In Silico Transcriptomic Expression of MSR1 in Solid Tumors Is Associated with Responses to Anti-PD1 and Anti-CTLA4 Therapies.

Immuno-oncology has gained momentum with the approval of antibodies with clinical activities in different indications. Unfortunately, for anti-PD (L)1 agents in monotherapy, only half of the treated population achieves a clinical response. For other agents, such as anti-CTLA4 antibodies, no biomarkers exist, and tolerability can limit administration. In this study, using publicly available genomic datasets, we evaluated the expression of the macrophage scavenger receptor-A (SR-A) (MSR1) and its association with a response to check-point inhibitors (CPI). MSR1 was associated with the presence of macrophages, dendritic cells (DCs) and neutrophils in most of the studied indications. The presence of MSR1 was associated with macrophages with a pro-tumoral phenotype and correlated with TIM3 expression. MSR1 predicted favorable overall survival in patients treated with anti-PD1 (HR: 0.56, FDR: 1%, p = 2.6 × 10-5), anti PD-L1 (HR: 0.66, FDR: 20%, p = 0.00098) and anti-CTLA4 (HR: 0.37, FDR: 1%, p = 4.8 × 10-5). When specifically studying skin cutaneous melanoma (SKCM), we observed similar effects for anti-PD1 (HR: 0.65, FDR: 50%, p = 0.0072) and anti-CTLA4 (HR: 0.35, FDR: 1%, p = 4.1 × 10-5). In a different dataset of SKCM patients, the expression of MSR1 predicted a clinical response to anti-CTLA4 (AUC: 0.61, p = 2.9 × 10-2). Here, we describe the expression of MSR1 in some solid tumors and its association with innate cells and M2 phenotype macrophages. Of note, the presence of MSR1 predicted a response to CPI and, particularly, anti-CTLA4 therapies in different cohorts of patients. Future studies should prospectively explore the association of MSR1 expression and the response to anti-CTLA4 strategies in solid tumors.

Understanding the Role of Scavenger Receptor A1 in Nanoparticle Uptake by Murine Macrophages.

Nanoparticles can be cleared from the circulation and taken up by tissue-resident macrophages. This property can be beneficial when drug or antigen delivery to macrophages is desired; however, rapid clearance of nanoparticles not intended for delivery to immune cells may reduce nanoparticle circulation time and affect the efficacy of nanoparticle-formulated drug products. Therefore, understanding nanoparticles' uptake by macrophages is an essential step in the preclinical development of nanotechnology-based drug products. Understanding the route of nanoparticle uptake by macrophages may also provide mechanistic insights into the immunotoxicity of nanomaterials. The protocol described herein can be used to assess the nanoparticles' uptake by macrophages and understand the involvement of scavenger receptor A1 to inform mechanistic studies.

Morinda officinalis polysaccharide promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via microRNA-210-3p/scavenger receptor class A member 3.

Morinda officinalis polysaccharide (MOP) is the bioactive ingredient extracted from the root of Morinda officinalis, and Morinda officinalis is applied to treat osteoporosis (OP). The purpose of this study was to determine the role of MOP on human bone marrow mesenchymal stem cells (hBMSCs) and the underlying mechanism. HBMSCs were isolated from bone marrow samples of patients with OP and treated with MOP. Quantitative real-time polymerase chain reaction was adopted to quantify the expression of microRNA-210-3p (miR-210-3p) and scavenger receptor class A member 3 (SCARA3) mRNA. Cell Counting Kit-8 assay was employed to detect cell viability; Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling assay and flow cytometry were adopted to detect apoptosis; Alkaline Phosphatase (ALP) activity assay kit was applied to detect ALP activity; Western blot was executed to quantify the expression levels of SCARA3, osteogenic and adipogenic differentiation markers. Ovariectomized rats were treated with MOP. Bone mineral density (BMD), serum tartrate-resistant acid phosphatase 5b (TRACP 5b), and N-telopeptide of type I collagen (NTx) levels were assessed by BMD detector and Enzyme-linked immunosorbent assay kits. It was revealed that MOP could promote hBMSCs' viability and osteogenic differentiation and inhibit apoptosis and adipogenic differentiation. MOP could also upregulate SCARA3 expression through repressing miR-210-3p expression. Treatment with MOP increased the BMD and decreased the TRACP 5b and NTx levels in ovariectomized rats. MOP may boost the osteogenic differentiation and inhibit adipogenic differentiation of hBMSCs by miR-210-3p/SCARA3 axis.

