InSug O-Sullivan

FoxO1 integrates direct and indirect effects of insulin on hepatic glucose production and glucose utilization

FoxO proteins are major targets of insulin action. To better define the role of FoxO1 in mediating insulin effects in the liver, we generated liver-specific insulin receptor knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice. Here we show that LIRKO mice are severely insulin resistant based on glucose, insulin and C-peptide levels, and glucose and insulin tolerance tests, and genetic deletion of hepatic FoxO1 reverses these effects. (13)C-glucose and insulin clamp studies indicate that regulation of both hepatic glucose production (HGP) and glucose utilization is impaired in LIRKO mice, and these defects are also restored in LIRFKO mice corresponding to changes in gene expression. We conclude that (1) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and utilization, and (2) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted.

Intratumoral injection of IL-secreting syngeneic/allogeneic fibroblasts transfected with DNA from breast cancer cells prolongs the survival of mice with intracerebral breast cancer.

Prior studies have revealed the immunotherapeutic properties of a vaccine prepared by transfer of genomic DNA from breast cancer cells into a highly immunogenic cell line. The rationale for this type of vaccine is that genes specifying an array of weakly immunogenic, unique tumor antigens associated with the malignant cells will be expressed in a highly immunogenic form by the transfected cells. Here, the immunotherapeutic properties of a vaccine prepared by transfection of mouse fibroblasts with DNA from a breast carcinoma (SB-5b) that arose spontaneously in a C3H/He mouse (H-2Kb) were tested in mice with intracerebral breast cancer. To augment their nonspecific immunogenic properties, before DNA transfer, the fibroblasts (of C3H/He mouse origin) were modified to express allogeneic MHC class I H-2Kb-determinants and to secrete IL-2, IL-18 or GM-CSF. The results indicate that C3H/He mice injected intracerebrally (i.c.) with the breast cancer cells and syngeneic/allogeneic-transfected fibroblasts modified to secrete IL-2 survived significantly longer (P<.005) than mice in various control groups, including mice injected i.c. with the breast cancer cells alone. The immunotherapeutic properties of transfected fibroblasts modified to secrete IL-18 or GM-CSF were less efficacious. The results of two independent in vitro cytotoxicity assays indicate that systemic cellular antitumor immunity was generated in mice injected i.c. with the transfected cells, and the immunity was mediated predominantly by CD8+ T cells.

Combined therapy of an established, highly aggressive breast cancer in mice with paclitaxel and a unique DNA‐based cell vaccine

Here, we describe the enhanced benefits of treating a highly aggressive breast cancer in mice with a combination of paclitaxel and immunization with a unique DNA‐based cell vaccine. An adenocarcinoma was isolated from a spontaneous neoplasm that arose in the mammary gland of a C3H/He mouse (H‐2k) (SB5b cells). The vaccine was prepared by transfer of genomic DNA‐fragments (25 kb) from the breast cancer cells into a mouse fibroblast cell line (LM), modified to enhance its immunogenic properties. As the transferred DNA is integrated, and replicated as the recipient cells divide, the vaccine could be prepared from relatively small numbers of cancer cells (107 = 4 mm tumor). SB5b cells were injected into the mammary fat pad of naïve C3H/He mice, which are highly susceptible to the growth of the cancer cells. When the tumors reached ˜3 mm, the mice were injected s.c. with a noncurative dose of paclitaxel. Six days later, when immune competence returned, the mice received the first of 3 weekly s.c. injections of the vaccine. The combined therapy induced robust cellular immunity to the breast cancer, mediated by CD8+ and NK/LAK cells, which resulted in prolonged survival. The immunity was specific, as immunization with a vaccine prepared by transfer of DNA from B16 melanoma cells into the fibroblasts failed to induce immunity to the breast cancer. This type of vaccine raises the possibility that an analogous strategy could be used in the treatment of breast cancer patients at an early stage of the disease.

Immunity to growth factor receptor-bound protein 10, a signal transduction molecule, inhibits the growth of breast cancer in mice.

This study describes the application of a unique strategy to identify breast cancer antigens [tumor-associated antigen (TAA)]. In a mouse model, the strategy led to the identification of growth factor receptor-bound protein 10 (Grb10) as a newly identified TAA. Grb10 is a signal transduction molecule associated with multiple transmembrane tyrosine kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice with nonenriched vaccines. The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line C3H/He origin (H-2(k)), into LM mouse fibroblasts (H-2(k)). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell population, however, incorporated cDNA fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was used, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were overrepresented in the enriched vaccines. One, the gene for Grb10, was approximately 100-fold overrepresented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor rejection.

