S100A4 Protein Research Articles

S100A10 promotes proliferation and invasion of lung adenocarcinoma cells by activating the Akt-mTOR signaling pathway

To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis. Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed. The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis (P < 0.05), but not with tumor differentiation or the patients' age or gender (P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients (P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro (P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation (P < 0.001). S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.

Glutamine supplementation accelerates functional recovery of EDL muscles after injury by modulating the expression of S100 calcium-binding proteins.

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1g/kg/day, via gavage) for 3 and 10days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1β and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.

Expression of S100A8 protein on B cells is associated with disease activity in patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an intractable disease characterized by autoantibody production and autoreactive B and T cell proliferation. Although several studies have revealed multiple genetic and environmental associations, the underlying mechanisms remain unknown.MethodsWe performed proteomics and transcriptomics using liquid chromatography-mass spectrometry and DNA microarray, using peripheral blood B cells from patients with SLE, and healthy controls (HC). We explored molecules associated with the pathophysiology of SLE by flow cytometry and B cell stimulation assay.ResultsWe identified for the first time that expression of both S100A8 protein and mRNA were markedly upregulated in SLE B cells. The results obtained using flow cytometry showed that S100A8 was highly expressed on the surface of B cells of patients with active SLE (MFI; HC 102.5 ± 5.97, stable SLE 111.4 ± 12.87, active SLE 586.9 ± 142.9), and S100A8 on the cell surface was decreased after treatment (MFI; pre-treat 1094.5 ± 355.38, post-treat 492.25 ± 247.39); therefore, it is suggested that S100A8 may be a marker for disease activity. The mRNA expression of S100A8 was particularly upregulated in memory B cells of SLE (56.68 fold higher than HC), suggesting that S100A8 may be mainly secreted by memory B cells in the pathogenesis of SLE.ConclusionsOur results imply that the S100A8 proteins secreted from memory B cells may stimulate granulocytes and monocytes through pattern recognition receptors, activate the innate immune system, and are involved in the pathogenesis of SLE.

Atrophic astrocytes in aged marmosets present tau hyperphosphorylation, RNA oxidation, and DNA fragmentation

Astrocytes perform multiple essential functions in the brain showing morphological changes. Hypertrophic astrocytes are commonly observed in cognitively healthy aged animals, implying a functional defense mechanism without losing neuronal support. In neurodegenerative diseases, astrocytes show morphological alterations, such as decreased process length and reduced number of branch points, known as astroglial atrophy, with detrimental effects on neuronal cells. The common marmoset (Callithrix jacchus) is a non–human primate that, with age, develops several features that resemble neurodegeneration. In this study, we characterize the morphological alterations in astrocytes of adolescent (mean 1.75 y), adult (mean 5.33 y), old (mean 11.25 y), and aged (mean 16.83 y) male marmosets. We observed a significantly reduced arborization in astrocytes of aged marmosets compared to younger animals in the hippocampus and entorhinal cortex. These astrocytes also show oxidative damage to RNA and increased nuclear plaques in the cortex and tau hyperphosphorylation (AT100). Astrocytes lacking S100A10 protein show a more severe atrophy and DNA fragmentation. Our results demonstrate the presence of atrophic astrocytes in the brains of aged marmosets.

Expression of S100A9 in adamantinomatous craniopharyngioma and its association with wet keratin formation.

Wet keratin is a hallmark of adamantinomatous craniopharyngioma (ACP), which is frequently infiltrated by inflammatory cells. S100 calcium-binding protein A9 (S100A9) has been confirmed to play a decisive role in the development of inflammation. However, the relationship between wet keratin (keratin nodules) and S100A9 in ACP is poorly understood. The objective of the present study was to explore the expression of S100A9 in ACP and its association with wet keratin formation. Immunohistochemistry and immunofluorescence were used to detect the expression of S100A9, β-catenin and Ki67 in 46 cases of ACP. A total of three online databases were used to analyze S100A9 gene expression and protein data. The results revealed that S100A9 was primarily expressed in wet keratin and some intratumoral and peritumoral cells, and its expression in wet keratin was upregulated in the high inflammation group (P=1.800x10-3). In addition, S100A9 was correlated with the degree of inflammation (r=0.6; P=7.412x10-3) and the percentage of Ki67-positive cells (r=0.37; P=1.000x10-2). In addition, a significant correlation was noted between the area of wet keratin and the degree of inflammation (r=0.51; P=2.500x10-4). In conclusion, the present study showed that S100A9 was upregulated in ACP and may be closely associated with wet keratin formation and the infiltration of inflammatory cells in ACP.

