Ravi N. Vellanki

AMPK regulates metabolism and survival in response to ionizing radiation

BACKGROUND AND PURPOSE AMPK is a metabolic sensor and an upstream inhibitor of mTOR activity. AMPK is phosphorylated by ionizing radiation (IR) in an ATM dependent manner, but the cellular consequences of this phosphorylation event have remained unclear. The objective of this study was to assess whether AMPK plays a functional role in regulating cellular responses to IR. METHODS The importance of AMPK expression for radiation responses was investigated using both MEFs (mouse embryo fibroblasts) double knockout for AMPK α1/α2 subunits and human colorectal carcinoma cells (HCT 116) with AMPK α1/α2 shRNA mediated knockdown. RESULTS We demonstrate here that IR results in phosphorylation of both AMPK and its substrate, ACC. IR moderately stimulated mTOR activity, and this was substantially exacerbated in the absence of AMPK. AMPK was required for IR induced expression of the mTOR inhibitor REDD1, indicating that AMPK restrains mTOR activity through multiple mechanisms. Likewise, cellular metabolism was deregulated following irradiation in the absence of AMPK, as evidenced by a substantial increase in oxygen consumption rates and lactate production. AMPK deficient cells showed impairment of the G1/S cell cycle checkpoint, and were unable to support long-term proliferation during starvation following radiation. Lastly, we show that AMPK proficiency is important for clonogenic survival after radiation during starvation. CONCLUSIONS These data reveal novel functional roles for AMPK in regulating mTOR signaling, cell cycle, survival and metabolic responses to IR.

OASIS/CREB3L1 Induces Expression of Genes Involved in Extracellular Matrix Production But Not Classical Endoplasmic Reticulum Stress Response Genes in Pancreatic β-Cells

Old astrocyte specifically induced substance (OASIS) has previously been shown to be a putative endoplasmic reticulum (ER) stress sensor in astrocytes with a mechanism of activation that is similar to ATF6. In this study we investigated the expression and activation of endogenous and overexpressed OASIS in pancreatic beta-cells. OASIS mRNA expression was detected in pancreatic beta-cell lines and rodent islets, and the expression level was up-regulated by ER stress-inducing compounds. Endogenous OASIS protein, however, is expressed at low levels in pancreatic beta-cell lines and rodent islets, possibly due to abundant levels of the micro-RNA miR-140 present in these cells. In contrast, expression of both full-length and cleaved (active) OASIS was readily detectable in the developing mouse pancreas (embryonic d 15.5). Microarray analysis after expression of an active nuclear-localized version of OASIS in an inducible INS-1 beta-cell line resulted in the up-regulation of many genes implicated in extracellular matrix production and protein transport but not classical ER stress response genes. Consistent with this, expression of active OASIS failed to induce glucose-regulated protein 78 kDa promoter activity in pancreatic beta-cells. These results suggest that the repertoire of genes induced by OASIS is cell type-dependent and that the OASIS protein may have a role in pancreas development.

New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1α signaling is essential for promoting adaptation and survival to hypoxia

BACKGROUND AND PURPOSE The unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. METHODS We utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. RESULTS Using knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. CONCLUSIONS Our results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts.

Contributions of AMPK and p53 dependent signaling to radiation response in the presence of metformin

BACKGROUND AND PURPOSE Metformin is commonly prescribed to treat type 2 diabetes, and has additional potential as a cancer prophylactic and therapeutic. Metformin activates AMPK that in turn can launch a p53-dependent metabolic checkpoint. Possible interactions between metformin and radiation are poorly understood. Since radiation-induced signaling also involves AMPK and p53, we investigated their importance in mediating responses to metformin and radiation. MATERIALS AND METHODS A549 cells, HCT116 cells wildtype or knockout for p53 or MEFs wildtype or double knockout for AMPKα1 and α2 were irradiated in the presence or absence of metformin. The impact of metformin on oxygen consumption and proliferation rates was determined, as well as clonogenic radiation survival. RESULTS Metformin resulted in moderate radiation protection in all cell lines, irrespective of AMPK and p53. Loss of AMPK sensitized cells to the anti-proliferative effects of metformin, while loss of p53 promoted both the growth inhibitory and toxic effects of metformin. Consequently, overall cell death after radiation was similar with and without metformin irrespective of AMPK or p53 genotype. CONCLUSIONS The anti-proliferative activity of metformin may confer benefit in combination with radiotherapy, and this benefit is intensified upon loss of AMPK or p53 signaling.

