A. Latif Kazim

Activation of MHC Class I, II, and CD40 Gene Expression by Histone Deacetylase Inhibitors

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-γ. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and β-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.

Cellular antitumor immune response to a branched lysine multiple antigenic peptide containing epitopes of a common tumor-specific antigen in a rat glioma model

Human malignant gliomas contain epidermal growth factor receptor (EGFR) gene mutations that encode tumor-associated antigens (TAAs) that can be targeted using immunological techniques. One EGFR mutant gene (EGFRvIII) encodes a protein with an epitope that is not found in normal tissues. A number of studies have focused on this unique epitope as a potential target for tumor vaccines. In the present study, we examined the cellular immune effects of a peptide containing multiple copies of the unique EGFRvIII epitope linked together by way of a lysine bridge. Fischer rats were vaccinated with an EGFRvIII multiple antigenic peptide (MAP). While vaccination produced a humoral immune response, anti-MAP antibody production was not accompanied by expression of the Th2 response cytokine IL-4. In MAP/GM-CSF vaccinated animals, a cellular immune response was detected in association with the appearance of CD4+ and CD8+ T cells at the tumor site. Splenocytes and CD8+ T cells from vaccinated rats produced the Th1 cytokine IFN-γ in vitro in response to stimulation by rat glioma cells expressing EGFRvIII, but not by those expressing wild-type EGFR. MAP vaccine also induced a specific lytic antitumor CTL immune response against F98 glioma cells expressing EGFRvIII, but not against F98 cells expressing either wild-type EGFR or no receptor. The in vivo growth of F98EGFRvIII cells was attenuated in vaccinated rats; whereas, growth of F98EGFR cells was not. The median survival of vaccinated rats was increased 72% over that of unvaccinated controls challenged with intracerebral F98EGFRvIII tumor implants. Therefore, MAP vaccination produced a predominantly cellular antitumor immune response directed against F98 gliomas expressing the EGFRvIII target antigen. The potent immunosuppressive effects of F98 glioma cells mimic the human disease and make this particular tumor model useful for studying immunotherapeutic approaches to malignant gliomas.

Peripheral blood mononuclear cells of patients with breast cancer can be reprogrammed to enhance anti-HER-2/neu reactivity and overcome myeloid-derived suppressor cells

Two major barriers in the immunotherapy of breast cancer include tumor-induced immune suppression and the establishment of long-lasting immune responses against the tumor. Recently, we demonstrated in an animal model of breast carcinoma that expanding and reprogramming tumor-sensitized lymphocytes, ex vivo, yielded T memory (Tm) cells as well as activated CD25+ NKT cells and NK cells. The presence of activated CD25+ NKT and NK cells rendered reprogrammed T cells resistant to MDSC-mediated suppression, and adoptive cellular therapy (ACT) of reprogrammed lymphocytes protected the host from tumor development and relapse. Here, we performed a pilot study to determine the clinical applicability of our protocol using peripheral blood mononuclear cells (PBMCs) of breast cancer patients, ex vivo. We show that bryostatin 1 and ionomycin combined with IL-2, IL-7, and IL-15 can expand and reprogram tumor-sensitized PBMCs. Reprogrammed lymphocytes contained activated CD25+ NKT and NK cells as well as Tm cells and displayed enhanced reactivity against HER-2/neu in the presence of MDSCs. The presence of activated NKT cells was highly correlated with the rescue of anti-HER-2/neu immune responses from MDSC suppression. Ex vivo blockade experiments suggest that the NKG2D pathway may play an important role in overcoming MDSC suppression. Our results show the feasibility of reprogramming tumor-sensitized immune cells, ex vivo, and provide rationale for ACT of breast cancer patients.

Emergence of immune escape variant of mammary tumors that has distinct proteomic profile and a reduced ability to induce "danger signals".

SummaryBreast tumors are shaped, in part, by a process termed immunoediting which selects for immunologically evasive phenotypes. In the present study we used the rat neu-transgenic mouse model of breast cancer and its congenic non-transgenic parental strain, FVB, to explore the phenotype of tumors that emerge in the presence of an immune response directed against the neu antigen. When inoculated into parental FVB mice, a neu-overexpressing mouse mammary carcinoma (MMC) cell line isolated from spontaneous breast tumors of the FVB neu (FVBN202) transgenic mouse, elicited a neu-specific immune response resulting in a tumor rejection because of the presence of the rat neu antigen. However, a neu negative variant (ANV) of MMC arose after a long latency in spite of the neu-specific immune response. We show that compared to MMC, ANV tumor cells have a significantly reduced ability to secrete pro-inflammatory cytokines and the CCL5 chemokine, to express immunostimulatory chaperones, and they have a distinct expression of proteins involved in cell motility, and metabolic and signal transduction pathways. These studies suggest that tumor escape through immunoediting can not be explained by the loss of a single tumor antigen, but rather by a selection process of a tumor variant that has a reduced ability to induce “danger signals” together with up-regulation of proteins involved in the tumor survival. Based on these findings, we propose to target novel antigens over-expressed in the escape variant of breast tumors to treat primary tumor and to prevent tumor relapse.