OA07 Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis

Abstract Background/Aims Preclinical studies suggest that the S100A4, a Damage Associated Molecular Pattern (DAMP) protein upregulated upon stress or injury, may be a target for antifibrotic therapy. We have studied expression and function of S100A4 in systemic sclerosis (SSc) to further explore its relevance as a driver of fibroblast activation. Methods S100A4 protein concentration was measured by ELISA (Cusabio, USA) in serum of SSc (n = 94) and healthy controls (n = 15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n = 6) and healthy controls (NF, n = 6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate activation of profibrotic molecular and functional phenotype in SSc and control fibroblasts. Results Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p = 0.027). There was significant association with SSc-ILD (p = 0.025, n = 55), scleroderma renal crisis (p = 0.026, n = 4) and trend of increase in pulmonary hypertension (p = 0.075, n = 7). There was a trend for S100A4 to fall in longer disease duration (r=-0.18, p = 0.09) and a weak (r = 0.28, p = 0.07) correlation with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF controls (0.28 [0.02-3.29]; p &amp;lt; 0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF = 0.50 [0.91]; SScF 8.16 [2.49]; p &amp;lt; 0.0001). S100A4 induced a fibrotic phenotype in NF with concentration dependent increased in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p &amp;lt; 0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR&amp;lt;0.001 and FC &amp;gt; 1.5) induced in NF by S100A4 were also constitutively overexpressed in SScF, and significantly downregulated by AX-202. Pathway mapping of these S100A4 dependent transcripts in SSc showed the most significant enriched Kegg® pathways (FDR&amp;lt;0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). Conclusion Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. Studies to elucidate the therapeutic potential of targeting S100A4 in SSc are warranted. Disclosure C.P. Denton: None. S. Xu: None. R.H. Maclean: None. K.E. Clark: None. S. Borchert: Corporate appointments; Employee of ARXX Therapeutics. R. Hussain: Corporate appointments; Employee of ARXX Therapeutics. J. Klingelhöfer: Corporate appointments; Employee of ARXX Therapeutics. J. Hallén: Corporate appointments; Employee of ARXX Therapeutics. V.H. Ong: None.

Proteome profiling of endogenous and potential S-nitrosylation in colorectal cancer.

As a common cancer with high incidence rate and mortality, colorectal cancer (CRC) is seriously threatening human health. S-nitrosylation (SNO) proteins mediated by nitric oxide (NO) has important implications in the genesis, progression, and apoptosis of CRC. It's worth noting that the SNO proteins also play an important role in the tumor endocrine and metabolic pathways of CRC. In this study, the protein extracts of human tissues and cell lines were treated by biotin switch technology and magnetic beads enrichment. The proteomic results of endogenous and potential SNO proteins were analyzed by mass spectrometry (MS). Through the comparison and analysis of MS results, Gene Ontology (GO) analysis, and literatures, some endogenous and potential SNO proteins were identified in CRC, which were closely related to the tumor endocrine and metabolic pathways, the apoptotic signaling pathways, protein maturation, and other biological processes of the proliferation and apoptosis of CRC cells. A total of 19 proteins containing potential or endogenous SNO sites were detected in both human cancer tissue and SW 480 cells. Through the cross validation of MS results, GO analysis, and literatures, several SNO proteins were identified frequently in CRC, such as the actin, cytoplasmic 1 (ACTB), peroxiredoxin-4 (PRDX4), protein S100A8 (S100A8), pyruvate kinase PKM (PKM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were closely related to the tumor endocrine and metabolic pathways and the apoptotic signaling pathways of CRC. Different CRC cells and tissues contained potential and endogenous SNO modified proteins. In addition, some SNO proteins could participate in the proliferation, metastasis and apoptosis of CRC by regulating the tumor endocrine and metabolic pathways.

