Activating Mutations in <i>PIK3CA</i> Lead to Widespread Modulation of the Tyrosine Phosphoproteome

Zahari, Muhammad Saddiq; Wu, Xinyan; Blair, Brian; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Jelinek, Christine; Malhotra, Radhika; Kim, Min‐Sik; Park, Ben Ho; Pandey, Akhilesh

doi:10.1021/acs.jproteome.5b00302

Cited by 8 publications

(5 citation statements)

References 42 publications

(81 reference statements)

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“…Following a label check to confirm incorporation of the heavy isotope, samples were lysed and protein concentration was measured. From each sample, 25 mg of lysate was taken, mixed, and digested in‐solution with trypsin and subjected to an antibody‐based phosphotyrosine enrichment and LC/MS–MS analysis (Figure 1) as described previously by our group (Syed et al., 2015; Zahari et al., 2015; Zhong et al., 2012). Peptides were identified using Mascot and Sequest run through Proteome Discoverer 2.0 and quantified using PyQuant.…”

Section: Resultsmentioning

confidence: 99%

Unbiased identification of substrates of protein tyrosine phosphatase ptp‐3 in C. elegans

et al. 2016

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A B S T R A C TThe leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans e PTPRF, PTPRD and PTPRS e that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2.

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Section: Resultsmentioning

confidence: 99%

Unbiased identification of substrates of protein tyrosine phosphatase ptp‐3 in C. elegans

et al. 2016

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“…This is in contrast to a favourable prognosis associated with these mutations in early-stage cancer ( Kalinsky et al , 2009 ; Sabine et al , 2014 ), no effect on prognosis in patients with MBC undergoing first-line hormonal therapy with tamoxifen and an improved outcome in patients with MBC with first-line aromatase inhibitor therapy ( Ramirez-Ardila et al , 2013 ). From a biologic perspective, mutation site-specific differences are noted for interacting proteins ( Zhao and Vogt, 2008 ; Hao et al , 2013 ), oncogenic potency and behaviour ( Bader et al , 2006 ; Pang et al , 2009 ), and the phosphoproteome ( Wu et al , 2014 ; Zahari et al , 2015 ) that may be clinically important and deserve further analysis in clinical studies and preclinical modelling.…”

Section: Discussionmentioning

confidence: 99%

Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR+, HER2− advanced breast cancer: results from BOLERO-2

Moynahan

Chen²,

He³

et al. 2017

Br J Cancer

118

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Background:The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation.Methods:PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm.Results:Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0.43 and 0.37, respectively). Everolimus also prolonged median PFS in patients with PIK3CA H1047R (HR, 0.37) and E545K/E542K mutations (HR=0.30) with a similar magnitude.Conclusions:Mutation analysis of plasma-derived cfDNA by ddPCR suggests that PFS benefit of everolimus was maintained irrespective of PIK3CA genotypes, consistent with the previous analysis of archival tumour DNA by next-generation sequencing.

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“…Antibodies employed for the migratory scWB study include rabbit anti-EphA2 19,20 (1:10, 6997S, Cell Signaling, with anti-rabbit secondary antibody conjugated with Alexa-Fluor 647), mouse anti-STAT3 21,22 (1:10, 9139S, Cell Signaling, with anti-mouse secondary antibody conjugated with Alexa-Fluor 488), mouse anti-Nestin 23,24 (1:10, MAB5326, EMD-Millipore, with anti-mouse secondary antibody conjugated with Alexa-Fluor 488), rabbit anti-β-tubulin 17,25 (1:10, ab6046, Abcam, with anti-rabbit secondary antibody conjugated with Alexa-Fluor 647).…”

Section: Methodsmentioning

confidence: 99%

Linking invasive motility to protein expression in single tumor cells

et al. 2018

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The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We identify protein-protein correlations within single TICs, which would be obscured with population-based assays. The integration of motility traits with single-cell protein analysis - on the same cell - offers a new means to identify druggable targets of invasive capacity.

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Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

Cited by 8 publications

References 42 publications

Unbiased identification of substrates of protein tyrosine phosphatase ptp‐3 in C. elegans

Unbiased identification of substrates of protein tyrosine phosphatase ptp‐3 in C. elegans

Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR+, HER2− advanced breast cancer: results from BOLERO-2

Linking invasive motility to protein expression in single tumor cells

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