Macrophage-expressed SRA ameliorates alcohol-induced liver injury by suppressing S-glutathionylation of Notch1 via recruiting thioredoxin.

Scavenger receptor A (SRA) is preferentially expressed in macrophages and implicated as a multifunctional pattern recognition receptor for innate immunity. Hepatic macrophages play a primary role in the pathogenesis of alcoholic liver disease. Herein, we observed that SRA expression was significantly increased in the liver tissues of mice with alcohol-related liver injury. SRA-deficient (SRA-/-) mice developed more severe alcohol-induced liver disease than wild-type mice. Enhanced liver inflammation existed in alcohol-challenged SRA-/- mice and was associated with increased Notch activation in hepatic macrophages compared with wild-type control animals. Mechanistically, SRA directly bound with Notch1 and suppressed its S-glutathionylation, thereby inhibiting Notch pathway activation. Further, we determined that the SRA interacted with thioredoxin-1 (Trx-1), a redox-active protein. SRA inhibited Trx-1 dimerization and facilitated the interaction of Trx-1 with Notch1. Application of a Trx-1-specific inhibitory agent during macrophage stimulation abolished SRA-mediated regulation of the Notch pathway and its downstream targets. In summary, our study revealed that SRA plays a critical role in macrophage inflammatory response by targeting Notch1 for its glutathionylation. SRA-mediated negative regulation of Notch activation might serve as a novel therapeutic strategy for alcohol-induced liver injury.

Cell-binding IgM in CSF is distinctive of multiple sclerosis and targets the iron transporter SCARA5.

Intrathecal IgM production in multiple sclerosis is associated with a worse disease course. To investigate pathogenic relevance of autoreactive IgM in multiple sclerosis, CSF from two independent cohorts, including multiple sclerosis patients and controls, were screened for antibody binding to induced pluripotent stem cell-derived neurons and astrocytes, and a panel of CNS-related cell lines. IgM binding to a primitive neuro-ectodermal tumour cell line discriminated 10% of multiple sclerosis donors from controls. Transcriptomes of single IgM producing CSF B cells from patients with cell-binding IgM were sequenced and used to produce recombinant monoclonal antibodies for characterization and antigen identification. We produced five cell-binding recombinant IgM antibodies, of which one, cloned from an HLA-DR + plasma-like B cell, mediated antigen-dependent complement activation. Immunoprecipitation and mass spectrometry, and biochemical and transcriptome analysis of the target cells identified the iron transport scavenger protein SCARA5 as the antigen target of this antibody. Intrathecal injection of a SCARA5 antibody led to an increased T cell infiltration in an experimental autoimmune encephalomyelitis (EAE) model. CSF IgM might contribute to CNS inflammation in multiple sclerosis by binding to cell surface antigens like SCARA5 and activating complement, or by facilitating immune cell migration into the brain.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

Methylated tumor suppressor gene SCARA5 inhibits the proliferation, migration and invasion of nasopharyngeal carcinoma.

