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• In recent years, various studies on the functions of S100 proteins in lung cancer emerged. • This paper presents an updated review of S100 proteins in lung cancer from 2017 to 2021. • This paper added a review on the correlation between S100A8/S100A9 and lung cancer. The S100 protein family is involved in the pathogenesis of several malignancies including lung cancer. Recent studies have shown that one member, S100A2, was over-expressed in advanced stage non-small cell lung cancer (NSCLC). Another, S100A6, demonstrated variable expression in different lung cancer subtypes. Research using NSCLC cell lines reported that SIX3 inhibited cell metastasis and proliferation via S100P down-regulation. This review represents an update on S100 proteins in lung cancer from 2017 to 2021 and includes the aforementioned as well as S100A4, S100A7, and S100B. Inconsistencies in mechanisms of action for S100A8/S100A9 are highlighted and a comprehensive evaluation of the most recent evidence for the S100 proteins in lung cancer is presented. [ABSTRACT FROM AUTHOR]

• Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important growth factor. • Dimeric Ca-loaded forms of S100A4, S100A6, and S100P proteins binds to GM-CSF. • Calcium removal prevents binding to GM-CSF. • S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. • A conserved S100A4/A6/P-binding site in the GM-CSF molecule was found by structural modelling. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, K d , values of 0.3–2 μM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the 'hinge' region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors. [Display omitted] [ABSTRACT FROM AUTHOR]

Patients diagnosed with lymph node (LN) metastatic liver cancer face an exceedingly grim prognosis. In-depth analysis of LN metastatic patients' characteristics and tumor cells' interactions with human lymphatic endothelial cells (HLECs), can provide important biological and therapeutic insights. Here we identify at the single-cell level that S100A6 expression differs between primary tumor and their LN metastasis. Of particular significance, we uncovered the disparity in S100A6 expression between tumors and normal tissues is greater in intrahepatic cholangiocarcinoma (ICC) patients, frequently accompanied by LN metastases, than that in hepatocellular carcinoma (HCC), with rare occurrence of LN metastasis. Furthermore, in the infrequent instances of LN metastasis in HCC, heightened S100A6 expression was observed, suggesting a critical role of S100A6 in the process of LN metastasis. Subsequent experiments further uncovered that S100A6 secreted from tumor cells promotes lymphangiogenesis by upregulating the expression and secretion of vascular endothelial growth factor-D (VEGF-D) in HLECs through the RAGE/NF-kB/VEGF-D pathway while overexpression of S100A6 in tumor cells also augmented their migration and invasion. Taken together, these data reveal the dual effects of S100A6 in promoting LN metastasis in liver cancer, thus highlighting its potential as a promising therapeutic target. • S100A6-rich tumor cells correlates with LN metastasis in liver cancer. • S100A6 secreted by tumor cells induces lymphangiogenesis in HLECs via RAGE/NF-kB/VEGF-D pathway. • High expression of S100A6 presents potential to initiate LN metastasis in liver cancer. • YY1 transcriptionally activates S100A6 expression and the expression level of YY1 is higher in ICC patients compared to HCC. [ABSTRACT FROM AUTHOR]

Abstract The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer's patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60–90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60–90 days), but later (90–150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60–90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120–150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120–150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis is a pathological process in which extracellular matrix excessively aggregates in an injured liver. Research on hepatic fibrosis is expanding, however, much information in this process is still unclear. Here, we examined the gene expression changes within the process of liver fibrosis, providing the first evidence that secreted S100A6 is a critical contributor. We discovered that expression of the S100 family is highly correlated with CCl 4 -induced liver fibrosis and post self-recovery in mice. Recombinant human S100A6 (rhS100A6) introduced to CCl 4 -induced mice was found to enhance liver fibrosis through the promotion of activated hepatic stellate cell (HSC) proliferation. More importantly, we showed that rhS100A6 can induce cell cycle transition from S to G2 stage and significantly elevate the level of ERK phosphorylation in the MARK pathway. In contrast to rhS100A6, recombinant human and soluble receptor for advanced glycation end products (sRAGE), a natural antagonist of the S100/RAGE pathway, was found to have a preventative effect on liver fibrosis in CCl 4 -induced mice. In conclusion, our study supports that S100A6 could be a novel therapeutic in liver fibrosis and its receptor antagonist, sRAGE, proofed to be effective for the treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways. [ABSTRACT FROM AUTHOR]

