A Mitochondrial VDAC1-Based Peptide Greatly Suppresses Steatosis and NASH-Associated Pathologies in a Mouse Model.

Non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH) are liver pathologies characterized by severe metabolic alterations due to fat accumulation that lead to liver damage, inflammation, and fibrosis. We demonstrate that the voltage-dependent anion channel 1 (VDAC1)-based peptide R-Tf-D-LP4 arrested steatosis and NASH progression, as produced by a high-fat diet (HFD-32) in a mouse model, and reversed liver pathology to a normal-like state. VDAC1, a multi-functional mitochondrial protein, regulates cellular metabolic and energetic functions and apoptosis and interacts with many proteins. R-Tf-D-LP4 treatment eliminated hepatocyte ballooning degeneration, inflammation, and liver fibrosis associated with steatosis, NASH, and hepatocarcinoma, and it restored liver pathology-associated enzyme and glucose levels. Peptide treatment affected carbohydrate and lipid metabolism, increasing the expression of enzymes and factors associated with fatty acid transport to mitochondria, enhancing ß-oxidation and thermogenic processes, yet decreasing the expression of enzymes and regulators of fatty acid synthesis. The VDAC1-based peptide thus offers a promising therapeutic approach for steatosis and NASH.

The long noncoding RNA TP73-AS1 promotes tumorigenicity of medulloblastoma cells.

Medulloblastoma is the most common malignant brain cancer in children. Since previous studies have mainly focused on alterations in the coding genome, our understanding of the contribution of long noncoding RNAs (lncRNAs) to medulloblastoma biology is just emerging. Using patient-derived data, we show that the promoter of lncRNA TP73-AS1 is hypomethylated and that the transcript is highly expressed in the SHH subgroup. Furthermore, high expression of TP73-AS1 is correlated with poor outcome in patients with TP53 wild-type SHH tumors. Silencing TP73-AS1 in medulloblastoma tumor cells induced apoptosis, while proliferation and migration were inhibited in culture. In vivo, silencing TP73-AS1 in medulloblastoma tumor cells resulted in reduced tumor growth, reduced proliferation of tumor cells, increased apoptosis and led to prolonged survival of tumor-bearing mice. Together, our study suggests that the lncRNA TP73-AS1 is a prognostic marker and therapeutic target in medulloblastoma tumors and serves as a proof of concept that lncRNAs are important factors in the disease.

Mitochondrial VDAC1 Silencing Leads to Metabolic Rewiring and the Reprogramming of Tumour Cells into Advanced Differentiated States.

Oncogenic properties, along with the metabolic reprogramming necessary for tumour growth and motility, are acquired by cancer cells. Thus, tumour metabolism is becoming a target for cancer therapy. Here, cancer cell metabolism was tackled by silencing the expression of voltage-dependent anion channel 1 (VDAC1), a mitochondrial protein that controls cell energy, as well as metabolic and survival pathways and that is often over-expressed in many cancers. We demonstrated that silencing VDAC1 expression using human-specific siRNA (si-hVDAC1) inhibited cancer cell growth, both in vitro and in mouse xenograft models of human glioblastoma (U-87MG), lung cancer (A549), and triple negative breast cancer (MDA-MB-231). Importantly, treatment with si-hVDAC1 induced metabolic rewiring of the cancer cells, reversing their oncogenic properties and diverting them towards differentiated-like cells. The si-hVDAC1-treated residual "tumour" showed reprogrammed metabolism, decreased proliferation, inhibited stemness and altered expression of genes and proteins, leading to cell differentiation toward less malignant lineages. These VDAC1 depletion-mediated effects involved alterations in master transcription factors associated with cancer hallmarks, such as highly increased expression of p53 and decreased expression of HIF-1a and c-Myc that regulate signalling pathways (e.g., AMPK, mTOR). High expression of p53 and the pro-apoptotic proteins cytochrome c and caspases without induction of apoptosis points to functions for these proteins in promoting cell differentiation. These results clearly show that VDAC1 depletion similarly leads to a rewiring of cancer cell metabolism in breast and lung cancer and glioblastoma, regardless of origin or mutational status. This metabolic reprogramming results in cell growth arrest and inhibited tumour growth while encouraging cell differentiation, thus generating cells with decreased proliferation capacity. These results further suggest VDAC1 to be an innovative and markedly potent therapeutic target.