Background: SCARA5 may play an important role in nasopharyngeal carcinoma. Materials & methods: PCR and immunohistochemistry were used to detect the expression and promoter methylation of SCARA5. Cell proliferation assays, spheroid culture, flow cytometry analysis, Transwell assays and xenotransplantation tests were utilized to determine the functional effects of SCARA5. RNA-sequencing, western blotting, immunofluorescence and dual-luciferase reporter assays were used to assess SCARA5-mediated outcomes. Results: SCARA5 was downregulated by promoter methylation. Overexpression of SCARA5 inhibited cell migration, invasion and proliferation. SCARA5 enhanced nasopharyngeal carcinoma cell sensitivity to chemotherapy with cisplatin and 5-fluorouracil. SCARA5 drives tumor apoptosis by downregulating HSPA2. Conclusion: SCARA5 may be a useful clinical marker in nasopharyngeal carcinoma.

m6A-mediated upregulation of lncRNA RMRP boosts the progression of bladder cancer via epigenetically suppressing SCARA5.

Aim: This study aimed to elucidate the relationship between SCARA5 and RMRP in bladder cancer and their underlying mechanism. Methods: Biological functions were evaluated using cell-counting kit 8 assay, 5-ethynyl-2'-deoxyuridine incorporation, wound healing and Transwell assays. RNA immunoprecipitation, RNA pull-down and chromatin immunoprecipitation were employed. A xenograft tumor model in nude mice was also conducted. Results & conclusion: RMRP and SCARA5 exhibited an inverse correlation. Downregulation of RMRP significantly suppressed bladder cancer cell proliferation, migration and invasion, which was reversed by SCARA5 overexpression. RMRP recruited DNA methyltransferases to the promoter region of SCARA5, thereby triggering the methylation of the SCARA5 promoter to epigenetically suppress its expression. Our findings elucidate the machinery by which RMRP, stabilized by METTL3, exerts a promoter role in bladder cancer tumorigenesis by triggering SCARA5 methylation.

SCARA5 as a downstream factor of PCAT29, inhibits proliferation, migration, and invasion of bladder cancer.

Scavenger receptor class A, member 5 (SCARA5) has been identified a novel tumor suppressor in several cancers. However, the functional and underlying mechanism of SCARA5 in bladder cancer (BC) need investigation. Here, we found SCARA5 expression was downregulated in both BC tissues and cell lines. Low SCARA5 in BC tissues was associated with a shorter overall survival. Moreover, SCARA5 overexpression reduced BC cell viability, colony formation, invasion, and migration. Further investigation demonstrated that the expression of SCARA5 was negatively regulated by miR-141. Furthermore, the long non-coding RNA prostate cancer associated transcript 29 (PCAT29) inhibited the proliferation, invasion, and migration of BC cells by sponging miR-141. Luciferase activity assays revealed that PCAT29 targeted miR-141 and miR-141 targeted SCARA5. In conclusion, SCARA5, as a downstream factor of the PCAT29/miR-141 axis, inhibited the proliferation, migration, and invasion of BC cells. These findings provide novel insights into the detailed molecular mechanisms of BC development.

SCARA5 in bone marrow stromal cell-derived exosomes inhibits colorectal cancer progression by inactivating the PI3K/Akt pathway.

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer worldwide. Bone marrow stromal cells (BMSCs) play an essential role in tumor development by secreting exosomes. Scavenger receptor class A member 5 (SCARA5) is a newly identified tumor suppressor. This study aimed to investigate the effects of BMSCs-derived exosomes (BMSCs-Exos) on CRC development and to explore their regulatory mechanisms. BMSCs-Exos showed an oval-shaped, bilayer membrane structure. BMSCs-Exos inhibited growth and motility of CRC cells, while BMSCs-Exos with SCARA5 knockdown significantly promoted cell proliferation and movement. Exosomal SCARA5 also effectively suppressed colorectal tumor growth in mouse xenografts. Further analysis revealed that exosomal SCARA5 inhibited the phosphorylation of protein kinase B and phosphoinositide 3-kinase in both CRC cells and tumors. In conclusion, SCARA5 in BMSCs-Exos inhibited CRC progression by inactivating PI3K/Akt, thus suggesting the potential clinical application of SCARA5-containing BMSCs-Exos for CRC treatment.