S100A6 is a calcium binding protein expressed in many types of epithelia including epidermis. S100A6 is a binding partner of a number of proteins engaged in cytoskeletal organization, cell cycle control, stress response or apoptosis. So far the effect of its overexpression or knock-down on cell physiology has been studied only at the cellular level. Here, we used an in vitro model of differentiating epidermis to study the role of S100A6 at the tissue level and in the context of tissue differentiation. First of all we have shown that S100A6 mRNA level diminished several fold during primary keratinocyte differentiation and investigated the epigenetic and transcriptional mechanisms involved in this tight expression control. Using bisulfite treatment, luciferase assay and chromatin immunoprecipitation we found that changes in S100A6 expression were DNA methylation independent but could be orchestrated by epidermal specific factors: the ΔNp63 transcription factor and retinoic acid. To investigate if the drop-down in S100A6 expression is indeed critical for keratinocyte differentiation we developed HaCaT cells with stable S100A6 knock-down or overexpression and tested them in 2- and 3-dimensional (organotypic) culture conditions. S100A6 overexpressing cells exhibited accelerated proliferation, enhanced adhesion properties and suppressed loricrin expression – features typical for undifferentiated keratinocytes. In organotypic culture these cells formed thicker epidermis with more Ki67 positive cells, keratin 10 expression spatially limited to the uppermost cell layers and non-detectable loricrin expression. Together, results obtained in both culture models proved that increased S100A6 content in keratinocytes dramatically changed the pace and extent of epidermal differentiation. [ABSTRACT FROM AUTHOR]

Objective A major challenge in muscle-invasive urothelial carcinoma (UC) is to identify biomarkers that can predict disease prognosis and treatment response after cystectomy. Therefore, we analyzed the potential prognostic value of the proteins vascular endothelial growth factor receptor 2 (VEGFR2), S100A4, and S100A6 in UC. Methods Retrospective outcome data and tumor specimens from 83 cystectomy patients with histologically confirmed invasive UC were included. Expression levels of VEGFR2 (also called flk-1 and KDR), S100A4, and S100A6 were analyzed in primary tumor tissue by immunohistochemistry. Results Immunohistochemical staining and analysis of VEGFR2, S100A4, and S100A6 showed localization mainly in tumor cell cytoplasm. High VEGFR2 expression and low tumor category were independent variables associated with longer overall survival (OS) and disease-free survival, revealed by a bivariate Cox proportional hazards regression model (both P <0.001). In addition, the univariate log-rank test and the Cox model demonstrated that OS beyond 2 years was significantly greater among patients with low S100A6 expression than in those with high S100A6 expression ( P = 0.017 and 0.022, respectively). Differences in tumor expression of S100A4 were not significantly associated with outcome. Conclusion In this study, VEGFR2 expression was significantly correlated with risk of disease relapse and OS in a defined cohort of patients with UC of the bladder treated by cystectomy. [ABSTRACT FROM AUTHOR]

S100A6 is a calcium binding protein belonging to the S100 family. In this work we examined the function of extracellular S100A6. Using mesenchymal stem cells isolated from Wharton's jelly of the umbilical cord (WJMS cells) we have shown that S100A6 is secreted by these cells, and when added to the medium, increases their adhesion and inhibits proliferation. The search for a potential target/receptor of S100A6 in the membrane fraction of WJMS cells allowed us to identify some proteins, among them integrin β1, which interacts with S100A6 in a calcium dependent manner. The interaction between S100A6 and integrin β1, was then confirmed by ELISA using purified proteins. Applying specific antibodies against integrin β1 reversed the effect on cell adhesion and proliferation observed in the presence of S100A6 which indicates that S100A6 exerts its function due to interaction with integrin β1. Since the data show the influence of extracellular S100A6 on cells isolated from Wharton's jelly, our results might help to establish molecular mechanisms leading to some pathologies characteristic for this tissue. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: In this work we compared the level, localization and binding partners of a calcium binding protein, S100A6, in extracellular matrix of Wharton's jelly of healthy and preeclamptic patients. Methods: Studies were performed on the umbilical cords taken from 10 newborns delivered by healthy and 10 newborns delivered by preeclamptic mothers. To characterize S100A6 in Wharton's jelly immunoblotting and immunohistochemistry were applied. For identification of S100A6 targets pull down assays and mass spectrometry were performed. Direct interaction of S100A6 with its targets was checked by ELISA while co-localization of these proteins was analyzed by immunofluorescence staining. Results: We have found that the level of S100A6 in Wharton's jelly is higher in patients with preeclampsia than in healthy ones and that post-translational modifications of S100A6 in preeclamptic tissue are different than those of S100A6 in control. We have identified several proteins that might interact with S100A6, among them are lumican and PRELP, found in Wharton's jelly of healthy and preeclamptic patients, and IGFBP-1 identified, as an S100A6 target, only in preeclamptic tissue. We have shown that the interactions between S100A6 and these proteins are direct and that IGF-1 competes with S100A6 for binding to IGFBP-1. Conclusion: In Wharton's jelly of preeclamptic tissue S100A6 is up-regulated and binds to different targets than in control. This suggests involvement of S100A6 in development of preeclampsia. [Copyright &y& Elsevier]