Targeting Liver Cancer and Associated Pathologies in Mice with a Mitochondrial VDAC1-Based Peptide.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Despite progress in identifying risk factors, the incidence of HCC is increasing. Moreover, therapeutic options are limited and survival is poor. Therefore, alternative and innovative therapeutic strategies are urgently required. R-Tf-D-LP4, a cell-penetrating peptide derived from the mitochondrial multifunctional protein the voltage-dependent anion channel (VDAC1), is identified here as a highly effective liver cancer treatment. Recently, we demonstrated that R-Tf-D-LP4 induced apoptosis and inhibited tumor growth in mouse models. We now demonstrate that R-Tf-D-LP4 induced apoptosis in cancer liver-derived cell lines and inhibited tumor growth in three different liver cancer mouse models. These included diethylnitrosamine (DEN)-induced HCC, metabolically high-fat diet-induced HCC, and using a subcutaneous HepG2 cell xenograft model. Intravenous injection of the peptide into tumor-carrying DEN-treated mice resulted in dose-dependent inhibition of tumor growth up to complete tumor elimination. TUNEL staining of liver sections demonstrated peptide-induced apoptosis. Hematoxylin/eosin and Sirius red staining of liver sections showed decreased fibrotic formation. Immunohistochemical staining demonstrated reduced numbers of α-SMA-expressing cells in R-Tf-D-LP4-treated mouse livers. Additionally, macrophage presence in liver tissue was reduced in R-Tf-D-LP4-treated mice. Liver sections from DEN-treated mice showed steatohepatic pathology, reflected as fatty liver, inflammation, ballooning degeneration, and fibrosis; all were eliminated upon peptide treatment. Peptide treatment also inhibited tumor development in a nonalcoholic steatohepatitis-hepatocellular carcinoma mouse model induced by HFD. In HepG2 subcutaneous tumor xenografts, R-Tf-D-LP4 inhibited tumor growth. CONCLUSION: These results show that the VDAC1-based peptide R-Tf-D-LP4 has multiple effects on liver cancer cells, leading to impairment of cell energy and metabolism homeostasis, induction of apoptosis, and elimination of liver cancer-associated processes, and thus represents a promising therapeutic approach for liver cancer.

A New Role for the Mitochondrial Pro-apoptotic Protein SMAC/Diablo in Phospholipid Synthesis Associated with Tumorigenesis.

The mitochondrial pro-apoptotic protein SMAC/Diablo participates in apoptosis by negatively regulating IAPs and activating caspases, thus encouraging apoptosis. Unexpectedly, we found that SMAC/Diablo is overexpressed in cancer. This paradox was addressed here by silencing SMAC/Diablo expression using specific siRNA (si-hSMAC). In cancer cell lines and subcutaneous lung cancer xenografts in mice, such silencing reduced cell and tumor growth. Immunohistochemistry and electron microscopy of the si-hSMAC-treated residual tumor demonstrated morphological changes, including cell differentiation and reorganization into glandular/alveoli-like structures and elimination of lamellar bodies, surfactant-producing organs. Next-generation sequencing of non-targeted or si-hSMAC-treated tumors revealed altered expression of genes associated with the cellular membrane and extracellular matrix, of genes found in the ER and Golgi lumen and in exosomal networks, of genes involved in lipid metabolism, and of lipid, metabolite, and ion transporters. SMAC/Diablo silencing decreased the levels of phospholipids, including phosphatidylcholine. These findings suggest that SMAC/Diablo possesses additional non-apoptotic functions related to regulating lipid synthesis essential for cancer growth and development and that this may explain SMAC/Diablo overexpression in cancer. The new lipid synthesis-related function of the pro-apoptotic protein SMAC/Diablo in cancer cells makes SMAC/Diablo a promising therapeutic target.

VDAC1 functions in Ca2+ homeostasis and cell life and death in health and disease.

