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Notch and Hedgehog activate cell-cycle progression of adult and cancer stem cells. Notch is activated by DLL and Jag presents on neighboring cells. We investigated the effects of density of the Notch-activating ligand, Jag-1, and targeting Gli-1, in activation of division of paclitaxel/taxol-resistant, (PTX Res) ovarian cancer cells SKOV3 (SKOV3). We used the specific gamma-presenilin inhibitor, DAPT, to identify the specificity of activating signals for Notch-1 and created 'butterfly-duplex-3548-Gli-1-inhibitory RNA' (i-Gli-1.RNA) to inhibit cell division. To accurately quantify kinetics of division, the expression of CD44 and CD24 was determined in each gated population of divided cells. CD44 High proliferated when activated by Jag-1 Low and poorly when activated by Jag-1 High. DAPT inhibited proliferation of cells activated by Jag-1 Low, and increased proliferation of cells activated by Jag-1 High. Only 5-10% of cells activated by Jag-1 High and Jag-1 Low divided fast, polynomial, and symmetric. i-Gli-1.RNA eliminated more than 50% of the small CD44 High/CD24 Neg cells in divisions 3 and 4. This effect appeared specific compared with cells transfected with negative control siRNA. i-Gli-1.RNA had no effect on large CD44 High/CD24 Neg cells, but inhibited the population of CD44 High/CD24 Low cells. Expansion of CD44 High inversely correlated with Jag-1 density on activating autologous tumor and fibrosarcoma cells. Created i-RNAs may decrease the resting CSC pool. Notch and Gli-1 signals play an important role in proliferation/division and survival of cancer stem cells. Targeting Notch-1 through its enhancer Gl-1, should be significant for novel treatments to eliminate taxol-resistant cancer stem cells (CSC). i.Gli-1 RNA should be more effective if used together with Taxol.

The transcription factor glioma-associated antigen-1 (Gli-1) mediates activation of the sonic hedgehog (Shh) pathway, a process that precedes the transformation of tissue stem cells into cancerous stem cells and that is involved in early and late epithelial tumorigenesis. Hypothesizing that targeting the 3'-untranslated region (3'-UTR) of Gli-1 mRNA would effectively inhibit epithelial tumor cell proliferation, we evaluated several complementary miRNA molecules for their ability to do so. The synthetic miRNAs and corresponding duplex/small temporal RNAs were introduced as 3-nucleotide (nt) loops into GU-rich portions of the 3'UTR Gli-1 sequence. One particular miRNA (miRNA Gli-1-3548) and its corresponding duplex (Duplex 3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells by delaying cell division and activating late apoptosis in MiaPaCa-2 cells. Here, we describe the design of effective miRNA sequences and their applications as anti-gene agents.

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-gamma 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-gamma, 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.

Notch is a plasma membrane receptor involved in the control of cell fate specification and in the maintenance of the balance between proliferation and differentiation in many cell lineages. Disruption of Notch has been implicated in a variety of hematological and solid cancers. Numb is also expressed in many adult mammalian cells. Adult cells divide symmetrically, and Numb is symmetrically partitioned at mitosis. The Numb-mediated regulation of Notch is believed to play a causative role in naturally occurring breast cancers. Reduction of Numb levels in breast tumors is regulated by proteasomal degradation. We reasoned that if the disregulated negative control of Notch by Numb protein is the consequence of Numb proteasomal degradation, then degradation of Numb can generate peptides which are transported, presented by MHC-I molecules. Surprisingly we found few candidate naturally processed peptides from Notch1, Notch2, and Numb1. CD8+ T cells expressing TCRs which specifically recognized peptides Notch1 (2112-2120) and Numb1 (87-95) were presented in the ascites of ovarian cancer patients. Many of these cells were differentiated and expressed high levels of Perforin. The natural immunogenicity of Notch1 and particularly of Numb1 suggests a mechanism of immunosurveillance which is overcome during tumor progression. Immunotherapy with tumor antigens from Notch and Numb should be important for treatment of cancer patients.

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.

MicroRNA (miRNA) are a class of non-coding RNAs found both in normal tissues and cancer cells. The natural miRNA, which target cancer genes for transcriptional repression are still unknown. Approaches for synthetic miRNA design targeting cancer genes have not yet been established. We designed miRNAs targeting the 3' UTR (nt 4350-4372) of HER-2 proto-oncogene. One miRNA (miR-14U) was designed by introducing a mutation in the nt 14 counting from the 5' end of anti-sense RNA. Two others (miR4350-10GGA and miR4350-11AAGCU) were designed by introducing either loop-forming nucleotides in position 10 or a part of the complementary sequence of the Brd-box consensus sequence, in position 11. miR4350-10GGA was more effective than the anti-sense strand in decreasing the numbers of ovarian tumor SKOV3 cells, which over-expressed HER-2 protein. Its inhibitory effects were lower than that of corresponding double-stranded (ds) RNA4350-4372. Inhibition of HER-2 expression mediated by miRNAs was higher in cells expressing higher levels of HER-2 protein than in cells expressing lower levels of HER-2 protein. This is the first demonstration of inhibition of expression of a constitutively over-expressed tumor protein by designed synthetic miRNA and its cor-responding dsRNA targeting 3' untranslated regions in mRNA. Our results also show that measuring the effects of miRNA and dsRNA in pooled populations of tumor cells, which express various levels of target oncogenes, may reflect the survival responses of siRNA resistant tumors rather than the growth inhibitory responses of siRNA-sensitive tumors.