Dysregulation of Iron Metabolism-Linked Genes at Myocardial Tissue and Cell Levels in Dilated Cardiomyopathy.

In heart failure, the biological and clinical connection between abnormal iron homeostasis, myocardial function, and prognosis is known; however, the expression profiles of iron-linked genes both at myocardial tissue and single-cell level are not well defined. Through publicly available bulk and single-nucleus RNA sequencing (RNA-seq) datasets of left ventricle samples from adult non-failed (NF) and dilated cardiomyopathy (DCM) subjects, we aim to evaluate the altered iron metabolism in a diseased condition, at the whole cardiac tissue and single-cell level. From the bulk RNA-seq data, we found 223 iron-linked genes expressed at the myocardial tissue level and 44 differentially expressed between DCM and NF subjects. At the single-cell level, at least 18 iron-linked expressed genes were significantly regulated in DCM when compared to NF subjects. Specifically, the iron metabolism in DCM cardiomyocytes is altered at several levels, including: (1) imbalance of Fe3+ internalization (SCARA5 down-regulation) and reduction of internal conversion from Fe3+ to Fe2+ (STEAP3 down-regulation), (2) increase of iron consumption to produce hemoglobin (HBA1/2 up-regulation), (3) higher heme synthesis and externalization (ALAS2 and ABCG2 up-regulation), (4) lower cleavage of heme to Fe2+, biliverdin and carbon monoxide (HMOX2 down-regulation), and (5) positive regulation of hepcidin (BMP6 up-regulation).

Activation of the RARα Attenuated CSF Hypersecretion to Inhibit Hydrocephalus Development via Regulating the MAFB/MSR1 Pathway.

Hydrocephalus has been observed in rats with spontaneous hypertension (SHRs). It has been demonstrated that activation of the oxidative stress related protein retinoic acid receptor alpha (RARα) has neuroprotective impacts. Our investigation aims to determine the potential role and mechanism of RARα in hydrocephalus. The RARα-specific agonist (Am80) and RARα inhibitor (AGN196996) were used to investigate the role of RARα in cerebrospinal fluid (CSF) secretion in the choroid plexus of SHRs. Evaluations of CSF secretion, ventricular volume, Western blotting, and immunofluorescent staining were performed. Hydrocephalus and CSF hypersecretion were identified in SHRs but not in Wistar-Kyoto rats, occurring at the age of 7 weeks. The RARα/MAFB/MSR1 pathway was also activated in SHRs. Therapy with Am80 beginning in week 5 decreased CSF hypersecretion, hydrocephalus development, and pathological changes in choroid plexus alterations by week 7. AGN196996 abolished the effect of Am80. In conclusion, activation of the RARα attenuated CSF hypersecretion to inhibit hydrocephalus development via regulating the MAFB/MSR1 pathway. RARα may act as a possible therapeutic target for hydrocephalus.

Long-term glucocorticoid treatment increases CD204 expression by activating the MAPK pathway and enhances modified LDL uptake in murine macrophages.

Atherosclerotic plaques develop from the accumulation of macrophage-derived foam cells via the uptake of modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Although glucocorticoids are suspected to exacerbate atherosclerosis, the precise mechanisms have not been fully elucidated. We investigated the effects of long-term treatment (2 weeks) with both a natural glucocorticoid (hydrocortisone, HC, 1 µM) and a synthetic glucocorticoid (dexamethasone, Dex, 100 nM) on murine bone marrow-derived macrophages using flow cytometry and western blotting. Treatment with HC and Dex enhanced CD204 expression but not CD36 expression and acetylated LDL (Ac-LDL) uptake. Treatment with HC and Dex also induced the phosphorylation of extracellular signal-regulated kinase (ERK). The Dex-induced enhancement in CD204 expression and Ac-LDL uptake were suppressed by an inhibitor of the mitogen-activated protein kinase (MAPK)/ERK kinase. These results suggest that glucocorticoids activate the MAPK/ERK pathway, which enhances CD204 expression and results in increased uptake of Ac-LDL in macrophages. The MAPK/ERK pathway in macrophages might be a key target to prevent atherosclerosis that is worsened by glucocorticoids.

