Contributed equally
Combined androgen deprivation therapy (ADT) and radiation therapy (RT) is the standard of care treatment for non-metastatic prostate cancer (NMPC). Despite the efficacy, treatment-related symptoms including fatigue greatly reduce the quality of life of cancer patients. The goal of the study is to examine the influence of combined ADT/RT on fatigue and understand its underlying mechanisms. A total of 64 participants with NMPC were enrolled. Fatigue was assessed using the Functional Assessment of Cancer Therapy-Fatigue. Mitochondrial function parameters were measured as oxygen consumption from peripheral blood mononuclear cells (PBMCs) extracted from participants' whole blood. An ADT/RT-induced fatigue mouse model was developed, with fatigue measured as a reduction in voluntary wheel-running activity (VWRA) in 54 mice. Mitochondrial function was assessed in the ADT/RT mouse brains using western blot analysis of glucose transporter 4 (GLUT4) and transcription factor A, mitochondrial (TFAM). The results demonstrated that fatigue in the ADT group was exacerbated during RT compared with the non-ADT group. This effect was specific to fatigue, as depressive symptoms were unaffected. PBMCs of fatigued subjects exhibited decreased ATP coupling efficiency compared to non-fatigued subjects, indicative of mitochondrial dysfunction. The ADT/RT mice demonstrated the synergistic effect of ADT and RT in decreasing VWRA. Brain tissues of ADT/RT mice exhibited decreased levels of GLUT4 and TFAM suggesting that impaired neuronal metabolic homeostasis may contribute to fatigue pathogenesis. In conclusion, these findings suggest that fatigue induced by ADT/RT may be attributable to mitochondrial dysfunction both peripherally and in the central nervous system (CNS). The synergistic effect of ADT/RT is behaviorally reproducible in a mouse model and its mechanism may be related to bioenergetics in the CNS.
Androgen-deprivation therapy (ADT) in combination with radiation therapy (RT) is the standard of care for locally advanced prostate cancer (
A number of large, randomized clinical trials in previous years have demonstrated improved long-term survival using combined ADT and RT compared to RT or ADT alone (
Despite these benefits, treatment with ADT can result in a multitude of iatrogenic conditions including increased risk for diabetes, cardiovascular diseases, sexual health dysfunction, depression, cognitive and mood dysfunction (
Various mechanisms have been hypothesized for the etiology of treatment-related fatigue; however, the exact underlying mechanism especially related to combined therapies remains unknown (
Given the prominent use of combined ADT and RT for localized prostate cancer, there is an increasing need to better understand adverse effects of the combined treatment. The goal of the current study is to explore the influence of ADT on fatigue progression and mitochondrial function during RT for localized prostate cancer. To examine mitochondrial function in human blood samples, a method previously published by the present group was used (
The present study (NCT00852111) was approved by the Institutional Review Board of the National Institutes of Health (NIH). All participants enrolled in this study were male, ≥18 years of age, diagnosed with non-metastatic prostate cancer with or without prior prostatectomy and scheduled to receive external beam radiation therapy (EBRT). Patient characteristics are presented in
Clinical and demographic data were obtained from chart review. Fatigue was measured using the frequently-used 13-item Functional Assessment of Cancer Therapy-Fatigue (FACT-F), a validated, reliable, stand-alone measure of fatigue in cancer therapy (questionnaire items and scoring method can be found at
Depressive symptoms were measured using the validated, 24-item Hamilton Depression Rating Scale (HAM-D) (
Blood cell counts were measured using standard procedures adapted by the Department of Laboratory Medicine, NIH. Anemia was defined as hemoglobin level <13 g/dl for men based on the World Health Organization guidelines (
Mitochondrial function parameters were measured as previously described (
This study was approved by the National Heart Lung and Blood Institute (NHLBI) Animal Care and Use Committee of the NIH. All investigators working with animals were properly trained by the NIH Office of Animal Care and Use and the NHLBI Murine Phenotyping Core. All interactions with animals in this study were in compliance with The Guide for the Care and Use of Laboratory Animals (
A total of 60 male C57Bl/6 mice were ordered from Charles River Laboratories and were 6-9 weeks old and 15-20 g at the beginning of each study. Mice were given
