Rational design of RAR-selective ligands revealed by RARbeta crystal stucture

The crystal structure of the ligand-binding domain of RARbeta, a suspect tumour suppressor, reveals important features that distinguish it from the two other RAR isotypes. The most striking difference is an extra cavity allowing RARbeta to bind more bulky agonists. Accordingly, we identified a ligand that shows RARbeta selectivity with a 100-fold higher affinity to RARbeta than to alpha or gamma isotypes. The structural differences between the three RAR ligand-binding pockets revealed a rationale explaining how a single retinoid can be at the same time an RARalpha, gamma antagonist and an RARbeta agonist. In addition, we demonstrate how to generate an RARbeta antagonist by gradually modifying the bulkiness of a single substitution. Together, our results provide structural guidelines for the synthesis of RARbeta-selective agonists and antagonists, allowing for the first time to address pharmacologically the tumour suppressor role of RARbeta in vitro and in animal models.     

Allele-specific amplification in cancer revealed by SNP array analysis

Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Apparent involvement of opioid peptides in stress-induced enhancement of tumor growth

Exposure to stress has been associated with alterations in both immune function and tumor development in man and laboratory animals. In the present study, we investigated the effect of a particular type of inescapable footshock stress, known to cause an opioid mediated form of analgesia, on survival time of female Fischer 344 rats injected with a mammary ascites tumor. Rats subjected to inescapable footshock manifested an enhanced tumor growth indicated by a decreased survival time and decreased percent survival. This tumor enhancing effect of stress was prevented by the opiate antagonist, naltrexone, suggesting a role for endogenous opioid peptides in this process. In the absence of stress, naltrexone did not affect tumor growth.     

Augmentation of tumor cell immunogenicity by viruses--an approach to specific immunotherapy of cancer

Several viruses have been evaluated as potential agents for cancer treatment using either their oncolytic properties, in order to lyse cancer cells, or their potential augmenting effects on the immune response to tumors. However, the direct oncolytic effect was found to be limited in time, in scope and in specificity, whereas the use of viral oncolysates to augment antitumor immunity was shown to be better than tumor cell homogenates or extracts but inferior to noninfected intact tumor cells, attesting for the importance of membrane architecture in preserving immunogenicity of tumor specific surface antigens. In order to get the maximum benefit from this approach we selected a nonlytic virus-tumor cell combination, using Newcastle disease virus as a nonpathogenic virus, to treat the experimental tumor model, Lewis lung carcinoma (3LL) in mice. The virus effectively infected 3LL cells without any cytopathic effect. The infected cells induced strong antitumor immunity, as judged by the appearance of immune cells in the spleen (Winn test and lymphocytotoxicity) and by the resistance to challenge with the 3LL cells after immunization. The antitumor immunity was superior to that obtained with intact noninfected tumor cells. We also designed a treatment protocol using the same virus-tumor cell preparation to treat mice after tumor inoculation. This treatment resulted in cure of 40% of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.     

Gene expression differences in bipolar disorder revealed by cDNA array analysis of post-mortem frontal cortex

Previous studies have implicated a number of biochemical pathways in the etiology of bipolar disorder (BD). However, the precise abnormalities underlying this disorder remain to be established. To investigate novel factors that may be important in the pathophysiology of BD, we utilized cDNA expression arrays to examine differences in expression of up to 1200 genes known to be involved in potentially relevant biochemical processes. This investigation was undertaken in post-mortem samples of frontal cortex tissue from patients with BD and matched controls, obtained (n = 10/group) from the Stanley Foundation Neuropathology Consortium. Results include significant (greater than 35% change in signal intensity) differences between BD and controls in a number of genes (n = 24). Selected targets were analyzed by RT-PCR, which confirmed a decrease in transforming growth factor-beta1 (TGF-beta 1), and an increase in both caspase-8 precursor (casp-8) and transducer of erbB2 (Tob) expression in BD. We further observed a significant decrease of TGF-beta 1 mRNA levels in BD by RT-PCR in individual post-mortem samples. Given the neuroprotective role attributed to this inhibitory cytokine, our results suggest that the down-regulation of TGF-beta 1 may lead to various neurotoxic insults potentially involved in the etiology of certain mood disorders.     