Abstract 2948: A humanized therapeutic antibody against S100A4 as a novel immune modulator and an inhibitor of cancer metastasis

Abstract Widespread cancer metastasis still contributes to ~ 90% of cancer-related deaths, and limited progress has been made in treating the metastatic disease. S100A4 is a highly conserved protein and has been demonstrated to promote cancer metastasis and stemness in multiple cancer types. Furthermore, our recent single cell RNA-sequencing analysis of cancer and stromal cells in human gliomas showed that S100A4 is expressed in both cancer and immune cells to promotes immune suppressive tumor microenvironment. Significantly deleting S100A4 function in stromal cells alone was sufficient to reverse the immune phenotypes of glioma associated T cells and myeloid cells, and resulted in significantly extend survival of two different mouse glioma models. Consistently, higher S100A4 expression is also associated with poor outcomes in many cancer types. Together these observations suggest that S100A4 is a promising therapeutic target for multiple cancer types, and we developed and characterized a blocking antibody against human S100A4. We have screened 114 monoclonal antibodies against S100A4 using S100A4-induced chicken neurite outgrowth ex vivo as a functional assay. Seven of the 114 monoclonals showed significant suppressive activity ex vivo and were selected for further analysis for their in vivo activity. We chose two well-characterized breast cancer metastasis models for in vivo screening: MMTV-PyMT spontaneous and 4T1 orthotopic transplantation tumor models. One of the clones, 4-11, showed the most consistent and dramatic lung metastasis suppressive activity in both models. To identify the potential mechanisms of action since the blocking antibody is anticipated to only affect soluble S100A4 function, we focused on potential immune modulatory effect, based on our own study and those of others. We performed flow cytometry to analyze the immune phenotypes in blood, bone marrow, spleen, breast tumor, and lung metastases. In S100A4-11 treated mice, circulating monocytes (CD11b+Ly6gloLy6chi) and tumor-associated CD163+ macrophage frequencies were significantly decreased in both models. In addition, circulating CD4+ T cells and neutrophils (CD11b+Ly6g+Ly6cint) frequencies were also decreased in the 4T1 model. Others have shown that myeloid-derived suppressive cells (MDSC: CD11b+, Ly6gloLy6chi,IAIE-) play an important role in cancer metastasis. Our result suggests that soluble S100A4 protein promotes MDSC polarization or survival in the tumor, peripheral blood, and metastatic lung tissues. These findings strongly suggest that S100A4-11 could be used to block metastasis. We are currently testing the humanized S1004-11 antibody in preparation for clinical studies with this novel immune modulator. Citation Format: Jia-Shiun Leu, Hui Deng, Han Nhat Tran, Jose Maria Salazar, Jose A. Maldonado, Carlo D. Cristobal, Xiong Wei, Hyun-Kyoung Lee, Zhiqiang An, Ningyan Zhang, Kyuson Yun. A humanized therapeutic antibody against S100A4 as a novel immune modulator and an inhibitor of cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2948.

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. A soluble recombinant S100A8 protein with a high yield and purity was produced usingEscherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

Decreased expression of S100A8/A9 in V30M related ATTRv amyloidosis

Introduction Hereditary Transthyretin Amyloidosis is a rare, progressive and life-threatening systemic disease with predominant peripheral and autonomic nervous system involvement caused by mutation of the transthyretin protein. The most common TTR mutation regarding to ATTRv is a substitution of a Methionine for a Valine at position 30 that predisposes TTR to form aggregates and fibrils. Methods S100A8 protein levels were measured in plasma samples from ATTRV30M patients and healthy donors. Additionally, S100A8/9 levels were measured in Schwann cells after incubation with human WT or V30M TTR. Moreover, bone marrow derived macrophages of either genetic background were generated and the expression of S100A8/9 was measured in response to toll like receptors agonists. Results S100A8/A9 mRNA levels are decreased in HSF V30M mice as compared with the WT. Moreover, S100A8 protein levels were found downregulated in plasma samples from ATTRV30M patients. Furthermore, we provide evidence for a dysregulated S100 expression by Schwann cells in response to TTRV30M and by mutated macrophages in response to toll like receptors agonists. Conclusion The presence of TTRV30M impacts S100 expression, possibly contributing to the impaired immune activation of Schwann cells in nerves from ATTRV30M patients. This may be linked to the diminished immune cellular infiltration in these nerves, contributing in this way for the neuronal dysfunction present in the disease.