In the outer mitochondrial membrane (OMM), the voltage-dependent anion channel 1 (VDAC1) serves as a mitochondrial gatekeeper, controlling the metabolic and energy cross-talk between mitochondria and the rest of the cell. VDAC1 also functions in cellular Ca2+ homeostasis by transporting Ca2+ in and out of mitochondria. VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis, contributing to the release of apoptotic proteins located in the inter-membranal space (IMS) and regulating apoptosis via association with pro- and anti-apoptotic members of the Bcl-2 family of proteins and hexokinase. VDAC1 is highly Ca2+-permeable, transporting Ca2+ to the IMS and thus modulating Ca2+ access to Ca2+ transporters in the inner mitochondrial membrane. Intra-mitochondrial Ca2+ controls energy metabolism via modulating critical enzymes in the tricarboxylic acid cycle and in fatty acid oxidation. Ca2+ also determines cell sensitivity to apoptotic stimuli and promotes the release of pro-apoptotic proteins. However, the precise mechanism by which intracellular Ca2+ mediates apoptosis is not known. Here, the roles of VDAC1 in mitochondrial Ca2+ homeostasis are presented while emphasizing a new proposed mechanism for the mode of action of pro-apoptotic drugs. This view, proposing that Ca2+-dependent enhancement of VDAC1 expression levels is a major mechanism by which apoptotic stimuli induce apoptosis, position VDAC1 oligomerization at a molecular focal point in apoptosis regulation. The interactions of VDAC1 with many proteins involved in Ca2+ homeostasis or regulated by Ca2+, as well as VDAC-mediated control of cell life and death and the association of VDAC with disease, are also presented.

A molecular signature of lung cancer: potential biomarkers for adenocarcinoma and squamous cell carcinoma.

Adenocarcinoma (AC) and squamous cell carcinoma (SCC), sub-types of non-small cell lung cancer (NSCLC), both present unique features at the genome, epigenome, transcriptome and proteome levels, as well as shared clinical and histopathological characteristics, but differ in terms of treatment. To ensure proper treatment, one must be able to distinguish between these sub-types. Here, we identify novel biomarker proteins in NSCLC, allowing for distinguishing between the AC and SCC sub-types. Proteomics analysis distinguished between healthy and tumor tissues, with the expression level of 1,494 proteins being altered, 378 of which showed a ≥|100|-fold change. Enrichment of proteins related to protein synthesis and degradation, and of proteins associated with mitochondria, metabolism, and apoptosis, was found. Network analysis defined groups of proteins, such as those associated with cell metabolic processes or with fatty acid/lipid metabolism and transport. Several biomarkers that enable for distinguishing between AC and SCC were identified here for the first time, and together with previous reports confirmed here, led us to propose a list of proteins differentially expressed in SCC and AC. Some of these biomarkers are clear signatures for AC or SCC and four of them are secreted proteins. The presence of the mitochondrial protein SMAC/Diablo in the nucleus was found to be a signature for SCC. Precise diagnosis of AC and SCC is essential for selecting appropriate treatment and thus, increasing patient life expectancy. Finally, the search for drugs that target some of these biomarkers may lead to new treatments for lung cancer.

Voltage-Dependent Anion Channel 1 As an Emerging Drug Target for Novel Anti-Cancer Therapeutics.

Cancer cells share several properties, high proliferation potential, reprogramed metabolism, and resistance to apoptotic cues. Acquiring these hallmarks involves changes in key oncogenes and non-oncogenes essential for cancer cell survival and prosperity, and is accompanied by the increased energy requirements of proliferating cells. Mitochondria occupy a central position in cell life and death with mitochondrial bioenergetics, biosynthesis, and signaling are critical for tumorigenesis. Voltage-dependent anion channel 1 (VDAC1) is situated in the outer mitochondrial membrane (OMM) and serving as a mitochondrial gatekeeper. VDAC1 allowing the transfer of metabolites, fatty acid ions, Ca2+, reactive oxygen species, and cholesterol across the OMM and is a key player in mitochondrial-mediate apoptosis. Moreover, VDAC1 serves as a hub protein, interacting with diverse sets of proteins from the cytosol, endoplasmic reticulum, and mitochondria that together regulate metabolic and signaling pathways. The observation that VDAC1 is over-expressed in many cancers suggests that the protein may play a pivotal role in cancer cell survival. However, VDAC1 is also important in mitochondria-mediated apoptosis, mediating release of apoptotic proteins and interacting with anti-apoptotic proteins, such as B-cell lymphoma 2 (Bcl-2), Bcl-xL, and hexokinase (HK), which are also highly expressed in many cancers. Strategically located in a "bottleneck" position, controlling metabolic homeostasis and apoptosis, VDAC1 thus represents an emerging target for anti-cancer drugs. This review presents an overview on the multi-functional mitochondrial protein VDAC1 performing several functions and interacting with distinct sets of partners to regulate both cell life and death, and highlights the importance of the protein for cancer cell survival. We address recent results related to the mechanisms of VDAC1-mediated apoptosis and the potential of associated proteins to modulate of VDAC1 activity, with the aim of developing VDAC1-based approaches. The first strategy involves modification of cell metabolism using VDAC1-specific small interfering RNA leading to inhibition of cancer cell and tumor growth and reversed oncogenic properties. The second strategy involves activation of cancer cell death using VDAC1-based peptides that prevent cell death induction by anti-apoptotic proteins. Finally, we discuss the potential therapeutic benefits of treatments and drugs leading to enhanced VDAC1 expression or targeting VDAC1 to induce apoptosis.