Mice were randomly split into two groups. Implant surgery took place over a two-day period, with half the animals in each group receiving implants on each of the two days. The 'ADT' group had a flutamide pellet (SA-152 5 mg/pellet, 60 Day Release; Innovative Research of America) surgically implanted subcutaneously on their backs. Mice were anesthetized using isoflurane anesthesia (3-5% isoflurane was used to induce anesthesia and 1-3% was used to maintain anesthesia) and placed atop a heating pad. A stab incision was made at the nape of the neck, the incision site was swabbed with disinfectant and a pellet was inserted subcutaneously after all disinfectant had completely dried. The wound was closed with tissue glue and a skin staple and mice were returned to their cages. The control (CTL) group underwent the same surgery as the ADT group, but no pellet was implanted. Skin staples were removed one week after surgery. When removing the staples, implanted pellets could be felt when touching the animal's back and could often be seen as a small bump underneath the skin; mice were removed from the study if they were in the ADT group but a pellet was not detected in this way.
Mice in each of the ADT and CTL groups were randomly subdivided into irradiated (Irrad) or sham (Sham) groups, resulting in four groups: Irrad-ADT, Irrad-CTL, Sham-ADT, and Sham-CTL. The procedure is described in detail by Wolff
Mice were housed in cages with a running wheel (Lafayette Instrument Neuroscience), which recorded wheel rotation in one-min intervals. After at least one week acclimating to the animal facility in standard plastic home cages, mice were housed in running wheel cages for at least 2 weeks before surgery, then for ~2 weeks prior to irradiation, then for 10-12 days after irradiation and before euthanasia. Mice were removed from their running wheel cages during two days of surgery and the three days of irradiation. Mice that did not consistently use the running wheels were removed from the study. Data were collected by the Lafayette Running Wheel software (Lafayette Instrument Neuroscience; version 11.16). The VWRA outcome measure was time spent using the wheel.
Mice were anesthetized with ketamine/xylazine (120/20 mg/kg) and decapitated immediately after exsanguination. The skull was opened and the brain was removed. The whole brain was placed in a petri dish with ice-cold PBS. Cortical tissues were extracted by removing the brain stem, cerebellum, midbrains and were immediately placed into ceramic bead tubes on ice. Modified radioimmunoprecipitation assay buffer (50 mm Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate and 150 mm NaCl) supplemented with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) was added to the samples for cell lysis using a bead-mill homogenizer (Thermo Fisher Scientific, Inc.). Lysates were centrifuged at 14,000 × g 15 min at 4°C. Supernatants were retained as the soluble lysate, boiled at 100°C for 10 min in the presence of Laemmli Sample Buffer (Bio-Rad Laboratories, Inc.) supplemented with dithiothreitol. All protein samples (30
Descriptive analyses were used to describe demographic characteristics of the sample. All data were expressed as the mean ± standard error of mean. To assess changes in clinical variables over time, a two-way repeated-measures analysis of variance (ANOVA) was employed. The presence or absence of ADT was defined as between-subject factors, while the within-subject factor was defined by study time points. The sphericity assumption was tested with Mauchly's test and fatigue differences at each time point were determined by non-directional Student's t-test with Bonferroni corrections for multiple comparisons. One-way ANOVA was used to determine significant differences in comparisons involving more than 2 groups. Post hoc non-directional Student's t-test with Bonferroni correction was used for between group comparisons. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed with SPSS statistics software version 23 (IBM Corps.). Behavioral data analysis was conducted in python using the statsmodels library for ANOVA and the scipy and pandas modules for all other tests. Two-way ANOVAs were used to test for main effects, Shapiro-Wilk tests were used for evaluating normality and post-hoc t-tests were used for pairwise comparisons with Holm-Sidak corrections for multiple comparisons. Pearson correlation coefficient analysis was performed to analyze correlations between variables. All experiments were performed with an n>8 and repeated three times. In all plots, error bars represent the standard error of the mean. Threshold α values were 0.05 for all tests.
