Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness.

BACKGROUND: We recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-ß receptor enhances its signaling and promotes breast cancer invasion and metastasis. However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown. METHOD: We performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, ingenuity pathway analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. RESULTS: Loss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity pathway analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvß5 or IL6ST might be crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial-mesenchymal transition and metastasis. CONCLUSIONS: Our report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis.

FUT8 promotes breast cancer cell invasiveness by remodeling TGF-ß receptor core fucosylation.

BACKGROUND: Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-ß signaling during breast cancer progression remains largely unknown. METHOD: In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-ß-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-ß1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. RESULTS: Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-ß-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-ß receptor complexes might be core fucosylated by FUT8 to facilitate TGF-ß binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. CONCLUSIONS: Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-ß signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.

SCUBE3 (signal peptide-CUB-EGF domain-containing protein 3) modulates fibroblast growth factor signaling during fast muscle development.

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

OBJECTIVE: Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. APPROACH AND RESULTS: We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. CONCLUSIONS: We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy.

Zebrafish scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis.

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.

Structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition.

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies.

A Comprehensive Analysis of FUT8 Overexpressing Prostate Cancer Cells Reveals the Role of EGFR in Castration Resistance.

The emergence of castration-resistance is one of the major challenges in the management of patients with advanced prostate cancer. Although the spectrum of systemic therapies that are available for use alongside androgen deprivation for treatment of castration-resistant prostate cancer (CRPC) is expanding, none of these regimens are curative. Therefore, it is imperative to apply systems approaches to identify and understand the mechanisms that contribute to the development of CRPC. Using comprehensive proteomic approaches, we show that a glycosylation-related enzyme, alpha (1,6) fucosyltransferase (FUT8), which is upregulated in CRPC, might be responsible for resistance to androgen deprivation. Mechanistically, we demonstrated that overexpression of FUT8 resulted in upregulation of the cell surface epidermal growth factor receptor (EGFR) and corresponding downstream signaling, leading to increased cell survival in androgen-depleted conditions. We studied the coregulatory mechanisms of EGFR and FUT8 expression in CRPC xenograft models and found that castration induced FUT8 overexpression associated with increased expression of EGFR. Taken together, our findings suggest a crucial role played by FUT8 as a mediator in switching prostate cancer cells from nuclear receptor signaling (androgen receptor) to the cell surface receptor (EGFR) mechanisms in escaping castration-induced cell death. These findings have clinical implication in understanding the role of FUT8 as a master regulator of cell surface receptors in cancer-resistant phenotypes.

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model.

BACKGROUND: Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration. METHODS: In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine. RESULTS: The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicity T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunity as indicated by depletion of lymphocytes in vivo. CONCLUSION: Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.

The opposing effects of lipopolysaccharide on the antitumor therapeutic efficacy of DNA vaccine.

DNA vaccine represents a novel method to elicit immunity against infectious disease. Lipopolysaccharide (LPS) copurified with plasmid DNA may affect therapeutic efficacy and immunological response. We aimed to study the effect of LPS on the therapeutic efficacy of HER-2/neu DNA vaccine in a mouse tumor animal model. Plasmid DNA purified from commercial EndoFree plasmid purification kits functioned as a better therapeutic DNA vaccine than that purified from Non-EndoFree purification kit, which contains >or=0.5 microg LPS per 100 mg DNA plasmid. To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine, increasing amount of LPS was added to endotoxin-free plasmid DNA, and inoculated on mice with established tumors. One mug of LPS significantly attenuated the therapeutic effect of neu DNA vaccine and increased Th2 immune responses bias with interleukin-4 cytokine production. In contrast, high amount (100 microg) of LPS enhanced the therapeutic efficacy of neu DNA vaccine with an increase of cytotoxic T lymphocyte response and Th1 immune response. The effect of LPS on DNA vaccine was diminished when the tumor was grown in toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice. Our results indicate that variation in the LPS doses exerts opposing effects on the therapeutic efficacy of DNA vaccine, and the observed effect is TLR4 dependent.

Transgenic overexpression of the secreted, extracellular EGF-CUB domain-containing protein SCUBE3 induces cardiac hypertrophy in mice.

