Benjamin J. Moeller

Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: Role of reoxygenation, free radicals, and stress granules

Through a poorly understood mechanism, tumors respond to radiation by secreting cytokines capable of inhibiting apoptosis in endothelial cells, thereby diminishing treatment response by minimizing vascular damage. We reveal here that this pathway is governed by a major angiogenesis regulator, HIF-1. Following radiotherapy, tumor reoxygenation leads to: (1) nuclear accumulation of HIF-1 in response to reactive oxygen, and (2) enhanced translation of HIF-1-regulated transcripts secondary to stress granule depolymerization. The resulting increase in HIF-1-regulated cytokines enhances endothelial cell radioresistance. Inhibiting postradiation HIF-1 activation significantly increases tumor radiosensitivity as a result of enhanced vascular destruction. These data describe novel pathways contributing significantly to our understanding of HIF-1 regulation which may be major determinants of tumor radiosensitivity, potentially having high clinical relevance.

Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response

Hypoxia and free radicals, such as reactive oxygen and nitrogen species, can alter the function and/or activity of the transcription factor hypoxia-inducible factor 1 (HIF1). Interplay between free radicals, hypoxia and HIF1 activity is complex and can influence the earliest stages of tumour development. The hypoxic environment of tumours is heterogeneous, both spatially and temporally, and can change in response to cytotoxic therapy. Free radicals created by hypoxia, hypoxia-reoxygenation cycling and immune cell infiltration after cytotoxic therapy strongly influence HIF1 activity. HIF1 can then promote endothelial and tumour cell survival. As discussed here, a constant theme emerges: inhibition of HIF1 activity will have therapeutic benefit.Hypoxia and free radicals, such as reactive oxygen and nitrogen species, can alter the function and/or activity of the transcription factor hypoxia-inducible factor 1 (HIF1). Interplay between free radicals, hypoxia and HIF1 activity is complex and can influence the earliest stages of tumour development. The hypoxic environment of tumours is heterogeneous, both spatially and temporally, and can change in response to cytotoxic therapy. Free radicals created by hypoxia, hypoxia–reoxygenation cycling and immune cell infiltration after cytotoxic therapy strongly influence HIF1 activity. HIF1 can then promote endothelial and tumour cell survival. As discussed here, a constant theme emerges: inhibition of HIF1 activity will have therapeutic benefit.

Hypoxia and radiotherapy: opportunities for improved outcomes in cancer treatment.

A large body of clinical evidence exists to suggest that tumor hypoxia negatively impacts radiotherapy. As a result, there has been longstanding active research into novel methods of improving tumor oxygenation, targeting hypoxic tumor cells, and otherwise modulating the effect hypoxia has on how tumors respond to radiation. Over time, as more has been learned about the many ways hypoxia affects tumors, our understanding of the mechanisms connecting hypoxia to radiosensitivity has become increasingly broad and complicated. This has opened up new potential avenues for interrupting hypoxia’s negative effects on tumor radiosensitivity. Here, we will review what is currently known about the spectrum of influence hypoxia has over the way tumors respond to radiation. Particular focus will be placed on recent discoveries suggesting that hypoxia-inducible factor-1 (HIF-1), a transcription factor that upregulates its target genes under hypoxic conditions, plays a major role in determining tumor radiosensitivity. HIF-1 and/or its target genes may represent therapeutic targets which could be manipulated to influence hypoxia’s impact on tumor radiosensitivity.

Hyperspectral imaging of hemoglobin saturation in tumor microvasculature and tumor hypoxia development

