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Schammim Ray Amith, Krista Marie Vincent, Jodi Marie Wilkinson, Lynne Marie Postovit, Larry Fliegel
Mounting evidence supports a major role for the Na(+)/H(+) exchanger NHE1 in cancer progression and metastasis. NHE1 is hyperactive at the onset of oncogenic transformation, resulting in intracellular alkalinization and extracellular microenvironmental acidification. These conditions promote invasion and facilitate metastasis. However, the signal pathways governing the regulation of exchanger activity are still unclear. This is especially important in the aggressively metastatic, triple-negative basal breast cancer subtype...
October 14, 2016: Cellular Signalling
Yiling Lu, Shiyun Ling, Apurva M Hegde, Lauren A Byers, Kevin Coombes, Gordon B Mills, Rehan Akbani
The majority of the targeted therapeutic agents in clinical use target proteins and protein function. Although DNA and RNA analyses have been used extensively to identify novel targets and patients likely to benefit from targeted therapies, these are indirect measures of the levels and functions of most therapeutic targets. More importantly, DNA and RNA analysis is ill-suited for determining the pharmacodynamic effects of target inhibition. Assessing changes in protein levels and function is the most efficient way to evaluate the mechanisms underlying sensitivity and resistance to targeted agents...
August 2016: Seminars in Oncology
C Petit, F Gouel, I Dubus, C Heuclin, K Roget, J P Vannier
BACKGROUND: Several studies show that bone marrow (BM) microenvironment and hypoxia condition can promote the survival of leukemic cells and induce resistance to anti-leukemic drugs. However, the molecular mechanism for chemoresistance by hypoxia is not fully understood. METHODS: In the present study, we investigated the effect of hypoxia on resistance to two therapies, methotrexate (MTX) and prednisolone (PRD), in two cell models for acute lymphoblastic leukemia (ALL)...
2016: BMC Cancer
Nathan Moerke, Mohammad Fallahi-Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies...
2016: Current Protocols in Chemical Biology
Merve Mutlu, Özge Saatci, Suhail A Ansari, Emre Yurdusev, Huma Shehwana, Özlen Konu, Umar Raza, Özgür Şahin
Dysregulation of PI3K and MAPK pathways promotes uncontrolled cell proliferation, apoptotic inhibition and metastasis. Individual targeting of these pathways using kinase inhibitors has largely been insufficient due to the existence of cross-talks between these parallel cascades. MicroRNAs are small non-coding RNAs targeting several genes simultaneously and controlling cancer-related processes. To identify miRNAs repressing both PI3K and MAPK pathways in breast cancer, we re-analyzed our previous miRNA mimic screen data with reverse phase protein array (RPPA) output, and identified miR-564 inhibiting both PI3K and MAPK pathways causing markedly decreased cell proliferation through G1 arrest...
2016: Scientific Reports
Toshihiro Yoneyama, Sumio Ohtsuki, Kazufumi Honda, Makoto Kobayashi, Motoki Iwasaki, Yasuo Uchida, Takuji Okusaka, Shoji Nakamori, Masashi Shimahara, Takaaki Ueno, Akihiko Tsuchida, Naohiro Sata, Tatsuya Ioka, Yohichi Yasunami, Tomoo Kosuge, Takashi Kaneda, Takao Kato, Kazuhiro Yagihara, Shigeyuki Fujita, Wilber Huang, Tesshi Yamada, Masanori Tachikawa, Tetsuya Terasaki
Pancreatic cancer is one of the most lethal tumors, and reliable detection of early-stage pancreatic cancer and risk diseases for pancreatic cancer is essential to improve the prognosis. As 260 genes were previously reported to be upregulated in invasive ductal adenocarcinoma of pancreas (IDACP) cells, quantification of the corresponding proteins in plasma might be useful for IDACP diagnosis. Therefore, the purpose of the present study was to identify plasma biomarkers for early detection of IDACP by using two proteomics strategies: antibody-based proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics...
2016: PloS One
Alaa Tarig Alshareeda, Ola H Negm, Mohammed A Aleskandarany, Andrew R Green, Christopher Nolan, Patrick J TigHhe, Srinivasan Madhusudan, Ian O Ellis, Emad A Rakha
Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group...
August 2016: Breast Cancer Research and Treatment
Xuexue Guo, Yihong Deng, Chenggang Zhu, Junlong Cai, Xiangdong Zhu, James P Landry, Fengyun Zheng, Xunjia Cheng, Yiyan Fei
In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies...
September 15, 2016: Analytical Biochemistry
Qingshan Yang, Lisa S Chen, Min Jin Ha, Kim-Anh Do, Sattva S Neelapu, Varsha Gandhi
PURPOSE: PI3 Kinase is a critical node in the B-cell receptor pathway which is responsible for survival and proliferation of B-cell malignancies. Idelalisib, a PI3Kδ-isoform specific inhibitor, has been approved to treat B-cell malignancies. While biological activity of the drug has been evaluated, molecular mechanisms and signaling pathway disruption leading to the biological effects of idelalisib are not yet well-defined. Prior laboratory reports have identified transcription and translation as the primary events for attenuation of PI3Kα isoform...
June 24, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Federica Frezzato, Benedetta Accordi, Valentina Trimarco, Cristina Gattazzo, Veronica Martini, Gloria Milani, Silvia Bresolin, Filippo Severin, Andrea Visentin, Giuseppe Basso, Gianpietro Semenzato, Livio Trentin
B cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B lymphocytes from proliferative activity and apoptosis resistance. The increased awareness of the importance of B cell receptor signaling in CLL has raised new opportunities for targeted intervention. Herein, we describe a study performed with the high-throughput RPPA (reverse phase protein array) technique, which allowed us to simultaneously study different molecules in a large series of patients. We analyzed B lymphocytes from 57 patients with CLL and 11 healthy subjects...