OBJECTIVE: The aim of this study was to investigate in a transgenic animal model the cardiac expression and function of a novel extracellular protein SCUBE3 [signal peptide-CUB (complement proteins C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor)-like domain-containing protein 3]. METHODS AND RESULTS: Real-time quantitative reverse transcriptase (RT)-PCR analysis showed that SCUBE3 is expressed in the ventricular myocardium. Male transgenic (TG) mice overexpressing SCUBE3 appeared normal during development, from birth to adulthood. However, echocardiography and histopathological examination revealed significant cardiac hypertrophy in TG animals as they aged, at 8 months. Interestingly, left-ventricle hypertrophy occurred more rapidly and more severely under pressure overload in TG mice, as compared to age-matched wild-type littermates. Induced SCUBE3 expression, together with elevated transforming growth factor (TGF)-beta1 level under pressure overload, may account for the accelerated onset and progression of cardiac hypertrophy in TG mice. Furthermore, biochemical and molecular studies revealed that the carboxyl-terminal portion of SCUBE3 could physically interact with TGF-beta1 and promote the TGF-beta1-mediated transcriptional activation. CONCLUSION: This report is the first demonstration that SCUBE3 may play a role in the regulation of cardiac growth and remodeling responses, possibly through the stabilization of the TGF-beta1 signaling pathway.

Localization and characterization of a novel secreted protein SCUBE1 in human platelets.

OBJECTIVE: The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1. METHODS AND RESULTS: Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the alpha-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively. CONCLUSION: These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.

Localization and characterization of an orphan receptor, guanylyl cyclase-G, in mouse testis and sperm.

We recently identified a novel testis-enriched receptor guanylyl cyclase (GC) in the mouse, designated mGC-G. To further investigate its protein expression and function, we generated a neutralizing antibody specifically against the extracellular domain of this receptor. RT-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both the mRNA and protein levels. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell surface protein restricted to the plasma membrane overlying the acrosome and midpiece of the flagellum in mature sperm. Interestingly, Western blot analysis demonstrates that testicular mGC-G is approximately 180 kDa but is subject to limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on the mature sperm surface. On Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that serum albumin-induced elevation of sperm intracellular Ca(2+) concentration, protein tyrosine phosphorylation, and progressive motility associated with capacitation are markedly reduced by preincubation of the anti-mGC-G neutralizing antibody. Together, these results indicate that mGC-G is proteolytically modified in mature sperm membrane and suggest that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology.

Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1(Delta cub/Delta cub)) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7-9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1(Delta cub/Delta cub) embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.

Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.

OBJECTIVES: This study investigates the potential application of plasma SCUBE1 [signal peptide-CUB (complement C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor)-like domain-containing protein 1] as a biomarker of platelet activation in acute coronary syndrome (ACS) and acute ischemic stroke (AIS). BACKGROUND: Platelet activation plays a crucial role in ACS and AIS. Platelet stimulation is associated with increased plasma concentration of SCUBE1, a novel platelet-endothelial secreted protein identified in our previous study. METHODS: Plasma concentrations of SCUBE1 from 40 ACS and 40 AIS patients were measured by enzyme-linked immunoadsorbent assay and compared with the levels of 40 healthy control subjects and 83 chronic coronary artery disease (CAD) patients. Two-dimensional electrophoresis followed by Western blotting was used to characterize SCUBE1 protein in patients' plasma. RESULTS: Plasma SCUBE1 concentration was virtually undetectable in healthy control subjects and CAD patients, but was significantly higher in ACS and AIS patients (median = 205 and 95.1 ng/ml, respectively, p < 0.01). The increase in plasma SCUBE1 was detectable in the plasma as early as 6 h after the onset of symptoms and remained detectable up to 84 h. Plasma SCUBE1 concentration is an independent predictor of stroke severity based on National Institutes of Health Stroke Scale (beta = 3.18, p < 0.001). Furthermore, smaller SCUBE1 fragments were detected in ACS patients' plasma, suggesting that plasma SCUBE1 might subject to a proteolytic regulation under pathological conditions. CONCLUSIONS: Plasma SCUBE1 concentration is significantly elevated in ACS and AIS but not CAD patients. Plasma SCUBE1 is a potential biomarker of platelet activation in acute thrombotic disease.

