Statin-induced metabolic reprogramming in head and neck cancer: a biomarker for targeting monocarboxylate transporters.

Prognosis of HPV negative head and neck squamous cell carcinoma (HNSCC) patients remains poor despite surgical and medical advances and inadequacy of predictive and prognostic biomarkers in this type of cancer highlights one of the challenges to successful therapy. Statins, widely used for the treatment of hyperlipidaemia, have been shown to possess anti-tumour effects which were partly attributed to their ability to interfere with metabolic pathways essential in the survival of cancer cells. Here, we have investigated the effect of statins on the metabolic modulation of HNSCC cancers with a vision to predict a personalised anticancer therapy. Although, treatment of tumour-bearing mice with simvastatin did not affect tumour growth, pre-treatment for 2 weeks prior to tumour injection, inhibited tumour growth resulting in strongly increased survival. This was associated with increased expression of the monocarboxylate transporter 1 (MCT1) and a significant reduction in tumour lactate content, suggesting a possible reliance of these tumours on oxidative phosphorylation for survival. Since MCT1 is responsible for the uptake of mitochondrial fuels into the cells, we reasoned that inhibiting it would be beneficial. Interestingly, combination of simvastatin with AZD3965 (MCT1 inhibitor) led to further tumour growth delay as compared to monotherapies, without signs of toxicity. In clinical biopsies, prediagnostic statin therapy was associated with a significantly higher MCT1 expression and was not of prognostic value following conventional chemo-radiotherapy. These findings provide a rationale to investigate the clinical effectiveness of MCT1 inhibition in patients with HNSCC who have been taking lipophilic statins prior to diagnosis.

LDHA in Neuroblastoma Is Associated with Poor Outcome and Its Depletion Decreases Neuroblastoma Growth Independent of Aerobic Glycolysis.

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB).Experimental Design: Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model.Results: Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed.Conclusions: High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Clin Cancer Res; 24(22); 5772-83. ©2018 AACR.

Comparative metabolic analysis in head and neck cancer and the normal gingiva.

OBJECTIVES: Chronic accumulation of lactate in malignant tumor tissue is associated with increased malignancy and radioresistance. For this study, biopsies of primary head and neck squamous cell carcinoma (HNSCC) and of the normal gingiva of the same patient were compared via metabolic profiling to the healthy gingiva from cancer-free patients. MATERIALS AND METHODS: Cryobiopsies of 140 HNSCC patients were used to determine ATP, lactate, and glucose concentrations of the tumor and normal gingiva via induced metabolic bioluminescence imaging (imBI). Additionally, these metabolites were quantified in a collective of 79 healthy (non-tumor-bearing) patients. Furthermore, tumor samples were analyzed via immunofluorescence imaging and quantitative real-time PCR for the expression of lactate and glucose transporters. RESULTS: There were significant differences in ATP concentrations detectable between the tumor, normal gingiva of tumor patients, and gingiva from healthy patients. Lactate concentrations were significantly increased in tumor tissue compared to the normal gingiva of tumor patients as well as the gingiva from healthy patients. Concerning glucose, there was a significant decrease in glucose concentrations detectable in the tumor biopsies compared to the normal gingiva of tumor patients. On the other hand, tumor samples from patients revealed significantly elevated relative expression levels of monocarboxylate transporters (MCT-1 and MCT-4), as well as glucose transporters (GLUT-1 and GLUT-3) compared to the corresponding normal gingiva of each patient. CONCLUSIONS: We could demonstrate that the lactate concentration in HNSCC correlates with primary tumor (T) stage. CLINICAL RELEVANCE: The aim of this study was to identify metabolic parameters to improve early cancer diagnosis, allow predictions on the degree of malignancy, and contribute to a personalized tumor therapy.

Uncovering Metabolic Effects of Anti-angiogenic Therapy in Tumors by Induced Metabolic Bioluminescence Imaging.