Immunological and inflammatory mapping of vascularized composite allograft rejection processes in a rat model.

BACKGROUND: Hand and face vascularized composite allotransplantation (VCA) is an evolving and challenging field with great opportunities. During VCA, massive surgical damage is inflicted on both donor and recipient tissues, which may contribute to the high VCA rejection rates. To segregate between the damage-induced and rejection phase of post-VCA responses, we compared responses occurring up to 5 days following syngeneic versus allogeneic vascularized groin flap transplantations, culminating in transplant acceptance or rejection, respectively. METHODS: The immune response elicited upon transplantation of a syngeneic versus allogeneic vascularized groin flap was compared at Post-operative days 2 or 5 by histology, immunohistochemistry and by broad-scope gene and protein analyses using quantitative real-time PCR and Multiplex respectively. RESULTS: Immune cell infiltration began at the donor-recipient interface and paralleled expression of a large group of wound healing-associated genes in both allografts and syngrafts. By day 5 post-transplantation, cell infiltration spread over the entire allograft but remained confined to the wound site in the syngraft. This shift correlated with upregulation of IL-18, INFg, CXCL9, 10 and 11, CCL2, CCL5, CX3CL1 and IL-10 in the allograft only, suggesting their role in the induction of the anti-alloantigen adaptive immune response. CONCLUSIONS: High resemblance between the cues governing VCA and solid organ rejection was observed. Despite this high resemblance we describe also, for the first time, a damage induced inflammatory component in VCA rejection as immune cell infiltration into the graft initiated at the surgical damage site spreading to the entire allograft only at late stage rejection. We speculate that the highly inflammatory setting created by the unique surgical damage during VCA may enhance acute allograft rejection.

VDAC1 as a Player in Mitochondria-Mediated Apoptosis and Target for Modulating Apoptosis.

BACKGROUND: The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane protein, functions as a mitochondrial governor, controlling transport of metabolites in and out of the mitochondria and energy production, while also coordinating glycolysis and oxidative phosphorylation. VDAC1 plays a key role in mitochondria-mediated apoptosis by functioning in the release of apoptotic proteins located in the inter-membranal space and due to its association with pro- and anti-apoptotic proteins. Thus, VDAC1 is considered as a promising target for controlling apoptosis. METHODS: We reviewed published data presenting accumulated evidence suggesting that VDAC1 oligomerization represents an important step in the intrinsic mitochondria-mediated apoptosis pathway. RESULTS: The published data support the proposal that VDAC1 oligomerization leads to the formation of a large pore that allows the release of pro-apoptotic proteins to the cytosol, thereby, activation of apoptosis. Evidence for the relationship between VDAC1 expression levels and induction of apoptosis are presented. This includes the finding that almost all apoptosis stimuli induce VDAC1 over-expression shifting VDAC1 from a monomeric to an oligomeric assembly, corresponding to the Cyto c release channel. Compounds or conditions inducing VDAC1 over-expression, VDAC1 oligomerization and apoptosis are presented. Likewise, VDAC1-interacting molecules, that inhibit both VDAC1 oligomerization and apoptosis are also presented. CONCLUSION: This review highlights the findings about VDAC1 oligomerization as a potential target for controlling apoptosis, specifically using drugs to induce apoptotic cell death in cancer and inhibit apoptosis in neurodegenerative diseases, as well as possible VDAC1-based therapeutic applications.

Mitochondrial VDAC1-based peptides: Attacking oncogenic properties in glioblastoma.

