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Emerging evidence has shown the role of mesenchymal stem cell-derived exosome (MSC-exo) in inducing resistance of cancer cells to chemotherapy. However, it remains unclear whether the change of MSC-exo in response to chemotherapy also contributes to chemoresistance. In this study, we investigated the effect of a standard-of-care chemotherapeutic agent, doxorubicin (Dox), on MSC-exo and its contribution to the development of Dox resistance in breast cancer cells (BCs). We found that the exosome secreted by Dox-treated MSCs (Dt-MSC-exo) induced a higher degree of Dox resistance in BCs when compared with non-treated MSC-exo. By analysis of the MSC-exo-induced transcriptome change in BCs, we identified S100A6 , a chemoresistant gene, as a top-ranked gene induced by MSC-exo in BCs, which was further enhanced by Dt-MSC-exo. Furthermore, we found that Dox induced the expression of miR-21-5p in MSCs and MSC-exo, which was required for the expression of S100A6 in BCs. Importantly, silencing of miR-21-5p expression in MSCs and MSC-exo abolished the resistance of BCs to Dox, indicating an exosomal miR-21-5p-regulated S100A6 in chemoresistance. Our study thus uncovered a novel mechanistic insight into the role of MSC-secreted exosome in the development of chemoresistance in the tumor microenvironment., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

The mammalian brain contains few niches for neural stem cells (NSCs) capable of generating new neurons, whereas other regions are primarily gliogenic. Here we leverage the spatial separation of the sub-ependymal zone NSC niche and the olfactory bulb, the region to which newly generated neurons from the sub-ependymal zone migrate and integrate, and present a comprehensive proteomic characterization of these regions in comparison to the cerebral cortex, which is not conducive to neurogenesis and integration of new neurons. We find differing compositions of regulatory extracellular matrix (ECM) components in the neurogenic niche. We further show that quiescent NSCs are the main source of their local ECM, including the multi-functional enzyme transglutaminase 2, which we show is crucial for neurogenesis. Atomic force microscopy corroborated indications from the proteomic analyses that neurogenic niches are significantly stiffer than non-neurogenic parenchyma. Together these findings provide a powerful resource for unraveling unique compositions of neurogenic niches., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Evidence has been accumulating that zinc ions can trigger β-amyloid (Aβ) deposition and senile plaque formation in the brain, a pathological hallmark of Alzheimer's disease (AD). Chelating zinc inhibits Aβ aggregation and may hold promise as a therapeutic strategy for AD. S100A6 is an acidic Ca 2+ /Zn 2+ -binding protein found only in a small number of astrocytes in the normal brain. However, in the AD brain, S100A6 is highly expressed in astrocytes around Aβ plaques. The role of the astrocytic S100A6 upregulation in AD is unknown. In the present study, we examined the effects of S100A6 on Aβ plaques and intracellular zinc levels in a mouse model of AD. Chronic exposure to zinc increased Aβ deposition and S100A6 expression, both reversible by the zinc chelator clioquinol, in the brains of amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice. To examine whether exogenous S100A6 could induce Aβ plaque disaggregation through competition for zinc in vitro, we incubated APP/PS1 mouse brain sections with recombinant human S100A6 protein or co-incubated them with human S100A6-expressing cells. Both treatments efficiently reduced the Aβ plaque burden in situ. In addition, treatment with exogenous S100A6 protected cultured COS-7 cells against zinc toxicity. Our results show for the first time that increased S100A6 levels correlate with both Aβ disaggregation and decrease of Aβ plaque-associated zinc contents in brain sections with AD-like pathology. Astrocytic S100A6 in AD may protect from Aβ deposition through zinc sequestration., Competing Interests: Conflict of Interest We have no conflicting interest to disclose.

Purpose: The objective of present study is to determine serum levels and placental distribution of two interacting proteins calcyclin and heat shock protein 90 in preeclampsia., Experimental Design: Maternal serum levels of calcyclin and heat shock protein 90 are compared throughout pregnancy from the first trimester till term among women with preeclampsia (n = 43) and age-matched normotensive pregnant controls (n = 46). A serum-based 2D LC-MS assay using Parallel Reaction Monitoring is applied to quantify both calcyclin and heat shock protein 90., Results: Serum levels of calcyclin are significantly lower in patients with preeclampsia in the second trimester of pregnancy as compared to controls (p < 0.05). Serum levels of heat shock protein 90 are significantly higher in patients with preeclampsia in the third trimester as compared to controls (p < 0.001)., Conclusion and Clinical Relevance: Both interacting proteins calcyclin and heat shock protein 90 are notably changed in preeclamptic patients compared to controls. Calcyclin is already decreased before the onset of preeclampsia in the second trimester and HSP90 is strongly increased in the third trimester. This suggests that these proteins may play a role in the pathogenesis of preeclampsia and ought to be investigated in large cohort studies as molecular biomarkers., (© 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

S100A6 is a member of the EF-hand Ca 2+ -binding protein family, which plays important roles in a wide variety of Ca 2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca 2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca 2+ -bound or Ca 2+ -free states with distinct aggregation propensities. That is the case among β-parvalbumins and S100 proteins, which under certain conditions undergo Ca 2+ -dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.

