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Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

University of Toronto

The chemo- and radioresponse of tumor cells can be determined by genetic factors (e.g., those that modify cell cycle arrest, DNA damage repair or cell death) and microenvironmental factors, such as hypoxia. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that rapidly recognizes and binds to DNA breaks to facilitate DNA strand break repair. Pre-clinical data suggest that PARP inhibitors (PARPi) may potentiate the effects of radiotherapy and chemotherapy. However, it is unclear as to whether PARPi are effective against hypoxic cells. We therefore tested the role for a novel PARPi, ABT-888, as a radiosensitizing agent under hypoxic conditions. Using human prostate (DU-145, 22RV1) and non-small cell lung (H1299) cancer cell lines, we observed that ABT-888 inhibited both recombinant PARP activity and intracellular PARP activity (86% to 92% decrease in all 3 cells lines following 2.5 microM treatment). ABT-888 was toxic to both oxic and hypoxic cells. When ABT-888 was combined with ionizing radiation (IR), clonogenic radiation survival was decreased by 40-50% under oxic conditions. Under acute hypoxia, ABT-888 radiosensitized malignant cells to a level similar to oxic radiosensitivity. To our knowledge, this is the first study to demonstrate that inhibition of PARP activity can sensitize hypoxic cancer cells and the combination of IR-PARPi has the potential to improve the therapeutic ratio of radiotherapy.

A novel poly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizes malignant human cell lines under hypoxia.

Major technical advances in radiotherapy, including IMRT and image-guided radiotherapy, have allowed for improved physical precision and increased dose delivery to the tumor, with better sparing of surrounding normal tissue. The development of inhibitors of the sensing and repair of DNA double-strand breaks (DSBs) is exciting and could be combined with precise radiotherapy targeting to improve local control following radiotherapy. However, caution must be exercised in order that DSB inhibitors are combined with radiotherapy in such a manner as to preserve the therapeutic ratio by exploiting repair deficiencies in malignant cells over that of normal cells. In this review, we discuss the rationale and current approaches to targeting DSB sensing and repair pathways in combined modality with radiotherapy. We also describe potential biomarkers that could be useful in detecting functional inhibition of DSB repair in a patient's tissues during clinical radiotherapy trials. Finally, we examine a number of issues relating to the use of DSB-inhibiting molecular agents and radiotherapy in the context of the tumor microenvironment, effects on normal tissues and the optimal timing and duration of the agent in relation to fractionated radiotherapy.

Biomarkers for DNA DSB inhibitors and radiotherapy clinical trials.

The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth.

Delta-like ligand 4-notch blockade and tumor radiation response.

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.

Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.

Radiation resistance poses a major clinical challenge in cancer treatment, but little is known about how microRNA (miR) may regulate this phenomenon. In this study, we used next-generation sequencing to perform an unbiased comparison of miR expression in PC3 prostate cancer cells rendered resistant to fractionated radiation treatment. One miR candidate found to be upregulated by ionizing radiation was miR-95, the enforced expression of which promoted radiation resistance in a variety of cancer cells. miR-95 overexpression recapitulated an aggressive phenotype including increased cellular proliferation, deregulated G2-M checkpoint following ionizing radiation, and increased invasive potential. Using combined in silico prediction and microarray expression analyses, we identified and validated the sphingolipid phosphatase SGPP1, an antagonist of sphingosine-1-phosphate signaling, as a target of miR-95 that promotes radiation resistance. Consistent with this finding, cell treatment with FTY720, a clinically approved small molecule inhibitor of S1P signaling, sensitized miR-95 overexpressing cells to radiation treatment. In vivo assays extended the significance of these results, showing that miR-95 overexpression increased tumor growth and resistance to radiation treatment in tumor xenografts. Furthermore, reduced tumor necrosis and increased cellular proliferation were seen after radiation treatment of miR-95 overexpressing tumors compared with control tumors. Finally, miR-95 expression was increased in human prostate and breast cancer specimens compared with normal tissue. Together, our work reveals miR-95 expression as a critical determinant of radiation resistance in cancer cells.

miRNA-95 mediates radioresistance in tumors by targeting the sphingolipid phosphatase SGPP1.

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4B(C74A)in vitro and in vivo, we show that the effects of ATG4B(C74A) are cell type, treatment, and context-dependent. ATG4B(C74A) expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.

Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy.

Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based 'collapse' and a significant tumour growth delay.

Dll4-notch signalling blockade synergizes combined ultrasound-stimulated microbubble and radiation therapy in human colon cancer xenografts.

