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1.
Detecting In-Situ oligomerization of engineered STIM1 proteins by diffraction-limited optical imaging.
Srinivasan, P
PloS one. 2019;(3):e0213655
Abstract
Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form 'puncta' upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1-6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.
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2.
A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl- channel ANO1 (TMEM16A).
Yu, K, Jiang, T, Cui, Y, Tajkhorshid, E, Hartzell, HC
Proceedings of the National Academy of Sciences of the United States of America. 2019;(40):19952-19962
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Abstract
ANO1 (TMEM16A) is a Ca2+-activated Cl- channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here, we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2 Three of these sites captured 85% of all ANO1-PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found 3 regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P2 Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl- ions. We propose a model consisting of a network of 3 PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.
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3.
Improving breast cancer sensitivity to paclitaxel by increasing aneuploidy.
Rodrigues-Ferreira, S, Nehlig, A, Moindjie, H, Monchecourt, C, Seiler, C, Marangoni, E, Chateau-Joubert, S, Dujaric, ME, Servant, N, Asselain, B, et al
Proceedings of the National Academy of Sciences of the United States of America. 2019;(47):23691-23697
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Abstract
Predictive biomarkers for tumor response to neoadjuvant chemotherapy are needed in breast cancer. This study investigates the predictive value of 280 genes encoding proteins that regulate microtubule assembly and function. By analyzing 3 independent multicenter randomized cohorts of breast cancer patients, we identified 17 genes that are differentially regulated in tumors achieving pathological complete response (pCR) to neoadjuvant chemotherapy. We focused on the MTUS1 gene, whose major product, ATIP3, is a microtubule-associated protein down-regulated in aggressive breast tumors. We show here that low levels of ATIP3 are associated with an increased pCR rate, pointing to ATIP3 as a predictive biomarker of breast tumor chemosensitivity. Using preclinical models of patient-derived xenografts and 3-dimensional models of breast cancer cell lines, we show that low ATIP3 levels sensitize tumors to the effects of taxanes but not DNA-damaging agents. ATIP3 silencing improves the proapoptotic effects of paclitaxel and induces mitotic abnormalities, including centrosome amplification and multipolar spindle formation, which results in chromosome missegregation leading to aneuploidy. As shown by time-lapse video microscopy, ATIP3 depletion exacerbates cytokinesis failure and mitotic death induced by low doses of paclitaxel. Our results favor a mechanism by which the combination of ATIP3 deficiency and paclitaxel treatment induces excessive aneuploidy, which in turn results in elevated cell death. Together, these studies highlight ATIP3 as an important regulator of mitotic integrity and a useful predictive biomarker for a population of chemoresistant breast cancer patients.
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Proteomic Approach to the Potential Role of Angiotensin II in Barrett Dysplasia.
Bratlie, SO, Wallenius, V, Edebo, A, Fändriks, L, Casselbrant, A
Proteomics. Clinical applications. 2019;(4):e1800102
Abstract
BACKGROUND AND AIM Dysplasia in Barrett's esophagus (BE) is regarded as a preneoplastic lesion. The renin-angiotensin system (RAS), known for its role in electrolyte homeostasis and hemodynamics, has also been shown to have tissue-based features linked to proliferation, inflammation, and cancer. RAS is associated with BE dysplasia. The aim of this study is to investigate possible effects of the RAS in BE dysplasia by using RAS-interfering pharmaceutical agents and by assessment of global protein expression in esophageal mucosal biopsies. METHODS Endoscopic biopsies are taken from 18 BE in patients with low-grade dysplasia before and after 3 weeks of treatment with either angiotensin-converting enzyme inhibitors (enalapril 5 mg; n = 6) or angiotensin II receptor type 1 blockers (candesartan 8 mg; n = 6), or no treatment (n = 6). A global proteomics analysis by 2D gel electrophoresis and mass spectrometry (MS) is then performed to identify proteins that are regulated after interference with RAS. RESULTS Three proteins are identified to show significant modulation of expression 60 kDa heat shock protein (downregulated), protein disulfide isomerase A3 (downregulated), and inorganic pyrophosphatase (upregulated). CONCLUSION Three proteins with no previously known links to esophageal RAS, but with possible relevance for the development of esophageal adenocarcinoma (EAC) are detected. Altered expression by interference with the RAS suggests an involvement of angiotensin II in the development of EAC in BE.