June 16, 2016: Journal of Leukocyte Biology
K Kalinsky, T Zheng, H Hibshoosh, X Du, P Mundi, J Yang, S Refice, S M Feldman, B Taback, E Connolly, K D Crew, M A Maurer, D L Hershman
PURPOSE: Reverse Phase Protein Array (RPPA) is a high-throughput antibody-based technique to assess cellular protein activity. The goal of this study was to assess protein marker changes by RPPA in tumor tissue from a pre-surgical metformin trial in women with operable breast cancer (BC). METHODS: In an open-label trial, metformin 1500-mg PO daily was administered prior to resection in 35 non-diabetic patients with stage 0-III BC, body mass index ≥25 kg/m(2)...
June 15, 2016: Clinical & Translational Oncology
Heather J Johnston, Sarah K Boys, Ashraff Makda, Neil O Carragher, Alison N Hulme
Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid-phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose-dependent response in IRS-1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed...
September 2, 2016: Chembiochem: a European Journal of Chemical Biology
Michele Signore, Mariachiara Buccarelli, Emanuela Pilozzi, Gabriele De Luca, Marianna Cappellari, Maurizio Fanciulli, Frauke Goeman, Elisa Melucci, Mauro Biffoni, Lucia Ricci-Vitiani
Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation...
June 6, 2016: Oncotarget
Anneli Andersson, Andrej Bluwstein, Nitin Kumar, Federico Teloni, Jens Traenkle, Michael Baudis, Matthias Altmeyer, Michael O Hottiger
Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing 'oxidative stress'. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca(2+)/PKCα signaling axis...
September 19, 2016: Nucleic Acids Research
Jennifer B Dennison, Maria Shahmoradgoli, Wenbin Liu, Zhenlin Ju, Funda Meric-Bernstam, Charles M Perou, Aysegul Sahin, Alana L Welm, Steffi Oesterreich, Matthew J Sikora, Robert E Brown, Gordon B Mills
PURPOSE: The current study evaluated associative effects of breast cancer cells with the tumor microenvironment and its influence on tumor behavior. EXPERIMENTAL DESIGN: Formalin-fixed paraffin embedded tissue and matched protein lysates were evaluated from two independent breast cancer patient data sets (TCGA and MD Anderson). Reverse-phase protein arrays (RPPA) were utilized to create a proteomics signature to define breast tumor subtypes. Expression patterns of cell lines and normal breast tissues were utilized to determine markers that were differentially expressed in stroma and cancer cells...
May 12, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Didier Meseure, Sophie Vacher, François Lallemand, Kinan Drak Alsibai, Rana Hatem, Walid Chemlali, Andre Nicolas, Leanne De Koning, Eric Pasmant, Celine Callens, Rosette Lidereau, Antonin Morillon, Ivan Bieche
BACKGROUND: Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known. METHODS: We used RT-PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome...
June 14, 2016: British Journal of Cancer
Noriko Kanaya, George Somlo, Jun Wu, Paul Frankel, Masaya Kai, Xueli Liu, Shang Victoria Wu, Duc Nguyen, Nymph Chan, Meng-Yin Hsieh, Michele Kirschenbaum, Laura Kruper, Courtney Vito, Behnam Badie, John H Yim, Yuan Yuan, Arti Hurria, Chu Peiguo, Joanne Mortimer, Shiuan Chen
The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2- and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2- tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2- tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake...
May 3, 2016: Journal of Steroid Biochemistry and Molecular Biology
E P Bastos, H Brentani, C A B Pereira, A Polpo, L Lima, R D Puga, F S Pasini, C A B T Osorio, R A Roela, M I Achatz, A P Trapé, A M Gonzalez-Angulo, M M Brentani
UNLABELLED: Breast cancer (BC) in young adult patients (YA) has a more aggressive biological behavior and is associated with a worse prognosis than BC arising in middle aged patients (MA). We proposed that differentially expressed miRNAs could regulate genes and proteins underlying aggressive phenotypes of breast tumors in YA patients when compared to those arising in MA patients. OBJECTIVE: Using integrated expression analyses of miRs, their mRNA and protein targets and stromal gene expression, we aimed to identify differentially expressed profiles between tumors from YA-BC and MA-BC...
2016: PloS One
Markus Loibl, Siegmund Lang, Alexander Hanke, Marietta Herrmann, Michaela Huber, Gero Brockhoff, Silvan Klein, Michael Nerlich, Peter Angele, Lukas Prantl, Sebastian Gehmert
BACKGROUND: Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue-derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. METHODS: Adipose tissue-derived mesenchymal stem cells were cultured in α-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum...
August 2016: Plastic and Reconstructive Surgery
Young Kwang Chae, Debora de Melo Gagliato, Sachin Gopalkrishna Pai, Benedito Carneiro, Nisha Mohindra, Francis Joseph Giles, Praveen Ramakrishnan-Geethakumari, Joohyuk Sohn, Shuying Liu, Huiqin Chen, Naoto Ueno, Gabriel Hortobagyi, Ana Maria Gonzalez-Angulo
EGFR and cMET cross-talk is involved in breast cancer (BC) progression and resistance to different targeted therapies, however little is known about the co-expression patterns of EGFR and cMET or its prognostic significance in BC. Protein levels of EGFR, cMET and their phosphorylated proteins were measured in 825 BC samples using reverse phase protein array (RPPA). Given unimodal distribution of proteins, the median was selected as a cut-off after sensitivity analyses. Kaplan-Meier survival curves were used to estimate relapse-free (RFS) and overall survival (OS)...
2016: PloS One
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