Radiotherapy alters expression of molecular targets in prostate cancer in a fractionation- and time-dependent manner

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.


Scientific Reports
| (2022) 12:3500 | https://doi.org/10.1038/s41598-022-07394-y www.nature.com/scientificreports/ radiation therapy, there is a high need for additional treatment options, since the possibilities to re-irradiate are often limited. In recent years, targeted therapy was implemented into multimodal cancer treatment regimens generally used before or simultaneously with radiation 7 . The underlying hypothesis is that while conventional chemotherapy impacts proliferation and survival of both malignant and normal tissue, targeted therapeutics aim to exploit the abnormal molecular signaling often found in cancer cells and to target and kill tumors more specifically [7][8][9] .
Targeted therapy is often directed against kinases overexpressed in cancer driving survival and proliferation such as receptor tyrosine kinases (RTK) and associated molecules. Pre-clinical studies show that inhibition of the epidermal growth factor receptor (EGFR) reduces tumor growth and results in radiosensitization of different cancer types [10][11][12][13][14][15] . Similar to EGFR, insulin-like growth factor type 1 receptor (IGF-1R) and platelet-derived growth factor receptor (PDGFR) also play a major role in tumor development and progression 16,17 . Therefore, it is not surprising that inhibitors of RTKs were among the first approved targeted therapeutics in radiation oncology 18,19 . Through activation by their ligands, growth factor receptors control a network of downstream signaling molecules. One central mediator is the serine kinase AKT which is linked to DNA repair, apoptosis, protein translation, and the cellular radiation response [20][21][22] . In lines with this, the AKT inhibitors Ipatasertib and Capivasertib increase radio-and chemosensitivity in in vivo studies and are currently in clinical trials for targeted cancer therapy [23][24][25] . Further, the MAPK pathway downstream of growth factor receptors also contains several promising targets for molecular inhibitors. Trametinib targeting MEK1/2 has been approved for metastatic melanoma and is under evaluation for solid tumors harboring BRAFV600 mutations 26 .
Some of the known key driver mutations in cancer cells which are targeted by molecular inhibitors have been shown to be maintained throughout the disease course and affect tumor characteristics, radiosensitivity, and the metastatic potential 4 . However, despite several early promising results, some clinical trials found no significant benefit in adding targeted therapy to the standard-of-care cancer treatment or even observed increased normal tissue side effects 27,28 . One potential reason for these negative results is that often pre-therapeutic molecular analyses are used to select molecular therapy for treatment without taking into consideration that irradiation can alter the expression and activity of molecular targets and thereby affect the efficacy of pharmacological inhibitors 22,29 . One research focus of our group is to examine if radiation-induced target expression can be exploited to increase the efficacy of targeted drugs 22,29,30 .
The modulation of target expression can occur at both the genetic and epigenetic levels. A recent study showed that radiotherapy can increase the histone H3 methylation and lead to a stable upregulation of stem cell markers in prostate cancer cells 31 . Histone deacetylase (HDAC) inhibitors such as Pabinostat and Vorinostat affecting histone acetylation and methylation reduce tumor radioresistance and are under clinical evaluation as anti-cancer drugs 32,33 .
Building on our work with radiation-inducible molecular targets 22,29,30 , here, we show that radiotherapy impacts gene expression and protein phosphorylation of molecular targets in a fractionation-and time-dependent manner and that some of these radiation-induced changes persist for several months. Further, a 10 Gy single dose of radiation leads to an upregulation of molecular targets and increases the sensitivity of prostate cancer cells to clinically used molecular inhibitors providing a potential novel approach to using radiation plus drug treatment.

Material and methods
Cell culture. PC3 cells were obtained from the NCI tumor bank and used up to a passage number of 15.
Asynchronously and exponentially growing cells were cultured at 37 °C and 5% CO 2 in RPMI 1640 containing GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Cells were regularly tested for mycoplasma contamination.
Radiation exposure and long-term cultures. Irradiation was performed at room temperature using single doses or multiple fractions of 320 kV X-rays with a dose-rate of 2.3 Gy/min (Precision X-Ray Inc.). Multifractionated radiation was carried out as described before with two times 1 Gy per day (with a 6 h time interval between both radiations) 34 . At 24 h after the final radiation dose or 6 d after the first radiation dose (Supplementary Figure S1), total RNA was extracted for short-term (ST) gene analysis (Fig. 1A). For long-term PC3 cultures, irradiated and unirradiated cells were passaged twice a week and cultured for at least 8 weeks after irradiation before the cells were used for long-term (LT) gene analysis or inhibitor experiments (Fig. 1A, Supplementary Figure S1).

Colony formation assay.
Colony formation assays were performed as previously described 35 . Briefly, cells were trypsinized, counted and seeded in six-well plates. Treatment with inhibitors (Table 1) was started at 24 h after plating. DMSO treated cells were used as control. The inhibitor was removed after 24 h incubation. Cells were cultured for a total of 12 days after plating. After fixation and staining with 0.4% crystal violet, cell clusters with > 50 cells were counted with a stereomicroscope (AmScope). Surviving fractions were calculated as follows: (colony number treated x cells plated untreated/ colony number untreated x cells plated treated).
Whole-genome gene expression analysis. Total RNA was extracted from three replicates using a QIA shredder spin column (Catalog no. 79654, Qiagen) as published previously 34 . The RNeasy mini kit (Qiagen) was used to purify the extracted RNA. Microarray analysis was done using CodeLink Whole Genome Bioarrays representing 55,000 probes. Scanned images from arrays (gridding and feature intensity) were processed with the CodeLink Expression Analysis software (GE Healthcare), and the data generated for each feature on the array Pathway analysis with IPA. Activation of molecular pathways was analyzed using Ingenuity Pathway Analysis (IPA) software (Qiagen) as described before 30 . Differentially expressed genes and corresponding p values were uploaded to the IPA platform. Each gene identifier was then mapped with its corresponding gene object in the Ingenuity Pathway Knowledge Base and an activation z-score was calculated which increased or decreased depending on the known activating or inhibiting function of pathway molecules.
Phospho-proteomic array. For analysis of the phospho-proteome, cells were plated and irradiated with 10 Gy SD, or with 10 fractions of 1 Gy dose per fraction with two fractions per day ( Fig. 1A). At 30 min (ST) and at 2 months (LT) after irradiation, the cells were lysed from plates in T-Per (ThermoFisher Scientific) mixed 1:1 with 2X SDS Tris-Glycine buffer (Invitrogen, Carlsbad, CA) + 2-mercaptoethanol (final concentration = 2.5%). Reverse phase protein microarrays were performed as previously published 22 . In brief, samples were diluted and printed in duplicates onto nitrocellulose slides. HeLa cell lysates (with or without pervanadate) were used as positive and negative controls (Supplementary Figure S2). Microarrays were stained with specific and validated antibodies and analyzed with a biotin-linked signal amplification system (DAKO). The total protein amount of the sample was determined with the SYPRO Ruby stain (ThermoFisher Scientific).

Fractionation impacts gene expression in a time-dependent manner.
To analyze the short-and long-term effects of fractionation on the gene expression of molecular targets, we irradiated PC3 prostate cancer cells with either a single dose of 10 Gy (10 Gy SD) or a multifractionated regimen of ten 1 Gy fractions (10 × 1 Gy MF) and performed whole genome microarrays at 24 h (short-term, ST) and 2 months (long-term, LT) after the final radiation dose (Fig. 1A). Interestingly, while immediately after irradiation (ST), 10 × 1 Gy MF irradiation had a stronger impact on gene expression, we found that 10 Gy SD irradiation resulted in more long-term (LT) expression changes (Fig. 1B). From the 669 genes that were significantly (P < 0.05) upregulated (fold change > 1.5) or downregulated (fold change < 0.66) at 24 h (ST) after radiotherapy compared to the unirradiated controls, only 26 were affected by both 10 Gy SD and 10 × 1 Gy MF irradiation (Fig. 1Ci). At 2 months (LT), the expression of 206 genes was changed by both regimens, 3188 genes only by 10 Gy SD and 190 genes by 10 × 1 Gy MF irradiation (Fig. 1Cii). Further, 10 Gy SD had an impact on 18 genes at both time points (24 h-ST, 2 months-LT) (Fig. 1Ciii) and 10 × 1 Gy MF on 40 genes (Fig. 1Civ). Results showed an overlap of 284 genes which changed shortly after MF irradiation and were also differentially expressed in the long-term SD cells.
Since multifractionated irradiation is delivered over 5 days in contrast to single-dose irradiation which is completed within minutes, we additionally examined the radiation-induced expression of selected genes at 6 days  Figure S1). The majority of genes showed similar expression but some genes for example IGFBP1 were strongly upregulated after 6 days but not after 24 h (Supplementary Figure S1).

SD results in a long-term upregulation of molecular targets. Next, we examined the short-and
long-term effects of irradiation on expression of genes regulating important cellular functions and pro-survival molecular pathways with Ingenuity Pathway Analysis. The pathways and cell functions which were most strongly affected by irradiation are presented in Fig. 2A and B. At 24 h (ST) after 10 × 1 Gy MF irradiation and at 2 months (LT) after 10 Gy SD irradiation, genes involved in cell movement, invasion, proliferation and survival were upregulated, while death-and apoptosis-related signaling was decreased in comparison to the unirradiated controls (0 Gy SD, 0 Gy MF)( Fig. 2A). Accordingly, growth factor-related pathways including IGF1, ErbB, PDGF, PI3K, and MAPK signaling were activated under these conditions (Fig. 2B). However, the expression of the actual target or receptor of these signaling pathways was only upregulated after 10 Gy SD but not after 10 × 1 Gy MF irradiation (Fig. 2C). It is important to note that although there were significant (P < 0.05) gene expression changes (fold change < 0.66 or > 1.5) at 24 h (ST) after 10 Gy SD irradiation compared to the unirradiated control, these did not substantially affect the activity of the selected pathways and cell functions shown in Fig. 2A and B.

Cell functions and pathways can be activated by different gene expression patterns.
To evaluate the similar changes in cell functions and pathways immediately after 10 × 1 Gy MF irradiation and at 2 months (LT) after 10 Gy SD further, we compared the affected genes under each condition. Although both types of irradiation led to a strong activation of cell movement, the genes causing this activation only partially overlapped (Fig. 3A, B). 214 genes were significantly (P < 0.05) altered under both conditions compared to the unirradiated controls, 152 genes were uniquely changed in PC3 cells shortly (ST) after an 10 × 1 Gy MF irradiation and 770 genes showed a differential expression in long-term (LT) 10 Gy SD cells (Fig. 3B). Similar results were obtained for other cell functions and pathways (Figs. 3B, 4).

SD leads to a long-term increase in phosphorylation of the molecular targets AKT and MET.
Since molecular targets are often regulated by protein modifications such as phosphorylation, we next examined the effects of 10 Gy SD and 10 × 1 Gy MF irradiation on the phospho-proteome (Fig. 5A). Interestingly, most of the changes in phosphorylation occurred during the first 24 h (ST) after irradiation with a peak at the 30 min time point (Fig. 5A, B, Supplementary Figure S4). Nevertheless, our analyses showed that the phosphorylation of AKT and MET was still enhanced at 2 months (LT) after 10 Gy SD indicating that irradiation can not only stably alter the expression but also the activity of molecular targets in a fractionation-dependent manner (Fig. 5C).
Prostate cancer cells surviving SD irradiation are more sensitive to molecular targeted drugs. To evaluate whether the observed overexpression and increased activation of molecular targets affects the efficacy of molecular inhibitors, we examined the sensitivity of long-term (LT) PC3 cultures to a panel of clinically used targeting drugs ( Table 1). As shown in Fig. 6, PC3 cells at 2 months (LT) after 10 Gy SD irradiation showed significantly (P < 0.05) decreased survival after treatment with the AKT inhibitors Capivasertib and Ipatasertib compared to the unirradiated controls (Fig. 6). Similar results were obtained when we targeted IGF-1R, MET, VEGFR2 or MEK signaling, while 10 Gy SD irradiation had no effect on the efficacy of Lapatinib (Fig. 6). It is important to note that treatment with inhibitors induced only minimal apoptosis indicating that there might be another form of cell death as underlying mechanism for the differential survival rates (Supplementary Figure S3).

SD irradiation reduces HDAC expression and increases the resistance of cancer cells to HDAC inhibitors.
As epigenetic modifications can impact gene expression, we next examined the expression of histones and HDACs after 10 Gy SD and 10 × 1 Gy MF irradiation. While at 2 months (LT) after 10 Gy SD irradiation several histone clusters were upregulated (Fig. 7A), HDAC levels were decreased (Fig. 7B, C). In parallel, long-term (LT) 10 Gy SD cells were more resistant to HDAC inhibition with Pabinostat or Vorinostat than the unirradiated controls (0 Gy SD) (Fig. 7D).

Discussion
Recurrent disease in prostate cancer patients after curatively-intended treatment can be clinically challenging 36,37 . While after total prostatectomy conventionally fractionated radiotherapy has been shown to be effective and safe 38 , re-irradiation after prior radiotherapy carries the risk for high toxicity rates 39 . Recently, the use of stereotactic ablative irradiation with one or a few high doses has increased for both recurrent prostate cancer after prostatectomy or primary radiotherapy, as well as for metastatic disease 37,40 . Still, there is clinical need for additional therapeutic strategies to improve patient outcome after disease relapse. During the last two decades, targeted therapy has evolved as promising approach for multiple cancer types for either monotherapy or in combination with irradiation or chemotherapy resulting in improved tumor response and patient survival 41 . However, some tumors have an intrinsic resistance to targeted therapy or develop resistance during treatment 41 . Since radiotherapy can modulate gene expression and protein phosphorylation, this effect can potentially be exploited to increase or restore the efficacy of targeted therapy 22,42 . Using the post-radiation adaptation of tumors to enhance efficacy of radiation therapy is different and complementary to using radiation and drugs simultaneously 30,42,43 . We demonstrate here that irradiation leads to long-term expression changes of multiple molecular targets and their associated pathways in surviving prostate cancer cells and that these adaptive changes are impacted by the   www.nature.com/scientificreports/ taking into account the dose-per-fraction, total dose and treatment time 45 . It has been shown that the BED can affect gene expression and therefore may contribute to the differential results between SD and MF irradiation which we observed 45 . Among others, IGF signaling was strongly activated at 2 months after 10 Gy SD but not after 10 × 1 Gy MF. Interestingly, high IGF-1R expression has been associated with high prostate cancer recurrence after primary radiotherapy indicating a potential role for IGF-1R for the adaptive tumor response 46 . Further, inhibition of IGF-1R sensitizes cancer cells to chemotherapy and irradiation identifying it as a promising target for molecular therapy 47,48 . Besides the higher IGF-1R expression, long-term 10 Gy SD cultures were also more sensitive to treatment with the IGF-1R inhibitor BMS-754807 which is in line with observations from Litzenburger and colleagues showing a correlation between IGF-1R expression and BMS-754807 efficacy in triple-negative breast cancer cell lines 49 . In contrast to targets such as IGF-1R and AKT3, SD irradiation reduced the expression of HDACs and in parallel increased the resistance to the HDAC inhibitors Pabinostat and Vorinostat. By modulating histone acetylation and methylation, targeting HDACs has been shown to radiosensitize cancer cells even when the inhibitors are applied up to 24 h after irradiation 32,50,51 . Interestingly, fractionated irradiation of 10 × 2 Gy doses results in elevated HDAC activity in breast cancer cells at 21 days after the final dose which correlated with enhanced cellular radioresistance indicating that HDACs are affected by irradiation depending on the fractionation regimen 52 .
Similar to gene expression, protein phosphorylation of the target also strongly affects the efficacy of targeted therapy and can be exploited to sensitize resistant cancer cells 22 . While we saw more pronounced modulation of gene expression at later time points, the radiation-induced alterations in protein phosphorylation occurred mainly within the first 24 h after irradiation and were more often transient than permanent. Targets showing www.nature.com/scientificreports/ increased long-term phosphorylation and activation included the kinases AKT and MET. Elevated phosphorylation of AKT promotes survival and radiation resistance and is a negative prognostic marker for poor clinical outcome in prostate cancer patients [20][21][22]53,54 . Combined treatment with the AKT inhibitor Ipatasertib and abiraterone significantly increased progression-free survival of metastatic castration-resistant prostate cancer patients compared to abiraterone alone especially in patients with PTEN-loss tumors and activated PI3K/AKT signaling 55 . Similar results were found in a randomized phase II trial examining AKT inhibition with Capivasertib Figure 6. Long-term SD tumor cells are more sensitive to molecular inhibitors when the target has been activated by irradiation. PC3 cells were irradiated with a single dose (SD) of 10 Gy and cultured for 2 months. Unirradiated cells were used as control. Colony formation assays were used to determine sensitivity to molecular targeted drugs. At 24 h after plating, cells were incubated with inhibitors at indicated concentrations or DMSO (0.1%) for 24 h. Results show mean ± STDEV (n = 3, *P < 0.05, **P < 0.01, Student's t-test).
Scientific Reports | (2022) 12:3500 | https://doi.org/10.1038/s41598-022-07394-y www.nature.com/scientificreports/ in combination with Paclitaxel for women with metastatic breast cancer indicating that stimulating AKT activity before targeting it may increase the drug efficacy 56 . Overall, our data show that radiotherapy especially large single doses can lead to stably elevated gene expression and activity of molecular targets and hereby sensitize cancer cells to the corresponding pharmacological inhibitors. Since the use of stereotactic ablative radiotherapy applying one or a few high doses has substantially increased, this may be a unique approach to improve the therapy outcome for recurrent and locally advanced prostate cancer patients.