Targeting FOXP3 Tumor-Intrinsic Effects Using Adenoviral Vectors in Experimental Breast Cancer.

The regulatory T cell master transcription factor, Forkhead box P3 (Foxp3), has been detected in cancer cells; however, its role in breast tumor pathogenesis remains controversial. Here we assessed Foxp3 tumor intrinsic effects in experimental breast cancer using a Foxp3 binder peptide (P60) that impairs Foxp3 nuclear translocation. Cisplatin upregulated Foxp3 expression in HER2+ and triple-negative breast cancer (TNBC) cells. Foxp3 inhibition with P60 enhanced chemosensitivity and reduced cell survival and migration in human and murine breast tumor cells. We also developed an adenoviral vector encoding P60 (Ad.P60) that efficiently transduced breast tumor cells, reduced cell viability and migration, and improved the cytotoxic response to cisplatin. Conditioned medium from transduced breast tumor cells contained lower levels of IL-10 and improved the activation of splenic lymphocytes. Intratumoral administration of Ad.P60 in breast-tumor-bearing mice significantly reduced tumor infiltration of Tregs, delayed tumor growth, and inhibited the development of spontaneous lung metastases. Our results suggest that Foxp3 exerts protumoral intrinsic effects in breast cancer cells and that gene-therapy-mediated blockade of Foxp3 could constitute a therapeutic strategy to improve the response of these tumors to standard treatment.

Mitochondrial Peptide Humanin Facilitates Chemoresistance in Glioblastoma Cells.

Humanin (HN) is a mitochondrial-derived peptide with robust cytoprotective effects in many cell types. Although the administration of HN analogs has been proposed to treat degenerative diseases, its role in the pathogenesis of cancer is poorly understood. Here, we evaluated whether HN affects the chemosensitivity of glioblastoma (GBM) cells. We found that chemotherapy upregulated HN expression in GBM cell lines and primary cultures derived from GBM biopsies. An HN analog (HNGF6A) boosted chemoresistance, increased the migration of GBM cells and improved their capacity to induce endothelial cell migration and proliferation. Chemotherapy also upregulated FPR2 expression, an HN membrane-bound receptor, and the HNGF6A cytoprotective effects were inhibited by an FPR2 receptor antagonist (WRW4). These effects were observed in glioma cells with heterogeneous genetic backgrounds, i.e., glioma cells with wild-type (wtIDH) and mutated (mIDH) isocitrate dehydrogenase. HN silencing using a baculoviral vector that encodes for a specific shRNA for HN (BV.shHN) reduced chemoresistance, and impaired the migration and proangiogenic capacity of GBM cells. Taken together, our findings suggest that HN boosts the hallmark characteristics of GBM, i.e., chemoresistance, migration and endothelial cell proliferation. Thus, strategies that inhibit the HN/FPR2 pathway may improve the response of GBM to standard therapy.

H3.3-G34R Mutation-Mediated Epigenetic Reprogramming Leads to Enhanced Efficacy of Immune Stimulatory Gene Therapy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are diffuse and highly aggressive CNS tumors which remain incurable, with a 5-year overall survival of less than 20%. Within glioma, mutations in the genes encoding the histones H3.1 and H3.3 have been discovered to be age-restricted and specific of pHGGs. This work focuses on the study of pHGGs harboring the H3.3-G34R mutation. H3.3-G34R tumors represent the 9-15% of pHGGs, are restricted to the cerebral hemispheres, and are found predominantly in the adolescent population (median 15.0 years). We have utilized a genetically engineered immunocompetent mouse model for this subtype of pHGG generated via the Sleeping Beauty-transposon system. The analysis of H3.3-G34R genetically engineered brain tumors by RNA-Sequencing and ChIP-Sequencing revealed alterations in the molecular landscape associated to H3.3-G34R expression. In particular, the expression of H3.3-G34R modifies the histone marks deposited at the regulatory elements of genes belonging to the JAK/STAT pathway, leading to an increased activation of this pathway. This histone G34R-mediated epigenetic modifications lead to changes in the tumor immune microenvironment of these tumors, towards an immune-permissive phenotype, making these gliomas susceptible to TK/Flt3L immune-stimulatory gene therapy. The application of this therapeutic approach increased median survival of H3.3-G34R tumor bearing animals, while stimulating the development of anti-tumor immune response and immunological memory. Our data suggests that the proposed immune-mediated gene therapy has potential for clinical translation for the treatment of patients harboring H3.3-G34R high grade gliomas.

In Vitro and In Vivo Efficacy of a Stroma-Targeted, Tumor Microenvironment Responsive Oncolytic Adenovirus in Different Preclinical Models of Cancer.

More than one million women are diagnosed annually worldwide with a gynecological cancer. Most gynecological cancers are diagnosed at a late stage, either because a lack of symptoms, such as in ovarian cancer or limited accessibility to primary prevention in low-resource countries, such as in cervical cancer. Here, we extend the studies of AR2011, a stroma-targeted and tumor microenvironment responsive oncolytic adenovirus (OAdV), whose replication is driven by a triple hybrid promoter. We show that AR2011 was able to replicate and lyse in vitro fresh explants obtained from human ovarian cancer, uterine cancer, and cervical cancer. AR2011 was also able to strongly inhibit the in vitro growth of ovarian malignant cells obtained from human ascites fluid. The virus could synergize in vitro with cisplatin even on ascites-derived cells obtained from patients heavily pretreated with neoadjuvant chemotherapy. AR2011(h404), a dual transcriptionally targeted derived virus armed with hCD40L and h41BBL under the regulation of the hTERT promoter, showed a strong efficacy in vivo both on subcutaneous and intraperitoneally established human ovarian cancer in nude mice. Preliminary studies in an immunocompetent murine tumor model showed that AR2011(m404) expressing the murine cytokines was able to induce an abscopal effect. The present studies suggest that AR2011(h404) is a likely candidate as a novel medicine for intraperitoneal disseminated ovarian cancer.

Breast cancer progression and kynurenine pathway enzymes are induced by hexachlorobenzene exposure in a Her2-positive model.

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor ß (ERß) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5 µM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERß while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERß and GPER levels, collaborating in HER2-positive breast cancer progression.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Potential of IDH mutations as immunotherapeutic targets in gliomas: a review and meta-analysis.

INTRODUCTION: Gliomas are stratified by the presence of a hotspot mutation in the enzyme isocitrate dehydrogenase genes (IDH1/2). While mutated IDH (mIDH) correlates with better prognosis, the role of this mutation in antitumor immunity and the response to immunotherapy is not completely understood. Understanding the relationship between the genetic features of these tumors and the tumor immune microenvironment (TIME) may help to develop appropriate therapeutic strategies. AREAS COVERED: In this review we discussed the available literature related to the potential role of IDH mutations as an immunotherapeutic target in gliomas and profiled the immune transcriptome of glioma biopsies. We aimed to shed light on the role of mIDH on the immunological landscape of the different subtypes of gliomas, taking into account the most recent WHO classification of tumors of the central nervous system (CNS). We also discussed different immunotherapeutic approaches to target mIDH tumors and to overcome their immunosuppressive microenvironment. EXPERT OPINION: Data presented here indicates that the TIME not only differs in association with IDH mutation status, but also within glioma subtypes, suggesting that the cellular context affects the overall effect of this genetic lesion. Thus, specific therapeutic combinations may help patients diagnosed with different glioma subtypes.

The role of the prolactin receptor pathway in the pathogenesis of glioblastoma: what do we know so far?

Introduction: Prolactin (PRL) and its receptor (PRLR) have been associated with the development of hormone-dependent tumors and have been detected in glioblastoma (GBM) biopsies. GBM is the most common and aggressive primary brain tumor in adults and the prognosis for patients is dismal; hence researchers are exploring the PRLR pathway as a therapeutic target in this disease. Areas covered: This paper explores the effects of PRLR activation on the biology of GBM, the correlation between PRL and PRLR expression and GBM progression and survival in male and female patients. Finally, we discuss how a better understanding of the PRLR pathway may allow the development of novel treatments for GBM. Expert opinion: We propose PRL and PRLR as potential prognosis biomarkers and therapeutic targets in GBM. Local administration of PRLR inhibitors using gene therapy may offer a beneficial strategy for targeting GBM cells disseminated in the non-neoplastic brain; however, efficacy and safety require careful and extensive evaluation. The data depicted herein underline the need to (i) improve our understanding of sexual dimorphism in GBM, and (ii) develop accurate preclinical models that take into consideration different hormonal contexts, specific genetic alterations, and tumor grades.

Humanin Promotes Tumor Progression in Experimental Triple Negative Breast Cancer.