Characterization of a Putative Ovarian Oncogene, Elongation Factor 1α, Isolated by Panning a Synthetic Phage Display Single-Chain Variable Fragment Library with Cultured Human Ovarian Cancer Cells

Purpose: In an effort to identify cell surface targets and single short-chain antibody (scFv) for ovarian cancer therapy, we used a phage display approach to isolate an antibody with high reactivity against ovarian cancer. Experimental Design: A phage scFv library was subjected to panning against human SK-OV-3 ovarian cancer cells. A clone with high reactivity was selected and tested in immunoperoxidase staining on a panel of normal tissues and ovarian carcinoma. Using immunoprecipitation, a differentially expressed band was analyzed by mass spectrometry. The antigen subclass was characterized with reverse transcription-PCR on cDNA library of normal tissues, and 91 ovarian cancer specimens, and correlated with clinicohistopathologic characteristics. Results: Ninety-six individual scFv clones were screened in ELISA following panning. scFv F7 revealed high reactivity with ovarian cancer cell lines and showed intense staining of 15 fresh ovarian cancer specimens and no staining of a panel of normal tissues. A 40-kDa protein was identified to be translation elongation factor 1α1 (EEF1A1; P < 0.05). The expression of EEF1A2, a highly homologous and functionally similar oncogene, was found to be restricted only to the normal tissues of the heart, brain, and skeletal muscle. Aberrant EEF1A2 mRNA expression was found in 21 of 91 (23%) of ovarian cancer specimens and significantly correlated with increased likelihood of recurrence (P = 0.021). Conclusions: scFv F7 may represent an ovarian cancer–specific antibody against translation EEF1A family of translational factors. We propose that EEF1A2 may be a useful target for therapy of human ovarian cancer.

Distinct oligoclonal T cells are associated with graft versus host disease after stem-cell transplantation.

Background In patients with hematologic malignancies who receive stem-cell transplantation, donors’ T cells can recognize minor histocompatibility antigens on recipient cells and generate an objective response against the tumor. However, a major side effect of such therapy is graft-versus-host disease (GVHD). The purpose of this study was to characterize distinct T-cell clones that were frequently and exclusively involved in GVHD or graft-versus-tumor (GVT) effects. Methods We hypothesized that distinct GVHD-associated T-cell clones can be identified during the disease progression. To test this, we conducted comparative analysis of T-cell receptor (TCR) V&bgr;s in donor-recipient pairs of patients with GVHD versus those with GVHD-free and relapse-free survival using quantitative reverse-transcriptase polymerase chain reaction and spectratyping analyses. Results We identified three sets of T-cell clones that were either frequently involved in GVHD (TCR V&bgr;4, 11, and 23) or GVT effect (TCR V&;9, 16, and 20), or were increased at the time of GVHD and GVT effects in a patient-specific manner (TCR V&bgr;2, 3, 7, 12, 15, and 17). Spectratyping analysis showed restricted clonality of the identified TCR V&bgr;s. Polymerase chain reaction analysis also confirmed the presence of GVHD-associated T-cell clones at the site of the disease. Conclusions These data suggest that GVHD- and GVT-associated clones can be distinguished by molecular analysis of TCR V&bgr; to develop targeted therapy for GVHD.

Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo

Purpose: We employed a grp170-secreting tumour cell system to determine whether tumour cells engineered to secrete grp170 generate an antitumour-specific immune response. Further, we examine the possibility that secreted grp170 can bind to and co-transport out of tumour cells full-length tumour antigens that may play a role in the anti-tumour immune response. Materials and methods: Wild type Colon-26 and Colon-26 engineered to secrete grp170 were subcutaneously inoculated into BALB/c mice. Tumour growth was monitored, and variations in immunoregulatory mechanisms were evaluated using immunohistochemistry, lymphocyte depletion, ELISpot assays, and Western blot analysis. Results: Immunisation of animals with grp170-secreting tumour cells results in rejection of the tumour by induction of antigen-specific, CD8-dependent immune responses. The secreted grp170 is able to deliver full-length tumour antigens to the tumour microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate tumour-specific immune responses. Conclusions: These data parallel our studies showing that hsp110 or grp170 are able to chaperone full-length proteins, and when complexed with protein antigens and used as vaccines, these complexes elicit immune responses in vivo against the protein antigens. This cell-based approach has the potential to be utilised as a tumour-specific vaccine in tumours of various histological origins.

Prolidase Directly Binds and Activates Epidermal Growth Factor Receptor and Stimulates Downstream Signaling

Background: All known ligands of EGF receptor (EGFR) are characterized by the EGF motif and generated from transmembrane precursors. Results: Prolidase, a cytosolic dipeptidase devoid of EGF motif, binds and activates EGFR independent of its dipeptidase activity when present outside of cell. Conclusion: Prolidase is a novel EGFR ligand. Significance: This shows a new function of prolidase and new mechanism of EGFR activation. Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV‐1 gag RNA. Named HRz‐W and HRz‐M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secondary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D20, following the imino protons. The data show that both HRz‐M and HRz‐W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy mimimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.