Bradly G. Wouters

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF‐1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant‐negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.

Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis

OBJECTIVESnWe sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis.nnnBACKGROUNDnAtherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF.nnnMETHODSnTo examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1 alpha, HIF2 alpha, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry.nnnRESULTSnPimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions.nnnCONCLUSIONnThis is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesis.

Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2α and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1–2 h and is due to phosphorylation of eIF2α. Continued hypoxic exposure activates a second, eIF2α‐independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E‐BP1 and transporter 4E‐T. Quantitative RT–PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2α. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

Low-Dose Hyper-radiosensitivity: A Consequence of Ineffective Cell Cycle Arrest of Radiation-Damaged G2-Phase Cells

Abstract Marples, B., Wouters, B. G., Collis, S. J., Chalmers, A. J. and Joiner, M. C. Low-Dose Hyper-radiosensitivity: A Consequence of Ineffective Cell Cycle Arrest of Radiation-Damaged G2-Phase Cells. Radiat. Res. 161, 247–255 (2004). This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.

Identification of residual metabolic-active areas within individual NSCLC tumours using a pre-radiotherapy 18Fluorodeoxyglucose-PET-CT scan

BACKGROUND AND PURPOSEnNon-small cell lung cancer (NSCLC) tumours are mostly heterogeneous. We hypothesized that areas within the tumour with a high pre-radiation (18)F-deoxyglucose (FDG) uptake, could identify residual metabolic-active areas, ultimately enabling selective-boosting of tumour sub-volumes.nnnMATERIAL AND METHODSnFifty-five patients with inoperable stage I-III NSCLC treated with chemo-radiation or with radiotherapy alone were included. For each patient one pre-radiotherapy and one post-radiotherapy FDG-PET-CT scans were available. Twenty-two patients showing persistent FDG uptake in the primary tumour after radiotherapy were analyzed. Overlap fractions (OFs) were calculated between standardized uptake value (SUV) threshold-based auto-delineations on the pre- and post-radiotherapy scan.nnnRESULTSnPatients with residual metabolic-active areas within the tumour had a significantly worse survival compared to individuals with a complete metabolic response (p=0.002). The residual metabolic-active areas within the tumour largely corresponded (OF>70%) with the 50%SUV high FDG uptake area of the pre-radiotherapy scan. The hotspot within the residual area (90%SUV) was completely within the GTV (OF=100%), and had a high overlap with the pre-radiotherapy 50%SUV threshold (OF>84%).nnnCONCLUSIONSnThe location of residual metabolic-active areas within the primary tumour after therapy corresponded with the original high FDG uptake areas pre-radiotherapy. Therefore, a single pre-treatment FDG-PET-CT scan allows for the identification of residual metabolic-active areas.

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response

IntroductionThe hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.MethodsA number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.ResultsUsing transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.ConclusionsThus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Low-Dose Hypersensitivity and Increased Radioresistance in a Panel of Human Tumor Cell Lines with Different Radiosensitivity

It is well known that cells of human tumor cell lines display a wide range of sensitivity to radiation, at least a part of which can be attributed to different capacities to process and repair radiation damage correctly. We have examined the response to very low-dose radiation of cells of five human tumor cell lines that display varying sensitivity to radiation, using an improved assay for measurement of radiation survival. This assay improves on the precision of conventional techniques by accurately determining the numbers of cells at risk, and has allowed us to measure radiation survival to doses as low as 0.05 Gy. Because of the statistical limitations in measuring radiation survival at very low doses, extensive averaging of data was used to determine the survival response accurately. Our results show that the four most resistant cell lines exhibit a region of initial low-dose hypersensitivity. This hypersensitivity is followed by an increase in radioresistance over the dose range 0.3 to 0.7 Gy, beyond which the response is typical of that seen in most survival curves. Mathematical modeling of the responses suggests that this phenomenon is not due to a small subpopulation of sensitive cells (e.g. mitotic), but rather is a reflection of the induction of resistance in the whole cell population, or at least a significant proportion of the whole cell population. These results suggest that a dose-dependent alteration in the processing of DNA damage over the initial low-dose region of cell survival may contribute to radioresistance in some cell lines.

An Association between the Radiation-Induced Arrest of G2-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism?

Abstract Marples, B., Wouters, B. G. and Joiner, M. C. An Association between the Radiation-Induced Arrest of G2-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism? Radiat. Res. 160, 38–45 (2003). The survival of asynchronous and highly enriched G1-, S- and G2-phase populations of Chinese hamster V79 cells was measured after irradiation with 60Co γ rays (0.1–10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G2-phase cells being the most radiosensitive and S-phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G2-phase enriched cell populations, with no evidence of HRS in the G1- and S-phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G2-phase cells. An association was discovered between the occurrence of HRS and the induction of a novel G2-phase arrest checkpoint that is specific for cells that are in the G2 phase of the cell cycle at the time of irradiation. Human T98G cells and hamster V79 cells, which both exhibit HRS in asynchronous cultures, failed to arrest the entry into mitosis of damaged G2-phase cells at doses less than 30 cGy, as determined by the flow cytometric assessment of the phosphorylation of histone H3, an established indicator of mitosis. In contrast, human U373 cells that do not show HRS induced this G2-phase checkpoint in a dose-independent manner. These data suggest that HRS may be a consequence of radiation-damaged G2-phase cells prematurely entering mitosis.

Low-dose radiation sensitivity and induced radioresistance to cell killing in HT-29 cells is distinct from the "adaptive response" and cannot be explained by a subpopulation of sensitive cells.

Several reports using two different improved assays of clonogenicity have indicated the presence of a hypersensitive region in the radiation survival response at low doses, followed by an increase in radioresistance, in many mammalian cell lines. Mathematical modeling of these responses has suggested that it is unlikely that this effect can be explained by the presence of a small subpopulation of sensitive cells; however, this possibility cannot be excluded solely on the basis of those results. A second explanation has been offered which hypothesizes that a radiation-induced mechanism causes an increase in cellular radioresistance. This proposal has led to speculation that the substructure observed at low doses in these cell lines is related to the adaptive response, which hypothesizes the induction of a repair mechanism after a small priming dose of radiation which can protect cells against a larger second dose given several hours later. We have investigated these proposals with a study of priming doses using human tumor HT-29 cells, which we have previously shown to exhibit low-dose hyper-radiosensitivity. Our results provide significant evidence that this effect cannot be explained by a subpopulation of sensitive cells. However, the results also suggest that the radiation-induced increase in radioresistance observed in this cell line is distinct from the adaptive response.

The mTOR target 4E-BP1 contributes to differential protein expression during normoxia and hypoxia through changes in mRNA translation efficiency.

Hypoxia causes a rapid and sustained inhibition in mRNA translation that is characterized by both a transient phosphorylation of eukaryotic initiation factor 2‐alpha (eIF2α) and by inhibition of the mRNA cap binding protein eIF4E via activation of two distinct inhibitory proteins, the mammalian target of rapamycin (mTOR) target 4E‐BP1 and the eIF4E transporter 4E‐T. Although the importance of eIF2α phosphorylation during hypoxia has been clearly demonstrated, there is little information on the potential relevance of eIF4E regulation. We generated HeLa cells stably expressing a short hairpin interfering RNA (shRNA) against 4E‐BP1 and found that despite efficient knockdown, no significant changes occurred in the overall inhibition of mRNA translation during hypoxia. However, using a proteomics approach we identified seven proteins that were exclusively expressed in the 4E‐BP1 knockdown cells during both normoxic and hypoxic conditions. Further investigation of the transcriptional and translational regulation of these genes by quantitative RT‐PCR indicated that the loss of 4E‐BP1 causes a significant increase in the rate of protein synthesis of S100 calcium‐binding protein A4 (S100A4) and transgelin 2. These 4E‐BP1 regulated proteins have previously been associated with tumor cell motility, invasion and metastasis and may thus contribute to an adverse tumor phenotype.