Glioblastoma multiforme (GBM), a primary brain malignancy characterized by high morbidity, invasiveness, proliferation, relapse and mortality, is resistant to chemo- and radiotherapies and lacks effective treatment. GBM tumors undergo metabolic reprograming and develop anti-apoptotic defenses. We targeted GBM with a peptide derived from the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), a key component of cell energy, metabolism and apoptosis regulation. VDAC1-based cell-penetrating peptides perturbed cell energy and metabolic homeostasis and induced apoptosis in several GBM and GBM-derived stem cell lines. We found that the peptides simultaneously attacked several oncogenic properties of human U-87MG cells introduced into sub-cutaneous xenograft mouse model, inhibiting tumor growth, invasion, and cellular metabolism, stemness and inducing apoptosis. Peptide-treated tumors showed decreased expression of all tested metabolism-related enzymes and transporters, and elevated levels of apoptotic proteins, such as p53, cytochrome c and caspases. Retro-Tf-D-LP4, containing the human transferrin receptor (TfR)-recognition sequence, crossed the blood-brain barrier (BBB) via the TfR that is highly expressed in the BBB to strongly inhibit tumor growth in an intracranial xenograft mouse model. In summary, the VDAC1-based peptides tested here offer a potentially affordable and innovative new conceptual therapeutic paradigm that might overcome GBM stemness and invasiveness and reduce relapse rates.

VDAC1 is a molecular target in glioblastoma, with its depletion leading to reprogrammed metabolism and reversed oncogenic properties.

BACKGROUND: Glioblastoma (GBM), an aggressive brain tumor with frequent relapses and a high mortality, still awaits an effective treatment. Like many cancers, GBM cells acquire oncogenic properties, including metabolic reprogramming, vital for growth. As such, tumor metabolism is an emerging avenue for cancer therapy. One relevant target is the voltage-dependent anion channel 1 (VDAC1), a mitochondrial protein controlling cell energy and metabolic homeostasis. METHODS: We used VDAC1-specific short interfering (si)RNA (si-VDAC1) to treat GBM cell lines and subcutaneous or intracranial-orthotopic GBM xenograft mouse models. Tumors were monitored using MRI, immunohistochemistry, immunoblotting, immunofluorescence, quantitative real-time PCR, transcription factor expression, and DNA microarray analyses. RESULTS: Silencing VDAC1 expression using si-VDAC1 in 9 glioblastoma-related cell lines, including patient-derived cells, led to marked decreases in VDAC1 levels and cell growth. Using si-VDAC1 in subcutaneous or intracranial-orthotopic GBM models inhibited tumor growth and reversed oncogenic properties, such as reprogrammed metabolism, stemness, angiogenesis, epithelial-mesenchymal transition, and invasiveness. In cells in culture, si-VDAC1 inhibits cancer neurosphere formation and, in tumors, targeted cancer stem cells, leading to their differentiation into neuronal-like cells. These VDAC1 depletion-mediated effects involved alterations in transcription factors regulating signaling pathways associated with cancer hallmarks. CONCLUSION: VDAC1 offers a target for GBM treatment, allowing for attacks on the interplay between metabolism and oncogenic signaling networks, leading to tumor cell differentiation into neuron- and astrocyte-like cells. Simultaneously attacking all of these processes, VDAC1 depletion overcame GBM heterogeneity and can replace several anticancer drugs that separately target angiogenesis, proliferation, or metabolism.

VDAC1 at the crossroads of cell metabolism, apoptosis and cell stress.

This review presents current knowledge related to VDAC1 as a multi-functional mitochondrial protein acting on both sides of the coin, regulating cell life and death, and highlighting these functions in relation to disease. It is now recognized that VDAC1 does not only play a crucial role in regulating the metabolic and energetic functions of mitochondria. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows the control of metabolic cross-talk between mitochondria and the rest of the cell and also enables its interaction with proteins involved in metabolic and survival pathways. Along with regulating cellular energy production and metabolism, VDAC1 is also involved in the process of mitochondria-mediated apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. VDAC1 functions in the release of apoptotic proteins located in the mitochondrion inter-membranal space via oligomerization to form a large channel that allows passage of cytochrome c and AIF and their release to the cytosol, subsequently apoptotic cell death. VDAC1 also regulates apoptosis via interactions with apoptosis regulatory proteins, such as hexokinase (HK), Bcl2 and Bcl-xL, some of which are also highly expressed in many cancers. This review also provide insight into VDAC1 function in Ca2+ homeostasis, oxidative stress, and presents VDAC1 as a hub protein interacting with over 100 proteins. Such interactions enable VDAC1 to mediate and regulate the integration of mitochondrial functions with cellular activities. VDAC1 can thus be considered as standing at the crossroads between mitochondrial metabolite transport and apoptosis and hence represents an emerging cancer drug target.