Abstract: The aetiology of pre-eclampsia is thought to originate from aberrant spiral artery remodelling and invasion evoking cellular oxidative stress. Previously, we discovered differentially expressed proteins in trophoblast cells of pre-eclamptic pregnancies. One of these proteins is calcyclin (S100A6); a Ca2+-binding protein associated with cellular stress response. By immunohistochemistry on formalin-fixed paraffin-embedded placental tissue, calcyclin expression was compared between women with early pre-eclampsia (n =72) and non-hypertensive control patients (n =66) (χ 2, p =0.006) blindly by two observers. Significantly more intense staining was seen in trophoblast cells of pre-eclamptic pregnancies compared to control placentas suggesting that trophoblast calcyclin is elevated in early pregnancy. [Copyright &y& Elsevier]

Highlights: [•] In the water maze, spatial memory was formed in Wister rats after 4-day training. [•] PCR revealed specific brain regional profiles of Ascl1, S100a6 and Casp3 expression. [•] These genes were associated with coupled neurogenesis/apoptosis/neuroplasticity. [•] An intra-structural prefrontal cortex/cerebellum correlation was shown to Casp3. [•] Hippocampal, prefrontal cortical and cerebellar integration was validated in memory. [ABSTRACT FROM AUTHOR]

Abstract: The calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein was shown to play a role in the organization of microtubules. In this work we have examined the neuronal distribution and possible function of CacyBP/SIP in cytoskeletal pathophysiology. We have used brain tissue from Alzheimer''s disease (AD) patients and from transgenic mice modeling 2 different pathologies characteristic for AD: amyloid and tau. In the brain from AD patients, CacyBP/SIP was found to be almost exclusively present in neuronal somata, and in control patients it was seen in the somata and neuronal processes. In mice doubly transgenic for amyloid precursor protein and presenilin 1 there was no difference in CacyBP/SIP neuronal localization in comparison with the nontransgenic animals. By contrast in tau transgenic mice, localization of CacyBP/SIP was similar to that observed for AD patients. To find the relation between CacyBP/SIP and tau we examined dephosphorylation of tau by CacyBP/SIP. We found that indeed it exhibited phosphatase activity toward tau. Altogether, our results suggest that CacyBP/SIP might play a role in AD pathology. [Copyright &y& Elsevier]

Abstract: In this paper we document an increased expression of S100A6, a calcium binding protein of the S100 family, and its co-localization with β-catenin in colorectal cancer tissues and in metastatic, SW620, versus non-metastatic, SW480, human colorectal cancer cell lines. Moreover, we show up-regulation of the S100A6 protein level in non-metastatic SW480 cells due to overexpression of β-catenin as well as the activation of the S100A6 gene promoter upon cell transfection with β-catenin and the TCF-Lef1 transcription factor. Since we found a high level of S100A6 in metastatic SW620 cells we searched for its interacting partners in the protein extract prepared from these cells. Using several methods we found that S100A6 interacts with lamin A/C, a protein known to be implicated in colon carcinogenesis. Our results reveal a novel and important network of relations and interactions between proteins potentially involved in colorectal cancer development and progression. [Copyright &y& Elsevier]

