Mariela A. Moreno Ayala

Dual activation of Toll-like receptors 7 and 9 impairs the efficacy of antitumor vaccines in murine models of metastatic breast cancer

PurposeSince combination of Toll-like receptor (TLR) ligands could boost antitumor immunity, we evaluated the efficacy of dendritic cell (DC) vaccines upon dual activation of TLR9 and TLR7 in breast cancer models.MethodsDCs were generated from mouse bone marrow or peripheral blood from healthy human donors and stimulated with CpG1826 (mouse TLR9 agonist), CpG2006 or IMT504 (human TLR9 agonists) and R848 (TLR7 agonist). Efficacy of antitumor vaccines was evaluated in BALB/c mice bearing metastatic mammary adenocarcinomas.ResultsCpG-DCs improved the survival of tumor-bearing mice, reduced the development of lung metastases and generated immunological memory. However, dual activation of TLRs impaired the efficacy of DC vaccines. In vitro, we found that R848 inhibited CpG-mediated maturation of murine DCs. A positive feedback loop in TLR9 mRNA expression was observed upon CpG stimulation that was inhibited in the presence of R848. Impaired activation of NF-κB was detected when TLR9 and TLR7 were simultaneously activated. Blockade of nitric oxide synthase (NOS) and indoleamine-pyrrole-2,3-dioxygenase (IDO) improved the activation of CpG-DCs. When we evaluated the effect of combined activation of TLR9 and TLR7 in human DCs, we found that R848 induced robust DC activation that was inhibited by TLR9 agonists.ConclusionsThese observations provide insight in the biology of TLR9 and TLR7 crosstalk and suggest caution in the selection of agonists for multiple TLR stimulation. Blockade of NOS and IDO could improve the maturation of antitumor DC vaccines. R848 could prove a useful adjuvant for DC vaccines in human patients.

Viral gene therapy for breast cancer: progress and challenges

ABSTRACT Introduction: Breast cancer is the most common cancer in women all over the world. Furthermore, up to one third of breast tumors develop metastases that are resistant to standard therapies. Gene therapeutic strategies have been developed in order to specifically target cancer cells either directly or through the stimulation of antitumor immunity. Areas covered: This review describes the therapeutic strategies that are currently under development to treat this disease using engineered viral vectors including: adenovirus, adeno-associated virus, lentivirus, poxvirus, reovirus, baculovirus, herpesvirus and oncolytic viruses. Advantages and disadvantages of these multiple gene therapy platforms are discussed in detail. Expert opinion: Metastatic breast cancer is a perfect candidate for gene therapy approaches due to the presence of several tumor antigens and the aberrant expression of many molecular pathways. Oncolytic vectors are able to attack tumor cells while sparing normal cells and their activity is often enhanced by the administration of chemotherapy. However, more efforts are needed in order to reduce toxicity and to achieve better transduction efficiency. Improved preclinical models and a more critical patient selection for clinical trials, along with advances in gene therapy regulations, will surely facilitate the evolution of gene therapy for the treatment of metastatic breast cancer.

Gene Therapy for the Treatment of Neurological Disorders: Central Nervous System Neoplasms.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of 16.2-21.2 months post diagnosis (Stupp et al., N Engl J Med 352(10): 987-996, 2005). Because of its location, complete surgical resection is impossible; additionally because GBM is also resistant to chemotherapeutic and radiotherapy approaches, development of novel therapies is urgently needed. In this chapter we describe the development of preclinical animal models and a conditionally cytotoxic and immune-stimulatory gene therapy strategy that successfully causes tumor regression in several rodent GBM models.

Immunotherapy for the treatment of breast cancer

ABSTRACT Introduction: Breast cancer is the most common cancer as well as the first cause of death by cancer in women worldwide. Although routine treatment improves the outcome of early stage breast cancer patients, there is no effective therapy for the disseminated disease. Immunotherapy has emerged as a powerful therapeutic strategy for the treatment of many cancers. Although traditionally conceived as a non-immunogenic tumor, breast cancer is now considered a potential target for immunotherapy. Areas covered: In this review, the authors discuss different immunotherapeutic strategies that are currently being tested for the treatment of breast cancer: These strategies include: (i) blockade of immunological checkpoints, (ii) antitumor vaccines, (iii) regulatory T cell blockade, (iv) adoptive T cell transfer therapy, (iv) adoptive immunotherapy with monoclonal antibodies, and (v) combination of immunotherapy with chemotherapy. Expert opinion: A growing body of evidence indicates that immunotherapeutic strategies can benefit a larger cohort of breast cancer patients than hitherto anticipated. Since breast tumors entail multiple mechanisms to impair antitumor immunity, the immunological characterization of individual tumors and the selection of suitable combinations of chemotherapeutic and immunotherapeutic approaches are required to achieve significant clinical benefit in these patients.

Lack of Oestrogenic Inhibition of the Nuclear Factor-κB Pathway in Somatolactotroph Tumour Cells

Activation of nuclear factor (NF)‐κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)‐α by inhibiting NF‐κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF‐κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17β‐oestradiol (E2) (10−9 m) increased the nuclear concentration of NF‐κB/p105, p65 and p50 in GH3 cells. However, E2 did not modify the expression of Bcl‐xL, a NF‐κB target gene. TNF‐α induced apoptosis of GH3 cells incubated in either the presence or absence of E2. Inhibition of the NF‐kB pathway using BAY 11‐7082 (BAY) (5 μm) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive GH3 cells. BAY also increased TNF‐α‐induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c‐Jun N‐terminal protein kinase pathway, SP600125 (10 μm). We also analysed the role of the NF‐κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL‐positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF‐κB pathway and that the pro‐apoptotic effect of TNF‐α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF‐κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF‐κB pathway could interfere with pituitary tumour progression.

