Ludwig Dubois

The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5

Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

Imaging of CA IX with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-oxygenated cells in a xenograft tumour model

BACKGROUND AND PURPOSE Carbonic anhydrase (CA) IX is suggested to be an endogenous marker of hypoxia. Fluorescent sulfonamides with a high affinity for CA IX (CAI) have been developed and shown to bind to cells only when CA IX protein was expressed and while cells were hypoxic. The aim of this study was to investigate the in vivo CAI binding properties in a xenograft tumour model using fluorescent imaging. MATERIALS AND METHODS NMRI-nu mice subcutaneously transplanted with HT-29 colorectal tumours were treated with 7% oxygen or with nicotinamide and carbogen and were compared with control animals. CAI accumulation was monitored by non-invasive fluorescent imaging. RESULTS Specific CAI accumulation could be observed in delineated tumour areas as compared with a non-sulfonamide analogue (P<0.01). Administration of nicotinamide and carbogen, decreasing acute and chronic hypoxia, respectively, prevented CAI accumulation (P<0.05). When treated with 7% oxygen breathing, a 3-fold higher CAI accumulation (P<0.01) was observed. Furthermore, the bound CAI fraction was rapidly reduced upon tumour reoxygenation (P<0.01). CONCLUSIONS Our in vivo imaging results confirm previous in vitro data demonstrating that CAI binding and retention require exposure to hypoxia. Fluorescent labelled sulfonamides provide a powerful tool to visualize hypoxia response. An important step is made towards clinical applicability, indicating the potential of patient selection for CA IX-directed therapies.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Disparity Between In Vivo EGFR Expression and 89Zr-Labeled Cetuximab Uptake Assessed with PET

The epidermal growth factor receptor (EGFR) is highly expressed in a significant number of human malignancies, and its expression is associated with tumor aggressiveness and overall treatment resistance. The monoclonal antibody cetuximab is increasingly used in clinical settings as a treatment modality in combination with more conventional therapies, such as radio- and chemotherapy. Currently, little is known about tumor-specific uptake and overall pharmacokinetics. Noninvasive quantification of cetuximab uptake could provide important diagnostic information for patient selection and therapy evaluation. To this end, we have developed and validated a novel probe using cetuximab labeled with the long-lived positron emitter 89Zr for PET imaging. Methods: Tumor cell lines with varying EGFR expression levels were used for in vivo tumor imaging experiments. PET with 89Zr-labeled cetuximab (3.75 ± 0.14 MBq) was performed on tumor-bearing NMRI-nu mice at multiple time points after injection (ranging from 1 to 120 h) and quantified by drawing regions of interest on selected tissues. Uptake was compared by biodistribution γ-counting, and ex vivo EGFR expression levels were quantified using Western blot analysis. Results: Uptake of 89Zr-labeled cetuximab was demonstrated in the EGFR-positive tumors. However, the EGFR levels measured in vivo did not correlate with the relative signal obtained by PET. Tumor-to-blood ratios were significantly higher in the cell lines with intermediate (compared with the high) EGFR expression starting from 24 h after injection. Normal tissue uptake was unaffected by the different tumor types. Ex vivo γ-counting experiments confirmed the observed in vivo PET results. A similar disparity was found between 89Zr-labeled cetuximab tumor uptake and in vivo EGFR expression levels as demonstrated by Western blotting. Conclusion: The 89Zr-labeled cetuximab imaging probe is a promising tool for noninvasive evaluation of cetuximab uptake. Our results demonstrate a disparity between in vivo EGFR expression levels and cetuximab uptake. In a general sense, the results indicate a disparity between antibody uptake and expression levels of a biologic target in a tumor, suggesting that additional pharmacokinetic or pharmacodynamic mechanisms influence tumor delivery of this therapy. These additional mechanisms may explain why receptor expression levels alone are not sufficient to predict patient response.

Specific inhibition of carbonic anhydrase IX activity enhances the in vivo therapeutic effect of tumor irradiation

BACKGROUND AND PURPOSE Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. The aim of this study was to evaluate the antitumor activity of a high CAIX-affinity indanesulfonamide (11c) combined with irradiation, compared with the general CA inhibitor acetazolamide (AZA). MATERIAL AND METHODS HT-29 carcinoma cells with or without (genetic knockdown, KD) CAIX expression were incubated with 11c/AZA under different oxygen levels and proliferation, apoptosis and radiosensitivity were evaluated. 11c/AZA was administered intravenously (1×/day; 5 days) to tumor-bearing mice and tumor irradiation (10 Gy) was performed at day 3 of the injection period. Tumor growth and potential treatment toxicity were monitored (3×/week). RESULTS Treatment with 11c/AZA alone resulted in tumor regression, which was further increased in CAIX expressing cells by combining 11c with irradiation. AZA demonstrated also an additional effect in the KD tumors when combined with irradiation. CAIX inhibition in vitro significantly reduced proliferation and increased apoptosis upon hypoxia exposure without affecting intrinsic radiosensitivity. CONCLUSIONS Specific inhibition of CAIX activity enhanced the effect of tumor irradiation and might, therefore, be an attractive strategy to improve overall cancer treatment.

