Sarah Hopmans

Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK.

Background:We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR).Methods:Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit−/− embryos (AMPKα1/2−/−-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC).Results:Metformin (2.5u2009μM–5u2009mM) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)–AMPK–p53/p21cip1 and inhibited the Akt–mammalian target of rapamycin (mTOR)–eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2−/−-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM–AMPK–p53/p21cip1 and inhibition of Akt–mTOR–4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers.Conclusion:Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM–AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.

Lovastatin Sensitizes Lung Cancer Cells to Ionizing Radiation: Modulation of Molecular Pathways of Radioresistance and Tumor Suppression

Introduction: In this study, we investigated the effect of the 3-hydroxy-3-methylgutaryl-CoA reductase inhibitor lovastatin, as a sensitizer of lung cancer cells to ionizing radiation (IR). Methods: A549 lung adenocarcinoma cells were treated with 0 to 50 &mgr;M lovastatin alone or in combination with 0 to 8 Gy IR and subjected to clonogenic survival and proliferation assays. To assess the mechanism of drug action, we examined the effects of lovastatin and IR on the epidermal growth factor (EGF) receptor and AMP-activated kinase (AMPK) pathways and on apoptotic markers and the cell cycle. Results: Lovastatin inhibited basal clonogenic survival and proliferation of A549 cells and sensitized them to IR. This was reversed by mevalonate, the product of 3-hydroxy-3-methylgutaryl-CoA reductase. Lovastatin attenuated selectively EGF-induced phosphorylation of EGF receptor and Akt, and IR-induced Akt phosphorylation, in a mevalonate-sensitive fashion, without inhibition on extracellular signal-regulated kinase 1/2 phosphorylation by either stimulus. IR phosphorylated and activated the metabolic sensor and tumor suppressor AMPK, but lovastatin enhanced basal and IR-induced AMPK phosphorylation. The drug inhibited IR-induced expression of p53 and the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, but caused a redistribution of cells from G1-S phase (control and radiated cells) and G2-M phase (radiated cells) of cell cycle into apoptosis. The latter was also evident by induction of nuclear fragmentation and cleavage of caspase 3 by lovastatin in both control and radiated cells. Conclusions: We suggest that lovastatin inhibits survival and induces radiosensitization of lung cancer cells through induction of apoptosis, which may be mediated by a simultaneous inhibition of the Akt and activation of the AMPK signaling pathways.

GnRH antagonist associates with less adiposity and reduced characteristics of metabolic syndrome and atherosclerosis compared with orchiectomy and GnRH agonist in a preclinical mouse model.

OBJECTIVESnObservational studies relate androgen deprivation therapy (ADT) to metabolic syndrome (MS) and cardiovascular disease, an association potentially subject to uncontrollable confounding factors, especially diet and genetic/metabolic risk factors. In the absence of prospective randomized clinical trials, causality remains unproven. We comparatively investigated the effects of different ADT modalities on the development of MS and atherosclerosis in a mouse model.nnnMATERIALS AND METHODSnLow-density lipoprotein receptor knockout mice underwent orchiectomy plus vehicle (2.5% mannitol), sham surgery plus vehicle (control), sham surgery plus gonadotropin-releasing hormone (GnRH) antagonist (degarelix), or sham surgery plus GnRH agonist (leuprolide) (n = 9-13/group) and were followed for 4 months. Visceral fat accumulation, lean body mass, adipocyte size, fasting blood glucose, glucose tolerance, serum levels of leptin, follicle-stimulating hormone, luteinizing hormone, and testosterone, along with atherosclerotic plaque size and characteristics were measured.nnnRESULTSnAll 3 modes of ADT decreased circulating testosterone levels in mice, although leuprolide treatment reached nadir levels of testosterone later. Orchiectomized and leuprolide-treated mice gained significantly more visceral fat compared with degarelix-treated mice. Improved glucose tolerance tests were recorded in degarelix-treated mice. The aortic atherosclerotic plaque area in leuprolide-treated and orchiectomized mice was larger than in control mice (P<0.005 and P = 0.002, respectively), but it was not significantly different from control in degarelix-treated mice. The necrotic core area in degarelix-treated mice was smaller compared with leuprolide-treated and orchiectomized mice (P = 0.011 and P = 0.002, respectively).nnnCONCLUSIONSnOur results suggest that ADT induced MS and atherosclerosis in a preclinical mouse model to a mode-specific extent. GnRH antagonist generated the least atherosclerosis and characteristics of MS compared with orchiectomy and GnRH agonist.

Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

IntroductionEarlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal transduction pathway involving ATM-AMPK-p53/p21cip1 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer.MethodsImmuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10u2009Gy, and left to grow for 8u2009weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis.ResultsIR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls.ConclusionIR inhibits epithelial cancer tumour growth and results in sustained expression and activation of ATM-Chk2, and AMPK-p53/p21cip1/p27kip1 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt-mTOR pathways by novel therapeutics can sensitize lung tumours to radiation.

Inhibition of carbonic anhydrase IX (CA9) sensitizes renal cell carcinoma to ionizing radiation

While normal kidneys are relatively sensitive to ionizing radiation (IR), renal cell carcinoma (RCC) is considered radioresistant. Carbonic anhydrase IX (CA9), an enzyme that maintains intracellular pH by carbon dioxide dissolution, is upregulated in the majority of RCC, but not in normal kidneys. Since regulation of intracellular pH may enhance radiation effects, we hypothesized that inhibition of CA9 may radiosensitize RCC. Clonogenic survival assay of human clear cell RCC 786-O and murine RCC RAG cells in the presence of a pharmacological CA9 inhibitor or with shRNA-mediated knockdown of CA9 was performed to investigate the response to IR in vitro (single dose or fractionated) and in vivo. Extracellular pH changes were measured in vitro. Treatment with AEBS [4-(2-aminoethyl)benzene sulfonamide], a sulfonamide, was used as a pharmacological inhibitor of the enzymatic activity of CA9. Nude mice bearing subcutaneous xenografts of 786-O cells stably expressing CA9 shRNA or scrambled control were irradiated (6 Gy). Tumor growth was followed longitudinally in the 786-O-bearing mice receiving AEBS (50-200 µg/ml drinking water) or control (vehicle only) which were irradiated (6 Gy) and compared with mice receiving either IR or AEBS alone. In vitro inhibition of CA9 activity or expression significantly sensitized RCC cells to the effects of IR (p<0.05), an effect even more significant when hypofractionated IR was applied. In vivo irradiated xenografts from RCC cells transfected with CA9 shRNA were significantly smaller compared to irradiated xenografts from the scrambled shRNA controls (p<0.05). RCC xenografts from mice treated with AEBS in combination with IR grew significantly slower than all controls (p<0.05). Inhibition of CA9 expression or activity resulted in radiation sensitization of RCC in a preclinical mouse model.

Abstract 2491: Metformin sensitizes human lung cancer xenografts to ionizing radiation: Response of the AMPK pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnIntroduction : Radiation therapy is a standard therapeutic modality in lung cancer (LC). Unfortunately, LC demonstrates radiation resistance and poor response to even high doses of chest radiotherapy. Therefore, there is an urgent need to develop effective and well tolerated radiation sensitizers in LC. Recently, we showed that ionizing radiation (IR) and metformin (MET) activate the energy sensor and tumor suppressor kinase AMP-activated kinase (AMPK). MET enhanced IR activation of AMPK in LC cells and increased the cytotoxicity of IR in clonogenic assays. In the present study we investigated, in human LC xenografts, the radio-sensitizing properties of MET and its effects on the activity of the AMPK pathway in intact tumors.nnMethods: A549 cells (2×106) were grafted in the franks of Balb/c immunodeficient athymic nude mice and tumors were left to grow to 100 mm3. MET supplementation was delivered in drinking water at a dose of 250 mg/kg daily, and IR of 10 Gy was delivered as a single fraction 7 days after initiation of metformin administration. Eight weeks later animals were euthanized and tumors where isolated. Half of each tumor was snap frozen for preparation of whole tumor lysates and immunoblotting analysis and the other half was fixed and embedded for immunohistochemistry analysis. Tumour growth kinetics and levels of total AMPK, phosphor (P)-AMPK (P-AMPK Thr172) and P-Acetyl-CoA Carboxylase (ACC) and P-Akt were evaluated.nnResults: MET and IR alone inhibited significantly A549 LC xenograft tumor growth. Furthermore, the combined treatment of MET and IR produce an additive effect and inhibited tumor growth more than each treatment alone. Both IR and MET enhanced AMPK phosphorylation and activity detected by ACC phosphorylation. Similar to tumor growth kinetics, the combined treatment of MET+IR enhanced AMPK activity and phosphorylation above the levels of each treatment alone. However, we also detected that MET and IR treatments also increased significantly the total AMPK α subunit levels in tumor tissues with a consistent potentiation of this effect when the two treatments were combined. On the other hand MET showed a tendency to inhibit basal and radiation-induced Akt phosphorylation levels in xenografts. Our immunoblotting results were verified with immunohistochemistry experiments.nnConclusion: Similar to our earlier observations in tissue culture models, the present studies suggest that MET inhibits LC tumour growth and sensitizes them to IR. The two agents mediate both expression and activation of AMPK which appears to be associated with inhibition of Akt. Activation of AMPK by MET and IR in-vivo does not appear to depend on LKB1 as A549 cells are LKB1 null. These results demonstrate the potential of MET to function as a radiation sensitizer in-vivo and support evaluation of this drug in LC clinical trials in combination with radiation.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2011-2491

