Tasleem Arif

Silencing VDAC1 Expression by siRNA Inhibits Cancer Cell Proliferation and Tumor Growth In Vivo

Alterations in cellular metabolism and bioenergetics are vital for cancer cell growth and motility. Here, the role of the mitochondrial protein voltage-dependent anion channel (VDAC1), a master gatekeeper regulating the flux of metabolites and ions between mitochondria and the cytoplasm, in regulating the growth of several cancer cell lines was investigated by silencing VDAC1 expression using small interfering RNA (siRNA). A single siRNA specific to the human VDAC1 sequence at nanomolar concentrations led to some 90% decrease in VDAC1 levels in the lung A549 and H358, prostate PC-3, colon HCT116, glioblastoma U87, liver HepG2, and pancreas Panc-1 cancer cell lines. VDAC1 silencing persisted 144 hours post-transfection and resulted in profound inhibition of cell growth in cancer but not in noncancerous cells, with up to 90% inhibition being observed over 5 days that was prolonged by a second transfection. Cells expressing low VDAC1 levels showed decreased mitochondrial membrane potential and adenoside triphosphate (ATP) levels, suggesting limited metabolite exchange between mitochondria and cytosol. Moreover, cells silenced for VDAC1 expression showed decreased migration, even in the presence of the wound healing accelerator basic fibroblast growth factor (bFGF). VDAC1-siRNA inhibited cancer cell growth in a Matrigel-based assay in host nude mice. Finally, in a xenograft lung cancer mouse model, chemically modified VDAC1-siRNA not only inhibited tumor growth but also resulted in tumor regression. This study thus shows that VDAC1 silencing by means of RNA interference (RNAi) dramatically inhibits cancer cell growth and tumor development by disabling the abnormal metabolic behavior of cancer cells, potentially paving the way for a more effective pipeline of anticancer drugs.

Ca2 +-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca(2+) across the OMM and because Ca(2+) has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca(2+) levels ([Ca(2)(+)]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca(2+) and induce VDAC1 over-expression. Direct elevation of [Ca(2+)]i by the Ca(2+)-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca(2+)]i using the cell-permeable Ca(2+)-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca(2+)]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca(2+)-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca(2+)]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca(2+) promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

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.

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.

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.

Novel Biomarker Proteins in Chronic Lymphocytic Leukemia: Impact on Diagnosis, Prognosis and Treatment

In many cancers, cells undergo re-programming of metabolism, cell survival and anti-apoptotic defense strategies, with the proteins mediating this reprogramming representing potential biomarkers. Here, we searched for novel biomarker proteins in chronic lymphocytic leukemia (CLL) that can impact diagnosis, treatment and prognosis by comparing the protein expression profiles of peripheral blood mononuclear cells from CLL patients and healthy donors using specific antibodies, mass spectrometry and binary logistic regression analyses and other bioinformatics tools. Mass spectrometry (LC-HR-MS/MS) analysis identified 1,360 proteins whose expression levels were modified in CLL-derived lymphocytes. Some of these proteins were previously connected to different cancer types, including CLL, while four other highly expressed proteins were not previously reported to be associated with cancer, and here, for the first time, DDX46 and AK3 are linked to CLL. Down-regulation expression of two of these proteins resulted in cell growth inhibition. High DDX46 expression levels were associated with shorter survival of CLL patients and thus can serve as a prognosis marker. The proteins with modified expression include proteins involved in RNA splicing and translation and particularly mitochondrial proteins involved in apoptosis and metabolism. Thus, we focused on several metabolism- and apoptosis-modulating proteins, particularly on the voltage-dependent anion channel 1 (VDAC1), regulating both metabolism and apoptosis. Expression levels of Bcl-2, VDAC1, MAVS, AIF and SMAC/Diablo were markedly increased in CLL-derived lymphocytes. VDAC1 levels were highly correlated with the amount of CLL-cancerous CD19+/CD5+ cells and with the levels of all other apoptosis-modulating proteins tested. Binary logistic regression analysis demonstrated the ability to predict probability of disease with over 90% accuracy. Finally, based on the changes in the levels of several proteins in CLL patients, as revealed from LC-HR-MS/MS, we could distinguish between patients in a stable disease state and those who would be later transferred to anti-cancer treatments. The over-expressed proteins can thus serve as potential biomarkers for early diagnosis, prognosis, new targets for CLL therapy, and treatment guidance of CLL, forming the basis for personalized therapy.

Selective induction of cancer cell death by VDAC1‐based peptides and their potential use in cancer therapy

Mitochondrial VDAC1 mediates cross talk between the mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca2+, and metabolites and serves as a key player in apoptosis. As such, VDAC1 is involved in two important hallmarks of cancer development, namely energy and metabolic reprograming and apoptotic cell death evasion. We previously developed cell‐penetrating VDAC1‐derived peptides that interact with hexokinase (HK), Bcl‐2, and Bcl‐xL to prevent the anti‐apoptotic activities of these proteins and induce cancer cell death, with a focus on leukemia and glioblastoma. In this study, we demonstrated the sensitivity of a panel of genetically characterized cancer cell lines, differing in origin and carried mutations, to VDAC1‐based peptide‐induced apoptosis. Noncancerous cell lines were less affected by the peptides. Furthermore, we constructed additional VDAC1‐based peptides with the aim of improving targeting, selectivity, and cellular stability, including R‐Tf‐D‐LP4, containing the transferrin receptor internalization sequence (Tf) that allows targeting of the peptide to cancer cells, known to overexpress the transferrin receptor. The mode of action of the VDAC1‐based peptides involves HK detachment, interfering with the action of anti‐apoptotic proteins, and thus activating multiple routes leading to an impairment of cell energy and metabolism homeostasis and the induction of apoptosis. Finally, in xenograft glioblastoma, lung, and breast cancer mouse models, R‐Tf‐D‐LP4 inhibited tumor growth while inducing massive cancer cell death, including of cancer stem cells. Thus, VDAC1‐based peptides offer an innovative new conceptual framework for cancer therapy.

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.

At the Crossroads Between Mitochondrial Metabolite Transport and Apoptosis: VDAC1 as an Emerging Cancer Drug Target

Many cancer cells undergo re-programing of metabolism and develop cell survival strategies involving anti-apoptotic defense mechanisms, a hallmark of a great majority of cancer types. The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane protein, serves as a mitochondrial gatekeeper, controlling the metabolic and energy cross-talk between mitochondria and the rest of the cell. VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis due to its association with pro- and anti-apoptotic members of the Bcl-2 family of proteins. At the same time, VDAC1 functions in the release of apoptotic proteins located in the inter-membranal space. Thus, VDAC1 is emerging 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 cancer therapy. We discuss the use of VDAC1-based strategies to attack the altered metabolism and apoptosis of cancer cells. These strategies include specific siRNA to impair energy and metabolic homeostasis, leading to arrest cancer cell growth and tumor development, as well as VDAC1-based peptides interacting with anti-apoptotic proteins and inducing 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.