Calcium-sensing Receptor, a Potential Biomarker Revealed by Large-scale Public Databases and Experimental Verification in Metastatic Breast Cancer.

INTRODUCTION: Breast cancer (BC) is a common cancer characterized by a high molecular heterogeneity. Therefore, understanding its biological properties and developing effective treatments for patients with different molecular features is imperative. Calcium-sensing receptor (CaSR) has been implicated in several regulatory functions in various types of human cancers. However, its underlying pathological mechanism in BC progression remains elusive. METHODS: We utilized The Cancer Genome Atlas and Gene Expression Omnibus databases to explore the function of CaSR in the metastasis of BC. Gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis of biological processes and cell signaling pathways revealed that CaSR could be activated or inhibited. Importantly, quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to verify the gene expression of the CaSR. Wound healing and transwell assays were conducted to assess the effect of CaSR on the migration of BC cells. RESULTS: We demonstrated that CaSR expression in metastatic BC was higher than that in non-metastatic BC. It is the first time that database information has been used to reveal the biological process and molecular mechanism of CaSR in BC. Moreover, the CaSR expression in normal breast epithelial cells was notably less compared to that in BC cells. The activation of CaSR by Cinacalcet (a CaSR agonist) significantly enhanced the migration of BC cells, whereas NPS-2143 (a CaSR antagonist) treatment dramatically inhibited these effects. CONCLUSION AND FUTURE PERSPECTIVE: Bioinformatics techniques and experiments demonstrated the involvement of CaSR in BC metastasis. Our findings shed new light on the receptor therapy and molecular pathogenesis of BC, and emphasize the crucial function of CaSR, facilitating the metastasis of BC.

Calcium-sensing receptor and NF-κB pathways in TN breast cancer contribute to cancer-induced cardiomyocyte damage via activating neutrophil extracellular traps formation.

Cardiovascular disorders are commonly prevalent in cancer patients, yet the mechanistic link between them remains poorly understood. Because neutrophil extracellular traps (NETs) have implications not just in cardiovascular diseases (CVD), but also in breast cancer (BC), it was hypothesized to contribute to CVD in the context of oncogenesis. We established a mouse model using nude mice to simulate liver metastasis of triple-negative BC (TNBC) through the injection of MDA-MB-231 cells. Multiple imaging and analysis techniques were employed to assess the cardiac function and structure, including echocardiography, HE staining, Masson staining, and transmission electron microscopy (TEM). MDA-MB-231 cells underwent treatment with a CaSR inhibitor, CaSR agonist, and NF-κB channel blocker. The phosphorylation of NF-κB channel protein p65 and the expression and secretion of IL-8 were assessed using qRT-PCR, Western Blot, and ELISA, respectively. In addition, MDA-MB-231 cells were co-cultured with polymorphonuclear neutrophils (PMN) under varying conditions. The co-localization of PMN extracellular myeloperoxidase (MPO) and DNA were observed by cellular immunofluorescence staining to identify the formation of NETs. Then, the cardiomyocytes were co-cultured with the above medium that contains NETs or not, respectively; the effects of NETs on cardiomyocytes apoptosis were perceived by flow cytometry. The ultrastructural changes of myocardial cells were perceived by TEM, and ELISA detected the levels of myocardial enzyme (LDH, MDA and SOD). Overall, according to our research, CaSR has been found to have a regulatory role in IL-8 secretion in MDA-MB-231 cells, as well as in the formation of NETs by PMN cells. These findings suggest CaSR-mediated stimulation in PMN can lead to increased NETs formation and subsequently to cytotoxicity in cardiomyocytes, which potentially via activation of the NF-κB signaling cascade of BC cell.

MiR-525-5p inhibits diffuse large B cell lymphoma progression via the Myd88/NF-κB signaling pathway.

Diffuse large B-cell lymphoma (DLBCL) is a B-cell lymphoma with a high degree of aggressiveness. Recently, evidence has shown that miR-525-5p is decreased in DLBCL, suggesting its possible involvement in tumor progression. In this study, miR-525-5p suppressed proliferation, invasion and clonogenicity, and increased apoptosis of U2932 cells, whereas miR-525-5p silencing enhanced tumor cell growth. Next, miR-525-5p targets the 3'-UTR of Myd88, and Myd88 protein was increased in lymphoma tissues. Similar to the miR-525-5p mimic, Myd88 siRNA suppressed proliferation, invasion, and clonogenicity, and enhanced apoptosis of U2932 cells. We observed that Myd88 reversed the inhibitory effect of miR-525-5p on tumor cell growth by transfecting cells with miR-525-5p mimics alone or together with Myd88 overexpression vector. In addition, in vivo studies have shown that compared to the control group, U2932 cells with upregulated miR-525-5p expression have a reduced ability to induce tumor formation. In conclusion, our results demonstrate that miR-525-5p inhibits the progression of DLBCL through the Myd88/NF-κB pathway, which largely fills the gap of previous studies, and our results may provide a new reference for the targeted treatment of DLBCL.

