Borivoj Vojnovic

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

Background Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. Methods and Findings In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Conclusions Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.

Low-Dose Studies of Bystander Cell Killing with Targeted Soft X Rays

Abstract Schettino, G., Folkard, M., Prise, K. M., Vojnovic, B., Held, K. D. and Michael, B. D. Low-Dose Studies of Bystander Cell Killing with Targeted Soft X Rays. Radiat. Res. 160, 505–511 (2003). The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 μm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0–2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (∼25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 keVmum(-1)are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keVmum(-1). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4MeV and 3He2+ ions in the energy range 3.4-6.9 MeV. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keVmum(-1) for protons, 18-50 keVmum(-1) for deuterons, and 59-106 keVmum(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keVmum(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells.

ABSTRACT While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.

A Focused Ultrasoft X-Ray Microbeam for Targeting Cells Individually with Submicrometer Accuracy

Abstract Folkard, M., Schettino, G., Vojnovic, B., Gilchrist, S., Michette, A. G., Pfauntsch, S. J., Prise, K. M. and Michael, B. D. A Focused Ultrasoft X-Ray Microbeam for Targeting Cells Individually with Submicrometer Accuracy. Radiat. Res. 156, 796–804 (2001). The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2–0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 μm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5–10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.

Measurement of Tumor Oxygenation: A Comparison between Polarographic Needle Electrodes and a Time-Resolved Luminescence-Based Optical Sensor

A novel oxygen sensor which does not rely on electrochemical reduction has been used to measure the oxygenation of the murine sarcoma F in a comparative study with an existing polarographic electrode that is available commercially. The prototype luminescence sensor yielded an oxygen distribution comparable with readings made using a pO2 histograph. The percentage of regions detected that had a pO2 less than 5 mm Hg was 79 and 75 using the Eppendorf pO2 histograph and the luminescence fiber optic sensor, respectively. These values were compatible with a measured radiobiologically hypoxic fraction of 67% in this tumor. The polarographic method detected more regions with a pO2 of 2.5 mm Hg or less (69%) compared with the optical sensor (50%) (P < 0.05). This could reflect differences in the oxygen use of the sensing devices. This initial assessment indicates the potential of a fiber-optic-based oxygen-monitoring system. Such a system should have several advantages including monitoring temporal oxygen changes in a given microregion and use with NMR procedures.

An image analysis-based approach for automated counting of cancer cell nuclei in tissue sections.

Semiquantitative evaluation and manual cell counting are the commonly used procedures to assess positive staining of molecular markers in tissue sections. Manual counting is also a laborious task in which consistent objectivity is difficult to achieve. Recently, image analysis has been explored, but the studies reported were limited to histological images acquired at high magnification and containing uniformly stained cells.

Low-Dose Hypersensitivity in Chinese Hamster V79 Cells Targeted with Counted Protons Using a Charged-Particle Microbeam

Abstract Schettino, G., Folkard, M., Prise, K. M., Vojnovic, B., Bowey, A. G. and Michael, B. D. Low-Dose Hypersensitivity in Chinese Hamster V79 Cells Targeted with Counted Protons Using a Charged-Particle Microbeam. Radiat. Res. 156, 526–534 (2001). The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-μm silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-μm-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-μm-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below 1 Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

Predictions regarding drug resistance mechanisms and treatment strategies in triple-negative breast cancer are confirmed in tumors from patients. From Models to Breast Cancer Treatments Patients with triple-negative breast cancer (TNBC), a particularly aggressive form, have few treatment options. Targeting either the phosphatidylinositol 3-kinase to Akt (PI3K-Akt) pathway or epidermal growth factor receptor (EGFR) inhibits tumor growth in some patients, but durable responses are rare. Modeling studies using cell lines predict that the EGFR family member HER3 (human epidermal growth factor receptor 3) may confer drug resistance. Now, Tao et al. provide evidence from patient tumors to support those predictions. Treatment with PI3K-Akt pathway inhibitors increased the abundance of both total and activated HER3 in TNBC cells in culture and TNBC xenografts in mice. Residual tumors from patients treated with EGFR inhibitors had increased abundance and activation of HER3. Combining inhibitors of the PI3K-Akt pathway with a dual inhibitor of EGFR and HER3 substantially suppressed tumor growth in mice with TNBC xenografts derived from either cell lines or patients, suggesting that this combined strategy may improve therapeutic outcome in TNBC patients. Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.