Abstract: Recently we have shown that the Sgt1 (suppressor of G2 allele of Skp1) protein translocates to the nucleus due to heat shock and that the Ca2+-bound form of S100A6 is required for Sgt1 translocation (). In this work we studied the influence of Sgt1 phosphorylation on nuclear translocation. By means of two-dimensional (2D) electrophoresis we showed that in the protein extract of heat-shocked human epidermoid carcinoma (HEp-2) cells a higher level of a basic, most probably non-phosphorylated, form of Sgt1 can be detected. Also, we found a more efficient translocation of Sgt1 induced by heat shock when casein kinase II inhibitor was added to the cells. To confirm the role of Sgt1 phosphorylation/dephosphorylation in its nuclear translocation we transfected cells with non-phosphorylable Sgt1 mutants (S249A, S299A, S249/299A) or a phosphorylation mimic S299D mutant. We found that the levels of S299A and S249/299A mutants were higher than the level of wild type Sgt1 in the nuclear fraction after heat shock. Accordingly, we found that the 139–333 fragment of Sgt1 harboring the mutated residues, but not the 1–138 fragment, translocated to the nucleus upon heat shock. Moreover, we show that S100A6 is required for translocation of the non-phosphorylable Sgt1 mutants and that upon heat shock S100A6 translocates to the nucleus together with Sgt1. In addition, we found that non-phosphorylable Sgt1 mutant interacts with S100A6 more efficiently and at the same time exhibits lower affinity for Hsp90 (heat shock protein 90) than wild type Sgt1. Altogether, our results suggest that S100A6-Ca2+-mediated Sgt1 dephosphorylation promotes its nuclear translocation, most likely due to disruption of the Sgt1-Hsp90 complex. [Copyright &y& Elsevier]

Abstract: Objective: We aim to check if serum levels of receptor for advanced glycation endproduct (RAGE) ligands S100B, S100A6 and S100P were related to myocardial injury in acute coronary syndrome (ACS). Methods: Serum levels of S100B, S100A6, S100P, and soluble RAGE (sRAGE) were analyzed in 882 patients. Based upon clinical and laboratory findings, they were assigned into control (n =251), stable angina (n =211), and ACS (n =420). To verify clinical data of ACS, forty Sprague-Dawley rats were subjected to cardiac ischemia–reperfusion (I/R) injury by occluding proximal (large infarct size; n =20) or distal (small infarct size; n =20) left anterior descending coronary artery, and another 20 rats were in sham-operation group. The expressions of S100B, S100A6, S100P and RAGE in the myocardium were analyzed. Results: Serum levels of S100B, S100A6 and S100P were higher in ACS group than in stable angina and control groups, and sRAGE levels were higher in ACS patients versus controls (all p <0.01). S100B and S100P levels correlated significantly with CK-MB and troponin I levels in ACS group (all p <0.05). In multivariable regression analysis, S100B, S100A6, S100P and conventional risk factors were independently associated with ACS. In animal models, the expressions of S100B, S100A6 and S100P were closely related to infarct size (all p <0.05). Conclusion: This study indicates that serum levels of S100B, S100A6 and S100P are associated with ACS, and serum levels and myocardial expression of these proteins are related to infarct size. [Copyright &y& Elsevier]

Abstract: Cigarette smoke has been widely investigated in terms of epidemiology and pathological endpoints in relation to human lung diseases and animal study. In this study we exposed Wistar rats to cigarette smoke at concentrations of 20% and 60% to explore potential molecular mechanisms at the protein level. Exposures were conducted twice a day, 5 days a week for 43 weeks. As a major metabolite of nicotine in cigarette, cotinine level in rat urine was determined by HPLC–MS. A dose-dependent analysis indicated that cotinine may be used as an exposure marker of cigarette smoke. Expression of receptor for advanced glycation endproducts (RAGE), an immunoglobulin super family that triggers the intracellular signal cascade reaction leading to inflammation and its ligand S100A6 (calgranulin) in bronchial epithelial cells and lung tissues of rats, were found to be positive correlated with cotinine levels, indicating that RAGE and S100A6 may be attributable to inflammation and oxidative damage caused by cigarette smoke. [Copyright &y& Elsevier]