Induced metabolic bioluminescence imaging (imBI) is an imaging technique which enables detection of various metabolites associated with glycolysis in tumor sections. Signals captured by imBI can be used to chart the topographic distribution of lactate, glucose, pyruvate, and ATP and quantify their absolute amount. ImBi can enable us to perform metabolic classification of tumors as well as to detect metabolic changes in the glycolytic pathway associated with certain therapies, such as anti-angiogenic drugs.

LDHA-Associated Lactic Acid Production Blunts Tumor Immunosurveillance by T and NK Cells.

Elevated lactate dehydrogenase A (LDHA) expression is associated with poor outcome in tumor patients. Here we show that LDHA-associated lactic acid accumulation in melanomas inhibits tumor surveillance by T and NK cells. In immunocompetent C57BL/6 mice, tumors with reduced lactic acid production (Ldhalow) developed significantly slower than control tumors and showed increased infiltration with IFN-γ-producing T and NK cells. However, in Rag2-/-γc-/- mice, lacking lymphocytes and NK cells, and in Ifng-/- mice, Ldhalow and control cells formed tumors at similar rates. Pathophysiological concentrations of lactic acid prevented upregulation of nuclear factor of activated T cells (NFAT) in T and NK cells, resulting in diminished IFN-γ production. Database analyses revealed negative correlations between LDHA expression and T cell activation markers in human melanoma patients. Our results demonstrate that lactic acid is a potent inhibitor of function and survival of T and NK cells leading to tumor immune escape.

Lactate-An Integrative Mirror of Cancer Metabolism.

The technique of induced metabolic bioluminescence imaging (imBI) has been developed to obtain a "snapshot" of the momentary metabolic status of biological tissues. Using cryosections of snap-frozen tissue specimens, imBI combines highly specific and sensitive in situ detection of metabolites with a spatial resolution on a microscopic level and with metabolic imaging in relation to tissue histology. Here, we present the application of imBI in human colorectal cancer. Comparing the metabolic information of one biopsy with that of 2 or 3 biopsies per individual cancer, the classification into high versus low lactate tumors, reflecting different glycolytic activities, based on a single biopsy was in agreement with the result from multiple biopsies in 83 % of all cases. We further demonstrate that the metabolic status of tumor tissue can be preserved at least over 10 years by storage in liquid nitrogen, but not by storage at -80 °C. This means that tissue banking with long-term preservation of the metabolic status is possible at -180 °C, which may be relevant for studies on long-term survival of cancer patients. As with other tumor entities, tissue lactate concentration was shown to be correlated with tumor development and progression in colorectal cancer. At first-time diagnosis, lactate values were low in rectal normal tissue and adenomas, were significantly elevated to intermediate levels in non-metastatic adenocarcinomas, and were very high in carcinomas with distant metastasis. There was an inverse behavior of tissue glucose concentration under corresponding conditions. The expression level of monocarboxylate transporter-4 (MCT4) was positively correlated with the tumor lactate concentration and may thus contribute to high lactate tumors being associated with a high degree of malignancy.

Feasibility of induced metabolic bioluminescence imaging in advanced ovarian cancer patients: first results of a pilot study.

PURPOSE: The precise determination of energy metabolites is challenged by the heterogeneity of their distribution, their rapid changes after surgical resection and the architectural complexity of malignancies. Induced metabolic bioluminescence imaging (imBI) allows to determine energy metabolites in tissue sections and to co-localize these with histological structures based on consecutive sections stained with HE. In this prospective pilot study patients with suspected advanced ovarian cancer (OC) were enrolled to prove the feasibility of imBI. METHODS: During surgery, suspicious peritoneal metastases were resected and transferred in liquid nitrogen within 30 s. ATP, glucose and lactate concentrations were measured. Furthermore, the expression of monocarboxylate transporters MCT1 and MCT4 was determined by immunofluorescence staining. RESULTS: 16 patients were screened, 12 entered the study. Final histological assessment revealed ten malignant and two benign peritoneal lesions. In all 12 cases high concentrations of ATP suggested that energy metabolism was not altered by the surgical and transport procedures (mean 0.56 µmol/g, range 0.24-1.21 µmol/g). The mean concentration of glucose was 1.95 µmol/g (range 0.58-4.71 µmol/g). The concentration of lactate was drastically higher in the ten OC cases (mean 24.79 µmol/g, range 17.51-37.16 µmol/g) compared to the benign samples (mean 5.98 µmol/g, range 5.43-6.54 µmol/g). Lactate concentrations seem to correlate with MCT1 (spearman rank correlation ρ = 0.624, 0.05 > p > 0.025), but not with MCT4 (spearman rank correlation ρ = 0.018, p > 0.1). CONCLUSIONS: ImBI is feasible in peritoneal metastases of OC and encourages further effort to elucidate the role of glucose, lactate, MCT1 and MCT4 in OC.

Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique.

Patients with malignant gliomas have a poor prognosis with average survival of less than 1 year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite that was discovered first in this tumor entity. D2HG is generated in large amounts due to various "gain-of-function" mutations in the isocitrate dehydrogenases IDH1 and IDH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (<0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the "oncometabolite" D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0-10 µmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models.

This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, (12)C(+6) in the plateau region, and (12)C(+6) in the Bragg peak, respectively. Similarly, a relative biological effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in ß1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during (12)C(+6) irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral mucosa, the superior effectiveness of heavy ions was confirmed by a twofold higher number of foci per nucleus. However, proinflammatory signs were similar for both treatment modalities, e.g., the activation of NFκB and the release of IL6 and IL8. The presence of peripheral blood mononuclear cell increased the radiation-induced release of the proinflammatory cytokines by factors of 2-3. Carbon ions are part of the cosmic radiation. Long-term exposure to such particles during extended space flights, as planned by international space agencies, may thus impose a medical and safety risk on the astronauts by a potential induction of mucositis. In summary, particle irradiation is superior to gamma-rays due to a higher radiobiological effectiveness, a reduced hypoxia-induced radioresistance, a multicellular radiosensitization, and the absence of a radiation-induced cell motility. However, the potential of inducing mucositis is similar for both radiation types.

Potential of Induced Metabolic Bioluminescence Imaging to Uncover Metabolic Effects of Antiangiogenic Therapy in Tumors.

Tumor heterogeneity at the genetic level has been illustrated by a multitude of studies on the genomics of cancer, but whether tumors can be heterogeneous at the metabolic level is an issue that has been less systematically investigated so far. A burning-related question is whether the metabolic features of tumors can change either following natural tumor progression (i.e., in primary tumors versus metastasis) or therapeutic interventions. In this regard, recent findings by independent teams indicate that antiangiogenic drugs cause metabolic perturbations in tumors as well as metabolic adaptations associated with increased malignancy. Induced metabolic bioluminescence imaging (imBI) is an imaging technique that enables detection of key metabolites associated with glycolysis, including lactate, glucose, pyruvate, and ATP in tumor sections. Signals captured by imBI can be used to visualize the topographic distribution of these metabolites and quantify their absolute amount. imBI can be very useful for metabolic classification of tumors as well as to track metabolic changes in the glycolytic pathway associated with certain therapies. Imaging of the metabolic changes induced by antiangiogenic drugs in tumors by imBI or other emerging technologies is a valuable tool to uncover molecular sensors engaged by metabolic stress and offers an opportunity to understand how metabolism-based approaches could improve cancer therapy.

Hypoxia-Associated Marker CA IX Does Not Predict the Response of Locally Advanced Rectal Cancers to Neoadjuvant Chemoradiotherapy.

Hypoxia-associated proteome changes have been shown to be associated with resistance to chemo- and radiotherapy. Our study evaluated the role of the hypoxia-inducible (HIF)-1 target gene carbonic anhydrase (CA) IX in the prediction of the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer (stages II and III). A total of 29 pretreatment biopsy specimens were stained for CA IX by immunohistochemistry, converted to digital images and evaluated in a quantitative fashion using image analysis software. Contrary to our expectations, a trend towards a correlation between better tumor regression (>50%) and higher expression of CA IX (p=0.056) was found. CA IX was also present more frequently in pathological tumor stage T1 (pT1) tumors (p=0.048). Conversely, no association with lymph node metastasis was identified. In conclusion, as a single marker, CA IX expression is not able to identify a hypoxia-related treatment resistant phenotype in rectal cancer.

