Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2–10 Gy) or a multifractionated regimen (2–10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, β1 and β4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of β1 and β4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target.

Implications:

Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.

Molecular targeted therapeutics have become a central part of multimodal cancer treatment (1). To date, a broad panel of these drugs such as kinase inhibitors or inhibitory antibodies has been developed and tested in clinical trials. By inhibiting specific pathways that are overactivated in tumors, these agents usually have a stronger impact on malignant cells than on normal tissue (2). Despite having a good efficacy in some patients, there are tumors that show no or limited response or become resistant to drug treatment. This can be due to low target expression, mutations leading to a constitutively active target or bypass signaling. Resistance to drug treatment can result from a selection of a drug-resistant population. One focus of our group is how to use radiation to induce targets and thereby sensitize cancer cells to a subsequent molecular targeted therapy or immunotherapy. We have recently shown that radiation can activate the mTOR/AKT pathway in cancer cells and increase the efficacy of mTOR and AKT inhibitors (3). In this study, we sought to examine the duration of the radiation-induced effect using the potential of targeting radiation-induced integrins in prostate cancer cells that survived clinical relevant single dose and multifraction (MF) radiotherapy.

Integrins are heterodimeric transmembrane receptors consisting of an alpha and a beta subunit (4). Serving as the main receptors for binding to the extracellular matrix, integrins control adhesion, migration, and invasion processes (5–8). To date, 18 alpha and 8 beta subunits have been discovered, which can form 24 different receptor types in humans. The receptors vary in their affinity to the different matrix components depending on their composition. One important integrin subunit is β1 integrin. Present in half of all known integrin receptor combinations including receptors for laminins, collagens, and RGD peptides such as fibronectin or vitronectin, β1 integrins are also linked to growth factor–related signaling and affect tumor proliferation and cellular survival (9–11). Moreover, β1 integrins are involved in the radiation stress response and impact the repair of radiation-induced DNA double-strand breaks (9, 12–14). In contrast to β1 integrin, β4 integrin can only bind to one alpha subunit, i.e., α6 integrin, forming a laminin receptor that is crucial for the adhesion of cells to the basal membrane. β4 integrin expression correlates with size and grade of breast tumors (15). Moreover, overexpression of β4 integrin promotes lung metastasis in a mouse model, while the loss of β4 integrin signaling impairs invasive tumor growth (16, 17). Therefore, both β1 and β4 integrins are promising molecular targets for radiation oncology.

We chose prostate cancer cells for our experiments because this disease has a high incidence in Western countries and can be curatively treated with radiotherapy. In addition, integrin signaling is deregulated in prostate cancer and known to promote prostate cancer progression and metastasis (18–20). Because we found in previous studies that there are fractionation-dependent differences in radiation-induced gene expression (21, 22), we irradiated with a single dose as well as with a fractionated regimen reflecting the different radiotherapeutic modalities used in the clinic for this tumor type.

Here, we show that integrin expression is upregulated after both single dose and multifractionated radiation and that prostate cancer cells that have been cultured for 2 months after radiation continue to exhibit elevated levels of β1 and β4 integrin. Further, treatment with inhibitory β1 and β4 integrin antibodies was significantly more effective in the radiation long-term survivors than in unirradiated control cells. These results indicate that increasing target expression with radiotherapy is a unique approach to improve the efficacy of molecular targeted therapy.

Antibodies

Antibodies for Western blotting included α7 integrin (Invitrogen), α5 integrin, α6 integrin, αv integrin, β1 integrin, β3 integrin, β4 integrin, β5 integrin (Cell Signaling Technology), α3 integrin (US Biologicals), β-actin (Millipore), and IRDye 800CW donkey anti-mouse and IRDye 680RD Donkey anti-rabbit antibodies (LI-COR). Antibodies for immunofluorescence staining included α3 integrin (US Biologicals), α5 integrin, β1 integrin, β4 integrin, αvβ3 integrin (Millipore), α6 integrin (Novus Biologicals), and Alexa594 anti-rabbit and Alexa488 anti-mouse antibodies (Invitrogen). Antibodies for immunoprecipitation included β1 integrin (clone AIIB2, Millipore), and rat immunoglobulin G (IgG; Santa Cruz Biotechnology). Antibodies for integrin inhibition included β1 integrin (clone AIIB2) and β4 integrin (clone ASC-8) from Millipore.