Autologous neu DNA vaccine can be as effective as xenogenic neu DNA vaccine by altering administration route.

We examined the therapeutic efficacy of xenogenic human N'-terminal neu DNA vaccine and autologous mouse N'-terminal neu DNA vaccine on MBT-2 tumor cells in C3H mice. Intramuscular injection of xenogenic and autologous neu DNA vaccines produced comparable therapeutic efficacies. Mouse and human N'-neu DNA vaccine induced tumor infiltration of CD8(+) T cells, while the human vaccine was less effective at stimulating natural killer cells. Depletion of CD8(+) T cells abolished the therapeutic efficacy of both types of DNA vaccines. On the other hand, xenogenic neu DNA vaccine showed significantly better therapeutic efficacy than autologous DNA vaccine with gene gun immunization. Increased infiltration of CD8(+) T cells was correlated with enhanced therapeutic efficacy in the human N'-neu group of mice. Therefore, intramuscular injection can enhance the therapeutic efficacy of autologous neu DNA vaccine.

Inhibitor of heat-shock protein 90 enhances the antitumor effect of DNA vaccine targeting clients of heat-shock protein.

Geldanamycin (GA), a heat-shock protein (HSP) 90 inhibitor, induces degradation of HSP90 client proteins, which may promote the presentation of degradation peptides with major histocompatibility complex class I on cancer cells. We hypothesized that GA may enhance the efficacy of DNA vaccination, and investigated the therapeutic effect of the combination of GA and a DNA vaccine against HSP90 clients p185(neu) and Met. The efficacy of various doses of GA combined with an N-terminal neu (N'-neu) DNA vaccine was investigated in a transplanted tumor constitutively overexpressing endogenous p185(neu). Low-dose (2.5 mug) but not high-dose (10 microg) GA enhanced the effect of N'-neu DNA vaccination on the inhibition of murine bladder tumor-2 tumors in syngeneic C3H mice. Anti-p185(neu) antibody titers were similar among all treated groups. Significantly increased infiltrations of CD8(+) T cells and NK cells were observed at tumor sites. GA sensitized tumor cells to the cytotoxic effects of lymphocytes. Depletion of CD8(+) T cells eliminated most of the therapeutic efficacy; in contrast, depletion of CD4(+) T cells enhanced the therapeutic efficacy. A similar enhancing effect was observed for the combination of GA and a DNA vaccine targeting the Met oncogene. Our results support the use of combination of GA and DNA vaccination against GA-targeted proteins.

Therapeutic HER2/Neu DNA vaccine inhibits mouse tumor naturally overexpressing endogenous neu.

The therapeutic efficacy of HER2/c-erbB-2/neu DNA immunization on mouse tumor cells expressing exogenous human or rat p185neu but not on mouse tumor cells naturally expressing mouse p185neu has been demonstrated. We investigated the feasibility of using N-terminal rat neu DNA immunization on mouse tumor overexpressing endogenous p185neu and enhancing the therapeutic efficacy of this vaccine by fusion to various cytokine genes, including interleukin-2 (IL-2), interleukin-4 (IL-4), or granulocyte-macrophage colony-stimulating factor. In a therapeutic model, N'-neu-IL-2 DNA vaccine was significantly better than N'-neu DNA vaccine, and N'-neu DNA vaccine was significantly better than control DNA or N'-neu-IL-4 DNA vaccine. The therapeutic efficacy of DNA vaccines was correlated with tumor infiltration of CD8+ T cells. Depletion of CD8+ T cells completely abolished the therapeutic effects of N'-neu-IL-2 DNA vaccine and N'-neu DNA vaccine. Depletion of CD4+ T cells after tumor implantation had no influence on N'-neu-IL-2 DNA vaccine, but enhanced the therapeutic efficacy of N'-neu DNA vaccine. Our results demonstrate that rat N'-neu DNA vaccine has a therapeutic effect on established tumor through the CD8+ T-cell-dependent pathway. Depletion of CD4+ T cells or fusion to the IL-2 gene can thus further enhance the therapeutic effects of N'-neu DNA immunization on mouse tumor expressing endogenous p185neu.