The clinical sample was predominantly Caucasian (62.50%) with an average age of 65.23±7.41 years and a body mass index (BMI) of 30.3±4.96 (
Subjects receiving ADT experienced significantly worse fatigue at the midpoint (P=0.00005) as well as completion of EBRT (P=0.0008), compared to subjects without ADT (
A 3-point longitudinal change in FACT-F score has been found to represent clinically important worsening of fatigue (
Hemoglobin levels decreased significantly over time in subjects treated with ADT (
Despite the similarity of depressive symptoms with fatigue in terms of the subjective experience and underlying physiological mechanism (
A schematic illustration of the mitochondrial function assay and coupling efficiency calculation is shown in
Due to limitations of sample availability (assessing brain mitochondrial function was not possible in human subjects), the present study decided to use a previously published mouse model of fatigue (
A total of two weeks after initiating ADT, mice received three days of irradiation targeted to the pelvic region. Previous studies indicate that lower-abdominal irradiation causes fatigue-like behavior that lasts about six days (
Hemoglobin levels were measured 9 days after irradiation to test whether the mice showed anemia-like conditions (
To investigate whether brain mitochondria are affected by the ADT and peripheral irradiation procedures, mice were euthanized 10-12 days after irradiation and whole brain protein lysates were collected and immunoblotted for glucose transporter GLUT4 and mitochondrial transcription factor TFAM. Representative bands from each group are shown in
Whether these levels of brain mitochondrial proteins correlated with either the anemia measure (hemoglobin) or the fatigue measure (VWRA) was looked at next. The present study found that hemoglobin levels were positively correlated with both GLUT4 (
The current study describes a novel finding that RT exacerbated the effects of ADT on fatigue in patients with non-metastatic localized prostate cancer. The present study showed that the combination of ADT and RT caused worsened fatigue that was associated with anemia and mitochondrial dysfunction. This combined effect of ADT and radiotherapy appeared to be specific to fatigue, as depressive symptoms were unaffected. As anemia can lead to hypoxia in brain tissues (
Although it has been shown that ADT and RT may independently cause anemia and fatigue, adverse effects of combined therapy have been less explored (
Mitochondria coupling efficiency represents the proportion of O2 consumed to drive ATP synthesis and is calculated as the fraction of basal mitochondrial respiration rate used for ATP production (
The present study showed that irradiation in the ADT/RT-induced fatigue mouse model resulted in the downregulation of GLUT4, a glucose transporter that is preferentially expressed in brain regions involved in the control of motor activity and is essential for maintaining neuronal metabolic homeostasis (
A limitation in the present study is the small clinical sample size, particularly in the non-ADT group. Future studies could validate findings from this study in a larger sample size. The current study did not detect any significant difference between fatigue scores and anemia status between subjects with or without ADT at baseline, at which point subjects had already received ADT but not RT. Future studies will examine the fatigue status in subjects with metastatic cancer that receive ADT monotherapy. PBMCs functional tests were used to assess the role of mitochondrial function in cancer-related fatigue. Even though as a heterogenous population, PBMCs serve as a useful tool for assessing global mitochondrial dysfunction. Future studies will further examine bioenergetics in each cell subpopulation. Additional efforts will also be made to establish baseline mitochondrial function test measurements in healthy individuals. In the current clinical study, the sample type available was a limitation. Blood collection is well tolerated by patients even at multiple time points. Future studies will examine markers in cerebral-spinal fluid to get a better picture of the CNS aspect of fatigue.