Tumor hypoxia has been shown to have prognostic value in clinical trials involving radiation, chemotherapy, and surgery. Tumor oxygenation studies at microvascular levels can provide understanding of oxygen transport on scales at which oxygen transfer to tissue occurs. To fully grasp the significance of blood oxygen delivery and hypoxia at microvascular levels during tumor growth and angiogenesis, the spatial and temporal relationship of the data must be preserved and mapped. Using tumors grown in window chamber models, hyperspectral imaging can provide serial spatial maps of blood oxygenation in terms of hemoglobin saturation at the microvascular level. We describe our application of hyperspectral imaging for in vivo microvascular tumor oxygen transport studies using red fluorescent protein (RFP) to identify all tumor cells, and hypoxia-driven green fluorescent protein (GFP) to identify the hypoxic fraction. 4T1 mouse mammary carcinoma cells, stably transfected with both reporter genes, are grown in dorsal skin-fold window chambers. Hyperspectral imaging is used to create image maps of hemoglobin saturation, and classify image pixels where RFP alone is present (tumor cells), or both RFP and GFP are present (hypoxic tumor cells). In this work, in vivo calibration of the imaging system is described and in vivo results are shown.

The type III TGF-β receptor suppresses breast cancer progression

TheTGF-β�signalingpathwayhasacomplexroleinregulatingmammarycarcinogenesis.�Herewedemon- stratethatthetypeIIITGF-β�receptor�(TβRIII,�orbetaglycan),�aubiquitouslyexpressedTGF-β�coreceptor,� regulatedbreastcancerprogressionandmetastasis.�MosthumanbreastcancerslostTβRIIIexpression,�with� lossofheterozygosityoftheTGFBR3�genelocuscorrelatingwithdecreasedTβRIIIexpression.�TβRIIIexpres- siondecreasedduringbreastcancerprogression,�andlowTβRIIIlevelspredicteddecreasedrecurrence-free� survivalinbreastcancerpatients.�RestoringTβRIIIexpressioninbreastcancercellsdramaticallyinhibited� tumorinvasivenessinvitroandtumorinvasion,�angiogenesis,�andmetastasisinvivo.�TβRIIIappearedto� inhibittumorinvasionbyundergoingectodomainsheddingandproducingsolubleTβRIII,�whichbinds� andsequestersTGF-β�todecreaseTGF-β�signalingandreducebreastcancercellinvasionandtumor-induced� angiogenesis.�OurresultsindicatethatlossofTβRIIIthroughallelicimbalanceisafrequentgeneticevent� duringhumanbreastcancerdevelopmentthatincreasesmetastaticpotential.

Prospective Risk-Adjusted [18F]Fluorodeoxyglucose Positron Emission Tomography and Computed Tomography Assessment of Radiation Response in Head and Neck Cancer

PURPOSE [(18)F]Fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT) imaging may improve assessment of radiation response in patients with head and neck cancer, but it is not yet known for which patients this is most useful. We conducted a prospective trial to identify patient populations likely to benefit from the addition of functional imaging to the assessment of radiotherapy response. PATIENTS AND METHODS Ninety-eight patients with locally advanced cancer of the oropharynx, larynx, or hypopharynx were prospectively enrolled and treated with primary radiotherapy, with or without chemotherapy. Patients underwent FDG-PET/CT and contrast-enhanced CT imaging 8 weeks after completion of treatment. Functional and anatomic imaging response was correlated with clinical and pathologic response. Imaging accuracy was then compared between imaging modalities. RESULTS Although postradiation maximum standard uptake values were significantly higher in nonresponders compared with responders, the positive and negative predictive values of FDG-PET/CT scanning were similar to those for CT alone in the unselected study population. Subset analyses revealed that FDG-PET/CT outperformed CT alone in response assessment for patients at high risk for treatment failure (those with human papillomavirus [HPV] -negative disease, nonoropharyngeal primaries, or history of tobacco use). No benefit to FDG-PET/CT was seen for low-risk patients lacking these features. CONCLUSION These data do not support the broad application of FDG-PET/CT for radiation response assessment in unselected head and neck cancer patients. However, FDG-PET/CT may be the imaging modality of choice for patients with highest risk disease, particularly those with HPV-negative tumors. Optimal timing of FDG-PET/CT imaging after radiotherapy merits further investigation.

