Strategies to improve radiocurability for prostate cancer: radiosensitizers and magnetic resonance imaging

External beam radiotherapy is one of the standard radical treatment options for men with clinically localized prostate cancer, being still the most common non-skin malignancy as well as the third cause of cancer-related mortality in men in developed countries. Despite technical advances in the field of radiation delivery and treatment planning, the rate of biochemical and clinical failure for these patients is still significant. New strategies to increase the therapeutic index, determined by the balance between tumor control probability and normal tissue complication probability, are therefore warranted. Since local tumor control after curative radiotherapy is influenced by several factors, such as the intrinsic radiosensitivity of tumor cells as well as adequate tumor targeting, two important means to improve the radiocurability for prostate cancer were studied in this dissertation: radiosensitizers that could attenuate the radioresistance of tumor cells and MR imaging techniques which could improve the accuracy of intra-prostatic tumor detection and localization, thereby allowing more adequate tumor targeting. In the first part of this work we explored the combination of radiotherapy with several molecular targeted agents (MTA) that could selectively enhance the lethal effects of ionizing radiation (i.e. radiosensitizers) in human prostate cancer cells. In order to achieve complete tumor control, all clonogenic cells capable of tumor regrowth should be eradicated, which depends on the sensitivity of these cells to killing by ionizing radiation. The combination of MTA with radiotherapy aims to modify the cellular response to radiotherapy by interfering with any cancer-related process which induces or supports radioresistance. Moreover, normal tissue toxicity should be minimal, since the molecular target proteins are often differentially expressed between tumor and normal cells. More specifically, the in vitro effects of three MTA on the radiosensitivity of both androgen-responsive (22Rv1) and androgen-irresponsive (PC3, DU145) human prostate cancer cells were investigated. Each of these agents acts on a different process relevant to the response to ionizing radiation: energy metabolism, mitogenic signaling and cell adhesion. The effect of every agent on cell proliferation and cell survival was determined using a bromodeoxyuridine (BrdU) assay and a sulforhodamine B (SRB) assay, respectively. Trypan blue exclusion assays were also performed in this context. The radiosensitizing capacity was tested using colony formation assays. Western blot analysis and immunoprecipitation were used to detect the expression level as well as the activation status of relevant proteins. Experiments to unravel the exact radiosensitization mechanism of these MTA included detection of apoptosis, determination of cell cycle distribution, reactive oxygen species production, double strand break (DSB) repair, etc. Transfectionexperiments with siRNA or plasmid DNA were used to determine the relative importance of key molecules in the radiosensitization effect of the agents being investigated. The first drug that was tested, was the low energy-mimetic and AMPK agonist AICAR that can affect the malignant phenotype by diminishing cancer cell anabolism. By doing so, we hypothesized that it could also enhance the cellular response to radiotherapy, which would effectively increase the dose-related effectiveness of AICAR. Incubation of PC3 human prostate cancer cells with AICAR inhibited cell proliferation and decreased viability in a dose- and time-dependent manner and sensitized them to ionizing radiation. Radiosensitization, however, did not correspond to the time- and dose-dependent activation pattern of AMPK. The AMPK independence of radiosensitization by AICAR was confirmed by the finding that siRNA knock down of AMPK also radiosensitized cells and synergized with AICAR. Drug agonist and antagonists showed that imbalanced deoxynucleotide pools due to ZMP accumulation after AICAR administration were involved in the radiosensitizing mechanism. Our findings on the favorable interaction between low doses of AICAR and ionizing radiation open new perspectives for the clinical use of this compound at relatively low doses that may minimize potential toxicities.Second, we explored the effect of the IGF-1R small molecule tyrosine kinase inhibitor NVP-AEW541 (Novartis) on the intrinsic radioresistance of prostate cancer cells. Although the important role of IGF-1R signaling in malignant transformation, progression and therapy resistance is well established, studies investigating the combination of IGF-1R targeted agents in combination with radiotherapy are relatively limited. Here, we showed that NVP-AEW541 inhibited cell proliferation and decreased cell viability in a time-and dose-dependent manner in all three cell lines. Radiosensitization, however, was observed in the PTEN wild-type cell lines DU145 and 22Rv1, but not in the PTEN-deficient PC3 cell line. NVP-AEW541-induced radiosensitization coincided with downregulation of phospho-Akt levels and high levels of residual DSB. Transfection of the PTEN wild-type cells DU145 and 22Rv1 with a constitutively active or a kinase-death form of Akt, attenuated or enhanced the radiosensitizing effect of NVP-AEW541, respectively. This demonstrated the importance of the PTEN status in the NVP-AEW541 induced radiosensitization effect. Therefore, we believe that proper patient selection based on the PTEN status of the tumor will be critical in order to achieve optimal results in clinical trials in which the combination of radiotherapy and this IGF-1R inhibitor is being explored.At last, we tested the capacity of the small molecule tyrosine kinase inhibitor Nilotinib, being used as an inhibitor of the cell adhesion receptor