Cell culture and radiation exposure

DU145 and PC3 were obtained from the NCI tumor bank in 2015, LNCaP were purchased from ATCC. The cells were used up to a passage number of 15. Asynchronously and exponentially growing cells were cultured in RPMI 1640 containing GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). On a monthly base, testing for mycoplasma was performed.

Cells were irradiated at room temperature using single doses or multiple fractions of 320 kV X-rays with a dose-rate of 2.3 Gy/minute (Precision X-Ray Inc.). Multifractionated radiation was carried out as described before with two times 1 Gy per day (with a 6-hour time interval between both radiations; ref. 3). Long-term survivor PC3 cells were passaged twice a week and cultured for at least 8 weeks after radiation before the cells were plated for experiments.

Animal experiments (short term)

All animal studies were conducted in accordance with the NIH Guide for Care and Use of Animals. PC3 human prostate cancer cells were injected subcutaneously into the flanks of the right hind legs of athymic nude mice (NCr nu/nu; NCI Animal Production Program, Frederick, MD) as described. On a daily basis, tumor volume was measured with a digital caliper. When tumors reached a size of 5 mm × 5 mm, randomization into 3 groups was performed: (i) nonirradiated controls; (ii) irradiation with a single dose of 10 Gy (SD); (iii) irradiation with 10 fractions of 1 Gy (MF1), twice a day for 5 days. A custom-designed lead jig was used to restrain the animals during radiation. At 24 hours after the final radiation dose, tumors were excised, snap frozen in liquid nitrogen, and stored at −80°C.

Animal experiments (long term)

Ten-week-old male athymic nude mice (NCr nu/nu; NCI Animal Production Program, Frederick, MD) were subcutaneously implanted in the right flank with 1 × 106 PC3 cells/100 μL PBS. PC3 cells were obtained from ATCC (CRL-1435) and transduced with virus 7079-MO1-685 (pFUGW-UbC-ffLuc-2-eGFP). Once tumors reached appropriate sorting size (170 mm3), mice were randomized into 0 Gy and 10 × 2 Gy (MF2). Mice were restrained for irradiation of the flank, with lead shielding the remainder of the body. Radiation was delivered with an X-RAD 320 (Precision X-Ray) with 2.0-mm Al filtration (300 kV peak) at 2.70 Gy/minute. Radiation was delivered from Monday to Friday for 2 weeks (2 Gy per day). Tumor growth was measured with a digital caliper until reaching >1,000 mm3. The growth times for the different tumors are controls: 319: 37 days, 311: 19 days; 10 × 2 Gy: 322: 68 days, 306: 77 days, 301: 77 days (Supplementary Fig. S1). Tumors from mice with delayed regrowth were collected in liquid nitrogen and stored at −80°C for further analysis. RNA from tumors exposed to 0 Gy and 10 × 2 Gy was extracted in TRIzol (Invitrogen) and purified with RNAeasy Plus kits (Qiagen). Protein from tumors exposed to 0 Gy and 10 × 2 Gy was extracted in RIPA (Thermo Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) by FastPrep-24 homogenizer (MP Biomedicals). Protein concentration was determined by BCA (Thermo Scientific).

3D colony formation assay

3D colony formation assays were carried out as published (23). After coating of 96-well plates with 1% agarose (Sigma), a Matrigel cell suspension with a final Matrigel (Corning) concentration of 0.5 mg/mL was plated in the agarose-precoated wells. Cell cultures were treated with β1 integrin antibody (AIIB2) or β4 integrin antibody (ASC-8) at 24 hours after plating. After 8 days (DU145, PC3) or 14 days (LNCaP), images of colonies were obtained using an EVOS microscope with a 2.5 × objective. ImageJ Cell Counter was used to count cell clusters with more than 50 cells. Results were confirmed by an automatic analysis using ImageJ and R (23). Surviving fractions were calculated as follows: (treated colony number/untreated colony number) (23).

