Role of metformin and AKT axis modulation in the reversion of hypoxia induced TMZ-resistance in glioma cells

Hypoxia is a key driver of tumor adaptation promoting tumor progression and resistance to therapy. Hypoxia related pathways might represent attractive targets for the treatment of Glioblastoma Multiforme (GBM), that up to date is characterized by a poor prognosis. Primary aim of this study was to investigate the role of hypoxia and hypoxia-related modifications in the effect of temozolomide (TMZ) given alone or in association with the antidiabetic agent Metformin (MET) or the PI3K/mTOR blocker, BEZ235. The study was conducted in the TMZ responsive U251 and resistant T98 GBM cells. Our results showed that during hypoxia, TMZ plus MET reduced viability of U251 cells affecting also CD133 and CD90 expressing cells. This effect was associated with a reduction of HIF-1\u3b1 activity, VEGF release and AKT activation. In T98 TMZ-resistant cells, TMZ plus MET exerted similar effects on HIF-1\u3b1. However, in this cell line, TMZ plus MET failed to reduce CD133 positive cells and AKT phosphorylation. Nevertheless, the administration of the dual PI3K/mTOR inhibitor BEZ235 potentiated the effect of TMZ plus MET on cell viability, inducing a pro-apoptotic phenotype during hypoxic condition also in T98 cells, suggesting the block of the PI3K/AKT/mTOR pathway as a complementary target to further overcome GBM resistance during hypoxia. In conclusion, we proposed TMZ plus MET as suitable treatment to revert TMZ-resistance also during hypoxia, an effect potentiated by the inhibition of PI3K/mTOR axis

Hypoxia-inducible factor-1α activity as a switch for glioblastoma responsiveness to temozolomide

Rationale: The activity of the transcription factor, hypoxia-inducible factor (HIF)-1\u3b1, is a common driver of a number of the pathways involved in the aggressiveness of glioblastomas (GBMs), and it has been suggested that the reduction in this activity observed, soon after the administration of temozolomide (TMZ), can be a biomarker of an early response in GBM models. As HIF-1\u3b1 is a tightly regulated protein, studying the processes involved in its downregulation could shed new light on the mechanisms underlying GBM sensitivity or resistance to TMZ. Methods: The effect of HIF-1\u3b1 silencing on cell responsiveness to TMZ was assessed in four genetically different human GBM cell lines by evaluating cell viability and apoptosis-related gene balance. LAMP-2A silencing was used to evaluate the contribution of chaperone-mediated autophagy (CMA) to the modulation of HIF-1\u3b1 activity in TMZ-sensitive and TMZ-resistant cells. Results: The results showed that HIF-1\u3b1 but not HIF-2\u3b1 activity is associated with GBM responsiveness to TMZ: its downregulation improves the response of TMZ-resistant cells, while blocking CMA-mediated HIF-1\u3b1 degradation induces resistance to TMZ in TMZ-sensitive cells. These findings are in line with the modulation of crucial apoptosis-related genes. Conclusion: Our results demonstrate the central role played by HIF-1\u3b1 activity in determining the sensitivity or resistance of GBMs to TMZ, and we suggest that CMA is the cellular mechanism responsible for modulating this activity after TMZ treatment

Optical imaging probes in oncology

Cancer is a complex disease, characterized by alteration of different physiological molecular processes and cellular features. Keeping this in mind, the possibility of early identification and detection of specific tumor biomarkers by non-invasive approaches could improve early diagnosis and patient management.Different molecular imaging procedures provide powerful tools for detection and non-invasive characterization of oncological lesions. Clinical studies are mainly based on the use of computed tomography, nuclear-based imaging techniques and magnetic resonance imaging. Preclinical imaging in small animal models entails the use of dedicated instruments, and beyond the already cited imaging techniques, it includes also optical imaging studies. Optical imaging strategies are based on the use of luminescent or fluorescent reporter genes or injectable fluorescent or luminescent probes that provide the possibility to study tumor features even by means of fluorescence and luminescence imaging. Currently, most of these probes are used only in animal models, but the possibility of applying some of them also in the clinics is under evaluation.The importance of tumor imaging, the ease of use of optical imaging instruments, the commercial availability of a wide range of probes as well as the continuous description of newly developed probes, demonstrate the significance of these applications. The aim of this review is providing a complete description of the possible optical imaging procedures available for the non-invasive assessment of tumor features in oncological murine models. In particular, the characteristics of both commercially available and newly developed probes will be outlined and discussed

