RIME proteomics of estrogen and progesterone receptors in breast cancer.

Nuclear receptors play an important role in transcriptional regulation of diverse cellular processes and is also relevant in diseases such as cancer. In breast cancer, the nuclear receptors - estrogen receptor (ER) and progesterone receptor (PR) are classical markers of the disease and are used to classify breast cancer subtypes. Using a recently developed affinity purification MS technique (RIME) [1], we investigate the protein interactors of ER and PR in breast cancer cell lines upon stimulation by the ligands - estrogen and progesterone. The data is deposited at proteomeXchange (PXD002104) and is part of a publication [2] that explains the link between the two nuclear receptors and potential consequences of this in breast cancer. In this manuscript, we describe the methodology used and provide details on experimental procedures, analysis methods and analysis of raw data. The purpose of this article is to enable reproducibility of the data and provide technical recommendations on performing RIME in hormonal contexts

Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment

Bcl-2 family member Bid is subject to autoinhibition; in the absence of stimuli, its N-terminal region sequesters the proapoptotic Bcl-2 homology 3 (BH3) domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C). Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly but not exclusively in helix 1, which is required for ubiquitination and degradation of tBid-N. Rescue of tBid-N from degradation blocked Bid's ability to induce mitochondrial outer membrane permeability but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C

C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signaling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. An assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification, which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the PI3K VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signaling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and mechanisms of immune failure in critical illness.AJTW was a Gates Cambridge Scholar supported by the Gates Cambridge Trust from 2015-2018. ACM is supported by a Clinical Research Career Development Fellowship from the Wellcome Trust (WT 2055214/Z/16/Z). Grants to ACM from the Academy of Medical Sciences and European Society for Intensive Care Medicine supported this work. The study was also supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre (IS-BRC-1215-20001) and the Medical Research Council SHIELD antimicrobial resistance consortium (MR/ N02995X/1

Progesterone receptor modulates ERα action in breast cancer.

Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.We would like to acknowledge the support of the University of Cambridge, Cancer Research UK and Hutchison Whampoa Limited. Research reported in this manuscript was supported by the National Cancer Institute of the National Institutes of Health under award number 5P30CA142543 (to UT Southwestern) and Department of Defense grants W81XWH-12-1-0288-03 (GVR). W.D.T. is supported by grants from the National Health and Medical Research Council of Australia (ID 1008349; ID 1084416) and Cancer Australia (ID 627229) T.E.H held a Fellowship Award from the US Department of Defense Breast Cancer Research Program (BCRP; #W81XWH-11-1-0592) and currently is supported by a Florey Fellowship from the Royal Adelaide Hospital Research Foundation. J.S.C is supported by an ERC starting grant and an EMBO Young investigator award.This is the accepted manuscript. The final version is available at www.nature.com/nature/journal/v523/n7560/full/nature14583.htm

Phosphorylation and Stabilization of PIN1 by JNK Promote Intrahepatic Cholangiocarcinoma Growth.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of the c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and therefore the key downstream effectors of this pathway, remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signaling in ICC that could open up therapeutic opportunities. APPROACH AND RESULTS: Using loss-of-function and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumors, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 through all-trans retinoic acid, a Food and Drug Administration-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation through PIN1 inhibition

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target.

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.This work was supported by a Cancer Research UK program grant (to DEN) and also by the US Department of Defense (Prostate Cancer Research Program Transformative Impact Award, grant ID W81XWH-13-2-0093; WDT and SMD), PCFA/Cancer Australia/Movember (grant IDs 1012337 and 1043482; WDT and LAS), Cancer Australia (grant ID 1043497; WDT and JC) and The Ray and Shirl Norman Cancer Research Trust (WDT and LAS). The Dame Roma Mitchell Cancer Research Laboratories were supported by an establishment grant from the PCFA (ID 2011/0452). FO was supported by a PhD project grant from Prostate Cancer UK (S10-10). LAS is supported by a Young Investigator Award from the Prostate Cancer Foundation (the Foundation 14 award)

Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction*

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3–7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions

Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins

Polyphosphoinositides (PPIns) play essential roles as lipid signaling molecules, and many of their functions have been elucidated in the cytoplasm. However, PPIns are also intranuclear where they contribute to chromatin remodeling, transcription, and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), has been mapped to the nucleus and nucleoli, but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5)P3, we applied a previously developed quantitative MS-based approach to identify the targets of PtdIns(3,4,5)P3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5)P3-interacting partners, and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA processes and DNA repair, including poly(ADP-ribose) polymerase 1 (PARP1, now referred as ADP-ribosyltransferase 1). PARP1 was found to interact directly with PPIn via three polybasic regions in the DNA-binding domain and the linker located N-terminal of the catalytic region. PARP1 was shown to bind to PtdIns(3,4,5)P3 as well as phosphatidylinositol 3,4-bisphosphate in vitro and to colocalize with PtdIns(3,4,5)P3 in the nucleolus and with phosphatidylinositol 3,4-bisphosphate in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5)P3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5)P3-mediated interactions in the nucleus and nucleolus

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer.

BACKGROUND: The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. RESULTS: The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. CONCLUSIONS: AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer