Albert J. van der Kogel

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.

RADIATION DOSE-VOLUME EFFECTS IN THE SPINAL CORD

Dose-volume data for myelopathy in humans treated with radiotherapy (RT) to the spine is reviewed, along with pertinent preclinical data. Using conventional fractionation of 1.8-2 Gy/fraction to the full-thickness cord, the estimated risk of myelopathy is <1% and <10% at 54 Gy and 61 Gy, respectively, with a calculated strong dependence on dose/fraction (alpha/beta = 0.87 Gy.) Reirradiation data in animals and humans suggest partial repair of RT-induced subclinical damage becoming evident about 6 months post-RT and increasing over the next 2 years. Reports of myelopathy from stereotactic radiosurgery to spinal lesions appear rare (<1%) when the maximum spinal cord dose is limited to the equivalent of 13 Gy in a single fraction or 20 Gy in three fractions. However, long-term data are insufficient to calculate a dose-volume relationship for myelopathy when the partial cord is treated with a hypofractionated regimen.

Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer

Activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (AKT) pathway is associated with three major radioresistance mechanisms: intrinsic radioresistance; tumour-cell proliferation; and hypoxia. Monitoring and manipulation of this signal-transduction pathway can have important implications for the management of head and neck cancer, because activation of the PI3-K/AKT pathway is a frequent event in these tumours. PI3-K/AKT signalling regulates cellular processes, including proliferation, invasion, apoptosis, and the upregulation of hypoxia-related proteins. Activation of this pathway can be caused by stimulation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). In clinical trials, a strong and independent association has been noted between expression of activated AKT and treatment outcome. Therefore, the search for molecular predictors of sensitivity to EGFR-directed treatment should be extended to markers of PI3-K/AKT activation. Another strategy might be the direct targeting and inhibition of this pathway. Such inhibition will enhance the efficacy of radiotherapy, by antagonising radiation-induced cellular defense mechanisms, especially in tumours that have activated the PI3-K/AKT cascade. Thus, the activation status of this pathway might be a key element for the prediction of treatment response and for therapeutic targeting in head and neck cancer.

Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers.

BACKGROUND AND PURPOSE Tumor oxygenation is recognized as an important determinant of the outcome of radiotherapy and possibly also of other treatment modalities in a number of tumor types and in particular in squamous cell carcinomas. The hypoxic status of various solid tumors has been related to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment. It has been demonstrated in head and neck cancer that hypoxic radioresistance can be successfully counteracted by hypoxia modifying approaches. The microregional distribution and the level of tumor hypoxia depend on oxygen consumption and temporal and spatial variations in blood supply. It is unclear if severely hypoxic cells can resume clonogenicity when O(2) and nutrients become available again as a result of (treatment related) changes in the tumor microenvironment. Non-terminally differentiated hypoxic cells that are capable of proliferation are important for outcome because of their resistance to radiotherapy and possibly other cytotoxic treatments. Various exogenous and endogenous markers for hypoxia are currently available and can be studied in relation to each other, the tumor architecture and the tumor microenvironment. Use of nitroimidazole markers with immunohistochemical detection allows studying tumor cell hypoxia at the microscopic level. Co-registration with other microenvironmental parameters, such as vascular architecture (vascular density), blood perfusion, tumor cell proliferation and apoptosis, offers the possibility to obtain a comprehensive functional image of tumor patho-physiology and to study the effects of different modalities of cancer treatment. CONCLUSION A number of functional microregional parameters have emerged that are good candidates for future use as indicators of tumor aggressiveness and treatment response. The key question is whether these parameters can be used as tools for selection of treatment strategies for individual patients. This requires testing of these markers in prospective randomized clinical trials comparing standard treatment against experimental treatments targeting the relevant microregional constituent.

ARCON: a novel biology-based approach in radiotherapy

Two mechanisms of radiotherapy resistance which are of major importance in various tumour types are tumour-cell repopulation and hypoxia. ARCON (accelerated radiotherapy with carbogen and nicotinamide) is a new therapeutic strategy that combines radiation treatment modifications, with the aim of counteracting these resistance mechanisms. To limit clonogenic repopulation during therapy, the overall duration of the radiotherapy is reduced, generally by delivering several fractions per day. This accelerated radiotherapy is combined with inhalation of hyperoxic gas to decrease diffusion-limited hypoxia, and nicotinamide, a vasoactive agent, to decrease perfusion-limited hypoxia. Preclinical studies have been done to test the enhancing effects of these three components of ARCON, individually and in combination, in several experimentally induced tumours and normal tissues. In a mouse mammary carcinoma, the tumour-control rate obtained with ARCON was the same as that with conventional treatment, but with a radiation dose almost 50% lower. Phase 1 and 2 clinical trials have shown the feasibility and tolerability of ARCON, and have produced promising results in terms of tumour control. In particular in cancers of the head and neck and bladder, the local tumour-control rates are higher than in other studies, and phase 3 trials for these tumour types are underway. In conjunction with these trials, hypoxia markers detectable by immunohistochemistry are being tested for their potential use in predictive assays to select patients for ARCON and other hypoxia-modifying therapies.

