Intrinsic neuronal excitability, defined by the balance between input and output signals, is crucial to neural function, and its disruption underlies various neurological diseases. Kv3.1 channels, encoded by KCNC1, are essential for high-frequency action potential firing. Variants in these channels are associated with several subtypes of epilepsy. We report a patient with developmental regression and epilepsy, meeting Rett syndrome criteria, who carries a KCNC1 variant encoding the S474C substitution in Kv3.1 (Kv3.1S474C). Electrophysiological and biochemical assays reveal that Kv3.1S474C reduces channel presence in the plasma membrane and is retained in the endoplasmic reticulum. In murine primary cortical neuron cultures expressing Kv3.1S474C, we observed reduced neuronal firing frequency and exclusion of the channel from the axon initial segment. Consistently, we found a decreased firing frequency using a conductance-based computational neuronal model. In summary, this study identifies a link between a KCNC1 variant and Rett syndrome, highlighting the importance of S474 residue in Kv3.1 channel trafficking and function in neurons.

Calcium signaling mediates fundamental cellular processes, including cell migration and cancer metastasis. Store-operated calcium entry (SOCE) is the main mechanism of endoplasmic reticulum calcium refilling after depletion in non-excitable cells, and its dysregulation is associated with diseases. DExH-Box Helicase 9 (DHX9) is a DNA/RNA helicase that catalyzes the ATP-dependent unwinding of DNA-RNA complexes or double strands. It has multiple canonic roles in transcriptional and posttranslational regulation of gene expression, and it has reported roles on cancer cell migration.

OBJECTIVE:To characterize the distribution of macrophages, neutrophils, NK cells, and blood vessels in peri-implantitis compared to healthy aged gingiva samples. MATERIALS AND METHODS:This observational study included eight gingival samples from peri-implantitis and eight from periodontally healthy individuals. By immunofluorescence were identified neutrophils, NK cells, macrophages, and their pro-inflammatory or pro-healing phenotypes, and blood vessels. Two ROIs were designated as zone 1, connective tissue closest to the epithelium and zone 2, connective tissue over 200 microns from the rete ridges. Immune cells and vascular structures were quantified and characterized according to their distribution in both zones. RESULTS:Two peri-implantitis zones were characterized by unique macrophage phenotypes and blood vessel architecture. Blood vessels were larger in zone 2 in peri-implantitis. A greater number of NK cells and macrophages were found in peri-implantitis compared to healthy aged samples. A higher presence of pro-inflammatory macrophages was found in zone 1 compared to zone 2. A similar proportion of pro-inflammatory and pro-healing macrophages were found in zone 2. CONCLUSION:A specific distribution for pro-inflammatory macrophages and vascular architecture is observed in peri-implantitis. TNF-α colocalizes with macrophages in the connective tissue near rete ridges. NK cells are more abundant in peri-implantitis than in healthy samples.

Cellular migration is a crucial process for several physiological and pathophysiological events. It depends on cyclical cytoskeletal reorganization, focal adhesion dynamics, and intracellular Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel permeable only to monovalent ions such as Na+ and K+. These channels play a role in diverse physiological events such as modulation of insulin secretion, immune response, and pressure-induced smooth muscle contraction. Additionally, TRPM4 promotes cellular migration and contractility, and its downregulation decreases global Ca2+ signals and focal adhesion disassembly.

The spatiotemporal regulation of intracellular Ca2+ levels has a crucial role in coordinating several physiological and pathophysiological processes. The Store-Operated Ca2+ Entry (SOCE) is critical for Ca2+ oscillatory signaling in non-excitable cells. This response occurs after endoplasmic reticulum Ca2+ depletion through the coupling between Orai-STIM proteins, which promotes the gating mechanism of the highly selective Orai Ca2+ channels. Given the relevance of SOCE in several physiological processes, identifying new mechanisms involved in regulating Orai1-STIM1 activity constitutes an important area of biomedical research.

Triple-negative breast cancer has a poor prognosis and is non-responsive to first-line therapies; hence, new therapeutic strategies are needed. Enhanced store-operated Ca2+ entry (SOCE) has been widely described as a contributing factor to tumorigenic behavior in several tumor types, particularly in breast cancer cells. SOCE-associated regulatory factor (SARAF) acts as an inhibitor of the SOCE response and, therefore, can be a potential antitumor factor. Herein, we generated a C-terminal SARAF fragment to evaluate the effect of overexpression of this peptide on the malignancy of triple-negative breast cancer cell lines. Using both in vitro and in vivo approaches, we showed that overexpression of the C-terminal SARAF fragment reduced proliferation, cell migration, and the invasion of murine and human breast cancer cells by decreasing the SOCE response. Our data suggest that regulating the activity of the SOCE response via SARAF activity might constitute the basis for further alternative therapeutic strategies for triple-negative breast cancer.

