Stochastic changes in gene expression promote chaotic dysregulation of homeostasis in clonal breast tumors.

Cells within tumors vary in phenotype as a result of changes in gene expression caused by a variety of mechanisms, permitting cancers to evolve under selective pressures from immune and other homeostatic processes. Earlier, we traced apparent losses in heterozygosity (LOH) of spontaneous breast tumors from first generation (F1) intercrossed mice to atypical epigenetic modifications in the structure of DNA across the tumor genomes. Here, we describe a parallel pattern of LOH in gene expression, revealed through quantitation of parental alleles across a population of clonal tumors. We found variegated patterns of LOH, based on allelic ratio outliers in hundreds of genes, enriched in regulatory pathways typically co-opted by tumors. The frequency of outliers was correlated with transcriptional repression of a large set of homozygous genes. These findings suggest stochastic losses in gene expression across the genome of tumors generate phenotypic variation among cells, allowing clonal selection during tumor development.

Exploring the effect of library preparation on RNA sequencing experiments.

RNA sequencing (RNA-seq) has become the widely preferred choice for surveying the genome-wide transcriptome complexity in many organisms. However, the broad adaptation of this methodology into the clinic still needs further evaluation of potential effect of sample preparation factors on its analytical reliability using patient samples. In this study, we examined the impact of three major sample preparation factors (i.e., cDNA library storage time, the quantity of input RNA, and cryopreservation of cell samples) on sequence biases, gene expression profiles, and enriched biological functions using RNAs isolated from primary B cell and CD4+ cell blood samples of healthy subjects. Our comprehensive comparison results suggested that different cDNA library storage time, quantity of input RNA, and cryopreservation of cell samples did not significantly alter gene transcriptional expression profiles generated by RNA-seq experiments. These findings shed new lights on the potential applications of RNA-seq technique to patient samples in a regular clinical setting.

Integrative Genome-Wide Analysis of Long Noncoding RNAs in Diverse Immune Cell Types of Melanoma Patients.

Genome-wide identification and characterization of long noncoding RNAs (lncRNA) in individual immune cell lineages helps us better understand the driving mechanisms behind melanoma and advance personalized patient treatment. To elucidate the transcriptional landscape in diverse immune cell types of peripheral blood cells (PBC) in stage IV melanoma, we used whole transcriptome RNA sequencing to profile lncRNAs in CD4+, CD8+, and CD14+ PBC from 132 patient samples. Our integrative computational approach identified 27,625 expressed lncRNAs, 2,744 of which were novel. Both T cells (i.e., CD4+ and CD8+ PBC) and monocytes (i.e., CD14+ PBC) exhibited differential transcriptional expression profiles between patients with melanoma and healthy subjects. Cis- and trans-level coexpression analysis suggested that lncRNAs are potentially involved in many important immune-related pathways and the programmed cell death receptor 1 checkpoint pathways. We also identified nine gene coexpression modules significantly associated with melanoma status, all of which were significantly enriched for three mRNA translation processes. Age and melanoma traits closely correlated with each other, implying that melanoma contains age-associated immune changes. Our computational prediction analysis suggests that many cis- and trans-regulatory lncRNAs could interact with multiple transcriptional and posttranscriptional regulatory elements in CD4+, CD8+, and CD14+ PBC, respectively. These results provide novel insights into the regulatory mechanisms involving lncRNAs in individual immune cell types in melanoma and can help expedite cell type-specific immunotherapy treatments for such diseases.Significance: These findings elucidate melanoma-associated changes to the noncoding transcriptional landscape of distinct immune cell classes, thus providing cell type-specific guidance to targeted immunotherapy regimens. Cancer Res; 78(15); 4411-23. ©2018 AACR.

Gene Expression Signatures Characterized by Longitudinal Stability and Interindividual Variability Delineate Baseline Phenotypic Groups with Distinct Responses to Immune Stimulation.

Human immunity exhibits remarkable heterogeneity among individuals, which engenders variable responses to immune perturbations in human populations. Population studies reveal that, in addition to interindividual heterogeneity, systemic immune signatures display longitudinal stability within individuals, and these signatures may reliably dictate how given individuals respond to immune perturbations. We hypothesize that analyzing relationships among these signatures at the population level may uncover baseline immune phenotypes that correspond with response outcomes to immune stimuli. To test this, we quantified global gene expression in peripheral blood CD4+ cells from healthy individuals at baseline and following CD3/CD28 stimulation at two time points 1 mo apart. Systemic CD4+ cell baseline and poststimulation molecular immune response signatures (MIRS) were defined by identifying genes expressed at levels that were stable between time points within individuals and differential among individuals in each state. Iterative differential gene expression analyses between all possible phenotypic groupings of at least three individuals using the baseline and stimulated MIRS gene sets revealed shared baseline and response phenotypic groupings, indicating the baseline MIRS contained determinants of immune responsiveness. Furthermore, significant numbers of shared phenotype-defining sets of determinants were identified in baseline data across independent healthy cohorts. Combining the cohorts and repeating the analyses resulted in identification of over 6000 baseline immune phenotypic groups, implying that the MIRS concept may be useful in many immune perturbation contexts. These findings demonstrate that patterns in complex gene expression variability can be used to define immune phenotypes and discover determinants of immune responsiveness.

Gene expression patterns in CD4+ peripheral blood cells in healthy subjects and stage IV melanoma patients.

Melanoma patients exhibit changes in immune responsiveness in the local tumor environment, draining lymph nodes, and peripheral blood. Immune-targeting therapies are revolutionizing melanoma patient care increasingly, and studies show that patients derive clinical benefit from these newer agents. Nonetheless, predicting which patients will benefit from these costly therapies remains a challenge. In an effort to capture individual differences in immune responsiveness, we are analyzing patterns of gene expression in human peripheral blood cells using RNAseq. Focusing on CD4+ peripheral blood cells, we describe multiple categories of immune regulating genes, which are expressed in highly ordered patterns shared by cohorts of healthy subjects and stage IV melanoma patients. Despite displaying conservation in overall transcriptome structure, CD4+ peripheral blood cells from melanoma patients differ quantitatively from healthy subjects in the expression of more than 2000 genes. Moreover, 1300 differentially expressed genes are found in transcript response patterns following activation of CD4+ cells ex vivo, suggesting that widespread functional discrepancies differentiate the immune systems of healthy subjects and melanoma patients. While our analysis reveals that the transcriptome architecture characteristic of healthy subjects is maintained in cancer patients, the genes expressed differentially among individuals and across cohorts provide opportunities for understanding variable immune states as well as response potentials, thus establishing a foundation for predicting individual responses to stimuli such as immunotherapeutic agents.

Widespread Non-Canonical Epigenetic Modifications in MMTV-NeuT Breast Cancer.

Breast tumors in (FVB × BALB-NeuT) F1 mice have characteristic loss of chromosome 4 and sporadic loss or gain of other chromosomes. We employed the Illumina GoldenGate genotyping platform to quantitate loss of heterozygosity (LOH) across the genome of primary tumors, revealing strong biases favoring chromosome 4 alleles from the FVB parent. While allelic bias was not observed on other chromosomes, many tumors showed concerted LOH (C-LOH) of all alleles of one or the other parent on sporadic chromosomes, a pattern consistent with cytogenetic observations. Surprisingly, comparison of LOH in tumor samples relative to normal unaffected tissues from these animals revealed significant variegated (stochastic) deviations from heterozygosity (V-LOH) in every tumor genome. Sequence analysis showed expected changes in the allelic frequency of single nucleotide polymorphisms (SNPs) in cases of C-LOH. However, no evidence of LOH due to mutations, small deletions, or gene conversion at the affected SNPs or surrounding DNA was found at loci with V-LOH. Postulating an epigenetic mechanism contributing to V-LOH, we tested whether methylation of template DNA impacts allele detection efficiency using synthetic oligonucleotide templates in an assay mimicking the GoldenGate genotyping format. Methylated templates were systematically over-scored, suggesting that the observed patterns of V-LOH may represent extensive epigenetic DNA modifications across the tumor genomes. As most of the SNPs queried do not contain standard (CpG) methylation targets, we propose that widespread, non-canonical DNA modifications occur during Her2/neuT-driven tumorigenesis.

Characterization of plant p23-like proteins for their co-chaperone activities.

The small acidic protein p23 is best described as a co-chaperone of Hsp90, an essential molecular chaperone in eukaryotes. p23 binds to the ATP-bound form of Hsp90 and stabilizes the Hsp90-client protein complex by slowing down ATP turnover. The stabilizing activity of p23 was first characterized in studies of steroid receptor-Hsp90 complexes. Earlier studies of the Hsp90 chaperone complex in plants suggested that a p23-like stabilizing activity was absent in plant cell lysates. Here, we show that p23-like proteins are present in plants and are capable of binding Hsp90, but unlike human p23 and yeast ortholog Sba1, the plant p23-like proteins do not stabilize the steroid receptor-Hsp90 complexes formed in wheat germ lysate. Furthermore, these proteins do not inhibit the ATPase activity of plant Hsp90. While transcripts of Arabidopsis thaliana p23-1 and Atp23-2 were detected under normal growing conditions, those of the closely related Brassica napus p23-1 were present only after moderate heat stress. These observations suggest that p23-like proteins in plants are conserved in their binding to Hsp90 but have evolved mechanisms of action different from their yeast and animal counterparts.

Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23.

Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.

Indirect recruitment of a CD40 signaling pathway in dendritic cells by B7-DC cross-linking antibody modulates T cell functions.

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40(-/-) DCs treated with B7-DC XAb (DC(XAb)) activated DAP12, but failed to activate NFkappaB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D(3). CD40(-/-) DC(XAb) also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DC(XAb) to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.

TREM-2 mediated signaling induces antigen uptake and retention in mature myeloid dendritic cells.

Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-gamma1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo.

Lymphocyte differentiation from naive CD4(+) T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DC(XAb) into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25(-) T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

GCUNC45 is the first Hsp90 co-chaperone to show alpha/beta isoform specificity.

Hsp90 is an essential molecular chaperone required for the normal functioning of many key regulatory proteins in eukaryotic cells. Vertebrates have two closely related isoforms of cytosolic Hsp90 (Hsp90alpha and Hsp90beta). However, specific functions for each isoform are largely unknown, and no Hsp90 co-chaperone has been reported to distinguish between the two isoforms. In this study, we show that the Hsp90 co-chaperone GCUNC45 bound preferentially to the beta isoform of Hsp90 in vitro. GCUNC45 efficiently blocked the progression of progesterone receptor chaperoning in an in vitro functional system when Hsp90beta was used, but did so with much less efficacy when Hsp90alpha was used. Knockdown experiments in HeLa cells showed that GCUNC45 is required for the normal cellular distribution of Hsp90beta, but not Hsp90alpha. This is the first example of a co-chaperone with isoform selectivity, and this approach may open novel avenues to understanding the functional differences between Hsp90 isoforms.

Cdc37 regulation of the kinome: when to hold 'em and when to fold 'em.

Although massive genome sequencing efforts have identified the protein kinases encoded by several eukaryotic genomes and proteomic analyses have begun to determine the kinases expressed in a cell, there is still much to learn about the additional cellular events that shape eukaryotic kinomes. Large-scale analyses in Saccharomyces cerevisiae have indicated that a relatively small subset of kinases requires chaperoning by heat shock protein 90 (Hsp90). However, new evidence suggests that most kinases do require chaperoning and, furthermore, that Cdc37, a chaperone that has Hsp90-dependent and -independent functions, serves as the chaperone for a large portion of the yeast kinome.

Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR).

Hsp90 is an abundant and highly conserved chaperone that functions at later stages of protein folding to maintain and regulate the activity of client proteins. Using a recently described in vitro system to fold a functional model kinase Chk1, we performed a side-by-side comparison of the Hsp90-dependent chaperoning of Chk1 to that of the progesterone receptor (PR) and show that these distinct types of clients have different chaperoning requirements. The less stable PR required more total chaperone protein(s) and p23, whereas Chk1 folding was critically dependent on Cdc37. When the 2 clients were reconstituted under identical conditions, each client folding was dose dependent for Hsp90 protein levels and was inhibited by geldanamycin. Using this tractable system, we found that Chk1 kinase folding was more effective if we used a type II Hsp40 cochaperone, whereas PR is chaperoned equally well with a type I or type II Hsp40. Additional dissection of Chk1-chaperone complexes and the resulting kinase activity suggests that kinase folding, like that previously shown for PR, is a dynamic, multistep process. Importantly, the cochaperones Hop and Cdc37 cooperate as the kinase transitions from immature Hsp70- to mature Hsp90-predominant complexes.

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones.

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.

p23, a simple protein with complex activities.

p23 is a small but important cochaperone for the Hsp90 chaperoning pathway. It appears to facilitate the adenosine triphosphate-driven cycle of Hsp90 binding to client proteins. It enters at a late stage of the cycle and enhances the maturation of client proteins. Although this role of p23 is fairly well established, recent studies suggest that it may have additional functions in the cell that merit further exploration.

Synthesis of novel fluorescent probes for the molecular chaperone Hsp90.

Heat shock protein 90 (Hsp90) is a molecular chaperone necessary for maintaining oncogenic transformation. There is substantial interest in developing novel agents that bind to the N-terminal of the chaperone. Here we report the synthesis and characterization of two fluorescent Hsp90 inhibitors and probe their use in an Hsp90 fluorescent polarization assay.