LESO: A ten-year ensemble of satellite-derived intercontinental hourly surface ozone concentrations.

This study presents a novel ensemble of surface ozone (O3) generated by the LEarning Surface Ozone (LESO) framework. The aim of this study is to investigate the spatial and temporal variation of surface O3. The LESO ensemble provides unique and accurate hourly (daily/monthly/yearly as needed) O3 surface concentrations on a fine spatial resolution of 0.1◦ × 0.1◦ across China, Europe, and the United States over a period of 10 years (2012-2021). The LESO ensemble was generated by establishing the relationship between surface O3 and satellite-derived O3 total columns together with high-resolution meteorological reanalysis data. This breakthrough overcomes the challenge of retrieving O3 in the lower atmosphere from satellite signals. A comprehensive validation indicated that the LESO datasets explained approximately 80% of the hourly variability of O3, with a root mean squared error of 19.63 µg/m3. The datasets convincingly captured the diurnal cycles, weekend effects, seasonality, and interannual variability, which can be valuable for research and applications related to atmospheric and climate sciences.

Characterization of Platelet Function-Related Gene Predicting Survival and Immunotherapy Efficacy in Gastric Cancer.

Immunotherapy is widely used to treat various cancers, but patients with gastric cancer (GC), which has a high mortality rate, benefit relatively less from this therapy. Platelets are closely related to GC progression and metastasis. This study aimed to find novel potential biomarkers related to platelet function to predict GC and immunotherapy efficacy. First, based on platelet activation, signaling, and aggregation (abbreviation: function)-related genes (PFRGs), we used the least absolute shrinkage and selection operator (Lasso) regression method to construct a platelet-function-related genes prognostic score (PFRGPS). PRFGPS was verified in three independent external datasets (GSE26901, GSE15459, and GSE84437) for its robustness and strong prediction performance. Our results demonstrate that PRFGPS is an independent prognostic indicator for predicting overall survival in patients with GC. In addition, prognosis, potential pathogenesis mechanisms, and the response to immunotherapy were defined via gene set enrichment analysis, tumor mutational burden, tumor microenvironment, tumor immune dysfunction and exclusion (TIDE), microsatellite instability, and immune checkpoint inhibitors. We found that the high-PRFGPS subgroup had a cancer-friendly immune microenvironment, a high TIDE score, a low tumor mutational burden, and relatively low microsatellite instability. In the immunophenoscore model, the therapeutic effect on anti-PD-1 and anti-CTLA-4 in the high-PRFGPS subgroup was relatively low. In conclusion, PRFGPS could be used as a reference index for GC prognosis to develop more successful immunotherapy strategies.

A Retrospective Clinicopathological Study of Osteosarcoma Patients with Metachronous Metastatic Relapse.

Purpose: Although osteosarcoma patients receive a standardized treatment, metachronous metastatic relapse still impairs the overall survival (OS). This study aimed to explore the clinicopathological features and prognostic factors of osteosarcoma patients with metachronous metastatic relapse. Patients and methods: We retrospectively analyzed 59 patients, between January 1st, 2004 and December 31st, 2013. Employed Chi-square test to recognize the differences in clinicopathological characteristics between early and late metastatic patients, and the differences between shorter and longer survival patients. Used the Kaplan-Meier method to evaluate the survival data, cox step proportional hazard test to analyze the prognostic factors associated with OS. Results: We found that early metastatic patients were prominently correlated with the male, tumor size ≥8 cm, histological grade G2, Enneking stages II, anatomic location of the distal femur, pathological of conventional types, and elevated alkaline phosphatase (ALP) level at diagnosis, (p<0.05). In parallel, the shorter survival patients were primarily linked to tumor size ≥8 cm, histological grade G2, Enneking stages II, early metastasis, multiple pulmonary metastases, lack of curative treatment after metastasis, increased level of ALP at diagnosis and LDH after metastasis, (p<0.05). The univariate analyses of the prognostic factors showed that patients who had these clinicopathological characteristics, such as male, tumor size ≥8 cm, Enneking stage IIB, multiple pulmonary metastases, lack of curative treatment after metastasis, the elevated ALP at diagnosis, elevated ALP and LDH after metastasis, had a worse OS in osteosarcoma patient with metachronous metastatic relapse, (p<0.05). The multivariate analyses showed that tumor size, type of metastasis and ALP level at diagnosis were independent factors for OS in osteosarcoma patient with metachronous metastatic relapse (p<0.05). Conclusion: These results indicated that osteosarcoma patients with metachronous metastatic relapse have special features which might be utilized to effectively predict the likelihood of early metastatic relapse and the prognosis.