An elevated level of S100A6 is associated with poor outcomes of many tumor types, but, how S100A6 contributes to nasopharyngeal carcinoma (NPC) progression remains unknown. Here, we investigated the expression and prognostic significance of S100A6 in NPC and explored the molecular mechanisms under-lying the role of S100A6 in NPC development. The results showed that S100A6 was markedly up-regulated in NPC tissues and cell lines compared to paired peritumoral normal tissues and a normal nasopharyngeal epithelial cell line, respectively. In tissues from 92 NPC patients, high S100A6 expression was associated with advanced N stage, locoregional failure and disease progression and was predictive of poor locoregional recurrence-free survival (LRRFS, P = 0.001) and progression-free survival (PFS, P = 0.001). Multivariate analysis showed that S100A6 is an independent prognostic factor for LRRFS and PFS. Silencing S100A6 using siRNA or shRNA significantly suppressed NPC cell proliferation, colony formation and p38/mitogen-activated protein kinase (MAPK) activity in vitro and inhibited tumor growth in a xenograft mouse model of NPC. In contrast, overexpressing S100A6 via plasmid transfection resulted in increased NPC cell proliferation and p38/MAPK activation. S100A6-induced proliferation was abolished by a p38 inhibitor. In summary, S100A6 may be a new prognostic marker of NPC and may promote NPC development via the activation of p38/MAPK signaling pathways. These findings suggest S100A6/p38/MAPK signaling as a potential therapeutic target for NPC. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Cardiomyocyte-specific transgenic mice overexpressing S100A6, a member of the family of EF-hand calcium-binding proteins, develop less cardiac hypertrophy, interstitial fibrosis, and myocyte apoptosis after permanent coronary ligation, findings that support S100A6 as a potential therapeutic target after acute myocardial infarction. Our purpose was to investigate S100A6 gene therapy for acute myocardial ischemia-reperfusion., Methods and Results: We first performed in vitro studies to examine the effects of S100A6 overexpression and knockdown in rat neonatal cardiomyocytes. S100A6 overexpression improved calcium transients and protected against apoptosis induced by hypoxia-reoxygenation via enhanced calcineurin activity, whereas knockdown of S100A6 had detrimental effects. For in vivo studies, human S100A6 plasmid or empty plasmid was delivered to the left ventricular myocardium by ultrasound-targeted microbubble destruction in Fischer-344 rats 2 days prior to a 30-minute ligation of the left anterior descending coronary artery followed by reperfusion. Control animals received no therapy. Pretreatment with S100A6 gene therapy yielded a survival advantage compared to empty-plasmid and nontreated controls. S100A6-pretreated animals had reduced infarct size and improved left ventricular systolic function, with less myocyte apoptosis, attenuated cardiac hypertrophy, and less cardiac fibrosis., Conclusions: S100A6 overexpression by ultrasound-targeted microbubble destruction helps ameliorate myocardial ischemia-reperfusion, resulting in lower mortality and improved left ventricular systolic function post-ischemia-reperfusion via attenuation of apoptosis, reduction in cardiac hypertrophy, and reduced infarct size. Our results indicate that S100A6 is a potential therapeutic target for acute myocardial infarction., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

S100A6 is involved in regulating the progression of cancer. S100A6 can regulate the dynamics of cytoskeletal constituents, cell growth and differentiation by interacting with binding or target proteins. The present study investigated whether S100A6 affects cell proliferation in gastric cancer cells by stimulating several downstream factors. Firstly, the expression and localization of S100A6 were investigated using immunohistochemical staining, an immunoelectron microscopy and laser confocal scanning. A ChIP-Chip assay was performed to determine the downstream factors of S100A6 using promoter Chip analysis, including approximately the -800 to +200 regions around the transcription starting point. Polymerase chain reaction analysis was performed to confirm this. It was found that the intensity of S100A6 staining was markedly higher in the cytoplasm and nucleus, and its expression level correlated with that of the Ki67 protein. The overexpression of S100A6 also promoted cell proliferation in AGS and BGC823 cell lines, detected using a Cell Counting-Kit 8 assay. In cells overexpressing S100A6, the expression levels of interleukin (IL)-8, cyclin-dependent kinase (CDK)5, CDK4, minichromosome maintenance complex component 7 (MCM7) and B-cell lymphoma 2 (Bcl2) were noticeably increased. In conclusion, the increased expression of S100A6 promoted cell proliferation by regulating the expression levels of IL-8, CDK5, CDK4, MCM7 and Bcl2 in gastric cancer cells.