Endoplasmic reticulum protein ERp46 in prostate adenocarcinoma

Endoplasmic reticulum (ER) protein ERp46 is a member of the protein disulfide isomerase family of oxidoreductases, which facilitates the reduction of disulfides in proteins and their folding. Accumulation of misfolded proteins has been implicated in cancer. The objectives of the present study were to investigate the role of ERp46 in prostate cancer, its expression and its effects on prostate cancer growth. A tissue microarray with human prostate cancer and normal prostate tissue samples was stained for ERp46 followed by image analysis. Human prostate adenocarcinoma 22Rv1 cells were stably transfected with short hairpin RNA (shRNA) specific for ERp46, a non-effective scrambled control or a plasmid containing full-length human ERp46 cDNA, and cell growth was determined. Subcloned cells were treated with thapsigargin or tunicamycin to induce ER stress and lysates were subjected to western blot analysis for ER stress proteins. Subcutaneous xenografts of parental 22Rv1, ERp46-overexpressing (ERp46+), shERp46 or scrambled control cells were established in male inbred BALB/c nude mice (n=10/group). Tumor growth curves of the xenografts were constructed over a period of 30 days and subsequently the mice were sacrificed and the amount of serum prostate-specific antigen was determined. The results demonstrated increased ERp46 expression levels in prostate cancer tissue samples of Gleason ≥7 compared with normal prostate tissue samples. When ERp46 was stably knocked down using shRNA or overexpressed in prostate carcinoma 22Rv1 cells, tumor growth in vitro and in BALB/c nude mice was inhibited and accelerated, respectively. ERp46 overexpression led to reduced sensitivity to ER stress as indicated by higher half maximal inhibitory concentrations for tunicamycin and thapsigargin in ERp46+ cells. The shERp46 cells lost the ability to upregulate protein disulfide isomerase following tunicamycin-induced ER stress. The present study suggests a role for ERp46 as a therapeutic target in prostate cancer, given its expression profile in human prostate cancer, and its effect on prostate cancer cell growth.

Abstract 4960: ERp46 (thioredoxin domain-containing protein 5, TXND5) promotes prostate cancer growth in vitro and in vivo

We have recently demonstrated that endoplasmic reticulum protein ERp46, a member of the protein disulfide isomerase family of oxidoreductases, TXND5, is overexpressed in human metastatic renal cell carcinoma. The expression and function of ERp46 in prostate cancer has not been studied. Using both in vitro and in vivo approaches, we explore the suitability of ERp46 as a potential therapeutic target in prostate cancer. Tissue microarray containing normal prostate epithelium (n = 9) and prostate cancer specimens from 57 patients was stained for ERp46 and the staining intensity (H-score) was determined. Human prostate adenocarcinoma 22Rv1 cells were used to generate gain- and loss-of-function models by stable ERp46 shRNA knockdown and ERp46 overexpression, respectively. In vitro, the doubling time and PSA production were determined. In vivo, xenografts of each subclone were established in nude mice (n = 10/group) to determine the longitudinal tumor growth and serum PSA values. Gene expression profiling of RNA isolated from 22Rv1 xenografts was performed using human whole genome HT-12 V4 BeadChip array (Illumina). Our results demonstrated that human prostate carcinoma samples of Gleason scores ≥7 showed strong cytoplasmic ERp46 staining which was significantly increased compared to normal prostatic tissue (p = 0.02). ERp46 staining in prostate tumors of Gleason scores ≤6, however, was not different compared to normal prostate tissue. The stably transfected human prostate carcinoma 22Rv1 cells expressed 89% knockdown of ERp46 protein expression (shERp46) or a 4-fold increase in ERp46 protein expression (ERp46+) compared to the respective control cells. In vitro, shERp46 cells proliferated slower, whereas ERp46+ cells exhibited accelerated growth compared to corresponding control cells (p Funding was provided by Prostate Cancer Canada and McMaster Surgical Associates (JHP and WCMD) Citation Format: Jehonathan H. Pinthus, Sarah N. Hopmans, Stephanie Federov, Wilhelmina C. Duivenvoorden. ERp46 (thioredoxin domain-containing protein 5, TXND5) promotes prostate cancer growth in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4960. doi:10.1158/1538-7445.AM2015-4960