Reducing VDAC1 expression induces a non-apoptotic role for pro-apoptotic proteins in cancer cell differentiation.

Proteins initially identified as essential for apoptosis also mediate a wide range of non-apoptotic functions that include cell cycle progression, differentiation and metabolism. As this phenomenon was mostly reported with non-cancer cells, we considered non-conventional roles for the apoptotic machinery in the cancer setting. We found that treating glioblastoma (GBM) tumors with siRNA against VDAC1, a mitochondrial protein found at the crossroads of metabolic and survival pathways and involved in apoptosis, inhibited tumor growth while leading to differentiation of tumor cells into neuronal-like cells, as reflected in the expression of specific markers. Although VDAC1 depletion did not induce apoptosis, the expression levels of several pro-apoptotic regulatory proteins were changed. Specifically, VDAC1 deletion led to up-regulation of caspases, p53, cytochrome c, and down-regulation of SMAC/Diablo, AIF and TSPO. The down-regulated group was highly expressed in U-87MG xenografts, as well as in GBMs from human patients. We also showed that the rewired cancer-cell metabolism resulting from VDAC1 depletion reinforced cell growth arrest and differentiation via alterations in the transcription factors p53, c-Myc, HIF-1α and NF-κB. The decrease in c-Myc, HIF-1α and NF-κB levels was in accord with reduced cell proliferation, whereas increased p53 expression promoted differentiation. Thus, upon metabolic re-programing induced by VDAC1 depletion, the levels of pro-apoptotic proteins associated with cell growth decreased, while those connected to cell differentiation increased, converting GBM cells into astrocyte- and neuron-like cells. The results reveal that in tumors, pro-apoptotic proteins can perform non-apoptotic functions, acting as regulators of cell growth and differentiation, making these molecules potential new targets for cancer therapy. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

The mitochondrial voltage-dependent anion channel 1 in tumor cells.

VDAC1 is found at the crossroads of metabolic and survival pathways. VDAC1 controls metabolic cross-talk between mitochondria and the rest of the cell by allowing the influx and efflux of metabolites, ions, nucleotides, Ca2+ and more. The location of VDAC1 at the outer mitochondrial membrane also enables its interaction with proteins that mediate and regulate the integration of mitochondrial functions with cellular activities. As a transporter of metabolites, VDAC1 contributes to the metabolic phenotype of cancer cells. Indeed, this protein is over-expressed in many cancer types, and silencing of VDAC1 expression induces an inhibition of tumor development. At the same time, along with regulating cellular energy production and metabolism, VDAC1 is involved in the process of mitochondria-mediated apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. The engagement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space involves VDAC1 oligomerization that mediates the release of cytochrome c and AIF to the cytosol, subsequently leading to apoptotic cell death. Apoptosis can also be regulated by VDAC1, serving as an anchor point for mitochondria-interacting proteins, such as hexokinase (HK), Bcl2 and Bcl-xL, some of which are also highly expressed in many cancers. By binding to VDAC1, HK provides both a metabolic benefit and apoptosis-suppressive capacity that offer the cell a proliferative advantage and increase its resistance to chemotherapy. Thus, these and other functions point to VDAC1 as an excellent target for impairing the re-programed metabolism of cancer cells and their ability to evade apoptosis. Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to both cancer development and therapy. In addressing the recently solved 3D structures of VDAC1, this review will point to structure-function relationships of VDAC as critical for deciphering how this channel can perform such a variety of roles, all of which are important for cell life and death. Finally, this review will also provide insight into VDAC function in Ca2+ homeostasis, protection against oxidative stress, regulation of apoptosis and involvement in several diseases, as well as its role in the action of different drugs. We will discuss the use of VDAC1-based strategies to attack the altered metabolism and apoptosis of cancer cells. These strategies include specific siRNA able to impair energy and metabolic homeostasis, leading to arrested cancer cell growth and tumor development, as well VDAC1-based peptides that interact with anti-apoptotic proteins to induce apoptosis, thereby overcoming the resistance of cancer cell to chemotherapy. Finally, small molecules targeting VDAC1 can induce apoptosis. VDAC1 can thus be considered as standing at the crossroads between mitochondrial metabolite transport and apoptosis and hence represents an emerging cancer drug target. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

The 106b∼25 microRNA cluster is essential for neovascularization after hindlimb ischaemia in mice.