SCARA5 induced ferroptosis to effect ESCC proliferation and metastasis by combining with Ferritin light chain.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) remains one of the most lethal cancers worldwide accompany with an extremely poor prognosis. Therefore, this study aims to screen for new molecules affecting ESCC and explore their mechanisms of action to provide ideas for targeted therapies for ESCC. METHODS: Firstly, we screened out the membrane protein SCARA5 by high-throughput sequencing of the ESCC patient tissues, and RT-qPCR and WB were used to verify the differential expression of SCARA5 in esophageal cell lines, and IHC analyzed the expression localization of SCARA5 in ESCC tissue. Then, flow cytometry, wound healing assay, Transwell assay and CCK-8 assay were used to explore the effects of SCARA5 on cell cycle, migration and invasion as well as cell proliferation activity of esophageal squamous carcinoma cells. Meanwhile, transmission electron microscopy was used to detect changes in cellular mitochondrial morphology, and flow cytometry were used to detect changes in intracellular reactive oxygen metabolism, and immunofluorescence and flow cytometry were used to detect changes in intracellular Fe2+. Mechanistically, co-immunoprecipitation was used to detect whether SCARA5 binds to ferritin light chain, and ferroptosis-related protein expression was detected by WB. Finally, the tumor xenograft model was applied to validation the role of SCARA5 tumor growth inhibition in vivo. RESULTS: We found that SCARA5 was aberrantly decreased in ESCC tissues and cell lines. Furthermore, we confirmed that SCARA5 suppressed the cell cycle, metastasis and invasion of ESCC cells. Meanwhile, we also found that overexpression of SCARA5 caused changes in mitochondrial morphology, accumulation of intracellular reactive oxygen species and increased intracellular Fe2+ in ESCC cells, which induced ferroptosis in ESCC cells. Mechanically, we validated that SCARA5 combined with ferritin light chain and increased intracellular Fe2+. As well as, overexpression SCARA5 induced ferroptosis by increasing ferritin light chain in nude mice subcutaneous tumors and inhibited the growth of nude mice subcutaneous tumors. CONCLUSION: Collectively, our findings demonstrated that SCARA5 suppressed the proliferation and metastasis of ESCC by triggering ferroptosis through combining with ferritin light chain.

The role of macrophage scavenger receptor 1 (MSR1) in inflammatory disorders and cancer.

Macrophage scavenger receptor 1 (MSR1), also named CD204, holds key inflammatory roles in multiple pathophysiologic processes. Present primarily on the surface of various types of macrophage, this receptor variably affects processes such as atherosclerosis, innate and adaptive immunity, lung and liver disease, and more recently, cancer. As highlighted throughout this review, the role of MSR1 is often dichotomous, being either host protective or detrimental to the pathogenesis of disease. We will discuss the role of MSR1 in health and disease with a focus on the molecular mechanisms influencing MSR1 expression, how altered expression affects disease process and macrophage function, the limited cell signalling pathways discovered thus far, the emerging role of MSR1 in tumour associated macrophages as well as the therapeutic potential of targeting MSR1.

SRA Suppresses Antiviral Innate Immune Response in Macrophages by Limiting TBK1 K63 Ubiquitination via Deubiquitinase USP15.

The innate immune system is the first line of host defense against microbial infections. During virus infection, pattern recognition receptors (PRRs) are engaged to detect specific viral components, such as viral RNA or DNA, and regulate the innate immune response in the infected cells or immune cells. Our previous study demonstrated that scavenger receptor A (SRA), an important innate PRR, impaired the anti-hepatitis B virus (HBV) response in hepatocytes. Given that SRA is primarily expressed in macrophages, here, we assessed the function of SRA expressed in macrophages in response to RNA or DNA viral infection. SRA-deficient (SRA-/-) mice showed reduced susceptibility to viral infection caused by vesicular stomatitis virus (VSV) or herpes simplex virus 1 (HSV-1). In the virus-infected SRA-/- mice, compared with their wild-type (WT) counterparts, we observed low amounts of virus accompanied by enhanced interferon (IFN) production. Furthermore, SRA significantly inhibited the phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). We provided biochemical evidence showing that SRA directly interacts with the N-terminal kinase domain (KD) of TBK1, resulting in the limitation of its K63-linked ubiquitination. Moreover, we demonstrated that SRA negatively regulates the activity of TBK1 by promoting the recruitment of ubiquitin-specific protease 15 (USP15) to deubiquitinate TBK1. In summary, we have identified the connection between SRA and the TBK1/IRF3 signaling pathway in macrophages, indicating a critical role of SRA in the regulation of host antiviral immunity. IMPORTANCE During virus infection, PRRs are engaged to detect specific viral components, such as viral RNA or DNA, and regulate the innate immune response in the infected cells or other immune cells. We reported that deficiency of SRA, an important innate PRR, promoted IRF3 activation, type I IFN production, and innate antiviral responses against RNA and DNA viruses in vivo and in vitro. Furthermore, the biochemical analysis showed that SRA directly interacts with the KD domain of TBK1 and limits its K63-linked polyubiquitination, reducing TBK1 activation. Further analyses determined that SRA is a modulator for TBK1 activation via the recruitment of USP15, which delineated a previously unrecognized function for SRA in innate antiviral immunity.

MSR1 characterized by chromatin accessibility mediates M2 macrophage polarization to promote gastric cancer progression.

BACKGROUND: M2 macrophages play an important role in gastric cancer progression and metastasis, but the underlying tumor-promoting mechanisms are largely unknown. METHODS: The TCIA database was used to identify the infiltration profile of macrophages. Integrated ATAC-seq, RNA-seq, and single-cell RNA sequencing (scRNA-seq) data from GC samples were used for the analysis. Using ATAC-seq profiles and RNA-seq datasets, combined with cox univariate survival analysis, we identified prognosis-related differentially expressed genes (DEGs) with chromatin accessibility, which were identified as hub genes. The CIBERSORTx algorithm was utilized to estimate the relative infiltration level of M2 macrophages, and Pearson correlation analysis was performed to investigate the relationship between hub genes and M2 macrophages. Multidimensional database validations were carried out to avoid biases. The expression level and function of hub genes in the clusters of macrophages were evaluated by using scRNA-seq data. The role of hub genes in the alternative activation of macrophages and gastric cancer malignant behaviors, as well as their potential regulatory mechanism in gastric cancer progression, were further explored. RESULTS: 17,334 genes were acquired with chromatin accessibility in promoter regions by ATAC-seq. 2,714 genes were identified with both chromatin accessibility and differential expression based on the gene expression profiles (RNA-seq). By performing Cox univariate survival analysis, 171 survival-related DEGs with chromatin accessibility were identified as hub genes. Through the CIBERSORTx algorithm and Pearson correlation analysis, the gene MSR1 most associated with M2 macrophages was screened out. According to the scRNA-seq analysis, MSR1 was highly expressed in the clusters of macrophages and may be involved in regulating M2 macrophage polarization. In vitro experiments confirmed that M2 macrophage polarization and its induced malignant behavior of gastric cancer cells were inhibited by knockdown of MSR1. Furthermore, MSR1 mediated M2 macrophage polarization by regulating arginine and proline metabolism, thereby activating the AMPK/mTOR pathway to promote gastric cancer progression. CONCLUSION: We identified a gene-MSR1-characterized by chromatin accessibility, associated with poor prognosis in gastric cancer. This gene dictates the progression of gastric cancer by facilitating M2 macrophage polarization.