Mass spectrometry-based proteomics methods are widely used to identify and quantify protein complexes involved in diverse biological processes. Specifically, tandem mass spectrometry methods represent an accurate and sensitive strategy for identifying protein-protein interactions. However, most of these approaches provide only lists of peptide fragments associated with a target protein, without performing further analyses to discriminate physical or functional protein-protein interactions. Here, we present the PPI-MASS web server, which provides an interactive analytics platform to identify protein-protein interactions with pharmacological potential by filtering a large protein set according to different biological features. Starting from a list of proteins detected by MS-based methods, PPI-MASS integrates an automatized pipeline to obtain information of each protein from freely accessible databases. The collected data include protein sequence, functional and structural properties, associated pathologies and drugs, as well as location and expression in human tissues. Based on this information, users can manipulate different filters in the web platform to identify candidate proteins to establish physical contacts with a target protein. Thus, our server offers a simple but powerful tool to detect novel protein-protein interactions, avoiding tedious and time-consuming data postprocessing. To test the web server, we employed the interactome of the TRPM4 and TMPRSS11a proteins as a use case. From these data, protein-protein interactions were identified, which have been validated through biochemical and bioinformatic studies. Accordingly, our web platform provides a comprehensive and complementary tool for identifying protein-protein complexes assisting the future design of associated therapies.

Breast cancer is one of the most frequent cancer types worldwide and the first cause of cancer-related deaths in women. Although significant therapeutic advances have been achieved with drugs such as tamoxifen and trastuzumab, breast cancer still caused 627,000 deaths in 2018. Since cancer is a multifactorial disease, it has become necessary to develop new molecular therapies that can target several relevant cellular processes at once. Ion channels are versatile regulators of several physiological- and pathophysiological-related mechanisms, including cancer-relevant processes such as tumor progression, apoptosis inhibition, proliferation, migration, invasion, and chemoresistance. Ion channels are the main regulators of cellular functions, conducting ions selectively through a pore-forming structure located in the plasma membrane, protein–protein interactions one of their main regulatory mechanisms. Among the different ion channel families, the Transient Receptor Potential (TRP) family stands out in the context of breast cancer since several members have been proposed as prognostic markers in this pathology. However, only a few approaches exist to block their specific activity during tumoral progress. In this article, we describe several TRP channels that have been involved in breast cancer progress with a particular focus on their binding partners that have also been described as drivers of breast cancer progression. Here, we propose disrupting these interactions as attractive and potential new therapeutic targets for treating this neoplastic disease.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.

Neurological and neuropsychiatric disorders are mediated by several pathophysiological mechanisms, including developmental and degenerative abnormalities caused primarily by disturbances in cell migration, structural plasticity of the synapse, and blood-vessel barrier function. In this context, critical pathways involved in the pathogenesis of these diseases are related to structural, scaffolding, and enzymatic activity-bearing proteins, which participate in Ca2+- and Ras Homologs (Rho) GTPases-mediated signaling. Rho GTPases are GDP/GTP binding proteins that regulate the cytoskeletal structure, cellular protrusion, and migration. These proteins cycle between GTP-bound (active) and GDP-bound (inactive) states due to their intrinsic GTPase activity and their dynamic regulation by GEFs, GAPs, and GDIs. One of the most important upstream inputs that modulate Rho GTPases activity is Ca2+ signaling, positioning ion channels as pivotal molecular entities for Rho GTPases regulation. Multiple non-selective cationic channels belonging to the Transient Receptor Potential (TRP) family participate in cytoskeletal-dependent processes through Ca2+-mediated modulation of Rho GTPases. Moreover, these ion channels have a role in several neuropathological events such as neuronal cell death, brain tumor progression and strokes. Although Rho GTPases-dependent pathways have been extensively studied, how they converge with TRP channels in the development or progression of neuropathologies is poorly understood. Herein, we review recent evidence and insights that link TRP channels activity to downstream Rho GTPase signaling or modulation. Moreover, using the TRIP database, we establish associations between possible mediators of Rho GTPase signaling with TRP ion channels. As such, we propose mechanisms that might explain the TRP-dependent modulation of Rho GTPases as possible pathways participating in the emergence or maintenance of neuropathological conditions.