Lactate as a predictive marker for tumor recurrence in patients with head and neck squamous cell carcinoma (HNSCC) post radiation: a prospective study over 15 years.

OBJECTIVES: Lactate as a key regulator of the glycolytic phenotype has been recently described in fueling tumor growth and metastatic spread in head and neck squamous cell carcinoma (HNSCC). However, in context of tumor recurrence following adjuvant radiation, the underlying mechanisms remain uncertain. We therefore investigate the role of lactate towards radioresistance in HNSCC in this prospective study for the first time in vivo. MATERIALS AND METHODS: Herein, we analyzed biopsies of primary squamous cell carcinoma after surgery and adjuvant irradiation in 17 patients. Tumor tissue levels of ATP, glucose, and lactate were detected using induced metabolic bioluminescence imaging (imBI) and correlated with clinical data within an observation period of up to 15 years. RESULTS: High amounts of lactate levels in tumors of HNSCC are significantly negatively correlated with overall patient survival. Moreover, high expression of lactate in a primary tumor site is significantly correlated with tumor recurrence post radiation, whereas ATP and/or glucose showed no such correlation. CONCLUSION: Lactate can be seen not only as a waste product of altered glycolytic metabolism but also as a key master of malignancy as well as resistance mechanism towards irradiation. CLINICAL RELEVANCE: High expression of lactate levels in tumor tissue, obtained by metabolic bioluminescence imaging, may therefore serve as a predictor for overall and recurrence-free survival and could represent a future biomarker in the validation of adjuvant irradiation.

Low density lipoprotein receptor-related protein 1 mediated endocytosis of ß1-integrin influences cell adhesion and cell migration.

The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with ß1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and ß1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of ß1-integrin and decreased ß1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with ß1-integrin mediating integrin internalization and thus correlates with downstream signaling of ß1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

Manipulation of tumor metabolism for therapeutic approaches: ovarian cancer-derived cell lines as a model system.

BACKGROUND: Malignant transformation of cells is often accompanied by up-regulation of glycolysis-related enzymes and transporters, as well as a distortion of mitochondrial respiration. As a consequence, most malignant tumors utilize high amounts of glucose and produce and accumulate high concentrations of lactate, even in the presence of oxygen. This phenomenon has been termed 'Warburg Effect'. Here, we aimed at resolving the interrelation between tumor metabolism, reactive oxygen species, double strand DNA breaks and radio-resistance in ovarian cancer-derived cells. METHODS: As a model system two ovarian cancer-derived cell lines, OC316 and IGROV-1, and its corresponding xenografts were used. First, the metabolic properties of the xenografts were tested to ensure that initial in vitro data might later be transferred to in vivo data. In parallel, three inhibitors of tumor cell metabolism, 2-deoxy-D-glucose, an inhibitor of glycolysis, oxamate, a pyruvate analogue and inhibitor of lactate dehydrogenase, and rotenone, a specific inhibitor of mitochondrial electron complex I, were tested for their effect on the metabolism and radio-sensitivity of the respective ovarian cancer-derived cell lines. RESULTS: We found that all three inhibitors tested led to significant changes in the tumor cell energy metabolism at non-cytotoxic concentrations. Furthermore, we found that inhibition of tumor glycolysis by 2-deoxy-D-glucose in combination with rotenone decreased the radio-resistance at a clinically relevant radiation dose. This apparent radio-sensitizing effect appears to be based on an increased level of double strand DNA breaks 1 h and 24 h after gamma irradiation. Both cancer-derived cell lines maintained their metabolic properties, as well as their protein expression profiles and levels of reactive oxygen species in xenografts, thus providing a suitable model system for further in vivo investigations. CONCLUSION: A combination of metabolic inhibitors and reactive oxygen species-generating therapies, such as irradiation, may effectively enhance the therapeutic response in particularly metabolically highly active (ovarian) tumors.

VEGF-targeted therapy stably modulates the glycolytic phenotype of tumor cells.

Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.

Carbon ions and X­rays induce pro­inflammatory effects in 3D oral mucosa models with and without PBMCs.