Morphological alterations in corneal nerves of patients with dry eye and associated biomarkers

The purposes of the present study were to (1) identify the relationship between dry eye symptoms and morphological changes in corneal subbasal nerves/ocular surfaces, and (2) discover tear film biomarkers indicating morphological changes in the subbasal nerves. This was a prospective cross-sectional study conducted between October and November 2017. Adults with dry eye disease (DED, n = 43) and healthy eyes (n = 16) were evaluated based on their subjective symptoms and ophthalmological findings. Corneal subbasal nerves were observed using confocal laser scanning microscopy. Nerve lengths, densities, branch numbers, and nerve fiber tortuosity were analyzed using ACCMetrics and CCMetrics image analysis systems; tear proteins were quantified by mass spectroscopy. Compared with the control group, the DED group had significantly lower tear breakup times (TBUT) and pain tolerance capacity, and significantly higher corneal nerve branch density (CNBD) and corneal nerve total branch density (CTBD). CNBD and CTBD showed significant negative correlations with TBUT. Six biomarkers (cystatin-S, immunoglobulin kappa constant, neutrophil gelatinase-associated lipocalin, profilin-1, protein S100-A8, and protein S100-A9) showed significant positive correlations with CNBD and CTBD. The significantly higher CNBD and CTBD in the DED group suggests that DED is associated with morphological alterations in corneal nerves. The correlation of TBUT with CNBD and CTBD further supports this inference. Six candidate biomarkers that correlate with morphological changes were identified. Thus, morphological changes in corneal nerves are a hallmark of DED, and confocal microscopy may help in the diagnosis and treatment of dry eyes.

Association between serum S100A11 levels and glucose metabolism in diabetic process

BackgroundDiabetes mellitus (DM) is a prevalent non-communicable metabolic disease, and S100A11 is a newly identified gene closely related to metabolism. The association of S100A11 with diabetes is unclear. This study aimed to assess the relationship between S100A11 and markers of glucose metabolism in patients with different glucose tolerance and gender.MethodsThis study included 97 participants. Baseline data were obtained, and the serum levels of S100A11 and metabolic markers (glycated hemoglobin [HbA1c], insulin release test, and oral glucose tolerance test) were measured. Linear and nonlinear correlations between serum S100A11 levels and HOMA-IR, HOMA of β, HbA1c, insulin sensitivity index (ISI), corrected insulin response (CIR), and oral disposition index (DIo) were analyzed. The expression of S100A11 was also detected in mice.ResultsSerum S100A11 levels increased in patients with impaired glucose tolerance (IGT) of both genders. S100A11 mRNA and protein expression increased in obese mice. There were nonlinear correlations between S10011 levels and CIR, FPI, HOMA-IR, whole-body ISI in the IGT group. S100A11 was nonlinearly correlated with HOMA-IR, hepatic ISI, FPG, FPI, and HbA1c in the DM group. In the male group, S100A11 was linearly correlated with HOMA-IR and nonlinearly correlated with DIo (derived from hepatic ISI) and HbA1c. In the female population, S100A11 was nonlinearly correlated with CIR.ConclusionsSerum S100A11 levels were highly expressed in patients with IGT and in the liver of obese mice. In addition, there were linear and nonlinear correlations between S100A11 and markers of glucose metabolism, demonstrating that S100A11 has a role in diabetes.Trial registration ChiCTR1900026990

Immunohistochemical Analysis of S100 Proteins in Normal and Irreversibly Inflamed Human Dental Pulps

IntroductionS100 proteins convey important roles in innate immune responses to infection and regenerative processes. However, their role in inflammatory or regenerative processes of the human dental pulp is poorly elucidated. The aim of the present study was to detect, localize, and compare the occurrence of 8 S100 proteins in normal, symptomatic, and asymptomatic irreversibly inflamed dental pulp specimens. MethodsHuman dental pulp specimens from 45 individuals were clinically assigned to 3 groups of pulpal diagnosis: normal pulp (NP, n = 17), asymptomatic irreversible pulpitis (AIP, n = 13), and symptomatic irreversible pulpitis (SIP, n = 15). The specimens were prepared and immunohistochemically stained for proteins S100A1, -A2, -A3, -A4, -A6, -A7, -A8, and -A9. Staining was classified using semiquantitative analysis and a 4-degree staining score (ie, no, decent, medium, and intense staining) at 4 different anatomic or functional regions (ie, the odontoblast layer [OL], pulpal stroma [PS], border area of calcifications [BAC], and vessel walls). The distribution of staining degrees between the 3 diagnostic groups was calculated using the Fisher exact text (P ≤ .05) at the 4 regions. ResultsSignificant differences in staining were observed mainly in the OL and PS and at the BAC. The most significant differences were detected in the PS and when comparing NP with 1 of the 2 irreversibly inflamed pulpal tissues (AIP or SIP). The inflamed tissues were then invariably stained more intensely than their normal counterparts at this location (S100A1, -A2, -A3, -A4, -A8, and -A9). In the OL, NP tissue was significantly stronger stained for S100A1, -A6, -A8, and -A9 compared with SIP and for S100A9 when compared with AIP. Differences between AIP and SIP in direct comparison were rare and were found only for 1 protein (S100A2) at the BAC. Also, at the vessel walls, only 1 statistical difference in staining was observed (SIP was stronger stained than NP for protein S100A3). ConclusionsThe occurrence of proteins S100A1, -A2, -A3, -A4, -A6, -A8, and -A9 is significantly altered in irreversibly inflamed compared with normal dental pulp tissue at different anatomic localizations. Some members of S100 proteins obviously participate in focal calcification processes and pulp stone formation of the dental pulp.

Abstract P6-03-07: Development of an at-home breast health assessment test, to increase compliance with screening mammography using proteins from tears

Abstract Anna E. Daily1,*, Prashanth Ravishankar1, Wanyi Wang3, Ryan Krone3, Steve Harms1,2, and V Suzanne Klimberg1,4,5 1Namida Lab Inc, Fayetteville, Arkansas; 2The Breast Center-Medical Associates of Northwest Arkansas, Fayetteville, Arkansas; 3Elite Research LLC, Irving, Texas; 4Department of Surgery, University of Texas Medical Branch, Galveston, Texas; 5Breast Surgical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas. *anna@namidalab.com Background: There is a growing body of evidence to support tears as a non-traditional biological fluid in clinical laboratory testing. In addition to the simplicity of tear fluid processing, the ability to access key cancer biomarkers in high concentrations quickly and inexpensively make them an attractive biofluid source. Here we report our biomarker discovery study on tears to identify and validate candidate biomarkers for breast cancer and develop a model that is significantly associated with a positive breast cancer diagnosis. Methods: Participants were recruited from individuals having a yearly screening mammogram, biopsy, and/or recently diagnosed with breast cancer. Imaging results were obtained from clinical sites and samples were then classified as: control (normal imaging no biopsy) or diagnosed breast cancer pre-treatment (diagnosed by biopsy). Biomarker discovery was conducted using 102 individual tear samples collected using the Schirmer strip collection method. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was performed to identify protein biomarker candidates with altered expression levels in breast cancer patients. ELISA assay to confirm LC-MS/MS trends for biomarkers of interest was conducted using 171 tear samples. An additional round of validation utilizing 848 samples was performed which included protein concentrations determined by ELISA and collection of demographic and clinical covariates. The resulting concentration data, combined with the demographic and clinical covariates, was analyzed using logistic regression analysis to build a model for classification of samples as positive or negative. Results: A total of 301 proteins were identified by LC-MS/MS and narrowed to a list of 14 proteins (p-value &amp;lt; 0.05) with potential significance in breast cancer patients. Three biomarkers, S100A8 (p-value = 0.0069), S100A9 (p-value = 0.0048), and Galectin-3 binding protein (p-value = 0.01) with an increased expression in breast cancer patients were selected for validation using ELISA. Logistic regression analysis produced three models, which were then evaluated on breast cancer cases and controls at two diagnostic thresholds and resulted in sensitivity ranging from 52% - 90% and specificity from 31% - 79%. Conclusions: Our results demonstrate clinical feasibility for tear proteins to detect breast cancer and includes the most extensive published data set of individually analyzed tear samples. This analysis suggests that models developed using tear fluid have clinical validity and could be used in further development of a biological assay. We envision positioning this assay as a tool for activation around breast health screening for low to average risk patients who may be screening avoidant or adverse to encourage participation in screening mammography. Citation Format: Anna Daily, Prashanth Ravishankar, Victoria S. Klimberg, Steve Harms. Development of an at-home breast health assessment test, to increase compliance with screening mammography using proteins from tears [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-03-07.