Single-cell RNA sequencing analysis revealed cellular and molecular immune profiles in lung squamous cell carcinoma

Although breakthroughs have been made in the treatment of non-small cell lung cancer, there are only a few choices for advanced-stage or recurrent lung squamous cell carcinoma (LUSC) patients. In our study, we identified 7 major cell types in thedepicted the immunolandscape of LUSC microenvironment using single-cell RNA sequencing. We found that an immunosuppressive receptor, T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), was highly expressed by regulatory T cells (Tregs) and exhausted CD8⁺T cells, suggesting that upregulation of TIGIT might promote an immunosuppressive microenvironment and inhibit the cytotoxic ability of CD8⁺T cells. We also identified tumor-associated neutrophil (TAN), characterized by CXCR2, CSF3R and CXCL8, in the tumor region, and TANs upregulated the expression of interleukin 1 receptor antagonist (IL1RN) which suggested that TAN might exert an immunosuppressive role via expressing IL1RN. Furthermore, the number of SPP1+ macrophages(SPP1⁺M) significantly increased in tumor microenvirnment, which was correlated with the poor survival of patients. Additionally, regulatory networks based on SPP1⁺M revealed that the disparities of several ligand-receptor pairs existed between tumor and normal tissues. Among these pairs, SPP1-CD44 showed the most interactions between SPP1⁺M and other cell types. Our results provided deep insight into the immune landscape of LUSC and an essential resource for drug discovery in the future.     

Defects in tumor cell immunogenicity: lymphokine signals in in vitro cytolytic responses to tumor alloantigens

The B16 melanoma of C57BL/6 mice illustrates a deficiency in immunostimulation which may be important in some host-tumor relationships. B16 immunizes very poorly, even against its own major histocompatibility complex (MHC) antigens. We have compared the anti-MHC cytolytic response induced in vitro by B16 and by other tumors of both lymphoid and nonlymphoid origin. We have also studied the role of indomethacin and exogenous lymphokines in facilitating these responses and examined the relationship of specific and nonspecific effector cells induced. In contrast to normal lymphoid cells and two lymphoid tumor cells (EL4 and WEHI-265), the three nonlymphoid tumors, B16, Lewis lung tumor (3LL), and MC-2 fibrosarcoma, failed to induce primary cytolytic responses by themselves. MC-2 and B16 represented two different defects in immunogenicity. MC-2, which we have shown previously to induce an in vivo cytolytic response, could also immunize in vitro provided that prostaglandin production was blocked with indomethacin. In contrast B16, which is poorly immunogenic in vivo, immunized in vitro only if a concanavalin A-induced lymphokine supernatant (CS) was added as an exogenous source of "signal 2." High concentrations of the interleukin 2-containing Con A-induced spleen cell culture supernatant-induced non-H-2b-specific lymphokine-activated killer (LAK) cells in the absence of B16 stimulator cells. However, lymphokine concentrations too low to induce LAK cells enabled the otherwise nonimmunogenic B16 cells to induce specific cytolytic activity.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line

We have recently demonstrated that a treatment combining the cell differentiating agent sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats. NaBut or IL2 treatment alone, never cured these tumour-bearing rats. In the present investigation, we report that NaBut-treatments induce apoptosis in the colonic cancer cells both in vitro and in vivo. We postulated that the significant therapeutic effect of NaBut/IL2 treatment can be mainly attributed to a NaBut-induced apoptosis of the tumoural cells increasing their immunogenicity. Indeed, treatment which combined apoptotic bodies (apobodies) as cell vaccine, plus IL2 immunotherapy significantly increased tumour remission and survival rate of the vaccinated rats, whereas IL2 treatment alone did not. We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells. This latter result suggests that these treatments generate an immune protection. This was confirmed by the presence, in the sera of the cured rats, of anti-tumoural antibodies directed against both the apobodies and the tumour cells, but not against normal colonocytes. In addition, we show that injections of apobodies before administration of the parental tumour cells results in a partial protection. We provide the first evidence that apobodies, derived from cancer cells after NaBut-treatment, induce a specific immune response against parental tumours cells. These data suggest that the distinctive immunologic properties of apobodies could provide a valuable tool in colorectal cancer immunotherapy.     

Integrated bioinformatics analysis revealed the regulation of angiogenesis by tumor cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, metastasis accounts for most of the cases. Angiogenesis plays an important role in cancer metastasis, but how tumor cells affect the function of endothelial cells by dictating their microRNA (miRNA) expression remains largely unknown.Differentially expressed miRNAs (DEMs) were identified through dataset downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. We then used online tools to obtain potential targets of candidate miRNAs and functional enrichment analysis, as well as the protein-protein interaction (PPI). Finally, the function of miR-302c-3p was validated through in vitro assay.In the current study, we found that HCC cells altered miRNA expression profiles of human umbilical vein endothelial cells (HUVECs) and miR-302c-3p was the most down-regulated miRNA in HUVECs when they were co-cultured with HCC-LM3 cells. Functional enrichment analysis of the candidate targets revealed that these genes were involved in epigenetic regulation of gene expression, in particular, cytosine methylation. In addition, PPI network demonstrated distinct roles of genes targeted by miR-302c-3p. Importantly, inhibition of angiogenesis, migration and permeability by the most down-regulated miR-302c-3p in HUVECs was confirmed in vitro. These findings brought us novel insight into the regulation of angiogenesis by HCC cells and provided potential targets for the development of therapeutic strategies.     