The ADT/RT mouse model mimicked clinical symptoms of human subjects going through the combined therapy. However, fatigue measured by VWRA lasted for days in mice, but lasts for months up to years in human subjects. Continued efforts are made to develop a mouse model with the more physiologically relevant 'persistent' fatigue. One limitation in the ADT model is that patients received ADT for on average 76 days prior to EBRT initiation, whereas mice in the animal model of fatigue received 12 days of ADT prior to irradiation. While it is difficult to determine what the 'equivalent' ADT treatment time would be for a mouse (
Another limitation in the ADT model is that patients received both an anti-androgen (Bicalutamide) and an LH-RH agonist, whereas mice received only an anti-androgen (flutamide). The flutamide-ADT mouse model was developed based on other studies showing that flutamide treatment reduced tumor incidence compared to a placebo in animal models (
In conclusion, the present study demonstrated that ADT exacerbated fatigue in subjects receiving RT. Anemia appeared to be a significant contributor of fatigue during EBRT, but not prior to treatment initiation, suggesting worsened hematologic toxicity with a combination of ADT and RT. Furthermore, self-reported fatigue was associated with mitochondrial dysfunction suggesting a physiological basis for the subjective phenomenon. Additionally, ADT/RT induced fatigue-like behaviors in a mouse model and mimicked clinical observations of human subjects receiving concomitant ADT and RT. Using the mouse model, alterations in markers of mitochondrial dysfunction and brain bioenergetics were found in mice receiving irradiation, suggesting the contribution of non-mitochondrial factors in the synergistic effect on fatigue exerted by combined ADT and RT. Results from this study will inform patients and clinicians of mechanisms related to the combined treatment and thus manage these symptoms more effectively.
androgen deprivation therapy
radiation therapy
non-metastatic prostate cancer
peripheral blood mononuclear cells
voluntary wheel-running activity
glucose transporter 4
transcription factor A mitochondrial
central nervous system
external beam radiation therapy
Gray
gonadotropin-releasing hormone agonist
Functional Assessment of Cancer Therapy-Fatigue
Hamilton Depression Rating Scale
control
irradiated
oxygen consumption rate
analysis of variance
The authors would like to thank Ms. Diane Cooper (National Institutes of Health Library Writing Center, Bethesda, MD, USA) for assistance with manuscript editing.
The present study is fully supported by the Division of Intramural Research of the National Institute of Nursing Research of the National Institutes of Health, Bethesda, MD, USA (grant no. ZIA NR000020-06).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
LRF, JMR, JL and LNS designed, collected, analyzed and interpreted patient data regarding the clinical aspect and mitochondrial function. BSW, SA and SR performed the mouse model experiments and analyzed the data. All authors contributed to writing the manuscript and approved the final version. All authors were involved in drafting the manuscript. All authors have agreed to be accountable for all aspects of the work.
The study was approved by the Institutional Review Board of the National Institutes of Health (approved protocol no. NCT00852111). Written informed consent was obtained prior to study participation. This study was approved by the National Heart Lung and Blood Institute Animal Care and Use Committee of the NIH.
Not applicable.
The authors declare that they have no competing interests.
ADT contributes to anemia during RT in men with non-metastatic prostate cancer. (A) ADT significantly affected fatigue development over time (
Mitochondrial dysfunction contributes to self-reported fatigue in men with non-metastatic prostate cancer. (A) Schematic diagram of OCR profile of the extracellular flux mito stress test. (B) ADT treatment during radiation therapy did not affect mitochondrial coupling efficiency (+ADT vs. −ADT P=0.633). (C) Non-fatigued subjects exhibited higher mitochondrial coupling efficiency compared to fatigued subjects (P=0.017). *P<0.05 vs. fatigued. OCR, oxygen consumption rate; ADT, androgen deprivation therapy.
ADT/radiation therapy-induced fatigue in a mouse model. (A) VWRA before and after implanting flutamide pellets on days 0 and 1. Data are normalized to the mean daily VWRA over the four days before surgery (n=12-15 mice per group). (B) There was no difference between groups in total activity over the 12 days post-surgery (
Brain mitochondrial effects in the mouse model of ADT/radiation therapy-induced fatigue. (A) Western blot analysis of GLUT4 and TFAM. Blots indicate representative bands. (B) Densitometry data were quantified (n=8 mouse samples per group for a total of 32 samples) and shown as bar graphs. Irradiation had a significant effect on GLUT4 levels in the mouse brain (
Demographics and clinical characteristics of sample population.