Abstract Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Indeed, EOC epidemiological scenario is partially due to its late diagnosis, when most of tumor cells have acquired metastatic and chemoresistant phenotype. Despite the initial satisfactory response of EOC cells to platinum/taxanes-based therapy, chemoresistance emerges in an uncontrolled pace, specifically when the patient carries a high-grade serous adenocarcinoma (HGS-AC). Therefore, EOC clinic urges for novel treatment possibilities, aiming the reversal of chemoresistance, the improvement of patients’ quality of life, and their survival rates. cDNA microarray data from our group demonstrated that chemokines secreted to the tumor microenvironment, as CXCL2, IL-6, and IL-8 likely play a crucial role in EOC cells chemoresistance. Thereafter, the present study focused on the role of CXCL2, which binds to its receptor CXCR2 in the membrane of target cells. Nonetheless its function in HGS-AC is unclear. Our study model was based in two HGS-AC lineages: chemosensitive A2780, and its pan-resistant derived ACRP (IC50 to cisplatin 7.3uM vs. 26,56uM, p<0.001; to doxuribicin 0.07uM vs. 1.5uM, p<0.0001, and to paclitaxel 0.22uM vs. 1.7uM, p<0.0001, respectively). CXCR2 was knockdown (KD), and its expression was decreased to 0.6-fold as compared to control cells (p= 0.0015). As compared to the control experimental condition (100%), CXCR2 KD HGS-AC cells described compromised cellular proliferation and metabolic viability (BrdU and Presto blue/SRB assays: A2780 21% vs. ACRP 23%, p<0.0001), and lower cell viability (clonogenic assay: A2780 70% vs. ACRP 49%, p<0.0001). Although angiogenesis does not seem to influence our observations, chicken embryo chorioallantoic membrane assay revealed that CXCR2 KD ACRP-induced tumors shrunk by 3.3-fold in comparison the control condition. Intriguingly, similar results were not observed when cells were treated with the pharmacological inhibitor of CXCR2. We lately discovered that the receptor was anomalously expressed in the cells nucleus, thus justifying our observations while opening novel avenues for future investigations in the field. We, then, argued whether CXCR2 KD would modulate carcinogenic classic signaling pathways. We observed a biological tendency of less P-AKT (0.5- fold, p=0.09), but not P-ERK (0.3-fold, p=0.7) when compared to control. We also investigated the involvement of EMT in our studied phenomena. Our findings demonstrated significative decline in the expression of SLUG (0.9-fold vs. control, p=0.022) and SNAIL (1.1-fold vs. control, p=0.005) in CXCR2 KD ACRP cells. Finally, in silico analysis including over 200 HGS-AC cases revealed that overexpression of CXCR2 is robustly correlated to the worse disease prognosis and patients’ overall survival rate, specially in late stage disease (p=0.035). Altogether, our results present innovative strategy to fight pan-chemoresistant HGS-AC by silencing anomalously overexpressed CXCR2. Citation Format: Taciane Henriques, Diandra dos Santos, Isabella Guimaraes, Nayara Tessarollo, Paulo Lyra, Patricia Mesquisa, Diana Padua, Ana Luisa Amaral, Luisa Pereira, Bruno Cavadas, Ian Silva, Raquel Almeida, Leticia Rangel. Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A133. doi:10.1158/1535-7163.TARG-19-A133

TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4-like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.