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A machine learning algorithm predicts molecular subtypes in pancreatic ductal adenocarcinoma with differential response to gemcitabine-based versus FOLFIRINOX chemotherapy.
Kaissis, G, Ziegelmayer, S, Lohöfer, F, Steiger, K, Algül, H, Muckenhuber, A, Yen, HY, Rummeny, E, Friess, H, Schmid, R, et al
PloS one. 2019;(10):e0218642
Abstract
PURPOSE Development of a supervised machine-learning model capable of predicting clinically relevant molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) from diffusion-weighted-imaging-derived radiomic features. METHODS The retrospective observational study assessed 55 surgical PDAC patients. Molecular subtypes were defined by immunohistochemical staining of KRT81. Tumors were manually segmented and 1606 radiomic features were extracted with PyRadiomics. A gradient-boosted-tree algorithm was trained on 70% of the patients (N = 28) and tested on 30% (N = 17) to predict KRT81+ vs. KRT81- tumor subtypes. A gradient-boosted survival regression model was fit to the disease-free and overall survival data. Chemotherapy response and survival were assessed stratified by subtype and radiomic signature. Radiomic feature importance was ranked. RESULTS The mean±STDEV sensitivity, specificity and ROC-AUC were 0.90±0.07, 0.92±0.11, and 0.93±0.07, respectively. The mean±STDEV concordance indices between the disease-free and overall survival predicted by the model based on the radiomic parameters and actual patient survival were 0.76±0.05 and 0.71±0.06, respectively. Patients with a KRT81+ subtype experienced significantly diminished median overall survival compared to KRT81- patients (7.0 vs. 22.6 months, HR 4.03, log-rank-test P = <0.001) and a significantly improved response to gemcitabine-based chemotherapy over FOLFIRINOX (10.14 vs. 3.8 months median overall survival, HR 2.33, P = 0.037) compared to KRT81- patients, who responded significantly better to FOLFIRINOX over gemcitabine-based treatment (30.8 vs. 13.4 months median overall survival, HR 2.41, P = 0.027). Entropy was ranked as the most important radiomic feature. CONCLUSIONS The machine-learning based analysis of radiomic features enables the prediction of subtypes of PDAC, which are highly relevant for disease-free and overall patient survival and response to chemotherapy.
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Sex-Related Differences in Impact on Safety of Pharmacogenetic Profile for Colon Cancer Patients Treated with FOLFOX-4 or XELOX Adjuvant Chemotherapy.
Ruzzo, A, Graziano, F, Galli, F, Galli, F, Rulli, E, Lonardi, S, Ronzoni, M, Massidda, B, Zagonel, V, Pella, N, et al
Scientific reports. 2019;(1):11527
Abstract
Polymorphisms contribute to inter-individual differences and show a promising predictive role for chemotherapy-related toxicity in colon cancer (CC). TOSCA is a multicentre, randomized, non-inferiority, phase III study conducted in high-risk stage II/stage III CC patients treated with 6 vs 3 months of FOLFOX-4 or XELOX adjuvant chemotherapy. During this post-hoc analysis, 218 women and 294 men were genotyped for 17 polymorphisms: TYMS (rs34743033, rs2853542, rs11280056), MTHFR (rs1801133, rs1801131), ERCC1 (rs11615), XRCC1 (rs25487), XRCC3 (rs861539), XPD (rs1799793, rs13181), GSTP1 (rs1695), GSTT1/GSTM1 (deletion +/-), ABCC1 (rs2074087), and ABCC2 (rs3740066, rs1885301, rs4148386). The aim was to assess the interaction between these polymorphisms and sex, on safety in terms of time to grade ≥3 haematological (TTH), grade ≥3 gastrointestinal (TTG) and grade ≥2 neurological (TTN) toxicity. Interactions were detected on TTH for rs1801133 and rs1799793, on TTG for rs13181 and on TTN for rs11615. Rs1799793 GA genotype (p = 0.006) and A allele (p = 0.009) shortened TTH in men. In women, the rs11615 CC genotype worsened TTN (co-dominant model p = 0.008, recessive model p = 0.003) and rs13181 G allele improved the TTG (p = 0.039). Differences between the two sexes in genotype distribution of rs1885301 (p = 0.020) and rs4148386 (p = 0.005) were found. We highlight that polymorphisms could be sex-specific biomarkers. These results, however, need to be confirmed in additional series.