AIMS: MicroRNAs (miRNAs, miR) are endogenous short RNA sequences that regulate a wide range of physiological and pathophysiological processes. Several miRNAs control the formation of new blood vessels either by increasing or by inhibiting angiogenesis. Here, we investigated the possible role of the miR-106b∼25 cluster in postnatal neovascularization and in regulation of the angiogenic properties of adult bone marrow-derived stromal cells. METHODS AND RESULTS: To study the effect of miR-106b∼25 deletion on neovascularization, we used a miR-106b∼25 knockout (KO) mouse model. After inducing hindlimb ischaemia, we showed that vascularization in ischaemic mice devoid of miR-106b∼25 is impaired, as evident from the reduced blood flow on laser Doppler perfusion imaging. The miR-106b∼25 cluster was also shown here to be an essential player in the proper functioning of bone marrow-derived stromal cells through its regulation of apoptosis, matrigel tube formation capacity, cytokine secretion, and expression of the stem-cell marker Sca-1. In addition, we showed that capillary sprouting from miR-106b∼25 KO aortic rings is diminished. CONCLUSION: These results show that the miR-106b∼25 cluster regulates post-ischaemic neovascularization in mice, and that it does so in part by regulating the function of angiogenic bone marrow-derived stromal cells and of endothelial cells.

Unique Versus Redundant Functions of IL-1α and IL-1ß in the Tumor Microenvironment.

Interleukin-1 (IL-1) is a major "alarm" upstream pro-inflammatory cytokine that also affects immunity and hematopoiesis by inducing cytokine cascades. In the tumor arena, IL-1 is produced by malignant or microenvironmental cells. As a pleiotropic cytokine, IL-1 is involved in tumorigenesis and tumor invasiveness but also in the control of anti-tumor immunity. IL-1α and IL-1ß are the major agonists of IL-1, while IL-1Ra is a physiological inhibitor of pre-formed IL-1. In their secreted form, IL-1α and IL-1ß bind to the same receptors and induce the same biological functions, but IL-1α and IL-1ß differ in their compartmentalization within the producing cell or the microenvironment. IL-1ß is only active in its processed, secreted form, and mediates inflammation, which promotes carcinogenesis, tumor invasiveness, and immunosuppression, whereas IL-1α is mainly cell-associated and in the tumor context, when expressed on the cell membrane, it stimulates anti-tumor cell immunity manifested by tumor regression. In the tumor milieu, extracellular levels of IL-1α are usually low and do not stimulate broad inflammation that promotes progression. Immunosuppression induced by IL-1ß in the tumor microenvironment, mainly through MDSC induction, usually inhibits or masks anti-tumor cell immunity induced by cell-associated IL-1α. However, in different tumor systems, redundant or unique patterns of IL-1α and IL-1ß expression and function have been observed. Recent breakthroughs in inflammasome biology and IL-1ß processing/secretion have spurred the development of novel anti-IL-1 agents, which are being used in clinical trials in patients with diverse inflammatory diseases. Better understanding of the integrative role of IL-1α and IL-1ß in distinct malignancies will facilitate the application of novel IL-1 modulation approaches at the bedside, in cancer patients with minimal residual disease (MRD), as an adjunct to conventional approaches to reduce the tumor burden.

Characterization of the melanoma brain metastatic niche in mice and humans.