Abstract P198: Associations Between Plasma Proteome and Leukocyte Mitochondrial DNA Copy Number: The Atherosclerosis Risk in Communities Study (ARIC)

Introduction: The number of copies of the mitochondrial genome, termed mitochondrial DNA copy number (MTCN), is a proxy for mitochondrial function and varies by cell. Critically, lower MTCN has been associated with aging and chronic diseases such as dementia and cancer. We conducted a large-scale proteomics analysis of leukocyte MTCN (L-MTCN) to elucidate pathways modulated by L-MTCN that may be involved in diseases of aging. Hypothesis: We hypothesize that L-MTCN is significantly associated to plasma proteins that have been implicated in biological pathways of L-MTCN and related diseases. Methods: Based on whole genome sequencing (WGS) data measured in samples collected in ARIC visits 1, 2, and 3 (i.e., baseline for the current study), L-MTCN was calculated as (2 * coverage of the mitochondrial genome) / coverage of the autosome, using the median coverage of 1000 base pair bins overlapping the mitochondrial genome (16 total) and the median coverage of ~142,000 higher quality autosomal bins. Only samples with read lengths of 150 or 151 were included in subsequent analyses, and L-MTCN estimates were inverse normalized within read length group and WGS group before being merged together. This normalized L-MTCN was then analyzed as a predictor for an association with 4,870 plasma proteins measured by SOMAscan v4 in ARIC visit 3 in race-specific generalized linear models, adjusting for the following covariates at baseline: WGS sample visit, age, gender, field center, smoking status, and a measure of kidney function. Bonferroni correction was applied to account for the number of proteins tested in 5,007 White participants (p≤1x10 -5 ). Associations were tested for replication in 1,178 Black participants and considered significant if protein estimates had the same direction of association as Whites at p-value ≤ 2.2x10 -4 . Results: Among White participants, 230 proteins were significantly associated with L-MTCN. Of these proteins S100A9, GP1Bα, EGFR, THBS2, and FCGR3B were significantly replicated among Black participants. Strongest associations with L-MTCN were seen with GP1Bα (Estimate: 0.121) and S100A9 (Estimate: -0.191). Biologically S100A9 is a calgranulin and together with S100A8 forms calprotectin, which have roles mediating inflammatory processes. GP1Bα helps mediate platelet activation and forms the glycoprotein Ib-IX-V complex which acts as a platelet surface receptor for von Willebrand factor facilitating platelet adhesion. Conclusion: This large proteomics analysis identified 230 plasma proteins significantly associated with L-MTCN. Some of the proteins have been connected to inflammatory diseases, cardiovascular disease, and cancer in the literature. These results provide insight into proteins involved in biological pathways related to L-MTCN.

Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes

BackgroundRecent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed.Results25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca2+ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells.ConclusionCollectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.

S100A6 Protein-Expression and Function in Norm and Pathology.

S100A6, also known as calcyclin, is a calcium-binding protein belonging to the S100 protein family. It was first identified and purified more than 30 years ago. Initial structural studies, focused mostly on the mode and affinity of Ca2+ binding and resolution of the resultant conformational changes, were soon complemented by research on its expression, localization and identification of binding partners. With time, the use of biophysical methods helped to resolve the structure and versatility of S100A6 complexes with some of its ligands. Meanwhile, it became clear that S100A6 expression was altered in various pathological states and correlated with the stage/progression of many diseases, including cancers, indicative of its important, and possibly causative, role in some of these diseases. This, in turn, prompted researchers to look for the mechanism of S100A6 action and to identify the intermediary signaling pathways and effectors. After all these years, our knowledge on various aspects of S100A6 biology is robust but still incomplete. The list of S100A6 ligands is growing all the time, as is our understanding of the physiological importance of these interactions. The present review summarizes available data concerning S100A6 expression/localization, interaction with intracellular and extracellular targets, involvement in Ca2+-dependent cellular processes and association with various pathologies.

IL-6 evoked biochemical changes in prostate cancer cells.

The pro-inflammatory cytokine IL-6 has been associated with the progression of PCa to a castration-resistant phenotype. In this work, we characterized the biochemical changes evoked by IL-6 in three different models of PCa cells, including LNCaP, C4-2, and PC3. The effect of IL-6 on PCa cells was compared with the effect obtained by co-stimulation with the cAMP-inducing agent forskolin (FSK). Stimulation of LNCaP cells with IL-6 or IL-6+FSK evoked increased expression of the neuroendocrine marker tubulin IIIβ and Cav3.2T-type Ca2+ channel subunit. PC3 cells, representing a more advanced state of PCa, had high levels of tubulin IIIβ expression without any further changes observed by treatment with IL-6 or IL-6+FSK. Elevated expression of the glucocorticoid receptor was observed in PC3, but not in LNCaP or C4-2 cells. Glucocorticoid receptor expression was not regulated by IL-6 stimulation of LNCaP or C4-2 cells. IL-6 acting alone or together with FSK evoked a significant reduction in the expression of the transcription factor REST and retinoblastoma tumor suppressor protein Rb1. In LNCaP cells, IL-6 acting alone or together with FSK had no effect on the expression of several biological markers of advanced PCa, including Aurora kinase A, valosin-containing protein, calcium-sensing receptor, calreticulin, S100A protein, and Protein S. In PC3 cells, co-treatment with IL-6+FSK evoked increased expression of REST and S100A proteins, as well as a reduction in Protein S levels. These findings reveal a complex pattern of biochemical changes in PCa cells under the influence of IL-6.

TARGETED THERAPY OF ANAPLASTIC THYROID CANCER

Introduction. Anaplastic thyroid cancer is an aggressive disease with meager patient survival. The tumors are often unresectable and insensitive to standard treatment methods (chemotherapy and radioiodine therapy). In recent years, much attention has been paid to new therapeutic strategies, mainly targeted therapy. The purpose of our study was to summarize the data on targeted drugs used to treat anaplastic thyroid cancer and establish the main side effects associated with their use. Materials and methods. A scientific search was conducted in Pubmed, Scopus, and Web of Science databases. The following search terms were used: "anaplastic thyroid cancer," "targeted therapy," and "clinical trials." Results. A literature search was conducted regarding targeted drugs to treat anaplastic thyroid cancer. The results of clinical trials using BRAF/MEK, RET, NTRK, mTOR, and TKI inhibitors were analyzed. Several clinical trials have demonstrated that dabrafenib, combined with trametinib, has a sufficiently high therapeutic effect and can effectively treat anaplastic thyroid cancer. Other targeted drugs show limited clinical response, such as NTRK inhibitors, TKIs, and other BRAF/MEK inhibitors. Some drugs (e.g., lenvatinib) may only be effective with other anticancer agents. RETi and mTORi are believed to have some therapeutic potential, but more than several clinical trials are needed to evaluate their effectiveness. Discussion. The main reason for the relapse of the disease and the ineffectiveness of targeted drugs is the appearance of new mutations. The RAS mutation is responsible for resistance to the combination of dabrafenib and trametinib; the S100A4 protein is responsible for the ineffectiveness of vemurafenib. Developing new drugs capable of overcoming resistance mechanisms is necessary to solve this problem. Conclusions. Targeted therapy is a promising direction in the treatment of anaplastic thyroid cancer. Side effects are common but mostly manageable.

Comparative Proteomic Analysis of Milk-Derived Extracellular Vesicles from Dairy Cows with Clinical and Subclinical Mastitis.

Extracellular vesicles (EVs) are membranous vesicles found in biological fluids with essential functions. However, milk-derived EV proteins from clinical mastitis (CM) and subclinical mastitis (SM) cows have yet to be studied in detail. In this study, milk-derived EVs of CM, SM, and Healthy cows were extracted using a combination of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a shotgun proteomic by data-independent acquisition mode. A total of 1253 milk exosome proteins were identified and quantified. Differently enriched (DE) proteins were identified as given a Benjamini−Hochberg adjusted p < 0.05 and a fold change of at least 2. There were 53 and 1 DE proteins in milk-derived EVs from CM and SM cows compared with healthy cows. Protein S100-A9, Protein S100-A8, Chitinase-3-like protein 1, Haptoglobin, Integrin beta-2, and Chloride intracellular channel protein 1 were more abundant in the CM group (adjusted p < 0.05). Still, their enrichment in the SM group was not significant as in the Healthy group. The enrichment of DE proteins between CM and Healthy group was consistent with elevated GO (Gene Ontology) processes—defense response, defense response to Gram-positive bacterium, granulocyte chemotaxis also contributed to Reactome pathways—neutrophil degranulation, innate immune system, and antimicrobial peptides in the CM group. These results provide essential information on mastitis-associated proteins in milk-derived EVs and indicate the biological functions of milk-derived EVs proteins require further elucidation.