Most cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Acute skin damage from cancer radiotherapy diminishes patients' quality of life, yet effective biological interventions for this damage are lacking. Protecting microvascular endothelial cells from irradiation-induced perturbations is emerging as a targeted damage-reduction strategy. Since Angiopoetin-1 signaling through the Tie2 receptor on endothelial cells opposes microvascular perturbations in other disease contexts, we used a preclinical Angiopoietin-1 mimic called Vasculotide to investigate its effect on skin radiation toxicity using a preclinical model.

Vasculotide, an Angiopoietin-1 mimetic, reduces acute skin ionizing radiation damage in a preclinical mouse model.

Sets of deregulated microRNAs (miRNAs), termed miRNA signatures, are promising biomarkers for cancer. Validation of miRNA signatures requires a technique that is accurate, sensitive, capable of detecting multiple miRNAs, fast, robust, and not cost-prohibitive. Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a capillary electrophoresis (CE)-based hybridization assay that was suggested as a methodological platform for validation and clinical use of miRNA signatures. While satisfying the other requirements, DQAMmiR is not sufficiently sensitive to detect low-abundance miRNAs. Here, we solve this problem by combining DQAMmiR with the preconcentration technique, isotachophoresis (ITP). The sensitivity improved 100 times (to 1 pM) allowing us to detect low-abundance miRNAs in an RNA extract. Importantly, ITP-DQAMmiR can be performed in a fully automated mode using a commercial CE instrument making it suitable for practical applications.

Highly-sensitive amplification-free analysis of multiple miRNAs by capillary electrophoresis.

To investigate the function of Pea3 in colorectal carcinoma (CRC) invasion and metastatic potential.

Pea3 expression promotes the invasive and metastatic potential of colorectal carcinoma.

Most cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Radiotherapy side-effects diminish patients' quality of life, yet effective biological interventions for normal tissue damage are lacking. Protecting microvascular endothelial cells from the effects of irradiation is emerging as a targeted damage-reduction strategy. We illustrate the concept of the microvasculature as a mediator of overall normal tissue radiation toxicity through cell death, vascular inflammation (hemodynamic and molecular changes) and a change in functional capacity. Endothelial cell targeted therapies that protect against such endothelial cell perturbations and the development of acute normal tissue damage are mostly under preclinical development. Since acute radiation toxicity is a common clinical problem in cutaneous, gastrointestinal and mucosal tissues, we also focus on damage in these tissues.

Endothelial perturbations and therapeutic strategies in normal tissue radiation damage.

MicroRNA contribute to tumor radiation resistance, which is an important clinical problem, and thus we are interested in identifying and characterizing their function. We demonstrate that miR-620 contributes to radiation resistance in cancer cells by increasing proliferation, and decreasing the G2/M block. We identify the hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (HPGD/15-PGDH) tumor suppressor gene as a direct miR-620 target, which results in increased prostaglandin E2 (PGE2) levels. Furthermore, we show that siRNA targeting of HPGD or administration of exogenous PGE2 recapitulates radioresistance. Targeting of the EP2 receptor that responds to PGE2 using pharmacological or genetic approaches, abrogates radioresistance. Tumor xenograft experiments confirm that miR-620 increases proliferation and tumor radioresistance in vivo. Regulation of PGE2 levels via targeting of HPGD by miR-620 is an innovative manner by which a microRNA can induce radiation resistance.

miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD).

Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact. This elevated temperature is used as the hybridization temperature. Practical implementation of this apparently trivial approach in DQAMmiR has two major challenges. First, melting temperatures of all mismatched hybrids should be similar to each other and should not reach the melting temperature of any of the matched hybrids. Second, the elevated hybridization temperature should not deteriorate CE separation of the hybrids from the excess probes and the hybrids from each other. The second problem is further complicated by the reliance of separation in DQAMmiR on single-strand DNA binding protein (SSB) whose native structure and binding properties may be drastically affected by the elevated temperature. These problems were solved by two approaches. First, locked nucleic acid (LNA) bases were incorporated into the probes to normalize the melting temperatures of all target miRNA hybrids allowing for a single hybridization temperature; binding of SSB was not affected by LNA bases. Second, a dual-temperature CE was developed in which separation started with a high capillary temperature required for proper hybridization and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids from excess probes and the hybrids from each other. The developed approach was sufficiently robust to allow its integration with sample preconcentration by isotachophoresis to achieve a limit of detection below 10 pM.

Stanley K. Liu

Department of Radiation Oncology,
Department of Medical Biophysics

University of Toronto

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Stanley K. Liu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Radiation Oncology,Department of Medical Biophysics

University of Toronto

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Department of Radiation Oncology,
Department of Medical Biophysics