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H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS.
Salatino, A, Aversa, I, Battaglia, AM, Sacco, A, Di Vito, A, Santamaria, G, Chirillo, R, Veltri, P, Tradigo, G, Di Cello, A, et al
Oxidative medicine and cellular longevity. 2019;:3461251
Abstract
Reactive oxygen species (ROS) mediates cisplatin-induced cytotoxicity in tumor cells. However, when cisplatin-induced ROS do not reach cytotoxic levels, cancer cells may develop chemoresistance. This phenomenon can be attributed to the inherited high expression of antioxidant protein network. H-Ferritin is an important member of the antioxidant system due to its ability to store iron in a nontoxic form. Altered expression of H-Ferritin has been described in ovarian cancers; however, its functional role in cisplatin-based chemoresistance of this cancer type has never been explored. Here, we investigated whether the modulation of H-Ferritin might affect cisplatin-induced cytotoxicity in ovarian cancer cells. First, we characterized OVCAR3 and OVCAR8 cells for their relative ROS and H-Ferritin baseline amounts. OVCAR3 exhibited lower ROS levels compared to OVCAR8 and greater expression of H-Ferritin. In addition, OVCAR3 showed pronounced growth potential and survival accompanied by the strong activation of pERK/pAKT and overexpression of c-Myc and cyclin E1. When exposed to different concentrations of cisplatin, OVCAR3 were less sensitive than OVCAR8. At the lowest concentration of cisplatin (6 μM), OVCAR8 underwent a consistent apoptosis along with a downregulation of H-Ferritin and a consistent increase of ROS levels; on the other hand, OVCAR3 cells were totally unresponsive, H-Ferritin was almost unaffected, and ROS amounts met a slight increase. Thus, we assessed whether the modulation of H-Ferritin levels was able to affect the cisplatin-mediated cytotoxicity in both the cell lines. H-Ferritin knockdown strengthened cisplatin-mediated ROS increase and significantly restored sensitivity to 6 μM cisplatin in resistant OVCAR3 cells. Conversely, forced overexpression of H-Ferritin significantly suppressed the cisplatin-mediated elevation of intracellular ROS subsequently leading to a reduced responsiveness in OVCAR8 cells. Overall, our findings suggest that H-Ferritin might be a key protein in cisplatin-based chemoresistance and that its inhibition may represent a potential approach for enhancing cisplatin sensitivity of resistant ovarian cancer cells.
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Influence of OATP1B1 and BCRP polymorphisms on the pharmacokinetics and pharmacodynamics of rosuvastatin in elderly and young Korean subjects.
Kim, Y, Yoon, S, Choi, Y, Yoon, SH, Cho, JY, Jang, IJ, Yu, KS, Chung, JY
Scientific reports. 2019;(1):19410
Abstract
A lack of information regarding whether genetic polymorphisms of SLCO1B1 and ABCG2 affect the pharmacokinetics (PKs)/pharmacodynamics (PDs) of rosuvastatin in elderly subjects prevents optimal individualized pharmacotherapy of rosuvastatin in clinical settings. This study aimed to investigate the effect of age and genetic polymorphisms and possible differences in genetic effects on the PKs/PDs of rosuvastatin between elderly and young subjects. Two separate clinical studies designed as open-label, one-sequence studies with multiple-dose administration for elderly (n = 20) and young (n = 32) subjects were conducted. All subjects received 20 mg of rosuvastatin once daily for 21 days. The exposure to rosuvastatin, characterized by the area under the time curve (AUC), increased by 23% in the elderly subjects compared with that of young subjects, which was not significant. When compared to the subjects with breast cancer resistance protein (BCRP) normal function, the exposure to rosuvastatin increased by 44% in young subjects (p = 0.0021) with BCRP intermediate function (IF) and by 35% and 59% (p > 0.05 for both) in elderly subjects with BCRP IF and low function, respectively. SLCO1B1 521T > C was also partially associated with a higher AUC of rosuvastatin in young subjects and a less pronounced increasing trend in elderly subjects (p > 0.05 for both). The lipid-lowering effect of rosuvastatin was less pronounced in the elderly subjects than in the young subjects, and genetic polymorphisms of neither SLCO1B1 nor ABCG2 significantly affected the PDs of rosuvastatin. The ABCG2 421C > A polymorphism was associated with the PKs of rosuvastatin and was identified as a more important determinant than the SLCO1B1 521T > C polymorphism in both elderly and young subjects.