DDR1</&gt;, to interfere with the process of cell adhesion-mediated radioresistance. We showed that DDR1 was activated in all cell lines. Incubation with nilotinib inhibited cell proliferation and decreased cell viability in a dose- and time-dependent manner. Cells grown on collagen I-coated flasks however were more resistant to nilotinib. Nilotinib treatment increased the radiosensitivity of the DU145 cells, but this effect was attenuated by collagen I stimulation. These results were in line with the expression profile of phospho-DDR1 after treatment with nilotinib and/or ionizing radiation. Similar effects on radiosensitivity were observed after siRNA targeting of DDR1. Our data thus demonstrated that DDR1 inhibition can induce radiosensitization in some prostate cancer cell lines, but collagen I can abolish this effect. This stresses the importance of the tumor cell microenvironment in mediating cellular radiosensitivity after targeted treatment. The second aim of this project was to evaluate different functional MRI techniques that could improve the detection and localization accuracy of tumor lesions within the prostate gland. While conventional T2w MRI mainly provides anatomical images, functional MRI modalities allow the spatial assessment of different physiological tissue characteristics, like perfusion (DCE MRI) or diffusion (DW MRI). The combination of both anatomical and physiological information should enable more accurate detection, localization and characterization of intra-prostatic tumor nodules, which is an indispensable prerequisite if one aims to escalate the radiation dose only to the intra-prostatic tumour regions. Such approach is an alternative to dose-escalation to the entire prostate, which is limited due to normal tissue toxicity. In the first study, 53 patients with biopsy-proven prostate cancer were examined by DCE-MRI at 1.5T. Cancerous and benign regions were delineated based on the histopathology of whole-mount sections and the accuracy of different semi-quantitative DCE MRI parameters to discriminate between cancerous and benign prostatic tissue was assessed. Within individual patients, a consistently higher Cpeak (maximal contrast enhancement) and faster Wash-in (speed of contrast uptake) were present in cancerous compared to benign prostatic tissue. Both the TTP (time to peak) and the Wash-out (contrast clearance rate) occurred more rapidly in tumor tissue than in normal prostatic tissue. Despite a considerable inter-patient overlap of parameter values between tumor and normal prostatic tissue, area under the ROC curve (AUC) analysis demonstrated that for the Wash-in a good sensitivity and specificity could be reached (AUC 0.82). Combination of the Wash-in and the Wash-out proved to be even more accurate to discriminate cancerous from benign prostatic tissue (AUC 0.87). These data showed that semi-quantitative parameters can be used for the identification of intra-prostatictumor nodules, with the Wash-in being the most accurate discriminator between malignant and benign prostatic tissue.In the second study in which 49 patients were enrolled, we prospectively evaluated the sensitivity and specificity of three MRI techniques or any combination of those to detect and localize intra-prostatic tumor nodules, i.e. T2w MRI, DCE MRI and DW MRI. This study demonstrated that the combination of T2W, DCE and DW MRI significantly improved prostate cancer detection and localization, irrespective of the reader experience. DW MRI had the highest sensitivity, but this could significantly be improved by the addition of T2w and DCE MRI information. Moreover, our results indicated that tumor volume could most accurately be assessed by means of DW MRI. Multi-modality MR imaging has thus the potential to guide focal escalation radiotherapy for prostate cancer. In conclusion, this work showed that both radiosensitization and multi-modality MR imaging could contribute to an improved radiocurability for patients with localized prostate cancer.Table of Contents Dankwoord 5 List of abbreviations 7 Table of Contents 9 1. General introduction 13 1.1. The prostate gland 13 1.2. Prostate cancer 14 1.3. Treatment options for clinically localized prostate cancer 15 1.4. Strategies to improve the response to radiotherapy 17 1.4.1. Therapeutic index for radiotherapy 17 1.4.2. Technical advances in the field of external beam radiotherapy 17 1.4.3. Strategies to improve radiocurability 19 1.5. Radiosensitizers 19 1.5.1. Molecular targeted agents in combination with radiotherapy 19 1.5.2. Energy metabolism 22 1.5.3. Mitogenic IGF-1R signaling 24 1.5.4. Cell adhesion 25 1.6. Multi-modality MR imaging to guide radiation oncology 27 1.6.1. Imaging in radiation oncology 27 1.6.2. MR imaging for prostate cancer 28 2. Objectives 39 2.1. General aim 39 2.2. Specific aims 39 2.2.1. Radiosensitizers 39 2.2.2. Multi-modality MR imaging to guide radiation oncology 40 3. 5-aminoimidazole-4-carboxamide riboside (AICAR) enhances the effect of ionizing radiation in PC3 prostate cancer cells 45 4. Insulin-like growth factor – type 1 receptor inhibitor NVP-AEW541 enhances radiosensitivity of PTEN wild-type but not PTEN-deficient human prostate cancer cells 71 5. The radiosensitizing effect of DDR1 inhibition in human prostate cancer cells is counteracted by collagen I 93 6. Evaluation of Semi-Quantitative Dynamic Contrast-Enhanced MRI Parameters for Prostate Cancer in Correlation to Whole-mount Histopathology 113 7. Multi-modality MR imaging for prostate cancer detection and localization in correlation to whole-mount histopathology 131 8. General discussion, conclusions and future perspectives 151 8.1. General discussion 151 8.1.1. Radiosensitizers 151 8.1.2. Multi-modality MR imaging to guide radiation oncology 155 8.2. Conclusions and future perspectives 158 Summary 173 Samenvatting 179 Curriculum vitae 185 Bibliography 187nrpages: 189status: publishe