Oral mucositis is a severe complication of radiotherapy. Hence, it may constitute a serious medical safety risk for astronauts during extended space flights, such as missions to Mars, during which they are exposed to heavy-ion irradiation. For risk assessment of developing radiation-induced mucositis, a three-dimensional (3D) organotypic oral mucosa model was irradiated with 12C heavy ions or X­rays. The present study focused mainly on early radiation­induced effects, such as the activation of nuclear factor κB (NFκB) and the expression or release of pro-inflammatory marker molecules. The 3D oral mucosa models with or without peripheral blood mononuclear cells (PBMCs) were irradiated with X­rays or 12C heavy ions followed by snap freezing. Subsequently, cryosections were derived from the specimens, which were immunostained for analysis of compactness, DNA double strand breaks (DSB) and activation of NFκB. Radiation­induced release of interleukin 6 (IL6) and interleukin 8 (IL8) was quantified by ELISA. Quantification of the DNA damage in irradiated mucosa models revealed distinctly more DSB after heavy-ion irradiation compared to X­rays at definite time points, suggesting a higher gene toxicity of heavy ions. NFκB activation was observed after treatment with X­rays or 12C particles. ELISA analyses showed significantly higher IL6 and IL8 levels after irradiation with X­rays and 12C particles compared to non-irradiated controls, whereas co­cultures including PBMCs released 2 to 3-fold higher interleukin concentrations compared to mucosa models without PBMCs. In this study, we demonstrated that several pro-inflammatory markers are induced by X­rays and heavy-ion irradiation within an oral mucosa model. This suggests that oral mucositis indeed poses a risk for astronauts on extended space flights.

MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts.

The oncogenes MYCN and survivin (BIRC5) maintain aggressiveness of diverse cancers including sarcomas. To investigate whether these oncogenes cooperate in initial malignant transformation, we transduced them into Rat-1 fibroblasts. Indeed, survivin enhanced MYCN-driven contact-uninhibited and anchorage-independent growth in vitro. Importantly, upon subcutaneous transplantation into mice, cells overexpressing both instead of either one of the oncogenes generated tumors with shortened latency, marked anaplasia and an increased proliferation-to-apoptosis ratio resulting in accelerated growth. Mechanistically, the increased tumorigenicity was associated with an enhanced Warburg effect and a hypoxia inducible factor 1α linked vascular remodeling. This cooperation between MYCN and survivin may be important in the genesis of several cancers.

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI.

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K (trans) and v e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K (trans) was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K (trans) is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K (trans) may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy.

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Impact of carbon ion irradiation on epidermal growth factor receptor signaling and glioma cell migration in comparison to conventional photon irradiation.

PURPOSE: Radiotherapy of malignant gliomas may be limited by an interference of radiation with the migratory potential of tumor cells. Therefore, the influence of conventional photon and modern carbon ion ((12)C) irradiation on glioblastoma cell migration and on epidermal growth factor receptor-related (EGFR) signaling was investigated in vitro. MATERIALS AND METHODS: EGFR overexpressing glioblastoma cell lines U87 EGFR++ and LN229 EGFR++ were irradiated with 0, 2 or 6 Gy photons or (12)C heavy ions. Migration was analyzed 24 h after treatment in a standardized Boyden Chamber assay. At different time points EGFR, protein kinase B (PKB/AKT) and extracellular signal-related kinases (ERK1/2) were analyzed by Western blotting. RESULTS: 2 Gy photon irradiation increased U87 EGFR++ migration and decreased motility of LN229 EGFR++ cells. Heavy ions decreased migration of both cell lines as a function of dose. There was a time-dependent increase of phosphorylation of EGFR, AKT and ERK1/2 in U87 EGFR++ after 2 Gy photon irradiation. After heavy ion irradiation EGFR, AKT or ERK1/2 remained unchanged. CONCLUSIONS: Results suggest that the impact of irradiation on tumor cell migration depends on radiation type and cell line. Photons, but not heavy ions, potentially contribute to treatment failure by increasing EGFR-related tumor cell migration.