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THADA_rs13429458 Minor Allele Increases the Risk of Polycystic Ovary Syndrome in Asian, but Not in Caucasian Women: A Systematic Review and Meta-Analysis.
Park, S, Liu, M, Zhang, T
Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme. 2019;(10):661-670
Abstract
Polycystic ovary syndrome (PCOS) is a highly prevalent disease in young women that also features increased insulin resistance. Genetic factors have a strong relationship with the etiology of PCOS. We assessed whether carrying THADA rs13429458 is associated with the development of PCOS by meta-analysis and whether the association is influenced by ethnicity. Articles were searched using PubMed, EMBASE, Cochrane Library, Korean scientific database, and Chinese and Indian medical databases to identify all eligible studies for evaluating the association of THADA rs13429458 and PCOS risk. The association was assessed in five genetic random effects models including the allelic (AG), recessive (RG), dominant (DG), homozygous (HMG), and heterozygous (HTG) genetic models. Subgroup analyses stratified by ethnicity (Asians and non-Asians) were assessed. Nine articles were selected and 1 association analysis of Korea PCOS study met Hardy-Weinberg equilibrium criteria. A set of 38 224 PCOS women and 120 173 healthy women were included. The AG and RG showed heterogeneity in the overall and Asian subjects, but the other genetic model did not exhibit heterogeneity in all subjects. AG, RG, DG, and HMG, but not HTG, exhibited publication bias in total subjects but there was no publication bias in all genetic models among Asians and non-Asians. The overall effect of THADA_rs13429458 on PCOS risk showed significant positive associations in pooling 10 studies. In sub-group analysis only Asians, but not non-Asians, had a positive association (AG: OR=1.24, p=0.001; RG: OR=1.32, p=0.002; DG: OR, 1.70, p<0.00001; HMG: OR, 1.71, p=0.002; HTG: OR=1.34, p=0,006). In conclusions, young Asian women with the minor allele (C) for THADA rs13429458 were at increased risk of PCOS.
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Relationship between Serum Vitamin D and Calcium Levels and Vitamin D Receptor Gene Polymorphisms in Colorectal Cancer.
Al-Ghafari, AB, Balamash, KS, Al Doghaither, HA
BioMed research international. 2019;:8571541
Abstract
BACKGROUND Many epidemiological studies have shown that vitamin D deficiency is associated with various types of human cancers. The biological action of vitamin D and its metabolites is mediated by the transcription factor vitamin D receptor (VDR). The VDR gene is highly expressed in the colon and is involved in many biological functions. The aim of the current study was to assess the relationship between serum vitamin D metabolite and calcium levels with VDR polymorphisms in normal and colorectal cancer (CRC) patients. METHODS Fifty Saudi CRC patients and fifty controls were enrolled in the study. The levels of total vitamin D, 25(OH)D3, and calcium were measured in serum. RESULTS The homozygous genotype (aa) of the ApaI VDR polymorphism (rs7975232) was found to correlate with total serum vitamin D levels of CRC patients, while the heterozygous (Tt) TaqI VDR polymorphism (rs731236) was associated with serum calcium levels. In contrast, the BsmI and FokI VDR polymorphisms (rs1544410 and rs2228570, resp.) did not affect the serum levels of total vitamin D, 25-hydroxyvitamin D3, and calcium. CONCLUSION Appropriate vitamin D levels were shown to be important in preventing the onset of CRC.