Total protein extracts and Western blotting

For Western blot analysis, asynchronously and exponentially growing cell cultures were used. Cell lysis was performed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (Complete; Roche) as previously described (24). Lysates were then passed through a 25-gauge needle for homogenization and centrifuged at 16,000xg for 20 minutes. Xenograft tumors (short term) were homogenized in 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 25 mmol/L NaF, 25 mmol/L β-glycerophosphate, 0.2% Triton X-100, 0.3% Tween20, and 0.5 mmol/L sodium orthovanadate, supplemented with phosphatase inhibitor cocktails II and III (Sigma), and HALT protease inhibitor cocktail (Thermo Fisher Scientific). Prior to centrifugation at 16,000 × g for 20 minutes, ultrasonic treatment was performed. Protein was quantified using the BCA protein assay (Bio-Rad), separated by SDS–PAGE, transferred to nitrocellulose membranes (Bio-Rad), and probed with indicated antibodies. For visualization of protein expression, fluorescence-labeled secondary antibodies (LI-COR) and an Odyssey CLx imager (LI-COR) were used.

Gene expression in in vitro and in vivo samples

PC3 cells or xenografts were irradiated with a single radiation dose of 10 Gy or multifractionated radiation with 10 fractions of 1 Gy (2 fractions per day) with a cumulative dose of 10 Gy as published (3). For in vitro analysis, total RNA was extracted at 6 and 24 hours after irradiation from three separate biological replicates using a QIA shredder spin column (catalog no. 79654, Qiagen) as previously published. All extracted RNAs were purified with an RNeasy mini kit (Qiagen). The microarray analysis was done using CodeLink Whole Genome Bioarrays representing 55,000 probes. CodeLink Expression Analysis software (GE Healthcare) was used to process the scanned images from arrays (gridding and feature intensity), and the data generated for each feature on the array were analyzed with GeneSpring software (Agilent Technologies). Raw intensity data for each gene on every array were normalized to the median intensity of the raw values from that array. For in vivo analysis, RNA was extracted from pulverized snap-frozen xenograft tissue as described (3). The mRNA microarray analysis was performed using Agilent Technologies Human Gene Expression 8 × 60K v2 arrays (design ID 039494) designed to target 30,326 annotated genes with Entrez Gene ID.

Real-time PCR

One microgram of total RNA was reverse transcribed using an RT2 First Strand synthesis kit (Qiagen, 330401). qPCR assays were performed using RT2 SYBR Green ROX qPCR Mastermix (Qiagen, 330520) and RT² qPCR Primer Assays (Qiagen; product no. 330001) for ITGA1, ITGA3, ITGA7, ITGA10, ITGA11, ITGB1, ITGB3, ITGB4, ITGB5, and ITGB7. GAPDH, 18S, and ACTB were used as normalizing genes. Real-time PCR reactions were performed in the Applied Biosystems' thermal cycler (Quant Studio 3). PCR steps included -the holding stage at 95°C for 15 minutes followed by 40 cycles of alternate denaturation at 95°C for 15 seconds, annealing/extension at 60°C for 1 minute. A melt curve analysis was performed to ensure the specificity of the corresponding RT-PCR reactions. Fold change = 2-ddCt, where ddCt = dCt (test) - dCt (control); dCt = Ct (gene) – Ct (mean of GAPDH, 18S, and ACTB); and Ct is the threshold cycle number. All assays were performed in triplicates. Statistical significance was calculated using an unpaired Student t test.

Immunoprecipitation

Immunoprecipitation of β1 integrin was accomplished as previously described (9). Cells were inserted into a 3D extracellular matrix. After 24 hours, cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with complete protease inhibitor cocktail (Roche). Protein-G-Agarose beads (Sigma) were incubated with β1 integrin antibodies (AIIB2) or nonspecific rat IgG antibody as a control. Prior to adding the 3D protein lysates, beads were washed twice with PBS. SDS–PAGE and Western blotting was performed to detect coprecipitating alpha integrin units.

Immunofluorescence staining

Immunofluorescence was performed as recently described (25). At 24 hours after plating, cells were fixed with 3% formaldehyde/PBS for 15 minutes, permeabilized with 0.25% Triton X-100/PBS for 10 minutes and blocked with 3% BSA/PBS for 30 minutes. Staining of integrin subunits was carried out with specific antibodies overnight at 4°C and with secondary antibodies for 1 hour at room temperature. After several washes with PBS, samples were covered with Vectashield/DAPI mounting medium (Alexis). Images were acquired using an AxioImager.Z1/ApoTome microscope (Zeiss). Integrin intensity of single cells was measured with ZEN imaging software (Zeiss). Overexpression of integrins in irradiated cells was defined as higher intensity than the mean plus standard deviation of the controls (> mean + 1× STDEV). Very high expression was defined as higher intensity than the mean plus 3 standard deviations of the controls (> mean + 3× STDEV).

Cell-cycle analysis

Cell-cycle distribution analysis was performed as published (26). Cells were incubated with 10 mmol/L BrdUrd for 10 minutes. After fixation with 80% EtOH and incubation with 2 N HCl and 0.1 M Na2B4O7, samples were stained with anti-BrdUrd antibody (BD) and anti-mouse AlexaFlour 647 (Invitrogen). Total DNA staining was performed with propidium iodide (Invitrogen) solution containing RNAse (Invitrogen). Data for 10,000 events were acquired on an LSR Fortessa flow cytometer (Becton Dickinson) with DIVA software. The distribution of cells in the different phases of the cell cycle was analyzed using FlowJo (TreeStar) software.

Data analysis

For data analysis, Microsoft Excel 2010 or R software was used. The measured values were normalized to the corresponding control to calculate the fold change. For survival analysis, a logarithmic transformation was performed before statistical significance was calculated (9). Statistical significance was tested with the unpaired, two-sided Student t test. Results were considered statistically significant if a P value of less than 0.05 was reached. For densitometric evaluation of protein expression, Image Studio Lite 4 (LI-COR) was used. “n” is the number of biological replicates.

Differential integrin receptor expression, composition, and localization in prostate cancer cell lines

Analysis of the basal expression of several integrin subunits showed a high expression of β1 integrin, β3 integrin, and α5 integrin in DU145 cells and a high expression of β4 integrin and α3 integrin in DU145 and PC3 cells while in LNCaP cells the expression of these integrins was very low. In contrast, LNCaP cells showed a higher expression of αV, α7, and β5 integrin (Fig. 1A). Next, we analyzed the basal mRNA expression of the different integrin subunits in vitro and in vivo and found that PC3 cells had high levels of α3, α6, αV, β1, β4, and β5 integrins under both conditions (Fig. 1B). Further, β1 integrin was more expressed in cultured cells and αV integrin in xenograft tumors which could be due to different matrix compositions. Immunoprecipitation of β1 integrin (Fig. 1C) with AIIB2 showed a strong coprecipitation of α5 and α6 integrin in all three prostate cancer cell lines as well as a weak coprecipitation of αV integrin in LNCaP cells (Fig. 1C). Whereas in DU145 and PC3 cells most of the integrin subunits had both a membranous and cytoplasmic localization, in LNCaP cells all except α3 integrin were localized in the cytoplasm and not in the membrane, which may indicate a limited functionality (Fig. 1D).

Figure 1.

Integrin expression, composition, and localization differ cell line dependently. A, Western blot and densitometric analysis of basal expression of different integrin subunits in DU145, PC3, and LNCaP prostate cancer cells. B, Relative mRNA expression of different integrin subunits in unirradiated PC3 cells (in vitro) or PC3 xenograft tumors (in vivo). C, Immunoprecipitation of β1 integrin in prostate cancer cell lysates. Nonspecific IgG was used as a control. D, Immunoflourescence staining of indicated integrin subunits or heterodimers in unirradiated prostate cancer cells. The nuclei were stained with DAPI. E, Schematic of treatment schedule and clonogenic survival of DU145, PC3, and LNCaP prostate cancer cells after treatment with different concentrations of inhibitory β1 integrin antibodies (AIIB2) or inhibitory β4 integrin antibodies (ASC-8). Results show mean ± STDEV (n = 3).