Abstract: To study the mechanisms underlying the linage commitment of CD4+CD8+ thymocytes and the skewed differentiation of CD4+CD8+ into CD4−CD8+ thymocytes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we stimulated with antigen DPK cells, a CD4+CD8+ thymic lymphoma cell line which can differentiate into CD4+CD8− thymocytes and performed a comparative proteomic analysis of DPK cells stimulated with antigen or not. Among the 10 up-regulated or induced proteins upon antigenic stimulation, S100A4, S100A6, and galectin-1 were highly up-regulated. Kinetic studies revealed that expression of S100A4, S100A6, and galectin-1 was dramatically increased as early as 10min after antigen stimulation, similar to that of cKrox and Runx3, transcription factors intimately associated with the lineage commitment. Among four thymocyte subpopulations of the thymus examined, S100A4, S1006, and galectin-1 were most prominently expressed in CD4+CD8+ thymocytes, but not at all in CD4−CD8+ and CD4−CD8− thymocytes. In the spleen, expression of S100A4, S1006, and galectin-1 was greater in CD4 than in CD8 splenocytes. When TCDD was added to antigen-stimulated DPK cells, antigen-induced up-regulation of S100A4, S1006, and galectin-1 were remarkably inhibited, probably partly accounting for the skewed differentiation of CD4+CD8+ into CD4−CD8+ thymocytes induced by TCDD. [Copyright &y& Elsevier]

Abstract: S100A6 (calcyclin) is a calcium-binding protein implicated in many cellular processes and often up-regulated in cancer. Its various biological effects possibly originate from the fact that it may bind to other proteins and modulate their function by inducing conformational changes or interfering with posttranslational modifications. Thus, to elucidate the biological role of S100A6 it is important to identify its targets. Here, we report, based on affinity chromatography and co-immunoprecipitation results that S100A6 interacts with p53 in the presence of calcium ions. We investigated functional implications of the S100A6–p53 interaction by comparing various aspects of p53 activity in HEp-2 cells with either unaltered or diminished S100A6 content due to stable expression of siRNA. We found that the presence of S100A6 results in higher p53 transcriptional activity which is also reflected by higher cell susceptibility to apoptosis evoked by hydrogen peroxide. As revealed by electrophoretic mobility shift assay (EMSA) S100A6 does not affect p53 binding to DNA. On the other hand, we observed that the presence of S100A6 coincides with more efficient nuclear accumulation of p53 under stress conditions. Collectively, our results indicate that S100A6 interacts with p53 and affects its biological activity. [Copyright &y& Elsevier]

Abstract: Background: In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC). Methods: S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens. Results: The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p =n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology (p <0.0001) and S100A4 expression (p =0.005), and inversely with p53 expression (p =0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases (p =0.07). This difference became significant when the analysis was restricted to p53-negative cases (n =72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27–0.81, p =0.017). Conclusions: In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins. [Copyright &y& Elsevier]

Background. Rise in cellular calcium is associated with acute tubular necrosis, the most common cause of acute renal failure (ARF). The mechanisms that calcium signaling induce in the quiescent tubular cells to proliferate and differentiate during acute tubular necrosis have not been elucidated. Methods. Acute tubular necrosis induced in mice by single intravenous injection of uranyl nitrate and examined after 1, 3, 7, and 14 days. Renal function was monitored and kidneys were evaluated by histology, immunohistochemistry, Western blotting, in situ hybridization, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Models of folic acid induced-ARF and ischemic/reperfusion (I/R) injury were similarly investigated. Results. Analysis of mRNA expression of intracellular calcium and phospholipid-binding proteins demonstrated selective expression of S100A6 and Annexin A2 (Anxa2) in the renal cortex with marked elevation on day 3, and gradually decline on day 7 and further attenuation on day 14. Similarly, the expression of both proteins, as demonstrated by immunohistochemistry and Western blot analysis, was increased and reached the peak level on day 7 and then gradually declined by day 14. Vimentin, a marker of dedifferentiated cells, was highly expressed during the recovery phase. Combined in situ hybridization immunohistochemistry revealed colocalization of both S100A6 and Anxa2 with proliferating cell nuclear antigen (PCNA). The universality of this phenomenon was confirmed in two other mouse acute tubular necrosis models, the ischemic-reperfusion injury and folic acid-induced ARF. Conclusion. Collectively, these findings demonstrate that S100A6 and Anxa2 expression, initiated in response to tubular injury, persist in parallel throughout the recovery process of tubular cells in acute renal failure. [ABSTRACT FROM AUTHOR]