Coupling between Nutrient Availability and Thyroid Hormone Activation

Background: Insulin/IGF-1 stimulates thyroid hormone action via type 2 deiodinase (D2). Results: Insulin/IGF-1-induced activation of the PI3K-mTORC2-Akt pathway transcriptionally up-regulates D2. Conclusion: FOXO1 represses DIO2 during fasting, and derepression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway. Significance: This mechanism explains how T3 production, serum T3 levels, and T3-dependent cellular metabolic rate are kept at a level proportionate to the availability of energy substrates. The activity of the thyroid gland is stimulated by food availability via leptin-induced thyrotropin-releasing hormone/thyroid-stimulating hormone expression. Here we show that food availability also stimulates thyroid hormone activation by accelerating the conversion of thyroxine to triiodothyronine via type 2 deiodinase in mouse skeletal muscle and in a cell model transitioning from 0.1 to 10% FBS. The underlying mechanism is transcriptional derepression of DIO2 through the mTORC2 pathway as defined in rictor knockdown cells. In cells kept in 0.1% FBS, there is DIO2 inhibition via FOXO1 binding to the DIO2 promoter. Repression of DIO2 by FOXO1 was confirmed using its specific inhibitor AS1842856 or adenoviral infection of constitutively active FOXO1. ChIP studies indicate that 4 h after 10% FBS-containing medium, FOXO1 binding markedly decreases, and the DIO2 promoter is activated. Studies in the insulin receptor FOXO1 KO mouse indicate that insulin is a key signaling molecule in this process. We conclude that FOXO1 represses DIO2 during fasting and that derepression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway.

Treatment of squamous carcinoma in mice with a vaccine enriched for cells that induce immunity to squamous carcinoma—A new vaccination strategy

We report a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA‐fragments (25 kb) from squamous carcinoma cells (KLN205, DBA/2 origin (H‐2d)) into LM mouse fibroblasts (C3H/He origin; H‐2k), a highly immunogenic cell line. To enhance their nonspecific immunogenic properties, the fibroblasts were modified before DNA transfer to secrete IL‐2 and to express additional allogeneic MHC class I determinants. As the transferred DNA integrates into the genome of the recipient cells, and is replicated as the cells divide, sufficient DNA to prepare the vaccine could be obtained from as few as 107 squamous carcinoma cells (4 mm tumor). Since only a small proportion of the transfected cell‐population was expected to have incorporated genes specifying antigens associated with the squamous carcinoma cells (TAA), we devised a novel approach to enrich the vaccine for cells that induce immunity to the SCC. Aliquots of the transfected population were divided into 10 small pools (initial inoculums = 1 × 103). We reasoned that if the starting inoculums were sufficiently small, then the distribution of highly immunogenic and weakly immunogenic cells in each pool would not be the same. Cells from individual pools were allowed to increase in number. A portion of the expanded cell populations were maintained frozen/viable for later recovery. The remaining portions were used to immunize naïve DBA/2 mice. Pools containing greater numbers of immunogenic cells were identified by 2 independent assays. Frozen aliquots of cells from the pool that stimulated immunity to the squamous carcinoma to the greatest extent were recovered and subdivided for additional rounds of immune selection. Enhanced immunity to squamous carcinoma mediated by CD8+ T cells was induced in tumor‐bearing mice treated solely by immunization with the enriched cell‐population.

Immunity to squamous carcinoma in mice immunized with dendritic cells transfected with genomic DNA from squamous carcinoma cells

Immunotherapy of squamous cell carcinoma (SCC) at an early stage of the disease increases the likelihood of success. We report a new vaccination strategy designed to prepare SCC vaccines from microgram amounts of tumor tissue, enabling the treatment of patients with minimal residual disease. The vaccine was prepared by transfer of sheared genomic DNA-fragments (25 kb) from KLN205 cells, an SCC cell line of DBA/2 mouse origin, into syngeneic bone marrow-derived mature dendritic cells (DCs). More than 90% of the transfected DCs took up DNA from the neoplasm and transferred genes were expressed as protein. The DCs expressed CD11c, CD11b, and the costimulatory molecules CD40, CD80 and CD86, characteristic of mature DCs. Syngeneic DBA/2J mice, highly susceptible to the growth of KLN205 cells, were injected intravenously (i.v.) with the transfected DCs, followed by a subcutaneous (s.c.) injection of the tumor cells. The strong immunogenic properties of the transfected cells were indicated by the finding that the survival of the tumor-bearing mice was prolonged (P<.001), relative to that of mice in various control groups. Enzyme-linked immuno spot (ELISPOT IFN-γ) assays revealed the activation of cell-mediated immunity directed toward the SCC in mice immunized with the transfected DCs. Two independent in vitro cytotoxicity assays indicated the presence of robust cell-mediated immunity directed toward the SCC in mice immunized with the transfected cells.