BMSC-derived exosomal lncRNA PTENP1 suppresses the malignant phenotypes of bladder cancer by upregulating SCARA5 expression.

LncRNAs can be transported to tumor cells where they exert regulatory effects by bone marrow mesenchymal stem cells (BMSC)-derived exosomes. Here, we aimed to investigate the functional mechanism of BMSC-derived exosomal lncRNA PTENP1 in the progression of bladder cancer (BC). Methods of BMSC were identified by detecting surface markers through flow cytometry. Exosomes from BMSC were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blot analysis of exosome markers. Cellular internalization of BMSC-derived exosomes (BMSC-Exo) into BC cells was detected by confocal microscopy. CCK-8, colony formation, flow cytometry, wound healing, and transwell assays were adopted to estimate cell proliferation, apoptosis, migration, and invasion abilities, respectively. Interplay between miR-17 and lncRNA PTENP1 or SCARA5 was verified by dual-luciferase reporter, RNA pull down, and/or RNA immunoprecipitation (RIP) assays. Tumor xenograft assay was conducted in nude mice to study the role of exosomal lncRNA PTENP1 in BC progression in vivo. We showed exosomal lncRNA PTENP1 can be delivered into and suppress the malignant phenotypes of BC cells. LncRNA PTENP1 was identified as a sponge of miR-17, and SCARA5 was identified as a target gene of miR-17. The exosomes derived from PTENP1-overexpressing BMSC (BMSCOE-PTENP1-Exo) abolished the promotive effects of miR-17 overexpression or SCARA5 knockdown on the malignant phenotypes of BC cells. Moreover, exosomal lncRNA PTENP1 was demonstrated to inhibit BC tumor growth in nude mice by miR-17/SCARA5 axis. In conclusion, BMSC-derived exosomal PTENP1 suppressed the BC progression by upregulating the expression of SCARA5 via sponging miR-17, offering a potential novel therapeutic target for BC therapy.

Synergistic anti-proliferative activity of JQ1 and GSK2801 in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801. METHODS: RNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies. RESULTS: Global expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation. CONCLUSION: JQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

Highly Expressing SCARA5 Promotes Proliferation and Migration of Esophageal Squamous Cell Carcinoma.

Background: Thrombospondin type 1 domain-containing 7A (THSD7A) was reported to play a procancer role in esophageal squamous cell carcinoma (ESCC). The aim of the study was to screen the downstream functional genes of THSD7A and explore their functions in ESCC, based on the reported research into THSD7A function and on gene microarrays. Methods: We adopted quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Celigo high-content screening (HCS) technology to screen the downstream genes of THSD7A. The expression level of target genes was examined by PCR, western blot, and immunohistochemistry (IHC). The effects of these target genes on ESCC malignant biological behavior were performed in vivo and in vitro. The Kaplan-Meier (K-M) survival analysis and Cox regression were used to analyze the prognostic significance of target genes in ESCC patients. Experiments in the literature on liver cancer (LC) were repeated to verify the functions of these genes in different tumors. We further explored the cancer-promoting mechanism of target genes in ESCC by sequencing of the genes' exons. Results: Scavenger receptor class A member 5 (SCARA5) was proved to be the downstream driving gene of THSD7A. SCARA5 promoted cell proliferation and migration but inhibited apoptosis in ESCC. IHC results confirmed that SCARA5 expression in ESCC exceeded that in normal tissues. The K-M survival analysis indicated that SCARA5 expression quantity was not related to prognosis, but tumor volume and T classification were both the independent prognostic factors. Repetition of experiments in LC in the literature confirmed that SCARA5 had exactly opposite functions in EC and LC. Conclusion: SCARA5 was related to the development and occurrence of ESCC. Our findings suggested that it was a potentially diagnostic individualized therapeutic target for ESCC in the future and that its application could possibly be combined with that of upstream THSD7A gene.