Abstract: While most osteosarcoma patients have metastatic or micrometastatic lesions, less than 15% of them have clinically detectable metastatic diseases at presentation. To identify potential markers that may predict osteosarcoma metastasis, we analyzed the expression of S100A6 in 50 osteosarcoma cases and found that 84% of the analyzed specimens stained positive for S100A6. There is a trend towards decreased clinically evident metastasis with increased S100A6 staining. Overexpression of S100A6 in osteosarcoma cells decreases cell motility and anchorage independent growth on collagen gels. Our findings provide evidence that, while S100A6 is commonly overexpressed in human osteosarcoma, loss of its expression correlates with a metastatic phenotype. [Copyright &y& Elsevier]

S100A6 is a member of the S100 family of Ca2+ binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca2+-free and Ca2+-bound states to resolutions of 1.15 A˚ and 1.44 A˚, respectively. Ca2+ binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca2+ sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca2+ binding in the S100 family. [Copyright &y& Elsevier]

• S100A6 binds to cofilin-1 in a Ca2+-dependent manner. • S100A6 increases affinity of cofilin-1 for F-actin. • S100A6 regulates actin filament dynamics by controlling activity of cofilin-1. S100A6 is a Ca2+-binding protein belonging to the S100 family. Many reports indicate that S100A6 is involved in actin filament organization, however the mechanism of S100A6 action in this process is not fully understood. By screening S100A6 binding partners in NIH3T3 mouse fibroblasts, we have found that S100A6 binds cofilin-1, a protein required for the dynamics of actin polymerization and depolymerization. By applying various biochemical and cell biology assays, we have shown that S100A6 bound to cofilin-1 in a Ca2+-dependent manner and increased cofilin-1 affinity for F-actin. Microscopic analysis indicated that S100A6 significantly decreased severing of the actin filaments induced by cofilin-1. Moreover, in the presence of cofilin-1, S100A6 stabilized the filaments by inhibiting their depolymerization. When S100A6 was present at sub-stoichiometric concentrations in relation to actin, polymerization of G-actin accelerated by cofilin-1 was increased. At higher S100A6:actin ratios the polymerization rate was decreased. Altogether, these results show that S100A6 regulates actin filament dynamics by controlling activity of cofilin-1 and suggest that this regulation is Ca2+ -dependent. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Genome-wide S100A2 interaction screening identifies multiple human S100A2 binding proteins. • TPPP is identified as a novel S100A2 target and is capable of interacting with S100A6 and S100B. • Ca2+/S100A2 interacts with the C-terminal (residues 111–160) of the central core domain of TPPP. • Direct binding of the S100 proteins with TPPP causes disassembly of TPPP dimer formation in a Ca2+-dependent manner. To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization. [ABSTRACT FROM AUTHOR]

Recent evidence has suggested the important implications of microRNAs (miRNAs) in the processes of proliferation and tissue remodeling in endometriosis (EMS). We therefore aim to determine the role of miR-202-3p in the pathophysiology of EMS and its underlying mechanisms. Experimental endometriosis was induced in ovariectomized mice implanted with a slow-release 17-β estradiol capsule. Eutopic endometrial stromal cells (euESCs) were isolated and assayed for proliferative, invasive and apoptotic properties by EdU staining, Transwell assays, and flow cytometry. The invasive and apoptotic features in the endometrium of mice with EMS in vivo were evaluated by using immunohistochemical staining and TUNEL assays. miR-202-3p was observed to be downregulated in the endometrial tissues of EMS patients. MiR-202-3p was also found to target YAP1 which resulted in reduced euESC proliferation and invasion and increased apoptosis. YAP1 was able to phosphorylated STAT3 which consequently upregulated S100A6 to promote the proliferative and invasive abilities of euESCs. MiR-202-3p was thereby proposed to act as an inhibitor of proliferation and tissue damage in the in vivo setting of EMS, its effects however, were able to be counteracted byS100A6, which reversed the effects of miR-202-3p on tissue injury and cell proliferation. Our data together evidenced that miR-202-3p targeted YAP1 to reduce STAT3-mediated S100A6 whereby preventing the progression of EMS. [ABSTRACT FROM AUTHOR]