetastatic mechanisms of ADQ. Results ADQ treatment by oral gavage significantly suppressed 4T1-Luc xenograft growth and lung metastasis in the orthotopic breast cancer mouse model, without noticeable hepatotoxicity, nephrotoxicity, or hematotoxicity. Meanwhile, ADQ remodeled the immunosuppressive tumor microenvironment (TME) by increasing the infiltration of tumor-infiltrating lymphocytes (TILs) and cytotoxic CD8+ T cells, and decreasing the infiltration of Tregs, naive CD4+ T cells, and tumor-associated macrophages (TAMs). Molecular mechanism studies revealed that ADQ remarkably inhibited CXCL1 expression and secretion from TAMs and thus suppressed the chemotaxis and differentiation of naive CD4+ T cells into Tregs, leading to the enhanced cytotoxic effects of CD8+ T cells. Mechanistically, TAM-derived CXCL1 promoted the differentiation of naive CD4+ T cells into Tregs by transcriptionally activating the NF-κB/FOXP3 signaling. Lastly, mouse 4T1-Luc xenograft experiments validated that ADQ formula inhibited breast cancer immune escape and lung metastasis by suppressing the TAM/CXCL1/Treg pathway. Conclusions This study not only provides preclinical evidence supporting the application of ADQ in inhibiting breast cancer metastasis but also sheds novel insights into TAM/CXCL1/NF-κB/FOXP3 signaling as a promising therapeutic target for Treg modulation and breast cancer immunotherapy.