Figure 1.

Integrin expression, composition, and localization differ cell line dependently. A, Western blot and densitometric analysis of basal expression of different integrin subunits in DU145, PC3, and LNCaP prostate cancer cells. B, Relative mRNA expression of different integrin subunits in unirradiated PC3 cells (in vitro) or PC3 xenograft tumors (in vivo). C, Immunoprecipitation of β1 integrin in prostate cancer cell lysates. Nonspecific IgG was used as a control. D, Immunoflourescence staining of indicated integrin subunits or heterodimers in unirradiated prostate cancer cells. The nuclei were stained with DAPI. E, Schematic of treatment schedule and clonogenic survival of DU145, PC3, and LNCaP prostate cancer cells after treatment with different concentrations of inhibitory β1 integrin antibodies (AIIB2) or inhibitory β4 integrin antibodies (ASC-8). Results show mean ± STDEV (n = 3).

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Inhibition of β1 integrin and β4 integrin decreases clonogenic survival dose- and cell line dependently

As integrins affect tumor cell survival, we evaluated the effect of integrin inhibition on prostate cancer cells using the anti-β1 integrin antibody AIIB2 and the anti-β4 integrin antibody ASC-8, which both have been shown to impair integrin function (13, 27). We found that AIIB2 reduced dose dependently the clonogenic survival of all three cell lines to a similar extent, although more antibodies bound to DU145 and PC3 cells (Supplementary Fig. S2). In contrast, ASC-8 had the highest effect in PC3 and LNCaP cells (Fig. 1E). These findings show that the efficacy of the inhibitors does not necessarily correlate with the basal integrin expression among the cell lines (Fig. 1A).

Radiation results in enhanced integrin expression

To examine the effect of radiation on integrin mRNA and protein expression, PC3 cells and xenografts were irradiated with a single dose of 10 Gy or a fractionated regimen of 10 times 1 Gy and subjected to a microarray analysis and PCR (Fig. 2A; Supplementary Fig. S3). As shown in Fig. 2B and C, β3 and β4 integrin mRNA expression was upregulated by both radiation regimens in vitro and in vivo, while α7 integrin expression was only affected by multifractionated radiation (Fig. 2B and C). Further, in xenograft tumors but not in cultured cells, α10 integrin mRNA expression was increased after 10 times 1 Gy (Fig. 2C). We have seen in previous studies that gene expression is not upregulated until fractionated radiation reaches a certain dose, in our experience 6 to 8 Gy, which we defined as the inflection point. Therefore, we also analyzed the integrin expression of β4 integrin, α7 integrin, and α10 integrin after 6 fractions of 1 Gy. The mRNA expression of all three integrins was enhanced to a similar extent as after 10 fractions (Fig. 2D). On the protein level, β1 and β4 integrins were upregulated after 6 and 10 fractions of 1 Gy (Fig. 2E). In contrast, β3 and α3 expression was unchanged after 6 fractions but increased after 10 fractions (Fig. 2E). These findings show that integrin mRNA and protein expression are differentially modulated by radiation. Because the inhibitory integrin antibodies and inhibitors target the integrin proteins and not the mRNA, analysis of the protein expression is crucial to identify them as radiation-inducible targets.

Figure 2.

Integrin gene expression is upregulated after radiation in vitro and in vivo. A, Schematic of treatment. B, PC3 cells or (C) PC3 xenografts were irradiated with either a single dose of 10 Gy or with 10 fractions of 1 Gy (2 Gy per day). At 6 and 24 hours after the final radiation dose, cells were lysed or tumors were excised and RNA was isolated. Integrin mRNA expression was determined by gene microarray analysis and normalized to unirradiated controls. Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). D, Integrin gene or (E) protein expression was analyzed in PC3 xenograft tumors at 24 hours after 6 fractions of 1 Gy or 10 fractions of 1 Gy and compared with unirradiated controls. Results show mean fold change ± STDEV (n = 2).