Abstract Background: We have demonstrated that 63% of primary epithelial ovarian cancer (EOC) anomalously overexpressed CLDN16 in the cell cytoplasm, regardless of tumor histological type or grade, thus suggesting that this is an early event in EOC development, and might serve as an EOC marker. The present work aimed to enlighten cellular mechanisms that modulate CLDN16 expression in EOC. Methods: PKA, PKC, and PI3K pathways affect CLDNs cellular localization and function, cancer development and progression. We investigated the effect of the pathways, and of intra-tumoral estrogen (0.5, 5, 50 and 500 nM), on CLDN16 expression in the serous and platinum-resistant EOC model, the OVCAR3 cell line. Immunofluorescence experiments were carried out to determine the cellular localization of CLDN16 in OVCAR3. CLDN16 expression was assessed by real-time RT-PCR, in the presence or absence of the pathway modulators cAMP (PKA activator, 10-6M), KT5720 (PKA inhibitor, 10-7M), PMA (PKC activator, 10-8M), and Wortmannin (WORT: PI3K activator, 10-7M). CLDN16 relative expression analyses followed the CT method of Pffalf, using the REST program (Version 2.0.13, 2009). Data, expressed as mean ± SE, were analyzed by Bonferroni and Dunnet multiple comparisons tests. Findings: OVCAR3 expressed CLDN16 exclusively in the cytoplasm compartment, resembling primary tumors, so being a reliable in vitro model to our expression study. While the PKA pathway did not affect CLDN16 expression in OVCAR3, the PKC pathway lead to an increase of the transcript by1.95-fold (PMA; p <0.001). Likewise, the PI3K pathway modulated CLDN16 expression, as seen by the 2.32-fold decrease on its expression in the presence of WORT (p <0.001). Whereas estrogen 0.5 and 5 nM did not change CLDN16 expression, but increased its expression by 6.57-fold at 50 nM. The stimulatory effect of estrogen was abolished at 500 nM, thus suggesting a biphasic effect of the hormone in CLDN16 expression in EOC. Estrogen effects in EOC result, in part, from the intra-tumoral production of the hormone by aromatases, as corroborated by the 10.31-fold inhibition of CLDN16 expression by the aromatase inhibitor anastrozole (IC50 2.5 x 10-4M). Conclusions: Our data strongly suggest that the expression of CLDN16 is stimulated by the PKC and PI3K, and intra-tumoral aromatase-produced estrogen in EOC. Whereas the anomalous cytoplasm overexpression of CLDN16 still puzzles us, we speculate that fragments of the molecule could be possibly secreted. If this is the case, screening for circulatory portions of the protein could serve as an EOC screening molecule, considering that it is overexpressed early in disease course. Despite the fact that more studies are necessary to elighten the mechanisms that control the expression and function of CLDN16 in EOC, our study opens a new avenue to overcome EOC dramatic epidemiological scenario, bringing hope to improve patients overall outcome and quality of life. Citation Format: Nayara G. Tessarollo, Marcela Paes, Murilo Cerri, Alice Herlinger, Klesia Madeira, Renata Daltoé, Ian Silva, Leticia Rangel. Anomalous expression of claudin 16 in ovarian cancer: Role of PKC, PI3K and estrogen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2014-3343

Abstract Background: Accurate pathological analysis of biopsies is crucial for conclusive and precise disease diagnosis, prognosis and therapeutic guidelines. Evaluation of estrogen receptor (ER) expression in breast cancer (BC) is imperative in determining the disease prognosis, and patients' eligibility for hormonotherapy. Herein, ERα status was evaluated in 61 BC cases using two different anti-ERα monoclonal antibodies, Sp1 (rabbit) and 1D5 (mouse). Methods: Primary BC cases registered in two reference hospitals, with correspondent written informed consent, were used to generate in house tissue microarray platforms. ERα expression was accessed by immunohistochemistry using the monoclonal antibodies anti-ERα, SP1 and 1D5. Staining scoring followed the Allred method (PS, proportion score or percentage of stained cells; IS, intensity score). Concordance and correlation parameters were calculated using Kappa factor; Pearson, Spearman's, or intraclass correlation coefficient (CF). Staining patterns were compared by paired T-Test and Wilcoxon test. Findings: SP1 and 1D5 provided equivalent results (Concordance rate, 96.7%; Kappa factor, 0.921), whereas SP1 was more prone for positive results than 1D5 (2 samples diverged). Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively); however, concordance between the antibodies seems more accurate in higher PS values. Excellent SI correlation between antibodies was observed throughout the population (Spearman's CF, α=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged; always pointing 1D5 to weaker staining than SP1. When calculating Spearman's CF of Total Score (TS) within the population, excellent correlation between both the antibodies (α=0.9238) was noted; nonetheless, results were less concordant result among the BC positive cases (α=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples, or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p<0.05). Interpretation: Despite the equivalence between the anti-ERα monoclonal antibodies, SP1 and 1D5, the former has proven superior to the latter with respect to the IS and TS. Therefore, we suggest the clinical use of SP1 while diagnosing BC, due to its higher accuracy in examining ER status; thus, enabling more accurate therapeutic decisions to treat the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3600. doi:1538-7445.AM2012-3600