Calcium­sensing receptors in human peripheral T lymphocytes and AMI: Cause and effect.

Acute myocardial infarction (AMI) is a disease associated with inflammation. T lymphocytes are involved by secreting cytokines and inflammatory factors. In our previous study, it was found that the T lymphocytes exhibited certain functional changes, the onset of which was induced by modulating calcium­sensing receptor (CaSR) in AMI. In the present study, western blotting was used to verified the expression of T lymphocyte CaSR and pathway proteins, including phosphorylated extracellular signal­regulated kinase (P­ERK)1/2 and phosphorylated c­Jun N­terminal kinase (P­JNK), and used cytometric bead array to detect the secretion of interleukin (IL)­4, IL­6, IL­10 and tumor necrosis factor (TNF)­α in AMI onset, the results demonstrated that they were all increased. In addition, the expression of T lymphocyte pathway proteins, including P­ERK1/2 and P­JNK, and the secretion of IL­4, IL­6, IL­10 and TNF­α decreased after T lymphocytes being transfected by CaSR small interfering RNA. By contrast, the neonatal mouse cardiomyocytes under hypoxia and hypoxia/re­oxygenation exhibited ultrastructural damage, increased apoptosis, increased production of lactate dehydrogenase (LDH) and malondialdehyde, and reduced superoxide dismutase; these indicators changed extensively when cardiomyocytes were co­cultured with T lymphocytes. However, the effects were reversed when the cardiomyocytes were co­cultured with CaSR­silenced T lymphocytes. These results indicated that CaSR may modulate T lymphocytes to release cytokines through mitogen­activated protein kinase pathways and affect cardiomyocyte injury. The relationship between AMI and T lymphocyte CaSR is reciprocal.

Expression and Role of the Calcium-Sensing Receptor in Rat Peripheral Blood Polymorphonuclear Neutrophils.

The calcium-sensing receptors (CaSRs) play an important role in many tissues and organs that are involved in inflammatory reactions. Peripheral blood polymorphonuclear neutrophils (PMNs) are important inflammatory cells. However, the expression and functions of CaSR in peripheral blood PMNs are still not reported. In this study, we collected rat peripheral blood PMNs to observe the relationship between CaSR and PMNs. From the results, we found first that the CaSR protein was expressed in PMNs, and it increased after PMNs were activated with fMLP. In addition, CaSR activator cincalcet promoted the expression of CaSR and P-p65 (NF-κB signaling pathway protein) and Bcl-xl (antiapoptosis protein), and it increased the secretion of interleukin-6 (IL-6) and myeloperoxidase (MPO); meanwhile, it decreased proapoptosis protein Bax expression and the production of IL-10 and reactive oxygen species (ROS). At the same time, cincalcet also decreased the PMN apoptosis rate analyzed by flow cytometry. However, CaSR inhibitor NPS-2143 and NF-κB signaling pathway inhibitor PDTC reverse the results cited earlier. All of these results indicated that CaSR can regulate PMN functions and status to play a role in inflammation, which is probably through the NF-κB signaling pathway.

Calcium-Sensing Receptor in Human Peripheral Blood T Lymphocytes Is Involved in the AMI Onset and Progression through the NF-κB Signaling Pathway.