Figure 2.

Integrin gene expression is upregulated after radiation in vitro and in vivo. A, Schematic of treatment. B, PC3 cells or (C) PC3 xenografts were irradiated with either a single dose of 10 Gy or with 10 fractions of 1 Gy (2 Gy per day). At 6 and 24 hours after the final radiation dose, cells were lysed or tumors were excised and RNA was isolated. Integrin mRNA expression was determined by gene microarray analysis and normalized to unirradiated controls. Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). D, Integrin gene or (E) protein expression was analyzed in PC3 xenograft tumors at 24 hours after 6 fractions of 1 Gy or 10 fractions of 1 Gy and compared with unirradiated controls. Results show mean fold change ± STDEV (n = 2).

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Integrin expression is stably upregulated in prostate cancer cells after radiation

Next, we analyzed whether certain integrins were still overexpressed in prostate cancer cells surviving radiotherapy. Therefore, we cultured the cells for 2 months after radiation (Fig. 3A) until there was no increased cell death compared with the unirradiated cells. As shown in Fig. 3B, PC3 cells irradiated with a single dose of 10 Gy had an increased mRNA expression of multiple integrin subunits including β1 and β4 integrins, while multifractionated radiation led only to an upregulation of α1 integrin mRNA in comparison to unirradiated cells. In contrast, protein expression of β1 and β4 integrins was significantly enhanced in both single dose and multifractionated long-term cell cultures (Fig. 3C and D). Additionally, cells after single dose radiation had increased β3 and β5 integrin protein levels (Fig. 3C and D). In contrast to the long-term in vitro results, analysis of integrin expression in PC3 xenografts more than 2 months after a multifractionated radiation of 10 fractions of 2 Gy (Fig. 3E) showed a pronounced heterogeneity (Fig. 3F and G). This heterogeneity was also present in unirradiated control tumors (Fig. 3G). Although we mostly found an upregulation of α7 integrin, α10 integrin, and β4 integrin mRNA and of β1 and β4 integrin protein expression in 2 of the 3 tested xenografts, the integrin expression of 1 tumor was unchanged in comparison to the unirradiated xenografts (Fig. 3F and G).

Figure 3.

Integrin mRNA and protein expression is upregulated in radiation long-term survivors. A, Schematic of the radiation schedule for long-term in vitro experiments. B, Gene expression of integrin subunits was analyzed in PC3 cells that had been cultured for 2 months after a single dose (SD) of 10 Gy or multifractionated radiation (MF) of 10 times 1 Gy. Unirradiated cells were used as controls. Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). C, Integrin protein expression and (D) densitometric analysis of PC3 cells at 2 months after a single-dose radiation (2, 6, 10 Gy) or multifractionated radiation (2 × 1 Gy, 6 × 1 Gy, 10 × 1 Gy). Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). E, Schematic of the radiation schedule for long-term in vivo experiments. F, Gene expression and (G) Western blots of integrin subunits were analyzed in PC3 xenografts after multifractionated radiation (MF) of 10 times 2 Gy. Unirradiated tumors were used as controls. Results show mean fold change ± STDEV (n = 3).

Figure 3.

Integrin mRNA and protein expression is upregulated in radiation long-term survivors. A, Schematic of the radiation schedule for long-term in vitro experiments. B, Gene expression of integrin subunits was analyzed in PC3 cells that had been cultured for 2 months after a single dose (SD) of 10 Gy or multifractionated radiation (MF) of 10 times 1 Gy. Unirradiated cells were used as controls. Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). C, Integrin protein expression and (D) densitometric analysis of PC3 cells at 2 months after a single-dose radiation (2, 6, 10 Gy) or multifractionated radiation (2 × 1 Gy, 6 × 1 Gy, 10 × 1 Gy). Results show mean fold change ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). E, Schematic of the radiation schedule for long-term in vivo experiments. F, Gene expression and (G) Western blots of integrin subunits were analyzed in PC3 xenografts after multifractionated radiation (MF) of 10 times 2 Gy. Unirradiated tumors were used as controls. Results show mean fold change ± STDEV (n = 3).