Antiapoptotic Factor Humanin Is Expressed in Normal and Tumoral Pituitary Cells and Protects Them from TNF-α-Induced Apoptosis

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.

Trypanosoma cruzi infection induces the expression of CD40 in murine cardiomyocytes favoring CD40 ligation-dependent production of cardiopathogenic IL-6

The inflammatory response in the myocardium is an important aspect of the pathogenesis of Chagas’ heart disease raised by Trypanosoma cruzi. CD40, a transmembrane type I receptor belonging to the tumor necrosis factor receptor (TNFR) family, is expressed in a broad spectrum of cell types and is crucial in several inflammatory and autoimmune diseases. Activation of CD40 through ligation to CD40L (CD154) induces multiple effects, including the secretion of proinflammatory molecules. In the present study, we examined the ability of T. cruzi to trigger the expression of CD40 in cardiac myocytes in vitro and in a murine model of chagasic cardiomyopathy. Our results indicate, for the first time, that T. cruzi is able to induce the expression of CD40 in HL-1 murine cardiomyocytes. Moreover, ligation of CD40 receptor upregulated interleukin-6 (IL-6), associated with inflammation. Furthermore, the induction of this costimulatory molecule was demonstrated in vivo in myocardium of mice infected with T. cruzi. This suggests that CD40-bearing cardiac muscle cells could interact with CD40L-expressing lymphocytes infiltrating the heart, thus contributing to inflammatory injury in chagasic cardiomyopathy.

Abstract 3132: Therapeutic blockade of Foxp3 in experimental breast cancer: Immune stimulation and direct antitumor effects

Our previous results indicate that systemic administration of a cell penetrating peptide (P60) that inhibits Foxp3, a transcription factor required for Treg function, improves the efficacy of antitumor vaccines in experimental breast cancer. We also found that P60 exerts direct antitumor effects, inhibiting tumor progression in Foxp3 + breast tumors. Although there is controversy over the role of Foxp3 in tumor cells, we found that P60 inhibits survival, proliferation and release of IL-10 in Foxp3 + breast tumor cells. Here we aimed to evaluate the pathways that control Foxp3 expression in murine LM3 breast tumor cells and to develop gene therapy vectors encoding P60 in order to improve the availability of this peptide in vivo. We assessed the role of TGF-β, mTOR and prostaglandins on the expression of Foxp3 in breast tumor cells, as these pathways have been shown to modulate Foxp3 expression in Tregs. Stimulation of LM3 cells with TGF-β and mTOR inhibitor rapamicyn upregulated Foxp3 expression, as assessed by flow cytometry, whereas indomethacin, an inhibitor of prostaglandin synthase cyclooxygenase, inhibited Foxp3 expression. These observations suggest that the regulatory mechanisms of Foxp3 expression are similar in breast tumor cells and Tregs. We next developed a plasmid that encodes P60 linked through an IRES sequence to Td.Tomato as a reporter gene (pCMV.P60.dTomato), as well a control plasmid. Transduction efficiency of these plasmids was evaluated in murine 4T1 breast tumor cells, which exhibit low expression of Foxp3 and thus are not affected by P60. Expression of Td.Tomato was readily detected by fluorescence microscopy. Conditioned media from pCMV.P60.dTomato-transfected 4T1 cells reduced the proliferation and the secretion of IL-10 in Foxp3 + LM3 cells when compared to control plasmid-transfected cells, which suggests that functional P60 is released from pCMV.P60.dTomato-transfected cells. In order to develop a gene therapeutic strategy to deliver P60 in vivo, we developed an adenoviral vector (Ad.P60.dTomato) encoding the therapeutic cassette, as well as a control vector (Ad.dTomato). These vectors successfully transduced breast tumor cells in vitro and in vivo, as evaluated by TdTomato expression. Our findings indicate that P60 could be delivered using gene therapy vectors, which could be useful for the treatment of breast cancer. Citation Format: Alejandro J. Nicola, Mariela A. Moreno Ayala, Maria F. Gottardo, Antonela S. Asad, Camila F. Zuccato, ELISA Bal de Kier Joffe, Adriana Seilicovich, Flavia Zanetti, Marianela Candolfi. Therapeutic blockade of Foxp3 in experimental breast cancer: Immune stimulation and direct antitumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3132.

Immunotherapies for Brain Cancer: From Preclinical Models to Human Trials

Glioblastoma Multiforme (GBM) is the most common and aggressive primary brain tumor. Every year ~22,000 patients are diagnosed with GBM in the US, and less than 5% survive 5 years post-diagnosis. Thus, novel therapeutic approaches are urgently needed to improve the outcome in these patients. Immunotherapy has the potential of stimulating the immune system to eliminate GBM cells that might have spread throughout the brain. Here we will discuss the latest advances in preclinical immunotherapy for glioma, which involve the local delivery of pro-inflammatory cytokines, such as Flt3L, Type I IFNs, IL-2, IL-4, and IL-12 using gene therapy vectors and neural stem cells, or the blockade of immune-suppressive mediators such as TGF-beta, FasL and phosphorylated STAT3. Novel immunotherapeutic approaches have also been assessed in clinical trials implemented in GBM patients. These involve the systemic or local adoptive transfer of autologous immune cells activated ex vivo back into the patient, and the administration of dendritic cell vaccines loaded with tumor peptides or cells, which induce active immunity against GBM. Preclinical and clinical findings so far indicate that immunotherapy improves anti-tumor immunity in preclinical GBM models and patients, which makes it a valuable adjuvant in the treatment of GBM.