Biochemical markers play a significant role in the diagnosis of lung cancer. Recent studies have demonstrated a link involving S100 Calcium Binding Proteins (S100A2, S100A6) and non-small cell lung cancer (NSCLC), but the expediency of their serum levels in NSCLC has not been established. In this study, we evaluate the potential of serum S100A2 and S100A6 levels as diagnostic markers for NSCLC. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of S100A2 and S100A6 in 141 NSCLC patients and 150 healthy subjects. Serum levels of the two proteins in patients with NSCLC were higher compared to healthy controls (P = 0.0002 for S100A2 and P < 0.0001 for S100A6). Moreover, the levels of S100A2 and S100A6 were higher in the sera of stage I/II NSCLC patients compared to healthy controls with P = 0.01 and <0.0001, respectively. Receiver operating characteristic (ROC) analysis showed that S100A2 could distinguish NSCLC patients from healthy controls (AUC = 0.646), and S100A6 could also identify NSCLC (AUC = 0.668). Meanwhile, these two proteins showed notable capabilities for distinguishing stage I/II NSCLC from healthy controls (AUC = 0.708 for S100A2 and AUC = 0.702 for S100A6). Our results indicate that serum levels of S100A2 and S100A6 are significantly elevated in early stage NSCLC and may have the potential for NSCLC biomarker. Further studies with large sample population would help validate our findings.

Hypertension frequently results in severe complications in cardiovascular system and histopathological changes in the heart. To better understand the cellular processes and signaling pathways responsible for the proper functioning of the heart, we decided to check whether doxazosin affects the density of structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats. The aim of this study is to find differences in the density of the structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats treated with doxazosin compared to untreated animals. Fragments of heart were collected from five spontaneously hypertensive rats and five spontaneously hypertensive rats receiving doxazosin for six weeks (dose 0.1 mg per 1 kg of body weight). On the paraffin sections S100A6 and atrial natriuretic factor peptides were localized in the heart using immunohistochemistry. Positive immunohistochemical reaction for S100A6 was observed in atrial and ventricular cardiomyocytes and in the coronary vasculature. In the heart of hypertensive rats treated with doxazosin the S100A6 immunoreactivity was significantly lower compared to untreated animals. Immunodetection of atrial natriuretic factor in the heart of rats confirmed presence of peptide in atrial myocardium. Delicate atrial natriuretic factor-immunoreactivity was observed also in few ventricular cardiomyocytes. The atrial natriuretic factor-immunosignal was significantly weaker in hearts of hypertensive rats receiving doxazosin compared to spontaneously hypertensive rats untreated. Since we found that doxazosin reduces the levels of S100A6 and atrial natriuretic factor peptides in the heart of spontaneously hypertensive rats, it can be assumed that cardiovascular disorders that occur in hypertension may be associated with disturbances of cellular processes and signaling pathways., (© 2016 by the Society for Experimental Biology and Medicine.)

In this work, we have found that casein kinase II (CKII) phosphorylates the CacyBP/SIP protein under in vitro conditions and have mapped the phosphorylation site to threonine 184. Moreover, we present evidence that S100A6, a CacyBP/SIP interacting protein, inhibits this phosphorylation in the presence of Ca(2+). CacyBP/SIP phosphorylation by CKII was also observed in neuroblastoma NB2a cells. Interestingly, we have found that the effect of DRB, a CKII inhibitor, on CacyBP/SIP phosphorylation state is similar to that of S100A6 overexpression. Phosphorylation at threonine 184 seems to have an effect on CacyBP/SIP phosphatase activity since the T184E phosphorylation mimic mutant overexpressed in NB2a cells has lower phosphatase activity toward p-ERK1/2 when compared to the non-phosphorylable T184A mutant or to the wild-type protein. In conclusion, our data suggest that S100A6 and Ca(2+), through inhibiting CacyBP/SIP phosphorylation on threonine 184, are important regulators of CacyBP/SIP phosphatase activity and of ERK1/2-Elk-1 signaling pathway., (© 2015 Wiley Periodicals, Inc.)

Clear cell renal cell carcinoma (ccRCC) is often resistant to existing therapy. We found elevated S100A6 levels in ccRCC tissues, associated with higher grade pathological features and clinical stages in ccRCC patients. Knockdown of S100A6 inhibited cell proliferation in vitro and tumor growth in vivo. Gene expression profiling suggests a novel function of S100A6 in suppressing apoptosis, as well as a relationship between S100A6 and CXCL14, a pro-inflammatory chemokine. We suggest that the S100A6/CXCL14 signaling pathway is a potential therapeutic target in ccRCC.

S100 proteins comprise a large family of Ca(2+)-binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions.