PDF - 1331K, Western Blot Analysis.

Subcellular localization of AKT and mTOR after radiation. A, Western blots of mTOR, phospho-AKT S473 and AKT 2 h after 3 fractions of 2 Gy in 3D-grown PC3 cells after CRISPR/Cas9-mediated depletion of mTOR (crMTOR) using different targeting sequences. A non-targeting sequence (NTS) was used as control. β-actin expression was evaluated to ensure equal sample loading. B, Colony formation data of 3D colony formation assays. Cells were irradiated either with 6 Gy single dose or with 3 fractions of 2 Gy (2 Gy per day). Results show mean {plus minus} SD (n = 3, ** P < 0.01, Student''s t test). (See also Figure 6)

Oligonucleotides used for CRISPR/Cas9 gene editing

PDF - 42K, miRNAs implicated in several cardiovascular events were also differentially expressed following fractionated irradiation in HCAEC.

Subcellular localization of AKT and mTOR after radiation. Immunofluorescence staining of AKT and mTOR in irradiated and unirradiated DU145 and PC3 cells. Nuclear staining was performed with DAPI.

PDF - 56K, Microarray data depicting gene expression fold changes respective to control group in genes from stress response category following SD and MF radiation exposure at 6h and 24h.

S1. Growth curves of PC3 xenograft tumors after radiation. S2. AIIB2 binds to prostate cancer cells. S3. Integrin expression is elevated after radiation in vitro and in vivo. S4. Cell cycle distribution is unchanged in PC3 long-term survivors (in vitro).

PDF - 64K, Supplementary Materials and Methods

PDF - 34K, Changes in selected differentially expressed genes identified by microarray analysis were confirmed by real-time RT-PCR.

Radiation schedule for the cell culture and xenograft experiments. Human prostate cancer cells were either plated or injected subcutaneously into the flanks of the right hind legs of athymic nude mice. After 24 h (in vitro) or tumors reached a tumor size of 5 mm x 5 mm (in vivo), tumors were irradiated with different fractionation regimens including a single dose of 10 Gy at day 5, 10 fractions of 1 Gy (2 fractions per day), or 5 fractions of 2 Gy (1 fraction per day). Unirradiated cells or tumors were used as control.

Metabolic data from PC3 cells 6h, 24h, and 48h after SD or MF radiation. Samples were sent to Metabolon for processing. Results indicate decrease in glycolysis intermediates in SD sample at 6h. No significant changes to TCA cycle intermediates. No significant changes to ATP at any time points measured.

PDF - 22K, Genes differentially expressed (> 2-fold change, P < 0.05) in response to SD and MF irradiations were classified into functional categories by gene ontology classification. Enrichment of individual functional gene categories was determined by hypergeometric distribution of P values obtained from comparison of the number of the number of genes differentially expressed to the number of annotated genes in each category.

PDF - 61K, Legends for Supplementary Tables

PDF - 46K, Microarray data depicting gene expression fold changes respective to control group in genes from immune response category following SD and MF radiation exposure at 6h and 24h.

PDF - 29K, Differentially expressed Target mRNAs and regulatory miRNAs (showing inverse correlation) from cardiovascular pathways in HCAEC at 24h following SD and MF irradiation were identified by Target Filter Analysis program.

Gene expression of mTOR/AKT related genes after single dose and multifractionated radiation. DU145 human prostate carcinoma cells were irradiated with 10 Gy single dose (SD) or 5 fractions of 2 Gy or 10 fractions of 1 Gy (2 Gy a day) as multifractionated (MF) radiotherapy. At indicated time points after radiation, cells were lysed and RNA was isolated.

Densitometric analysis of basal protein expression and phosphorylation in DU145 and PC3 cells. Western blots of 3D-grown prostate cancer cell lines were evaluated with ImageJ. Phospho-protein expression was normalized to total protein expression. Results show mean {plus minus} SD (n = 3) (See also Figure 3A).

The efficacy of molecular targeted therapy depends on expression and enzymatic activity of the target molecules. As radiotherapy modulates gene expression and protein phosphorylation dependent on dose and fractionation, we analyzed the long-term effects of irradiation on the post-radiation efficacy of molecular targeted drugs. We irradiated prostate cancer cells either with a single dose (SD) of 10 Gy x-ray or a multifractionated (MF) regimen with 10 fractions of 1 Gy. Whole genome arrays and reverse phase protein microarrays were used to determine gene expression and protein phosphorylation. Additionally, we evaluated radiation-induced pathway activation with the Ingenuity Pathway Analysis software. To measure cell survival and sensitivity to clinically used molecular targeted drugs, we performed colony formation assays. We found increased activation of several pathways regulating important cell functions such as cell migration and cell survival at 24 h after MF irradiation or at 2 months after SD irradiation. Further, cells which survived a SD of 10 Gy showed a long-term upregulation and increased activity of multiple molecular targets including AKT, IGF-1R, VEGFR2, or MET, while HDAC expression was decreased. In line with this, 10 Gy SD cells were more sensitive to target inhibition with Capivasertib or Ipatasertib (AKTi), BMS-754807 (IGF-1Ri), or Foretinib (VEGFR2/METi), but less sensitive to Panobinostat or Vorinostat (HDACi). In summary, understanding the molecular short- and long-term changes after irradiation can aid in optimizing the efficacy of multimodal radiation oncology in combination with post-irradiation molecularly-targeted drug treatment and improving the outcome of prostate cancer patients.

Abstract Radiotherapy is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This systemic therapy can be utilized before radiotherapy as an adjuvant or induction treatment, during radiotherapy as a radiation “sensitizer,” or following radiotherapy as a part of combined modality therapy. As part of a unique concept of using radiation as “focused biology,” we investigated how tumors and normal tissues adapt to clinically relevant multifraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 × 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of radiotherapy. In addition, we identified inducible changes in integrin expression 2 months following cessation of radiotherapy that differ between MF (1 Gy × 10) and SD (10 Gy) that remain targetable compared with preradiotherapy. Adaptation is reflected across different “omics” studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multimodality cancer treatment.

Abstract Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals. Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice. Methods: Mice were whole body irradiated with X-rays (2 Gy–15 Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature. Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates. Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.