Identification of Hypoxic Cells Using an Organotellurium Tag Compatible with Mass Cytometry

Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody-metal-chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity-based probe designed specifically for use with the technology. A compact MC-detectable telluroether is linked to a bioreductively sensitive 2-nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O2-deprived cells. A proof-of-concept experiment employing metal-bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O2 can be effectively discriminated by the quantity of tellurium-labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC-compatible probes for activity-based profiling of single cells.

Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents.

CHCHD2 Is Coamplified with EGFR in NSCLC and Regulates Mitochondrial Function and Cell Migration

Coiled-coil-helix-coiled-coil-helix domain-containing 2, a mitochondrial protein, encoded by CHCHD2 is located at chromosome 7p11.2 and proximal to the EGFR gene. Here, bioinformatic analyses revealed that CHCHD2 is consistently coamplified with EGFR in non–small cell lung carcinoma (NSCLC). In addition, CHCHD2 and EGFR protein expression levels were positively correlated and upregulated relative to normal lung in NSCLC tumor-derived xenografts. Knockdown of CHCHD2 expression in NSCLC cells attenuated cell proliferation, migration, and mitochondrial respiration. CHCHD2 protein–protein interactions were assessed by the complementary approaches of affinity purification mass spectrometry and in vivo proximity ligation. The CHCHD2 interactome includes the apparent hub proteins C1QBP (a mitochondrial protein) and YBX1 (an oncogenic transcription factor), and an overlapping set of hub-associated proteins implicated in cell regulation. Implications: CHCHD2 influences mitochondrial and nuclear functions and contributes to the cancer phenotype associated with 7p11.2 amplification in NSCLC. Mol Cancer Res; 13(7); 1119–29. ©2015 AACR.

OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87) and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium-containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)-technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

MATE2 Expression Is Associated with Cancer Cell Response to Metformin.

Background There is great interest in repurposing the commonly prescribed anti-diabetic drug metformin for cancer therapy. Intracellular uptake and retention of metformin is affected by the expression of organic cation transporters (OCT) 1–3 and by multidrug and toxic compound extrusion (MATE) 1–2. Inside cells, metformin inhibits mitochondrial function, which leads to reduced oxygen consumption and inhibition of proliferation. Reduced oxygen consumption can lead to improved tumor oxygenation and radiation response. Purpose Here we sought to determine if there is an association between the effects of metformin on inhibiting oxygen consumption, proliferation and expression of OCTs and MATEs in a panel of 19 cancer cell lines. Results There was relatively large variability in the anti-proliferative response of different cell lines to metformin, with a subset of cell lines being very resistant. In contrast, all cell lines demonstrated sensitivity to the inhibition of oxygen consumption by metformin, with relatively small variation. The expression of OCT1 correlated with expression of both OCT2 and OCT3. OCT1 and OCT2 were relatively uniformly expressed, whereas expression of OCT3, MATE1 and MATE2 showed substantial variation across lines. There were statistically significant associations between resistance to inhibition of proliferation and MATE2 expression, as well as between sensitivity to inhibition of oxygen consumption and OCT3 expression. One cell line (LNCaP) with high OCT3 and low MATE2 expression in concert, had substantially higher intracellular metformin concentration than other cell lines, and was exquisitely sensitive to both anti-proliferative and anti-respiratory effects. In all other cell lines, the concentration of metformin required to inhibit oxygen consumption acutely in vitro was substantially higher than that achieved in the plasma of diabetic patients. However, administering anti-diabetic doses of metformin to tumor-bearing mice resulted in intratumoral accumulation of metformin and reduced hypoxic tumor fractions. Conclusions All cancer cells are susceptible to inhibition of oxygen consumption by metformin, which results in reduced hypoxic tumor fractions beneficial for the response to radiotherapy. High MATE2 expression may result in resistance to the anti-proliferative effect of metformin and should be considered as a negative predictive biomarker in clinical trials.