Preclinical evaluation and validation of [18F]HX4, a promising hypoxia marker for PET imaging

Hypoxia has been shown to be an important microenvironmental parameter influencing tumor progression and treatment efficacy. Patient guidance for hypoxia-targeted therapy requires evaluation of tumor oxygenation, preferably in a noninvasive manner. The aim of this study was to evaluate and validate the uptake of [18F]HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [18F]HX4 was found within rat rhabdomyosarcoma tumors that was significantly (P < 0.0001) higher compared with the surrounding tissues, with temporal increasing tumor-to-blood ratios reaching a plateau of 7.638 ± 0.926 and optimal imaging properties 4 h after injection. [18F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [18F]HX4 tumor uptake was analyzed based on 16 regions within the tumor according to the different orthogonal planes at the largest diameter. Validation of heterogeneous [18F]HX4 tumor uptake was shown by a strong and significant relationship (r = 0.722; P < 0.0001) with the hypoxic fraction as calculated by the percentage pimonidazole-positive pixels. Furthermore, a causal relationship with tumor oxygenation was established, because combination treatment of nicotinamide and carbogen resulted in a 40% reduction (P < 0.001) in [18F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [18F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.

PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

A Comparative Study of the Hypoxia PET Tracers [18F]HX4, [18F]FAZA, and [18F]FMISO in a Preclinical Tumor Model

PURPOSE Several individual clinical and preclinical studies have shown the possibility of evaluating tumor hypoxia by using noninvasive positron emission tomography (PET). The current study compared 3 hypoxia PET tracers frequently used in the clinic, [(18)F]FMISO, [(18)F]FAZA, and [(18)F]HX4, in a preclinical tumor model. Tracer uptake was evaluated for the optimal time point for imaging, tumor-to-blood ratios (TBR), spatial reproducibility, and sensitivity to oxygen modification. METHODS AND MATERIALS PET/computed tomography (CT) images of rhabdomyosarcoma R1-bearing WAG/Rij rats were acquired at multiple time points post injection (p.i.) with one of the hypoxia tracers. TBR values were calculated, and reproducibility was investigated by voxel-to-voxel analysis, represented as correlation coefficients (R) or Dice similarity coefficient of the high-uptake volume. Tumor oxygen modifications were induced by exposure to either carbogen/nicotinamide treatment or 7% oxygen breathing. RESULTS TBR was stabilized and maximal at 2 hours p.i. for [(18)F]FAZA (4.0 ± 0.5) and at 3 hours p.i. for [(18)F]HX4 (7.2 ± 0.7), whereas [(18)F]FMISO showed a constant increasing TBR (9.0 ± 0.8 at 6 hours p.i.). High spatial reproducibility was observed by voxel-to-voxel comparisons and Dice similarity coefficient calculations on the 30% highest uptake volume for both [(18)F]FMISO (R = 0.86; Dice coefficient = 0.76) and [(18)F]HX4 (R = 0.76; Dice coefficient = 0.70), whereas [(18)F]FAZA was less reproducible (R = 0.52; Dice coefficient = 0.49). Modifying the hypoxic fraction resulted in enhanced mean standardized uptake values for both [(18)F]HX4 and [(18)F]FAZA upon 7% oxygen breathing. Only [(18)F]FMISO uptake was found to be reversible upon exposure to nicotinamide and carbogen. CONCLUSIONS This study indicates that each tracer has its own strengths and, depending on the question to be answered, a different tracer can be put forward.

How do changes in the mtDNA and mitochondrial dysfunction influence cancer and cancer therapy? Challenges, opportunities and models

Several mutations in nuclear genes encoding for mitochondrial components have been associated with an increased cancer risk or are even causative, e.g. succinate dehydrogenase (SDHB, SDHC and SDHD genes) and iso-citrate dehydrogenase (IDH1 and IDH2 genes). Recently, studies have suggested an eminent role for mitochondrial DNA (mtDNA) mutations in the development of a wide variety of cancers. Various studies associated mtDNA abnormalities, including mutations, deletions, inversions and copy number alterations, with mitochondrial dysfunction. This might, explain the hampered cellular bioenergetics in many cancer cell types. Germline (e.g. m.10398A>G; m.6253T>C) and somatic mtDNA mutations as well as differences in mtDNA copy number seem to be associated with cancer risk. It seems that mtDNA can contribute as driver or as complementary gene mutation according to the multiple-hit model. This can enhance the mutagenic/clonogenic potential of the cell as observed for m.8993T>G or influences the metastatic potential in later stages of cancer progression. Alternatively, other mtDNA variations will be innocent passenger mutations in a tumor and therefore do not contribute to the tumorigenic or metastatic potential. In this review, we discuss how reported mtDNA variations interfere with cancer treatment and what implications this has on current successful pharmaceutical interventions. Mutations in MT-ND4 and mtDNA depletion have been reported to be involved in cisplatin resistance. Pharmaceutical impairment of OXPHOS by metformin can increase the efficiency of radiotherapy. To study mitochondrial dysfunction in cancer, different cellular models (like ρ(0) cells or cybrids), in vivo murine models (xenografts and specific mtDNA mouse models in combination with a spontaneous cancer mouse model) and small animal models (e.g. Danio rerio) could be potentially interesting to use. For future research, we foresee that unraveling mtDNA variations can contribute to personalized therapy for specific cancer types and improve the outcome of the disease.