The current modalities for treating cancer employ not only single but multiple approaches involving surgery, radiotherapy and chemotherapy. Unfortunately, the survival outcome is not promising even with these approaches. Alternative approaches for cancer therapy are now emerging. Immunotherapy is aiming at both increasing the power, and in redirecting the specificity of the patients' immune system to attack the tumor cells. Recently, many studies using tumor associated lymphocytes (TAL) isolated from malignant ascites cultured in a media containing interleukin-2 exhibit antitumor responses. IL-2 is a lymphokine produced by T-cells. It facilitates activation, sustained growth and rescue from apoptosis. Lately, newly developed IL-15 has also exhibited antitumor activity similar to IL-2. IL-15 is a newly described cytokine produced from monocytes-marcrophages and T-cells. It has a different molecular structure but it functions like IL-2 by binding to the IL-2R beta and gammac chain. These antitumor responses are mediated by the cytotoxic T lymphocytes (CTL) that recognize the antigen in the context of the MHC molecules using the T cell receptors. CD8+-CTL recognize the peptide epitopes that are processed from the cellular proteins in the context of the MHC class I molecules. These peptides have a restricted length of 8-11 amino acids. The folate binding protein (FBP) is overexpressed in over 90% of ovarian and 20-50% in breast cancers. The FBP is the source of the antigenic peptides that are recognized by a number of these CTL-TAL, and is antigenic to both ovarian and breast cancer in vivo. To define the antitumor response of IL-15 and its' FBP immunogenicity, a peptide defining epitope E39 and E75 were presented by the PMBC derived dendritic cells (DC) from healthy donors isolated by the CD14 method to ovarian and breast CTL-TAL. Stimulating both ovarian and breast CTL- TAL by E39 or E75 pulsed DC (DC-E39, DC-E75), in the presence of IL-15 and IL-2 can rapidly enhance or induce the E39 or E75 specific CTL activity. The antitumor activities were measured by a chromium release assay for the tumor specific lysis activity using the ovarian and breast cancer cell lines. The tumor specific lysis activity for the ovarian TALs for IL-15 vs IL-2 were 28.6 +/- 3.9% and 30.3 +/- 3.2%, respectively and in the breast TALs, they were 14.8 +/- 3.1% vs 13.5 +/- 2.9%, respectively. Using autologous tumor cells, a slightly higher tumor specific lysis activity was obtained for the ovarian TALs cultured in IL-15 compared to IL-2 (72.0 +/- 8.2% vs 68.5 +/- 3.6%). However, for the breast TALs, they were 39.5 +/- 4.2% vs 41.5 +/- 3.3%, respectively. IL-15 is a newly developed cytokine that shows promising antitumor activity similar to IL-2. However, it requires lower dosage and is less toxic. Therefore, IL-15 might be a potential anticancer immunotherapeutic agent.

To determine the toxicity and immunogenicity of the HER-2/neu, HLA-A2-restricted peptide E75 in patients with metastatic breast and ovarian cancer, 14 patients were vaccinated with escalating amounts of E75 (100, 500, and 1000 microg) mixed with 250 microg granulocyte macrophage colony-stimulating factor as adjuvant. Each vaccine dose was administered in a total volume of 1.5 ml divided into four intradermal injections and administered weekly for 4 weeks, followed by monthly boosts for a total of 10 injections. Vaccinations were well tolerated without significant toxicity. Blood was drawn before, at 8 weeks, and up to 13-16 months after vaccination for measurement of cellular immunity. Seven of 8 patients tested had significant delayed type hypersensitivity to E75 defined as >5 mm induration. Peripheral blood mononuclear cells from 5 of 9 patients tested proliferated to E75 with a stimulation index of > or = 2.0. Of 8 vaccinated patients tested for induction of a CTL response, 4 responded to stimulation by autologous dendritic cells plus cytokines by eliciting E75-specific lytic activity consistent with the presence of activated/memory cells, 2 others after in vitro stimulation with E75 + interleukin-12 +/- anti-CD152(33KD), whereas 2 others did not respond. Four patients with E75-specific CTLs present specifically recognized E75 on indicator tumors as demonstrated by cold-target inhibition of tumor lysis. These 4 patients showed E75-specific IFN-gamma production. peripheral blood mononuclear cell from 3 of these patients proliferated to E75, but stimulation indices were higher in the prevaccine samples. All 4 of the patients showed DTH responses to E75. These results demonstrate that vaccination with E75+ granulocyte macrophage colony-stimulating factor can induce both peptide-specific IFN-gamma and epitope specific CTLs, which lyse HER-2/neu+ tumors in stage IV patients.