Humanin (HN) is a mitochondrial-derived peptide with cytoprotective effect in many tissues. Administration of HN analogs has been proposed as therapeutic approach for degenerative diseases. Although HN has been shown to protect normal tissues from chemotherapy, its role in tumor pathogenesis is poorly understood. Here, we evaluated the effect of HN on the progression of experimental triple negative breast cancer (TNBC). The meta-analysis of transcriptomic data from The Cancer Genome Atlas indicated that HN and its receptors are expressed in breast cancer specimens. By immunohistochemistry we observed up-regulation of HN in TNBC biopsies when compared to mammary gland sections from healthy donors. Addition of exogenous HN protected TNBC cells from apoptotic stimuli whereas shRNA-mediated HN silencing reduced their viability and enhanced their chemo-sensitivity. Systemic administration of HN in TNBC-bearing mice reduced tumor apoptotic rate, impaired the antitumor and anti-metastatic effect of chemotherapy and stimulated tumor progression, accelerating tumor growth and development of spontaneous lung metastases. These findings suggest that HN may exert pro-tumoral effects and thus, caution should be taken when using exogenous HN to treat degenerative diseases. In addition, our study suggests that HN blockade could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer.

Immunotherapy for gliomas: shedding light on progress in preclinical and clinical development.

INTRODUCTION: Gliomas are infiltrating brain tumors associated with high morbidity and mortality. Current standard of care includes radiation, chemotherapy, and surgical resection. Today, survival rates for malignant glioma patients remain dismal and unchanged for decades. The glioma microenvironment is highly immunosuppressive and consequently this has motivated the development of immunotherapies for counteracting this condition, enabling the immune cells within the tumor microenvironment to react against this tumor. AREAS COVERED: The authors discuss immunotherapeutic strategies for glioma in phase-I/II clinical trials and illuminate their mechanisms of action, limitations, and key challenges. They also examine promising approaches under preclinical development. EXPERT OPINION: In the last decade there has been an expansion in immune-mediated anti-cancer therapies. In the glioma field, sophisticated strategies have been successfully implemented in preclinical models. Unfortunately, clinical trials have not yet yielded consistent results for glioma patients. This could be attributed to our limited understanding of the complex immune cell infiltration and its interaction with the tumor cells, the selected time for treatment, the combination with other therapies and the route of administration of the agent. Applying these modalities to treat malignant glioma is challenging, but many new alternatives are emerging to by-pass these hurdles.

Prospects of biological and synthetic pharmacotherapies for glioblastoma.

Introduction: The field of neuro-oncology has experienced significant advances in recent years. More is known now about the molecular and genetic characteristics of glioma than ever before. This knowledge leads to the understanding of glioma biology and pathogenesis, guiding the development of targeted therapeutics and clinical trials. The goal of this review is to describe the state of basic, translational, and clinical research as it pertains to biological and synthetic pharmacotherapy for gliomas.Areas covered: Challenges remain in designing accurate preclinical models and identifying patients that are likely to respond to a particular targeted therapy. Preclinical models for therapeutic assessment are critical to identify the most promising treatment approaches.Expert opinion: Despite promising new therapeutics, there have been no significant breakthroughs in glioma treatment and patient outcomes. Thus, there is an urgent need to better understand the mechanisms of treatment resistance and to design effective clinical trials.

Prolactin and its receptor as therapeutic targets in glioblastoma multiforme.

Although prolactin (PRL) and its receptor (PRLR) have been detected in glioblastoma multiforme (GBM), their role in its pathogenesis remains unclear. Our aim was to explore their contribution in GBM pathogenesis. We detected PRL and PRLR in all GBM cell lines tested. PRLR activation or overexpression using plasmid transfection increased proliferation, viability, clonogenicity, chemoresistance and matrix metalloproteinase activity in GBM cells, while PRLR antagonist ∆1-9-G129R-hPRL reduced their proliferation, viability, chemoresistance and migration. Meta-analysis of transcriptomic data indicated that PRLR was expressed in all grade II-III glioma (GII-III) and GBM samples. PRL was upregulated in GBM biopsies when compared to GII-III. While in the general population tumour PRL/PRLR expression did not correlate with patient survival, biological sex-stratified analyses revealed that male patients with PRL+/PRLRHIGH GBM performed worse than PRL+/PRLRLOW GBM. In contrast, all male PRL+/PRLRHIGH GII-III patients were alive whereas only 30% of PRL+/PRLRLOW GII-III patients survived after 100 months. Our study suggests that PRLR may be involved in GBM pathogenesis and could constitute a therapeutic target for its treatment. Our findings also support the notion that sexual dimorphism should be taken into account to improve the care of GBM patients.

