Dataset Information

Phosphoproteomic analysis of breast epithelial cells with isogenic knockin of PIK3CA mutations

ABSTRACT: The PIK3CA gene is frequently mutated in human cancers. To study the signaling mechanisms responsible for cell growth and invasion phenotypes induced by mutant PIK3CA molecules, we carried out a SILAC-based quantitative phosphoproteomic analysis of MCF10A, a spontaneously immortalized normal mammary epithelial cell line, and two MCF10A knockin cell lines containing different activating mutations of the PIK3CA gene. MCF10A and PIK3CA mutation knock in cells were propagated in DMEM/F12 SILAC media deficient in both L-lysine and L-arginine and supplemented with light lysine (K) and arginine (R) for light, 2H4-K and 13C6-R for medium state and 13C615N2-K and 13C615N4-R for heavy state labeling. Cell lysates were prepared in urea lysis buffer containing 20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate and 5mM sodium fluoride. The lysates were reduced, alkylated and digested by trypsin. Tryptic peptides were desalted by C18 reverse phase column and followed by strong cation exchange (SCX) fractionation. Fractionated peptides were subjected to TiO2-based phosphopeptide enrichment. LC-MS/MS analysis of enriched phosphopeptides was carried out using a reverse-phase liquid chromatography system interfaced with an LTQ-Orbitrap Velos mass spectrometer. Proteome Discoverer (v 1.3) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using Mascot (2.2.0) and SEQUEST search algorithms against a Human RefSeq database supplemented with frequently observed contaminants.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD000599 | Pride | 2015-07-28

REPOSITORIES: Pride

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Publications

Activation of diverse signalling pathways by oncogenic PIK3CA mutations.

Wu Xinyan X Renuse Santosh S Sahasrabuddhe Nandini A NA Zahari Muhammad Saddiq MS Chaerkady Raghothama R Kim Min-Sik MS Nirujogi Raja S RS Mohseni Morassa M Kumar Praveen P Raju Rajesh R Zhong Jun J Yang Jian J Neiswinger Johnathan J Jeong Jun-Seop JS Newman Robert R Powers Maureen A MA Somani Babu Lal BL Gabrielson Edward E Sukumar Saraswati S Stearns Vered V Qian Jiang J Zhu Heng H Vogelstein Bert B Park Ben Ho BH Pandey Akhilesh A

Nature communications 20140923

The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream ...[more]

PMID: 25247763

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Project description:Individuals with Urea Cycle Disorders (UCD) cannot remove ammonia, a waste product, from the blood. At present there is little information on the use of sodium phenylbutyrate (Buphenyl™) in children with argininosuccinic aciduria (ASA). It is hoped that this drug may help lower ammonia and argininosuccinic acid levels in patients with ASA. Through this study we hope to learn whether the use of sodium phenylbutyrate (Buphenyl™) in patients with ASA, in addition to diet and arginine therapy, will decrease liver damage and the number of periods during which ammonia levels are high. The primary study objective is to determine if the treatment of ASA patients with sodium phenylbutyrate (Buphenyl™) in conjunction with lowered doses of arginine improves liver function as measured by short-term assessment of synthetic activity and the use of stable isotope tracers to assess ureagenesis and nitric oxide production. Twelve patients with ASA will be studied using a randomized, cross-over design. Medical records will be reviewed to confirm the diagnosis prior to enrollment. Patients will be on the each arm of the study for one week each preceded by a three day washout period. Patients will come to the Baylor College of Medicine General Clinical Research Center in Texas Children's Hospital for an inpatient hospitalization. This hospitalization is for diet control, clinical evaluation, and stable isotope infusions for measurement of in vivo rates of ureagenesis and nitric oxide production. During the washout periods Buphenyl™ or other alternative route medications, e.g., benzoate, will be discontinued and arginine will be administered at the standard therapeutic dose of 500 mg/kg/day or 10 grams/m2. Following randomization, subjects will be maintained on either high-dose arginine (500 mg/kg/day or 10 grams/m2) alone, or the alternative of low-dose arginine (100 mg/kg/d or 2 grams/m2) along with sodium phenylbutyrate (500 mg/kg/day or 10 grams/m2). For the second week they will be crossed over to the other arm of the study preceded by another three day washout. Previous medications and dietary protein intake (0.6 g/kg/day or current metabolic diet) will be continued during both arms of the study. The two arms may be performed as separate ten-day admissions. The decision to design the study with two arms, high-dose arginine and low-dose arginine/Buphenyl™, was reached after weighing several considerations. First, the current accepted practice for treatment of ASA is high-dose arginine. Based on metabolic flux studies, we have quantified the effects of this dose of arginine as compared to standard doses of Buphenyl™/phenylacetate on ureagenesis. Since the study is not designed or intended to address the independent effects of Buphenyl™ as compared to arginine alone, Buphenyl™ was added for safety reasons to accommodate loss of clearance of nitrogen by decreasing the arginine dose. Moreover, based on our experience with recruiting for previous versions of this study, most ASA patients with liver dysfunction are on an alternative route ammonia scavenger medication in addition to arginine. We found that this has in fact become an accepted practice. Hence, when considering study design from both a safety and recruitment perspective, it was most reasonable to design the study to investigate specifically the hypothesis that the production and presence of argininosuccinic acid leads to liver dysfunction. Since both low-dose arginine with Buphenyl™ and high-dose arginine alone lead to a decreased production of argininosuccinic acid, and both are currently accepted therapies in ASA and UCDs in general, the need to address their effects distinctly is not being investigated at this time. Sodium phenylbutyrate dosage will be 500 mg/kg/day in patients weighing less than 20kg and 10 g/m2/day in larger patients. Daily dosage will be given in equally divided doses 4 times per day. In order to ensure that the study remains double-blind to investigators, study staff and subjects, the Investigational Pharmacy Service at Texas Children's Hospital will dispense both the Buphenyl™ and arginine in powder form which is compounded into capsules or will be mixed with syrpalta (up to 5 grams will dissolve in 10cc liquid) and administered orally or through nasogastric or gastrostomy tube. No nasogastric tube should be placed or gastrostomy performed solely for the purpose of inclusion in this study. Because the reported adverse effects of sodium phenylbutyrate include gastritis, participants will be started on a standard dose of ranitidine (Zantac™), or comparable drug, during both arms of the study.

Dataset Information

Phosphoproteomic analysis of breast epithelial cells with isogenic knockin of PIK3CA mutations

Publications

Activation of diverse signalling pathways by oncogenic PIK3CA mutations.

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