HIF-1 and tumour radiosensitivity

Hypoxia-inducible factor-1 (HIF-1) plays important roles in regulating radiosensitivity, making it a potentially promising target for tumour radiosensitisation. Here, we discuss the rationale for, and the potential pitfalls of, combining HIF-1 blockade with radiotherapy. In doing so, we describe clinical scenarios in which HIF-1 inhibition might optimise tumour radiosensitivity.

Palliative thoracic radiotherapy in lung cancer: An American Society for Radiation Oncology evidence-based clinical practice guideline

Purpose To provide guidance to physicians and patients with regard to the use of external beam radiotherapy, endobronchial brachytherapy, and concurrent chemotherapy in the setting of palliative thoracic treatment for lung cancer, based on available evidence complemented by expert opinion. Methods and Materials A Task Force authorized by the American Society for Radiation Oncology (ASTRO) Board of Directors synthesized and assessed evidence from 3 systematic reviews on the following topics: (1) dose fractionation in thoracic external beam radiotherapy (EBRT); (2) clinical utility of initial and salvage endobronchial brachytherapy (EBB); and (3) use of concurrent chemotherapy (CC) with palliative thoracic radiotherapy. Practice guideline recommendations were produced and are contained herein. Results Studies suggest that higher dose/fractionation palliative EBRT regimens (eg, 30 Gy/10 fraction equivalent or greater) are associated with modest improvements in survival and total symptom score, particularly in patients with good performance status. As these improvements are associated with an increase in esophageal toxicity, various shorter EBRT dose/fractionation schedules (eg, 20 Gy in 5 fractions, 17 Gy in 2 weekly fractions, 10 Gy in 1 fraction), which provide good symptomatic relief with fewer side effects, can be used for patients requesting a shorter treatment course and/or in those with a poor performance status. No defined role for EBB in the routine initial palliative treatment of chest disease has been demonstrated; however, EBB can be a reasonable option for the palliation of endobronchial lesions causing obstructive symptomatology including lung collapse, or for hemoptysis after EBRT failure. The integration of concurrent chemotherapy with palliative intent/fractionated radiotherapy is not currently supported by the medical literature. Conclusion This Guideline is intended to serve as a guide for the use of EBRT, EBB, and CC in thoracic palliation of lung cancer outside the clinical trial setting. Further prospective clinical investigations with relevant palliative endpoints into the respective roles of EBB and CC/targeted therapy in the thoracic palliation of lung cancer are warranted, given the current state of the medical literature in these areas.

Observation of Incipient Tumor Angiogenesis That Is Independent of Hypoxia and Hypoxia Inducible Factor-1 Activation

It is well established that hypoxia potently stimulates tumor angiogenesis by activating hypoxia inducible factor-1 (HIF-1)-induced proangiogenic factors, such as vascular endothelial growth factor. However, very little is known about the role of hypoxia in incipient angiogenesis in avascular tumors during their early stages of growth. To noninvasively investigate the functional significance of hypoxia and HIF-1 activation in incipient tumor angiogenesis, we genetically engineered HCT116 human colon carcinoma cells and 4T1 mouse mammary carcinoma cells with constitutively expressed red fluorescence protein as a tumor marker and green fluorescence protein (GFP) as a reporter for hypoxia and HIF-1 activation. The accuracy of GFP fluorescence in reporting hypoxia was confirmed by flow cytometry analysis and by immunohistochemical comparison with pimonidazole, a well-established hypoxia marker drug. Mouse dorsal skin-fold window chambers showed that incipient angiogenesis preceded a detectable level of hypoxia. The detectable levels of hypoxia were spatially and temporally related with more intensive secondary angiogenesis following the initial onset of new vessel formation. Selective killing of hypoxic cells by tirapazamine efficiently eliminated or delayed the detection of hypoxic cells, but it did not significantly delay the onset of incipient angiogenesis. These findings provide the first in vivo evidence that incipient tumor angiogenesis may not depend on hypoxia or HIF-1 activation. This is in contrast to the clear role of hypoxia in driving angiogenesis once initial tumor microvessel formation has occurred.

DNA repair biomarker profiling of head and neck cancer: Ku80 expression predicts locoregional failure and death following radiotherapy

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years. Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.