Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats.

Hepatic mitochondrial function, APOC-III, and LPL are potential targets for triglyceride (TG)-lowering drugs. After 3 weeks of dietary treatment with the compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), the hepatic mitochondrial FA oxidation increased more than 5-fold in male Wistar rats. Gene expression analysis in liver showed significant downregulation of APOC-III and upregulation of LPL and the VLDL receptor. This led to lower hepatic (53%) and plasma (73%) TG levels. Concomitantly, liver-specific biomarkers related to mitochondrial biogenesis and function (mitochondrial DNA, citrate synthase activity, and cytochrome c and TFAM gene expression) were elevated. Interestingly, 1-triple TTA lowered plasma acetylcarnitine levels, whereas the concentration of ß-hydroxybutyrate was increased. The hepatic energy state was reduced in 1-triple TTA-treated rats, as reflected by increased AMP/ATP and decreased ATP/ADP ratios, whereas the energy state remained unchanged in muscle and heart. The 1-triple TTA administration induced gene expression of uncoupling protein (UCP)2 and UCP3 in liver. In conclusion, the 1-triple TTA-mediated clearance of blood TG may result from lowered APOC-III production, increased hepatic LPL gene expression, mitochondrial FA oxidation, and (re)uptake of VLDL facilitating drainage of FAs to the liver for ß-oxidation and production of ketone bodies as extrahepatic fuel. The possibility that UCP2 and UCP3 mediate a moderate degree of mitochondrial uncoupling should be considered.

GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development.

Glioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12-15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis.

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

A 3D Spheroid Model for Glioblastoma.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Upregulated PDK4 expression is a sensitive marker of increased fatty acid oxidation.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly three-fold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation.

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was ß-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that ß-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of ß-sitosterol was linked to mitochondrial interference. Mechanistically, ß-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either ß-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, ß-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.

Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: correlation of nestin with prognosis of patient survival.

BACKGROUND: The IF protein nestin and the RNA-binding protein musashi are expressed by neural progenitor cells during CNS development. Their expression in glial tumors was evaluated by immunohistochemistry, and the histopathological scores correlated with levels of cysteine cathepsins that are known prognostic markers in several tumors. METHODS: The levels of nestin, musashi, and cathepsins B and L were assessed by immunohistochemical analysis of biopsies from 87 patients with primary CNS tumors. To confirm the immunohistochemical data, nestin expression was analyzed by real-time PCR in 12 brain tumor biopsies. The exact location of nestin-positive cells was determined by mapping the distribution of nestin in a highly invasive human glioma xenograft model. RESULTS: Immunostaining revealed nestin to be expressed in 95.8% and musashi in 80% of the patient biopsies. The total IHC score for nestin was significantly higher in high- than in low-grade tumors (P < .0001). No difference was observed for musashi (P = .11). Real-time PCR of nestin expression confirmed the immunohistochemical data. Nestin expression was shown to be a strong prognostic marker for decreased overall survival (P = .0001), whereas musashi expression has no prognostic significance. Moreover, nestin was shown by Cox regression analysis to be a stronger prognostic marker than cathepsins B and L. IHC staining of nestin in a xenograft model showed that its expression is localized mainly in the invasive tumor cells at the tumor periphery. CONCLUSIONS: Nestin is shown to be a strong prognostic marker for glioma malignancy. The presented data links the invasive glioma cells to CNS precursor cells, indicating that the most malignant cells in the gliomas may well be closely related to the glioma stem cells.

Novel points of attack for targeted cancer therapy.

New molecular insight reveals novel points of attack for targeted cancer therapy. The recent advances in cancer genomics and novel insight into the complex biology of cancer make the promise of personalized, targeted cancer medicine closer than ever. The massive parallel sequencing endeavours performed by The Cancer Genome Atlas, the International Cancer Genome Consortium and by numerous individual investigators have provided a comprehensive genomic characterization of a wide range of cancers. The joint efforts enabled by the improved sequencing technology have demonstrated that individual cancers comprise mutational repertoires with only a few frequently recurrent driver genes. Thus, the identification of new drug targets and novel drugs have accelerated and renewed the hopes of personalized cancer therapy achieving clinical reality for a wider range of cancers. Together with cost-effective sequencing technology to perform comprehensive mutational profiling of each individual cancer, this provides the basis for a personalized cancer medicine revolution within the next few years. The aim of this MiniReview is to provide an overview of the history and evolution of targeted cancer therapy, exemplified by molecularly targeted drugs successfully implemented in the clinic. Furthermore, we aim to highlight novel molecular targets for therapeutic intervention, as well as the main present challenges including inter- and intratumor heterogeneity and cellular plasticity in addition to the importance of the tumor micro-environment. Many cancer patients already receive some form of tailored therapy, and recent evidence suggests that novel and highly innovative, targeted approaches are on their way into the clinic.

Increased lymphatic vascular density is seen before colorectal cancers reach stage II and growth factor FGF-2 is downregulated in tumor tissue compared with normal mucosa.

Lymphangiogenesis is an important event in progression of colorectal cancer (CRC), and the estimated lymphatic vascular density (LVD) probably indicates facilitated lymphatic tumor cell invasion and metastasis. However, at what time point during tumor progression this process is triggered, is unclear. The aim of this study was twofold. Firstly, to examine LVD in paired samples of CRC tissue and normal mucosa with specific emphasis on possible difference in LVD between tumors stages II and III, and secondly, the expression of the lymphangiogenic growth factor fibroblast growth factor-2 (FGF-2). Eighteen patients were studied. Immunostaining for podoplanin was performed to highlight lymphatic vessels. FGF-2 mRNA expression was determined by quantitative real-time RT-PCR, whereas protein expression was quantitatively assessed by densitometric analysis of Western blot signal intensity. The immunoblots were further validated by FGF-2 immunostaining of histological sections. LVD was significantly increased in tumor tissue compared with the normal mucosa but no changes in LVD between stages II and III CRC was observed. FGF-2 was found to be downregulated both at the mRNA and protein level in tumor tissues compared with normal mucosa. Lymphangiogenesis was triggered early in tumor development. An increased LVD was established before the tumor reached stage II. FGF-2 was downregulated in tumor tissue. The importance of this finding remains unclear.

Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation.

Human mesenchymal stem cells (hMSC) aid in tissue maintenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequisite to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that hMSC may contribute to cancer development and progression either by acting as cancer-initiating cells or through interactions with stromal elements. If spontaneous transformation ex vivo occurs, this may jeopardize the use of hMSC as therapeutic tools. Whereas murine MSC readily undergo spontaneous transformation, there are conflicting reports about spontaneous transformation of hMSC. We have addressed this controversy in a two-center study by growing bone marrow-derived hMSC in long-term cultures (5-106 weeks). We report for the first time spontaneous malignant transformation to occur in 45.8% (11 of 24) of these cultures. In comparison with hMSC, the transformed mesenchymal cells (TMC) showed a significantly increased proliferation rate and altered morphology and phenotype. In contrast to hMSC, TMC grew well in soft agar assays and were unable to undergo complete differentiation. Importantly, TMC were highly tumorigenic, causing multiple fast-growing lung deposits when injected into immunodeficient mice. We conclude that spontaneous malignant transformation may represent a biohazard in long-term ex vivo expansion of hMSC. On the other hand, this spontaneous transformation process may represent a unique model for studying molecular pathways initiating malignant transformation of hMSC.