Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

Overexpression of HER-2 via immunohistochemistry in canine urinary bladder transitional cell carcinoma - A marker of malignancy and possible therapeutic target.

Transitional cell carcinoma (TCC) is the most commonly diagnosed neoplasm in the urinary bladder. Distant metastases to the regional lymph nodes, lungs, abdominal organs or bones are noted in up to 50% of dogs at time of death. Surgical excision is often not practical as TCC typically involve the trigone of the bladder and/or occurs multifocally throughout the bladder with field cancerization. Therapeutic approaches are very challenging and the requirement to evaluate alternative therapeutic protocols that may prolong survival times in dogs bearing these tumours is compelling. We assessed the immunohistochemical expression of HER-2 in 23 cases of canine TCCs of the urinary bladder and compare it with non-neoplastic urothelium in order to evaluate a rationale for targeted therapies and gene-based vaccines. HER-2 positivity was recorded in 13/23 (56%) neoplastic lesions. The receptor was significantly overexpressed in neoplastic than in non-neoplastic samples (P = .015). According to our preliminary results, it would be of interest to further evaluate the role of HER-2 in canine TCCs as a marker of malignancy and a therapeutic target for cancer vaccine and antibodies. Moreover, the significantly different overexpression of HER-2 in TCCs than in non-neoplastic urothelium further supports to investigate its role in the progression toward malignancy of non-neoplastic lesions.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge.

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

Electroporation in veterinary oncology.

Cancer treatments in veterinary medicine continue to evolve beyond the established standard therapies of surgery, chemotherapy and radiation therapy. New technologies in cancer therapy include a targeted mechanism to open the cell membrane based on electroporation, driving therapeutic agents, such as chemotherapy (electro-chemotherapy), for local control of cancer, or delivery of gene-based products (electro-gene therapy), directly into the cancer cell to achieve systemic control. This review examines electrochemotherapy and electro-gene therapy in veterinary medicine and considers future directions and applications.

Decreased LRIG1 in fulvestrant-treated luminal breast cancer cells permits ErbB3 upregulation and increased growth.

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the antiestrogen fulvestrant enhances PI3K/mTOR-mediated cell survival. However, the mechanism by which ErbB3 is upregulated in fulvestrant-treated cells is unknown. We found that ErbB3 protein levels and cell surface presentation were increased following fulvestrant treatment, focusing our attention on proteins that regulate ErbB3 at the cell surface, including Nrdp1, NEDD4 and LRIG1. Among these, only LRIG1 correlated positively with ERα, but inversely with ErbB3 in clinical breast cancer data sets. LRIG1, an estrogen-inducible ErbB downregulator, was decreased in a panel of fulvestrant-treated luminal breast cancer cells. Ectopic LRIG1 expression from an estrogen-independent promoter uncoupled LRIG1 from estrogen regulation, thus sustaining LRIG1 and maintaining low ErbB3 levels in fulvestrant-treated cells. An LRIG1 mutant lacking the ErbB3 interaction motif was insufficient to downregulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, whereas cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα downregulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in antiestrogen-treated breast cancer patients. These data suggest that ERα-dependent expression of LRIG1 dampens ErbB3 signaling in luminal breast cancer cells, and by blocking ERα activity with fulvestrant, LRIG1 is decreased thus permitting ErbB3 accumulation, enhanced ErbB3 signaling to cell survival pathways and blunting therapeutic response to fulvestrant.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Lactose intolerance. Recognizing the link between diet and discomfort.

Lactose intolerance is a common disorder encountered in clinical practice. Evaluation involves obtaining a thorough nutritional history and recognizing associations between diet and gastrointestinal complaints. Lack of suspicion of the problem can lead to expensive and invasive diagnostic procedures, which may further aggravate patients' anxiety and result in iatrogenic complications. Once the diagnosis is confirmed, simple dietary management may resolve symptoms completely.

Electro-gene-transfer as a new tool for cancer immunotherapy in animals.

The concept of vaccines based on the direct inoculation of plasmid DNA gained initial proof-of-concept in small rodent species. Further development was hampered by the difficulty to confirm immunogenicity and efficacy in large animal species and, most importantly, in human clinical trials. These negative findings led to the search of complementary technologies which, in combination with intradermal or intramuscular plasmid DNA injection would result in more robust delivery, decreased interindividual variability, clear evidence of clinical efficacy and which would eventually lead to market approval of new vaccine products. The use of high-pressure, needleless devices as an enhancing tool for plasmid DNA delivery led to recent approval by USDA of Oncept™, a therapeutic cancer vaccine directed against tyrosinase for the therapy of melanoma in dogs. An alternative approach to improve plasmid DNA delivery is electro-gene-transfer (EGT). In this article, we briefly review the principles of DNA-EGT and the evidences for efficacy of a telomerase reverse transcriptase vaccine in a dog clinical trial, and provide perspectives for the use of this technology for broader applications in pet animals.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells.

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

An oral TLR7 agonist is a potent adjuvant of DNA vaccination in transgenic mouse tumor models.

In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines resulting in increased DNA uptake, enhanced protein expression and increased immune responses to the target antigen in a variety of species. To further enhance the efficacy of DNA-EP, we have evaluated the toll-like receptor7 (TLR7) agonist-2, 9, substituted 8-hydroxyadenosine derivative or SM360320--as an adjuvant to vaccines against HER2/neu and CEA in BALB-neuT and CEA transgenic mice (CEA.Tg), respectively. SM360320 induced in vivo secretion of interferon alpha (IFNalpha) and exerted a significant antitumor effect in CEA.Tg mice challenged with a syngenic tumor cell line expressing CEA and an additive effect with a CEA vaccine. Additionally, combination of SM360320 with plasmid encoding the extracellular and transmembrane domain of ratHER2/neu affected the spontaneous tumor progression in BALB-neuT mice treated in an advanced disease setting. The antitumor effect in mice treated with DNA-EP and SM360320 was associated with an anti-CEA and anti-p185(neu) antibody isotype switch from IgG1 to IgG2a. These data demonstrate that SM360320 exerts significant antitumor effects and can act in association with DNA-EP for CEA-positive colon cancer and HER2-positive mammary carcinoma. These observations therefore emphasize the potential of SM360320 as immunological adjuvant for therapeutic DNA vaccines.

A multi-DNA preventive vaccine for p53/Neu-driven cancer syndrome.

The highly aggressive cancer syndrome of female mice carrying a p53 knockout allele and a rat HER-2/neu (Neu) transgene (BALB-p53Neu) can be prevented by a cell vaccine presenting three components: Neu, interleukin (IL)-12 production, and allogeneic major histocompatibility complex (MHC) alleles (Triplex cell vaccine). Here we tested a second-generation Triplex DNA-based vaccine (Tri-DNA), consisting of the combination of three gene components (a transmembrane-extracellular domain fragment of the Neu gene, IL-12 genes, and the H-2D(q) allogeneic MHC gene), carried by separate plasmids. The Tri-DNA vaccine was at least as effective as the Triplex cell vaccine for cancer immunoprevention, giving a similar delay in the onset of mammary cancer and complete protection from salivary cancer. Both vaccines induced anti-Neu antibodies of the murine IgG2a isotype at similar levels. The Tri-DNA vaccine gave more restricted immunostimulation, consisting of a fully helper T cell type 1 (Th1)-polarized response, with effective production of interferon (IFN)-gamma in response to the vaccine but no spontaneous production, and no induction of anti-Neu IgG3 antibodies. On the other hand, the Triplex cell vaccine induced both Th1 and Th2 cytokines, a strong increase in spontaneous IFN-gamma production, and high levels of IgG3 antibodies recognizing Neu-positive syngeneic cells. In conclusion, the Tri-DNA vaccine is as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Identification of the thyroid transcription factor-1 as a target for rat MST2 kinase.

Thyroid transcription factor-1 (TTF-1) is a homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase genes as well as for lung-specific expression of the surfactant protein A, B, and C and the CC10 and the HNF-3 alpha genes. TTF-1 is a phosphoprotein, and the phosphorylation of TTF-1 has been studied already. However, the kinase(s) that could be responsible for this phosphorylation have not been known. In this paper we report the identification by in-gel kinase assay of a 56-kDa serine/threonine kinase that is able to phosphorylate TTF-1 in thyroid cells. The cloning of this kinase revealed that we had identified the rat homolog of the human MST2 kinase. The pathway in which human MST2 functions is not known; however, it does not appear to involve either mitogen-activated protein kinases such as Erk1 and Erk2 nor the stress-activated protein kinases such as JNK and p38. We show that the activity responsible for TTF-1 phosphorylation is constitutive in thyroid cells. Furthermore, we demonstrate that TTF-1 is phosphorylated in vivo by rMST2 at the same residues that had been identified previously as the major phosphorylation sites. Thus, TTF-1 represents the first identified target of this class of protein kinases.

Tamarin alpha-interferon is active in mouse liver upon intramuscular gene delivery.

BACKGROUND: The hepatitis C virus (HCV) is responsible for a severe and widespread form of hepatitis for which a durable and effective therapy has not yet been established. The only approved therapy against hepatitis C, alpha-interferon protein intramuscular administration, presents numerous drawbacks that might be overcome by adopting a gene therapy approach. HCV exclusively infects humans and chimpanzees, hence an acceptable animal model for hepatitis C pharmacological studies is not available. Recently, tamarins infected by GB virus B (GBV-B) have been proposed as a surrogate animal model for HCV infection. The aim of the present study was the production of tamarin interferon (tIFN) through delivery of tIFN-coding DNA to evaluate the feasibility of a gene therapy approach based on IFN electro-gene transfer (EGT) in future studies with primates. METHODS: Production and biological activity of cloned tamarin interferon was monitored in cultured cells upon transfection and in mice upon muscle EGT of the corresponding plasmid DNA, respectively. RESULTS: A tamarin gene encoding a protein homologous to human interferon-alpha2 (hIFN-alpha2) has been cloned. The tamarin IFN-alpha (tIFN-alpha) protein shows antiviral activity in a cell-based assay. Upon EGT of the corresponding gene in mouse muscles, tIFN-alpha is detectable at high levels in serum for at least 4 months. Most important, activity of tIFN, measured as enhancement of mRNA levels of genes induced by type I IFNs, is also detectable in the liver of EGT-treated mice. CONCLUSION: The present study demonstrates that the delivery of tIFN-alpha DNA via intramuscular injection yields a functional protein able to produce biological effects inside a remote target organ, the liver. This finding, besides the specific purpose of the present study, is of general relevance with a view to establishing therapeutic protocols based on EGT.

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis.

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

Regulated and prolonged expression of mIFN(alpha) in immunocompetent mice mediated by a helper-dependent adenovirus vector.

A major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. This objective is becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. The ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. The helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. The most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. To explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the HD-TET-IFN vector, containing the mIFN(alpha) gene under the control of the tetracycline inducible transactivator rtTA2(s)-S2. Mice injected with HD-TET-IFN showed high levels of serum mIFN(alpha) only upon transcriptional activation. The transgene expression was reinducible to the same high level up to 3 months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. Transcriptional activation of mIFN(alpha) induced by doxycycline resulted in prolonged survival and reduced liver damage in HD-TET-IFN-injected mice challenged with a lethal dose of coronavirus. Activation of antiviral genes mediated by doxycycline-dependent mIFN(alpha) expression was also observed at low HD-TET-IFN doses. The possibility of controlling gene expression by the combination of HD vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models.

Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2(s)-M2 tetracycline transactivator and repressor system.

Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2(s)-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.