Brain metastases occur in 15% of patients with melanoma and are associated with a dismal prognosis. Here, we investigate the architectural phenotype and stromal reaction of melanoma brain metastasis in mice and humans. A syngeneic, green fluorescence protein (GFP)-expressing murine B16-F1 melanoma clone was introduced via intracardiac injection, and was examined in vivo in comparison with human specimens. Immunofluorescence analyses of the brain metastases revealed that F4/80(+) macrophages/microglia were most abundant at the tumor front, but rare in its core, where they were found only around blood vessels (P = 0.01). Similar pattern of infiltration was found in CD3(+) T cells (P < 0.01). Infiltrating T cells were prominently CD4(+) compared with CD8(+) T cells (P < 0.001). Blood vessels (CD31(+)) were less abundant at the tumor front than in its center (12 ± 1 vs. 4 ± 0.6 vessels per high-power field [HPF], P < 0.001). In contrast, there were few vessels at the tumor front, but their diameter was significantly larger at the front (8236 µm(2) vs. 4617 µm(2) average cross-sectional area, P < 0.005). This is the first comparative analysis of melanoma brain metastases showing similar stromal reaction in murine models and human specimens. Our results validate the utility of this murine model of melanoma brain metastases for investigating the mechanism of the human disease.

Association between tumorigenic potential and the fate of cancer cells in a syngeneic melanoma model.

The self-renewal potential of a cancer cell can be estimated by using particular assays, which include xenotransplantation in immunocompromised animals or culturing in non-adherent serum-free stem-cells media (SCM). However, whether cells with self-renewal potential actually contribute to disease is unknown. Here we investigated the tumorigenic potential and fate of cancer cells in an in-vivo melanoma model. We examined cell lines which were derived from the same parental line: a non-metastatic cell line (K1735/16), a metastatic cell line (K1735/M4) and a cell line which was selected in non-adherent conditions (K1735/16S). All cell lines exhibited similar proliferation kinetics when grown on culture plates. K1735/16 cells grown in soft agar or in suspension non-adherent conditions failed to form colonies or spheroids, whereas the other cell lines showed prominent colonogenicity and spheroid formation capacity. By using sphere limiting dilution analysis (SLDA) in serum-free media, K1735/16S and K1735/M4 cells grown in suspension were capable of forming spheroids even in low frequencies of concentrations, as opposed to K1735/16 cells. The tumorigenic potential of the cell lines was determined in SCID mice using intra footpad injections. Palpable tumors were evident in all mice. In agreement with the in-vitro studies, the K1735/M4 cell line exhibited the highest growth kinetics, followed by the K1735/16S cell line, whereas the K1735/16 cell line had the lowest tumor growth potential (P<0.001). In contrast, when we repeated the experiments in syngeneic C3H/HeN mice, the K1735/16 cell line produced macroscopic tumors 30-100 days after injection, whereas K1735/M4 and K1735/16S derived tumors regressed spontaneously in 90-100% of mice. TUNEL analysis revealed significantly higher number of apoptotic cells in K1735/16S and K1735/M4 cell line-derived tumors compared to K1735/16 tumors (P<0.001). The models we have examined here raised the possibility, that cells with high-tumorigenic activity may be more immunogenic and hence are more susceptible to immune-regulation.

Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor.

Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDA), prostate, or head and neck cancers. These patients undergo palliative rather than curative treatment due to dissemination of cancer along nerves, well beyond the extent of any local invasion. Although CPNI is a common source of distant tumor spread and a cause of significant morbidity, its exact mechanism is undefined. Immunohistochemical analysis of specimens excised from patients with PDAs showed a significant increase in the number of endoneurial macrophages (EMΦ) that lie around nerves invaded by cancer compared with normal nerves. Video microscopy and time-lapse analysis revealed that EMΦs are recruited by the tumor cells in response to colony-stimulated factor-1 secreted by invading cancer cells. Conditioned medium (CM) of tumor-activated EMΦs (tEMΦ) induced a 5-fold increase in migration of PDA cells compared with controls. Compared with resting EMΦs, tEMΦs secreted higher levels of glial-derived neurotrophic factor (GDNF), inducing phosphorylation of RET and downstream activation of extracellular signal-regulated kinases (ERK) in PDA cells. Genetic and pharmacologic inhibition of the GDNF receptors GFRA1 and RET abrogated the migratory effect of EMΦ-CM and reduced ERK phosphorylation. In an in vivo CPNI model, CCR2-deficient mice that have reduced macrophage recruitment and activation showed minimal nerve invasion, whereas wild-type mice developed complete sciatic nerve paralysis due to massive CPNI. Taken together, our results identify a paracrine response between EMΦs and PDA cells that orchestrates the formation of cancer nerve invasion.