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Irradiated cells with increased integrin expression are more susceptible to anti-integrin therapy

For an effective molecular therapy, it is important that the target is upregulated homogenously in the tumor cell population. To examine the integrin expression in the long-term radiation survivors, we performed immunofluorescence staining of β1 and β4 integrins (Fig. 4A). We found that 2 months after radiation β1 integrin was overexpressed (> mean + 1× STDEV) in 82% of the cells after a 10 Gy single dose (58% with a very high expression, > mean + 3× STDEV) and in 79% of the cells after 10 times 1 Gy fractionated radiotherapy (39% with a very high expression), while β4 integrin was overexpressed in 81% of the cells after a 10 Gy single dose (32% with a very high expression) and in 76% of the cells after 10 times 1 Gy fractionated radiotherapy (27% with a very high expression; Fig. 4A and B). Correlating with the increased protein expression, treatment with inhibitory β1 and β4 integrin antibodies (Fig. 4C) was significantly more effective in the long-term surviving cells than in unirradiated controls (Fig. 4D). Further, long-term radiation survivors after a single dose of 10 Gy had a reduced plating efficiency and showed a greater heterogeneity in cell size and morphology while cell-cycle distribution was similar to unirradiated cells (Supplementary Fig. S4). In summary, our findings indicate that exploiting radiation-induced changes for targeted therapy is possible even after a long period of time (Fig. 4E).

Figure 4.

Radiation-induced integrin expression sensitizes cells to integrin inhibition. A, Fluorescence staining and (B) intensity analysis of β1 integrin and β4 integrin in long-term surviving PC3 cells irradiated with a single dose of 10 Gy (SD) or with a multifractionated radiation of 10 times 1 Gy (MF). Unirradiated cells served as control. The nuclei were stained with DAPI. C, Schematic of the treatment schedule for clonogenic assays. D, Clonogenic survival of irradiated long-term surviving cells after treatment with an inhibitory β1 integrin (AIIB2) or β4 integrin (ASC-8) antibody. Results show mean ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). E, Schematic of radiation-induced integrin targeting.

Figure 4.

Radiation-induced integrin expression sensitizes cells to integrin inhibition. A, Fluorescence staining and (B) intensity analysis of β1 integrin and β4 integrin in long-term surviving PC3 cells irradiated with a single dose of 10 Gy (SD) or with a multifractionated radiation of 10 times 1 Gy (MF). Unirradiated cells served as control. The nuclei were stained with DAPI. C, Schematic of the treatment schedule for clonogenic assays. D, Clonogenic survival of irradiated long-term surviving cells after treatment with an inhibitory β1 integrin (AIIB2) or β4 integrin (ASC-8) antibody. Results show mean ± STDEV (n = 3, *, P < 0.05; **, P < 0.01, Student t test). E, Schematic of radiation-induced integrin targeting.

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Large and extensive tumors and persistent and recurrent cancer are clinically relevant problems for both cure and quality of life. The risk of tumor persistence or relapse depends not only on the tumor type and disease stage but also on molecular characteristics, for example, the ability of the tumor cells to adapt to cytotoxic stress and thereby survive cancer treatment. A tumor relapse after a completed radiotherapy is very challenging because the treatment options are limited. The focus of our group is to identify molecular changes in irradiated cancer cells and to exploit them for molecular therapy (3, 22). This relates to both an initial treatment plan built on using radiotherapy to enhance the efficacy of molecular targeted agents and also interrogating persistent and recurrent tumors to molecular/biochemical adaptations that may be targetable. In this study, we showed that prostate cancer cells had a radiation-induced overexpression of β1 and β4 integrin after both single dose and fractionated radiation that persisted for 2 months after radiotherapy. Upregulation of beta1 integrins after radiation has been found in different tumor types including glioblastoma and pancreatic cancer (6, 28). However, all these studies examined the integrin expression during the first hours and days after radiation while our study focused on the long-term effects in the surviving cells and the possibility to target these changes.

We saw that both in vitro and in vivo, α3, α6, αv, β1, and β4 integrins had the highest basal mRNA expression in PC3 cells, while at 24 hours after radiation mRNA expression of α7 and β4 integrin was increased compared with unirradiated controls showing that the initial assessment of the tumor does not predict the molecular changes induced by radiation (29). These results call into question that in clinical practice, pretherapeutic biopsies are often used to choose the molecular drugs for treatment after or during radiochemotherapy without knowing if the target expression has changed.

As shown in previous studies, tumor cells react to radiation with the activation of prosurvival molecules as part of the stress response (3, 28, 30). Depending on which pathways are involved, these adaptive changes can enable the cell to escape death and hereby lower the efficacy of conventional anticancer treatment (30, 31). The extent of the radiation-induced molecular modulations has been shown to be affected by the radiation dose, the number of fractions, and the time after therapy (29, 32).

Interestingly, single-dose and fractionated radiation had similar effects on the β1 and β4 integrin protein expression in the long-term cultures, while mRNA expression was only enhanced after single-dose radiation. These results indicate that the molecular mechanisms causing the observed upregulation of β1 and β4 integrin are different for both radiation regimens. In contrast to the gene overexpression after single-dose radiation, the cells that survived fractionated radiation may have a decreased integrin degradation or an enhanced integrin recycling leading to the high β1 and β4 integrin protein levels (33). Because the protein and not the mRNA expression of the target is the determining factor for the efficacy of a molecular drug, a proteomic approach may therefore be more accurate to identify the changes after fractionated radiation.

In line with previous studies, when comparing the 3 prostate cancer cell lines, neither the protein levels of β1 or β4 integrin nor the membranous localization correlated with the efficacy of inhibitory antibodies, whereas upregulation of the target resulted in increased susceptibility. Similar results have been found for cetuximab in head and neck tumors (9, 34) and may be due to a different genetic background and bypass signaling of the cell lines.

Although we found an upregulation of specific integrins in the majority of irradiated long-term xenografts, the data showed a strong heterogeneity in integrin expression between tumors. This heterogeneity was not limited to the irradiated tumors but was also present in the unirradiated controls and may be caused by microenvironmental factors such as the composition of the extracellular matrix, which can differ from mouse to mouse. Analyzing the integrin expression of multiple tumors with different doses and radiation schedules before and after radiation as well as targeting regrowing irradiated xenografts with inhibitory β1 and β4 integrin antibodies will elucidate the translational potential of target induction using radiation and its applicability to radio-oncologic patients in clinical practice.

Overall, our findings show that adaptive changes in tumor cells after radiation can remain for a long time and may be exploited to increase the efficacy of molecular targeted drugs. Besides small-molecule inhibitors of the AKT/mTOR pathway as demonstrated previously, inhibitory β1 and β4 integrin antibodies also induce more cell death when the target expression has been enhanced by a single dose or fractionated radiotherapy. Therefore, activating prosurvival pathways by radiation before targeting them is a promising novel and unique approach that may be applicable for primary tumors, metastases, or recurrent cancer after radiotherapy.

No potential conflicts of interest were disclosed.

Conception and design: I. Eke, J.L. Reedy, C.N. Coleman

Development of methodology: M.J. Aryankalayil, J.L. Reedy, D.E. Citrin, C.N. Coleman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I. Eke, A.Y. Makinde, M.J. Aryankalayil, J.L. Reedy, D.E. Citrin

Writing, review, and/or revision of the manuscript: I. Eke, A.Y. Makinde, M.J. Aryankalayil, J.L. Reedy, D.E. Citrin, M.M. Ahmed, C.N. Coleman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Chopra

Study supervision: I. Eke, C.N. Coleman

This study was supported by the NIH Intramural Research Program, National Cancer Institute, Center for Cancer Research (grant ZIA BC 010670). The authors thank Barbara H. Rath, Joel Levin, and David Cerna (NCI/NIH) for excellent technical assistance.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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