Method for quantitative mapping of dynamic MRI contrast agent uptake in human tumors.

A method is presented for the acquisition and analysis of dynamic contrast‐enhanced (DCE) MRI data, focused on the characterization of tumors in humans. Gadolinium (Gd) contrast was administered by bolus injection, and its effect was monitored in time by fast T1‐weighted MRI. A simple algorithm was developed for automatic extraction of the arterial input function (AIF) from the DCE‐MRI data. This AIF was used in the pixelwise pharmacokinetic determination of physiological vascular parameters in normal and tumor tissue. Maps were reconstructed to show the spatial distribution of parameter values. To test the reproducibility of the method 11 patients with different types of tumors were measured twice, and the rate of contrast agent uptake in the tumor was calculated. The results show that normalizing the DCE‐MRI data using individual coregistered AIFs, instead of one common AIF for all patients, substantially reduces the variation between successive measurements. It is concluded that the proposed method enables the reproducible assessment of contrast agent uptake rates. J. Magn. Reson. Imaging 2001;14:457–463.

Angiogenesis-independent tumor growth mediated by stem-like cancer cells

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

Accelerated Radiotherapy With Carbogen and Nicotinamide for Laryngeal Cancer: Results of a Phase III Randomized Trial

PURPOSE To report the results from a randomized trial comparing accelerated radiotherapy (AR) with accelerated radiotherapy plus carbogen inhalation and nicotinamide (ARCON) in laryngeal cancer. PATIENTS AND METHODS Patients with cT2-4 squamous cell laryngeal cancer were randomly assigned to AR (68 Gy within 36 to 38 days) or ARCON. To limit the risk of laryngeal necrosis, ARCON patients received 64 Gy on the laryngeal cartilage. The primary end point was local control. Secondary end points were regional control, larynx preservation, toxicity, disease-free survival, and overall survival. In a translational side study, the hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies. RESULTS From April 2001 to February 2008, 345 patients were accrued. After a median follow-up of 44 months, local tumor control rate at 5 years was 78% for AR versus 79% for ARCON (P = .80), with larynx preservation rates of 84% and 87%, respectively (P = .48). The 5-year regional control was significantly better with ARCON (93%) compared with AR (86%, P = .04). The improvement in regional control was specifically observed in patients with hypoxic tumors and not in patients with well-oxygenated tumors (100% v 55%, respectively; P = .01). AR and ARCON produced equal levels of toxicity. CONCLUSION Despite lack of benefit in local tumor control for advanced laryngeal cancers, a significant gain in regional control rate, with equal levels of toxicity, was observed in favor of ARCON. The poor regional control of patients with hypoxic tumors is specifically countered by ARCON treatment.

Dynamics of tumor hypoxia measured with bioreductive hypoxic cell markers.

Abstract Ljungkvist, A. S. E., Bussink, J., Kaanders, J. H. A. M. and van der Kogel, A. J. Dynamics of Tumor Hypoxia Measured with Bioreductive Hypoxic Cell Markers. Radiat. Res. 167, 127–145 (2007). Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1α and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.

Hedgehog: An attribute to tumor regrowth after chemoradiotherapy and a target to improve radiation response

Purpose: Despite aggressive chemotherapy, radiotherapy, surgery, or combination approaches, the survival rate of patients with esophageal cancer remains poor. Recent studies have suggested that constitutive activation of the Hedgehog (Hh) pathway in cancers of the digestive tract may contribute to the growth and maintenance of cancer. However, the relationship between Hh signaling and therapeutic response is unknown. Experimental Design: The expression and temporal kinetics of Hh signaling and proliferation biomarkers after chemoradiotherapy were examined in esophageal tumor xenografts. Additionally, immunohistochemical analysis of Sonic Hh (Shh) and Gli-1 expression were done on residual tumors from patients who received neoadjuvant chemoradiotherapy followed by surgery. The ability of Shh signaling to induce proliferation in esophageal cell lines was determined. Expression of cell cycle checkpoint proteins was analyzed in cells in which Hh signaling was activated or inhibited. We further determined the effect of inhibiting Hh signaling in sensitizing esophageal tumors to radiation. Results: We showed that the Shh signaling pathway was extensively activated in esophageal cancer xenografts and residual tumors after chemoradiotherapy and the temporal kinetics of Hh signaling preceded increases in proliferation biomarker expression and tumor size during tumor regrowth. We further showed that Hh pathway activity influences proliferation rates of esophageal cancer cell lines through up-regulation of the G1-cyclin-Rb axis. Additionally, we found that blocking Hh signaling enhanced radiation cytotoxicity of esophageal cancer cells. Conclusions: These results suggest that activation of the Hh pathway may promote tumor repopulation after chemoradiotherapy and contribute to chemoradiation resistance in esophageal cancers.