Ankit Sinha

Onco-proteogenomics: cancer proteomics joins forces with genomics

The complexities of tumor genomes are rapidly being uncovered, but how they are regulated into functional proteomes remains poorly understood. Standard proteomics workflows use databases of known proteins, but these databases do not capture the uniqueness of the cancer transcriptome, with its point mutations, unusual splice variants and gene fusions. Onco-proteogenomics integrates mass spectrometry–generated data with genomic information to identify tumor-specific peptides. Linking tumor-derived DNA, RNA and protein measurements into a central-dogma perspective has the potential to improve our understanding of cancer biology.

Tumor-derived exosomes and microvesicles in head and neck cancer: Implications for tumor biology and biomarker discovery

Exosomes and microvesicles (MVs) are nanometer‐sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro‐environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro‐RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)‐associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC‐derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV‐associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio‐fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.

In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine

Expressed prostatic secretions (EPS) are proximal fluids of the prostate that are increasingly being utilized as a clinical source for diagnostic and prognostic assays for prostate cancer (PCa). These fluids contain an abundant amount of microvesicles reflecting the secretory function of the prostate gland, and their protein composition remains poorly defined in relation to PCa. Using expressed prostatic secretions in urine (EPS‐urine), exosome preparations were characterized by a shotgun proteomics procedure. In pooled EPS‐urine exosome samples, ∼900 proteins were detected. Many of these have not been previously observed in the soluble proteome of EPS generated by our labs or other related exosome proteomes. We performed systematic comparisons of our data against previously published, prostate‐related proteomes, and global annotation analyses to highlight functional processes within the proteome of EPS‐urine derived exosomes. The acquired proteomic data have been deposited to the Tranche repository and will lay the foundation for more extensive investigations of PCa derived exosomes in the context of biomarker discovery and cancer biology.

In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome.

Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery.

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2+ xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2+ BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2+ BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2+ BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2+ BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

VennDIS: A JavaFX-based Venn and Euler diagram software to generate publication quality figures

Venn diagrams are graphical representations of the relationships among multiple sets of objects and are often used to illustrate similarities and differences among genomic and proteomic datasets. All currently existing tools for producing Venn diagrams evince one of two traits; they require expertise in specific statistical software packages (such as R), or lack the flexibility required to produce publication‐quality figures. We describe a simple tool that addresses both shortcomings, Venn Diagram Interactive Software (VennDIS), a JavaFX‐based solution for producing highly customizable, publication‐quality Venn, and Euler diagrams of up to five sets. The strengths of VennDIS are its simple graphical user interface and its large array of customization options, including the ability to modify attributes such as font, style and position of the labels, background color, size of the circle/ellipse, and outline color. It is platform independent and provides real‐time visualization of figure modifications. The created figures can be saved as XML files for future modification or exported as high‐resolution images for direct use in publications.

Proteomic analysis of human fetal atria and ventricle.

In this study we carried out a mass spectrometry-based proteome analysis of human fetal atria and ventricles. Heart protein lysates were analyzed on the Q-Exactive mass spectrometer in biological triplicates. Protein identification using MaxQuant yielded a total of 2754 atrial protein groups (91%) and 2825 ventricular protein groups (83%) in at least 2 of the 3 runs with ≥ 2 unique peptides. Statistical analyses using fold-enrichment (>2) and p-values (≤ 0.05) selected chamber-enriched atrial (134) and ventricular (81) protein groups. Several previously characterized cardiac chamber-enriched proteins were identified in this study including atrial isoform of myosin light chain 2 (MYL7), atrial natriuretic peptide (NPPA), connexin 40 (GJA5), and peptidylglycine alpha-amidating monooxygenase (PAM) for atria, and ventricular isoforms of myosin light chains (MYL2 and MYL3), myosin heavy chain 7 (MYH7), and connexin 43 (GJA1) for ventricle. Our data was compared to in-house generated and publicly available human microarrays, several human cardiac proteomes, and phenotype ontology databases.

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors

PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate β-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2− and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.

HLA-DP 84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway

Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with β-chains encoding Gly84 (DP84Gly) constitutively present endogenous peptides. DP84Gly does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP84Gly from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP84Gly via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP84Gly constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP84Gly, found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.

Divergent evolution of temozolomide resistance in glioblastoma stem cells is reflected in extracellular vesicles and coupled with radiosensitization

Background Glioblastoma (GBM) is almost invariably fatal due to failure of standard therapy. The relapse of GBM following surgery, radiation, and systemic temozolomide (TMZ) is attributed to the ability of glioma stem cells (GSCs) to survive, evolve, and repopulate the tumor mass, events on which therapy exerts a poorly understood influence. Methods Here we explore the molecular and cellular evolution of TMZ resistance as it emerges in vivo (xenograft models) in a series of human GSCs with either proneural (PN) or mesenchymal (MES) molecular characteristics. Results We observed that the initial response of GSC-initiated intracranial xenografts to TMZ is eventually replaced by refractory growth pattern. Individual tumors derived from the same isogenic GSC line expressed divergent and complex profiles of TMZ resistance markers, with a minor representation of O6-methylguanine DNA methyltransferase (MGMT) upregulation. In several independent TMZ-resistant tumors originating from MES GSCs we observed a consistent diminution of mesenchymal features, which persisted in cell culture and correlated with increased expression of Nestin, decline in transglutaminase 2 and sensitivity to radiation. The corresponding mRNA expression profiles reflective of TMZ resistance and stem cell phenotype were recapitulated in the transcriptome of exosome-like extracellular vesicles (EVs) released by GSCs into the culture medium. Conclusions Intrinsic changes in the tumor-initiating cell compartment may include loss of subtype characteristics and reciprocal alterations in sensitivity to chemo- and radiation therapy. These observations suggest that exploiting therapy-induced changes in the GSC phenotype and alternating cycles of therapy may be explored to improve GBM outcomes.