Immunohistochemical expression of RARalpha, RARbeta, and Cx43 in breast tumor cell lines after treatment with lycopene and correlation with RT-QPCR.

Lycopene, the major carotenoid found in tomatoes, is a potent antioxidant associated with the prevention of degenerative diseases such as breast cancer. This effect could be due to the interaction between lycopene and retinoic acid receptors as well as the stimulation of gap junction communication and synthesis of connexin 43. The expression of the RARalpha, RARbeta, and Cx43 proteins was analyzed using immunohistochemistry in two breast cancer cell lines, MCF-7 and MDA-MB-231, and in a fibrocystic dystrophy cell line, MCF-10a, after a 48-hr exposure to 10 microM lycopene. A real-time quantitative PCR analysis was then performed to measure mRNA expression. RARalpha and Cx43 expression were increased at both mRNA and protein levels in two breast cell lines.     

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

Lysate array analyses of signal transduction inhibitors in tumor cell lines

We probed the ability of reverse lysate array technology to help explain potential differences in the responses of cancer cells to various small-molecule kinase inhibitors. To understand the antitumor potential of signal transduction inhibitors and their effects on signaling pathways downstream of Src, we used reverse lysate array technology to study SIG11293, a selective inhibitor of Src and LcK kinases, and AEE788, a selective inhibitor of Kdr (VEGFR1) and epidermal growth factor receptor/ErbB-2 that also has affinity for Src, c-abl, c-fms, and Flt-1. We observed the effects of drug dose on cell killing and expression and phosphorylation of various signal transduction proteins in MDA435 and MDA231 human breast cancer cells and U251HF glioblastoma cells. After 24 h, SIG11293 induced the least amount of cell killing in MDA435 cells; decreased Stat3(pY705) and Src(pY529) in all cell lines; decreased Src(pY418) and total Src in MDA231 and MDA435 cells, but not U251 cells; and in U251 cells, uniquely increased activated caspase 3, Src(pY418), panSrc, and p70S6K. AEE788 decreased Src(pY529) and Stat3(pY705) in U251HF and MDA435 cells. In regard to Src phosphorylation, both drugs decreased the negative regulatory site, Src (pY529), more than the positive regulatory site, Src(pY418), relative to total Src. These observations suggest that the two drugs have complex and different effects on Src signaling pathways. Although this general conclusion could be predicted, we believe that these studies exemplify the ability and robustness of reverse lysate arrays to measure signaling pathway modulation in tumor cells. Our hope is that these techniques will help to develop more robust preclinical and, eventually, clinical treatment paradigms.     

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.     

Differential tumor immunogenicity of L1210 and its sublines. I. Effect of an increased antigen density on tumor cell surfaces on primary B cell responses in vitro.

The differential immunogenicity of DBA/2 lymphoma L1210 and three L1210 sublines, each resistant to a different anti-leukemic agent (guanazole, methylglyoxal-bis-guanylhydrazone, and 4,4-diacetyldiphenylurea-bis-guanylhydrazone), was evaluated in vitro. Syngeneic spleen cells from nonimmunized DBA/2 mice were cultured in the presence of graded numbers of irradiated cells of L1210 or its sublines. The stimulation elicited a T-independent primary antibody response in vitro which was measured by determining the number of plaque-forming cells by using the immunizing lymphoma cells as target. Cells of all three sublines exhibited an increased immunogenicity, as compared to that of the parental L1210 cells, in eliciting the response directed to tumor-associated antigens which were common to all sublines. Dose-response experiments showed that high doses of the parental cells did stimulate responses which were detectable with subline cells as target. The results indicated that the differential immunogenicity of L1210 and its sublines, as demonstrated in the present assay system, is primarily quantitative, and was apparently due to increased amount or density of common tumor-associated antigens on the subline cells. The implications of these observations are discussed in relation to the possible mechanisms underlying the emergence of highly immunogenic drug-resistant sublines.     

Passive enhancement of mouse tumor allografts by alloantibodies is Fc-dependent.

Enhancement of growth of a B10.D2 fibrosarcoma in B6AF1 recipients could be induced by administration of B6AF1 anti-B10.D2 lymphocyte serum. The role of the Fc part in this phenomenon was studied by treatment of the B6AF1 recipients with F(ab')2 fragments of enhancing alloantibodies. A highly purified F(ab')2 preparation was used to exclude any effects of undigested IgG. Administration of F(ab')2 did not lead to enhancement of the tumor allografts, not even when given in a dose that was 22 times the molar amount of the lowest enhancing dose of undigested IgG. We therefore conclude that passive enhancement of mouse tumor allografts by alloantibodies is Fc dependent.