Papel del péptido mitocondrial humanina como blanco terapéutico en cáncer y neurodegeneración / Role of mitochondrial-derived peptide humanin as a therapeutic target in cancer and neurodegeneration

Resumen La humanina es un péptido derivado de la mitocondria con efectos protectores robustos contra una gran variedad de estímulos citotóxicos en diversos tipos celulares. Esto la convierte en un blanco terapéutico interesante para muchas enfermedades, como el cáncer y enfermedades neurodegenerativas, entre otras. Además, este péptido podría utilizarse como un biomarcador en estas enfermedades. Durante la última década, han sido desarrollados análogos y péptido-miméticos de la humanina que muestran resultados prometedores en modelos preclínicos. A su vez, también se está explorando el potencial terapéutico de vectores de terapia génica que puedan sobreexpresar o silenciar la humanina endógena. Varios puntos importantes a considerar antes de trasladar estas estrategias terapéuticas a la clínica son su posible papel en la progresión del cáncer y la eventual generación de quimiorresistencia. Todos estos temas serán abordados en este artículo de revisión.

Abstract Humanin is a mitochondrial-derived peptide which shows robust protective effects against large series of cytotoxic stimuli in many cell types. This makes it an interesting therapeutic target for many diseases, including cancer and neurodegenerative diseases, among others. Furthermore, this peptide could be used as a biomarker for such diseases. Over the last decade, humanin analogs and peptide mimetics have been developed, which exert highly promising results in preclinical models. Besides, the therapeutic potential of gene therapy vectors that overexpress or silence endogenous humanin is under evaluation. Nonetheless, its possible role in cancer progression and chemoresistance are critical issues to be addressed before translating these therapeutic approaches to the clinic. All these matters will be covered in this review.

Mitochondrial-derived peptide humanin as therapeutic target in cancer and degenerative diseases.

INTRODUCTION: Mitochondrial-derived peptides (MDPs) are encoded within the mitochondrial genome. They signal within the cell or are released to act as autocrine/paracrine/endocrine cytoprotective factors playing a key role in the cellular stress response. The first reported and better characterized MDP is humanin (HN), which exerts robust protective effects against a myriad of cytotoxic stimuli in many cell types. These effects have led to the evaluation of HN and its analogs as therapeutic targets for several chronic diseases. Areas covered: We describe the latest findings on the mechanism of action of HN and discuss the role of HN as therapeutic target for neurodegenerative and cardiovascular diseases, diabetes, male infertility, and cancer. Since HN can be detected in circulation, we also depict its value as a biomarker for these diseases. Expert opinion: HN analogs and peptide mimetics have been developed over the last decade and show promising results in preclinical models of degenerative diseases. Local administration of gene therapy vectors that overexpress or silence endogenous HN could also hold therapeutic potential. Controversy on the role of HN in cancer progression and chemoresistance should be addressed before the translation of these therapeutic approaches.

Oncolytic Adenovirus-Loaded Menstrual Blood Stem Cells Overcome the Blockade of Viral Activity Exerted by Ovarian Cancer Ascites.

Patients with ovarian cancer present peritoneal ascites at recurrence as a marker of disseminated disease and dismal prognosis. Oncolytic immunotherapy is an emerging approach for the treatment of disseminated cancer. In the present work, we constructed a novel oncolytic adenovirus, AR2011, to target malignant ovarian tumors. AR2011 exhibited a clear lytic effect in vitro in human ovarian cancer cell lines and malignant cells obtained from ascitic fluids (AFs) of patients with ovarian cancer. AR2011 activity was neutralized by antibodies present in 31 samples of patient-derived AFs. However, this blockade was overridden by preloading menstrual blood stem cells (MenSCs) with AR2011 (MenSC-AR), since AFs exerted no in vitro inhibitory effect on viral lytic activity under these conditions. Moreover, soluble factors present in AFs act as MenSC chemoattractants. MenSC-AR treatment of nude mice carrying established peritoneal carcinomatosis following administration of human ovarian cancer cells was able to inhibit tumor growth at levels similar to those observed with AR2011 alone. This study demonstrates that MenSCs can be used to override the blockade that AFs exert on viral oncolytic effects.

Viral gene therapy for breast cancer: progress and challenges.

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.

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

PURPOSE: Since 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. METHODS: DCs 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. RESULTS: CpG-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. CONCLUSIONS: These 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.