Theranostic application of miR-429 in HER2+ breast cancer

Human epidermal growth factor receptor 2 (HER2) is overexpressed/amplified in one third of breast cancers (BCs), and is associated with the poorer prognosis and the higher metastatic potential in BC. Emerging evidences highlight the role of microRNAs (miRNAs) in the regulation of several cellular processes, including BC. Methods: Here we identified, by in silico approach, a group of three miRNAs with central biological role (high degree centrality) in HER2+ BC. We validated their dysregulation in HER2+ BC and we analysed their functional role by in vitro approaches on selected cell lines and by in vivo experiments in an animal model. Results: We found that their expression is dysregulated in both HER2+ BC cell lines and human samples. Focusing our study on the only upregulated miRNA, miR-429, we discovered that it acts as an oncogene and its upregulation is required for HER2+ cell proliferation. It controls the metastatic potential of HER2+ BC subtype by regulating migration and invasion of the cell. Conclusions: In HER2+ BC oncogenic miR-429 is able to regulate HIF1\u3b1 pathway by directly targeting VHL mRNA, a molecule important for the degradation of HIF1\u3b1. The overexpression of miR-429, observed in HER2+ BC, causes increased proliferation and migration of the BC cells. More important, silencing miR-429 succeeds in delaying tumor growth, thus miR-429 could be proposed as a therapeutic probe in HER2+ BC tumors

Validation of an Engineered Cell Model for In Vitro and In Vivo HIF-1 alpha Evaluation by Different Imaging Modalities

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Mesenchymal stem cells from tumor microenvironment favour breast cancer stem cell proliferation, cancerogenic and metastatic potential, via ionotropic purinergic signalling

Interaction between tumor cells and the microenvironment is key in initiation, progression, and invasiveness of cancer. In particular, mesenchymal stem cells (MSCs) are recruited to the sites of developing tumors, thus promoting metastasis formation. Although it is well known that MSCs migrate and integrate in the tumor microenvironment (TME), their fate and function inside the tumor is still not clear. In this study, we analyzed the role played by MSCs in breast cancer oncogenesis. Data indicate that interaction of breast cancer cells with MSCs results in an increased proliferation and metabolic activity of breast cancer cells, partially due to MSC-derived microvesicles that are shed in the TME. Moreover, we addressed the question of whether we could modulate such interaction by acting on P2X-mediated intercellular communication. By inhibiting P2X-mediated purinergic signaling, we succeeded in reducing both the cancerogenic as well as the metastatic potential of breast cancer cells co-cultured with MSCs, in 2D as well as in 3D in vitro models. Data obtained demonstrate for the first time that the trophic effect of MSCs on breast cancer cell growth is exerted via ionotropic purinergic signaling, thus suggesting the inhibition of the purinergic signaling system as a potential target for therapeutic intervention

Specific V-ATPase expression sub-classifies IDHwt lower-grade gliomas and impacts glioma growth in vivo

Background: Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods: V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients\u2019 cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings: GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0\ub703, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation: Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund: This work was supported by Fondazione Cariplo (2014\u20131148 to VV), Fondazione IRCCS Ca\u2019 Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV)

Development of clinical simultaneous SPECT/MRI

There is increasing clinical use of combined positron emission tomography (PET) and magnetic resonance imaging (MRI) but to date there has been no clinical system developed capable of simultaneous single photon emission computed tomography (SPECT) and MRI. There has been development of preclinical systems, but there are several challenges faced by researchers who are developing a clinical prototype including the need for the system to be compact and stationary with MRI-compatible components. The limited work in this area is described with specific reference to the Integrated SPECT/MRI for Enhanced stratification in Radio-chemo Therapy (INSERT) project, which is at an advanced stage of developing a clinical prototype. Issues of SPECT/MRI compatibility are outlined and the clinical appeal of such a system is discussed, especially in the management of brain tumour treatment

Real-Time Dynamic Imaging of Virus Distribution In Vivo

The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR) fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection

A GBM-like V-ATPase signature directs cell-cell tumor signaling and reprogramming via large oncosomes

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF)