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Supplementary Figures 1-3, Tables 1-3 from Synthetic MicroRNA Designed to Target Glioma-Associated Antigen 1 Transcription Factor Inhibits Division and Induces Late Apoptosis in Pancreatic Tumor Cells more...

Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA.Experimental Design: We hypothesized that targeting the 3′-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3′-untranslated region Gli-1 sequence of high GU content.Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA.Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers. more...

Supplementary Figures 1-3 from Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)+ melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11–30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11–30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11–30 VH chain, VH (3–11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76–84). Lymphocytes from HLA-A2+-immunized patients proliferated to VH (3–11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76–84). Significant increase in IFN-γ production but not in interleukin 10 production in response to VH (3–11) and to variant HMW-MAA peptide (76–84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2+ patients with the two peptides induced CTL, which lysed HMW-MAA+/HLA-A2+ A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences. more...

It has been proposed that chemotherapy enhances tumor antigen (TA)–specific immunity. The molecular form of TA from ovarian tumor that activates cellular immunity is unknown. We report here identification of a novel molecular form of immunogenic TA for CD8+ cells named self-immune stimulatory multimolecular complexes (ISMMC). ISMMC consist of a molecular complex of polyosome/ribosome-bound ubiquitinated nascent HER-2 polypeptides. This complex is chaperoned by heat shock protein Gp96, which mediates ISMMC uptake by antigen-presenting cells through the scavenger receptor CD91. RNAs in ISMMC stimulate immature dendritic cells to secrete interleukin 12 and induce IFN-γ in peripheral blood mononuclear cells. ISMMC dissociate, retrotranslocate from the lysosome to cytoplasm, and are processed to peptides by the proteasome. At subpharmacologic doses, Taxol increased the amount of ISMMC by three to four times and modified their composition by inducing the attachment of cochaperones of HSP70, such as the mitotic-phase phosphoprotein 11J. On a total protein basis, Taxol induced ISMMC, expanded more CD8+ cells, activated more CD56+ NKG2D+ cells to produce IFN-γ, and were more potent inducers of high T-cell receptor density Perforin+ cells than native ISMMC and peptide E75. Elucidation of the composition of ISMMC and identification of adducts formed by Taxol should be important for developing molecular cancer vaccines. [Cancer Res 2007;67(17):8378–87] more...

Supplementary Tables 1-5, Figure Legends 1-3 from Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape. more...

Supplementary Methods from Fine Specificity of High Molecular Weight-Melanoma-Associated Antigen-Specific Cytotoxic T Lymphocytes Elicited by Anti-Idiotypic Monoclonal Antibodies in Patients with Melanoma more...

Supplementary Figure 1 from BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells