Rainer Blaesius

Intratumor Heterogeneity (ITH) is a functionally important property of tumor tissue and may be involved in drug resistance mechanisms. Although descriptions of ITH can be traced back to very early reports about cancer tissue, mechanistic investigations are still limited by the precision of analysis methods and access to relevant tissue sources. PDX models have provided a reproducible source of tissue with at least a partial representation of naturally occurring ITH. We investigated the properties of phenotypically distinct cell populations by Fluorescence activated cell sorting (FACS) tissue derived cells from multiple tumors from a triple negative breast cancer patient derived xenograft (PDX) model. We subsequently subjected each population to in depth gene expression analysis. Our findings suggest that process related gene expression changes (caused by tissue dissociation and FACS sorting) are restricted to Immediate Early Genes (IEGs). This allowed us to discover highly reproducible gene expression profiles of distinct cellular compartments identifiable by cell surface markers in this particular tumor model. Within the context of data from a previously published model our work suggests that gene expression profiles associated with hypoxia, stemness and drug resistance may reside in tumor subpopulations predictably growing in PDX models. This approach provides a novel opportunity for prospective mechanistic studies of ITH.

Flow based single cell analysis of the immune landscape distinguishes Barretts esophagus from adjacent normal tissue

Cancer tissue functions as an ecosystem of a diverse set of cells that interact in a complex tumor microenvironment. Genomic tools applied to biopsies in bulk fail to account for this tumor heterogeneity, whereas single-cell imaging methods limit the number of cells which can be assessed or are very resource intensive. The current study presents methods based on flow cytometric analysis and cell sorting using known cell surface markers (CXCR4/CD184, CD24, THY1/CD90) to identify and interrogate distinct groups of cells in triple-negative breast cancer clinical biopsy specimens from patient-derived xenograft (PDX) models. The results demonstrate that flow cytometric analysis allows a relevant subgrouping of cancer tissue and that sorting of these subgroups provides insights into cancer cell populations with unique, reproducible, and functionally divergent gene expression profiles. The discovery of a drug resistance signature implies that uncovering the functional interaction between these populations will lead to deeper understanding of cancer progression and drug response. Implications: PDX-derived human breast cancer tissue was investigated at the single-cell level, and cell subpopulations defined by surface markers were identified which suggest specific roles for distinct cellular compartments within a solid tumor. Mol Cancer Res; 15(4); 429–38. ©2016 AACR.

Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Gα is complexed with the Gβγ dimer, and an active state in which GTP-bound Gα is freed of its Gβγ binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise ‘switch regions’ of the Gα subunit. Discrete differences in conformation between GDP- and GTP-bound Gα underlie its nucleotide-dependent protein-protein interactions (e.g., with Gβγ/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Gα (and free Gβγ) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Gα and Gβγ signaling, and serving as biosensor tools in assays for Gα activation including high throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.

G-Proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind Gα subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069−1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP·AlF₄^-- and GTPγS-bound states of Gα_i subunits. KB-1753 blocks interaction of Gα_transducin with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated Gα in vitro. The crystal structure of KB-1753 bound to Gα_i1·GDP·AlF₄^- reveals binding to a conserved hydrophobic groove between switch II and α3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for Gα_i subunits.

Signaling via G‐protein coupled receptors is initiated by receptor‐catalyzed nucleotide exchange on Gα subunits normally bound to GDP and Gβγ. Activated Gα · GTP then regulates effectors such as adenylyl cyclase. Except for Gβγ, no known regulators bind the adenylyl cyclase‐stimulatory subunit Gα_s in its GDP‐bound state. We recently described a peptide, KB‐752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase‐inhibitory subunit Gα_i. Herein, we report that KB‐752 binds Gα_s · GDP yet slows its rate of nucleotide exchange. KB‐752 inhibits GTPγS‐stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Gα_i and Gα_s.

P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y₁ receptor and the human P2Y₁₂ receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 μg/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.