Acute myocardial infarction (AMI) is a condition triggered by an inflammatory process that seriously affects human health. Calcium-sensing receptor (CaSR) in T lymphocytes is involved during the inflammation reaction. However, the relationship between them is not very clear. In this study, we collected human peripheral blood T lymphocytes from patients with AMI and in different stages of percutaneous coronary intervention (PCI) (at the onset of AMI, the first day after PCI (PCI-1), PCI-3, and PCI-5) to study the CaSR and NF-κB pathway protein expression, cytokine release and T cell apoptosis. The results showed that the expressions of CaSR, P-p65, Caspase-12, and the secretions of Th-1 and Th-2 type cytokines were increased at the onset of AMI, especially on the PCI-1. Meanwhile, the apoptosis rate of CD(3+), CD(4+) and CD(8+) T lymphocytes also increased. However, from PCI-3, all the indicators began to decline. In addition, we also found that positive CaSR small interfering RNA (siRNA) transfection in T lymphocytes and NF-κB pathway blocker Bay-11-7082 reversed the increased expressions of CaSR, P-p65, Caspase-12, reduced the secretions of Th-1 and Th-2 type cytokines, and decreased T lymphocytes apoptosis rate not only in the AMI patients but also in the normal controls. All of these results indicated that CaSR in the human peripheral blood T lymphocytes were involved in the AMI onset and progression, which probably was related to the NF-κB pathway. Our study demonstrated the relationship between AMI and CaSR, and will provide new effective prevention theory and new targets for drug treatment.

Activation of calcium-sensing receptor increases TRPC3/6 expression in T lymphocyte in sepsis.

Sepsis is a systemic inflammatory response syndrome induced by infection. T Lymphocytes play an important role in this disease. Transient receptor potential (TRP) channels and calcium-sensing receptors (CaSR) are expressed in lymphocytes to promote intracellular Ca(2+) release. However, data about the link between CaSR and TRP channels in septic T lymphocytes are few. In this study, by Ca(2+) imaging and Western blotting, we found that in septic rat peripheral blood T lymphocytes expressions of TRPC3 and TRPC6 proteins are higher. The SR/ER Ca(2+) ATPase inhibitor thapsigargin (TG) and CaSR agonist NPS R-568 also increased expressions of TRPC3 and TRPC6 proteins, which were reversed by PLC-IP3 channel blocker U73122 and TRPC channels inhibitor SKF96365. By Ca(2+) imaging, we found that the depletion of ER Ca(2+) stores by TG elicited a transient rise in cytoplasmic Ca(2+), followed by sustained increase depending on extracellular Ca(2+). But, SKF96365, not Verapamil (L-type channels inhibitor) and NiCl2 (Na(+)/Ca(2+) exchanger inhibitor), inhibited the relatively high [Ca(2+)]i. NPS R-568 also resulted in the same effect, and the duration of [Ca(2+)]i increase was eliminated completely by U73122 and was reduced in the absence of [Ca(2+)]o. NPS R-568 and TG increased the apoptotic ratio of septic T lymphocytes, which can be suppressed by SKF96365 and U73122. These results suggested that CaSR activation promoted the expression of TRPC3 and TRPC6 and enhanced T lymphocytes apoptosis through PLC-IP3 signaling pathway in sepsis.

Calcium-sensing receptor in the T lymphocyte enhanced the apoptosis and cytokine secretion in sepsis.

Calcium-sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily that existed in lymphocytes and promoted cytokine secretion. Lymphocytes are also involved in sepsis. However, the role of CaSR in lymphocytes in sepsis is unclear. In this study, we want to examine whether the CaSR in lymphocytes in sepsis is involved in the cytokine secretions and apoptosis and make clear the relationship between NF-κB and MAPK signal transduction pathways. We investigated the issues mentioned earlier using Western blotting, ELISA, and Flow Cytometry. The sepsis was remodeled by cecal ligation and puncture (CLP). We found that CaSR protein expression increased in the peripheral blood T lymphocytes in CLP rats. The calcimimetic R568 (NPS R568) promoted, whereas the calcilytic NPS 2143 attenuated, signaling pathways proteins P65 (subunit of NF-κB), ERK1/2, and JNK (one subgroup of MAPKs) phosphorylation. However, P-P38 and P-JAKs exhibit no significant changes. Furthermore, the production TNF-α and IL-4 was greater in CLP rats than in normal rats, and NPS R568 promoted secretion of these cytokines. Simultaneously, the apoptotic ratio of T cells in CLP increased, and NPS R 568 exacerbated the apoptosis degree. However, these effects could also be inhibited by U0126 or SP600125 (MAPKs pathway inhibitor) or Bay-11-7082 or (NF-κB pathway inhibitor). From these results, we can conclude that, in the sepsis, CaSR activation promoted T-cell apoptosis and the secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokines IL-4 probably through NF-κB and partial MAPK signal transduction pathways.