A DNA adjuvant encoding a fusion protein between anti-CD3 single-chain Fv and AIMP1 enhances T helper type 1 cell-mediated immune responses in antigen-sensitized mice

T helper type 1 (Th1) cell‐mediated immune responses contribute to host defences against intracellular pathogen infections and cancer. Previously, we found that aminoacyl tRNA synthetase‐interacting multifunctional protein 1 (AIMP1) activated macrophages and dendritic cells to enhance Th1 responses. Herein, we manipulated this property to improve Th1 immune responses in vivo by constructing a mammalian expression plasmid (pAnti‐CD3sFv/AIMP1) encoding AIMP1 fused to the anti‐CD3 single‐chain Fv (sFv), the smallest unit of the antibody that interacts with the CD3ε region of the T‐cell receptor. Intramuscular injection of ovalbumin (OVA)‐sensitized BALB/c mice with pAnti‐CD3sFv/AIMP1 DNA adjuvant increased the OVA‐specific, interferon‐γ production by their CD4+ T cells and the levels of anti‐OVA immunoglobulin G2a (IgG2a) isotype in their sera. Furthermore, the pAnti‐CD3sFv/AIMP1 DNA adjuvant decreased interleukin‐4 production and anti‐OVA IgE levels in the OVA‐injected mice. Importantly, the pAnti‐CD3sFv/AIMP1 was more efficient than a mixture of pAnti‐CD3sFv and pAIMP1 in inducing OVA‐specific Th1 immune responses and also in inhibiting OVA‐specific Th2 responses during antigen priming. These studies indicated that the pAnti‐CD3sFv/AIMP1 fusion DNA adjuvant enhanced Th1 immune responses in antigen‐sensitized mice.

New strategy for the identification of squamous carcinoma antigens that induce therapeutic immune responses in tumor-bearing mice

This study describes a new strategy for the identification of squamous carcinoma antigens tumor-associated antigens (TAA). The antigens were discovered by comparing microarrays of squamous carcinoma vaccines highly enriched for immunotherapeutic cells with non-enriched vaccines. The vaccines were prepared by transferring sheared genomic DNA fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 mouse origin (H-2d) into LM fibroblasts (C3H/He origin, H-2k). The transferred tumor DNA segments integrate spontaneously into the genome of the recipient cells, replicate as the cells divide and are expressed. As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying TAA (the vast majority specify normal cellular constituents), a novel strategy was employed to enrich the vaccine for TAA-positive cells. Microarrays were used to compare genes expressed by enriched and non-enriched vaccines. Seventy-five genes were overexpressed in cells from the enriched vaccine. One, the gene for Cytochrome P450 (family 2, subfamily e, polypeptide 1) (Cyp2e1), was overexpressed in the enriched but not the non-enriched vaccine. A vaccine for squamous carcinoma was prepared by transfer of a 357 bp fragment of the gene for Cyp2e1 into the fibroblast cell line. Robust immunity, sufficient to result in indefinite survival, was induced in tumor-bearing mice immunized with cells transfected with this gene fragment.

T-regulatory cells are relatively deficient in squamous carcinomas undergoing regression in mice immunized with a squamous carcinoma vaccine enriched for immunotherapeutic cells

In a prior report (Int J Cancer 2006; 119: 339–348), we described a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA-fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 origin; H-2d) into LM cells, a highly immunogenic mouse fibroblast cell line (C3H/He origin; (H-2k)). As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying antigens associated with the squamous carcinoma cells, we devised a novel strategy to enrich the vaccine for immunotherapeutic cells. Enhanced immunity to squamous carcinoma was induced in tumor-bearing mice treated solely by immunization with the enriched vaccine, which translated into prolonged survival without toxicity. Here, we describe the characteristics of the cell populations infiltrating established squamous carcinomas undergoing regression in mice immunized with vaccines enriched for immunotherapeutic cells. The results indicated that CD8+ T cells were predominant and that T-regulatory cells (FoxP3+, CD4/CD25+, CD4/CD62Lhigh, CD4/CTLA-4e) were relatively deficient in the regressing tumors. Inflammatory infiltrates were not detected in various organs and tissues of mice immunized with the DNA-based vaccine.