Characteristics | Total (n=64) | +ADT all time points (n=27) | +ADT during EBRT (n=20) | No ADT (n=17) |
---|---|---|---|---|
Age, years | 65.41±7.78 | 66.37±8.10 | 65.25±7.06 | 64.06±8.31 |
BMI, kg/m2 | 30.26±4.91 | 30.15±4.51 | 31.20±5.86 | 29.32±4.34 |
Ethnicity, % | ||||
Asian | 6.25 | 7.41 | 0.00 | 11.76 |
Black | 26.56 | 25.93 | 30.00 | 23.53 |
Hispanic | 3.13 | 0.00 | 0.00 | 11.76 |
White | 64.06 | 66.67 | 70.00 | 11.76 |
Education, % | ||||
Did not complete high school | 6.25 | 3.70 | 3.70 | 0.00 |
High school grad/GED | 10.94 | 11.11 | 10.00 | 11.76 |
Associate degree/some college | 7.81 | 11.11 | 5.00 | 5.88 |
Bachelor's degree | 46.88 | 48.15 | 40.00 | 52.94 |
Advanced degree | 26.56 | 22.22 | 30.00 | 29.41 |
No answer | 1.56 | 3.70 | 0.00 | 0.00 |
T stage, % | ||||
T1c | 29.69 | 18.52 | 45.00 | 29.41 |
T2 | 1.56 | 0.00 | 0.00 | 5.88 |
T2a | 29.69 | 29.63 | 35.00 | 23.53 |
T2b | 6.25 | 7.41 | 0.00 | 11.76 |
T2c | 10.94 | 11.11 | 5.00 | 17.65 |
T3 | 21.88 | 33.33 | 15.00 | 11.76 |
Gleason score, % | ||||
3+3=6 | 6.25 | 3.70 | 0.00 | 17.65 |
3+4=7 | 26.56 | 0.00 | 50.00 | 41.18 |
3+5=8 | 1.56 | 3.70 | 0.00 | 0.00 |
4+3=7 | 14.06 | 14.81 | 10.00 | 17.65 |
4+4=8 | 32.81 | 44.44 | 25.00 | 23.53 |
4+5=9 | 14.06 | 29.63 | 5.000 | 0.00 |
5+4=9 | 4.69 | 3.70 | 10.00 | 0.00 |
CBC, 1,000/ |
||||
WBC | 6.52±1.74 | 6.03±1.68 | 6.77±1.92 | 7.01±1.47 |
RBC | 4.59±0.42 | 4.46±0.43 | 4.54±0.32 | 4.83±0.42 |
Neutrophils absolute | 3.86±1.42 | 3.54±1.49 | 3.96±1.36 | 4.27±1.32 |
Lymphocytes absolute | 1.91±0.63 | 1.79±0.60 | 2.06±0.79 | 1.92±0.45 |
Monocytes absolute | 0.54±0.23 | 0.46±0.13 | 0.61±0.34 | 0.57±0.16 |
Eosinophils absolute | 0.19±0.15 | 0.19±0.18 | 0.19±0.11 | 0.19±0.16 |
Basophils absolute | 0.03±0.02 | 0.03±0.02 | 0.04±0.02 | 0.03±0.01 |
PSA baseline, ng/ml | 5.99±13.59 | 4.32±6.64 | 9.77±22.76 | 4.19±3.83 |
PSA completion of EBRT, ng/ml | 0.41±0.92 | 0.07±0.10 | 0.07±0.07 | 1.35±1.43 |
Data are presented as mean ± standard deviation or percentages. BMI, body mass index; EBRT, external beam radiation therapy; ADT, androgen deprivation therapy; CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; PSA, prostate specific antigen.