The αvβ3 integrin is involved in various physiologic and pathologic processes such as wound healing, angiogenesis, tumor growth, and metastasis. The impact of αvβ3 integrin on the radiosensitivity of prostate cancer cells and the molecular mechanism controlling cell survival in response to ionizing radiation (IR) was investigated. Both LNCaP cells stably transfected with αvβ3 integrin and PC-3 cells that contain endogenous β3 integrin were used. This study demonstrated that αvβ3 integrin increases survival of αvβ3-LNCaP cells upon IR while small hairpin RNA (shRNA)-mediated knockdown of αvβ3 integrin in PC-3 cells sensitizes to radiation. Expression of αvβ3 integrin in LNCaP cells also enhances anchorage-independent cell growth while knockdown of αvβ3 integrin in PC-3 cells inhibits anchorage-independent cell growth. The αvβ3 antagonist, cRGD, significantly increases radiosensitivity in both αvβ3-LNCaP and PC-3 cells. Moreover, αvβ3 integrin prevents radiation-induced downregulation of survivin. Inhibition of survivin expression by siRNA or shRNA enhances IR-induced inhibition of anchorage-independent cell growth. Overexpression of wild-type survivin in PC-3 cells treated with αvβ3 integrin shRNA increases survival of cells upon IR. These findings reveal that αvβ3 integrin promotes radioresistance and regulates survivin levels in response to IR.

Implications:

Future translational research on targeting αvβ3 integrin and survivin may reveal novel approaches as an adjunct to radiotherapy for patients with prostate cancer.

Prostate cancer is the most common noncutaneous malignant disease and the second expected cause of cancer-related death among men in the United States in 2018 (1). Radiotherapy is an important primary treatment modality for localized prostate cancer, and recent advances in radiosurgery and intensity-modulated radiotherapy have allowed dose escalation (i.e., 76–80 Gy) to improve biochemical failure rate and decrease metastasis (2). Despite these advances, intermediate- and high-risk populations of patients with prostate cancer continue to relapse after definitive radiotherapy (3). One possible reason for failure after radiotherapy may be due to intrinsic radioresistance of a small subpopulation of prostate tumor clonogen within the primary tumor. Therefore, the research on the influence of specific tumor signal response to radiation and cell survival is important for advancing the care of patients with prostate cancer (4, 5).

Integrin belongs to a family of at least 24 heterodimeric cell surface receptors that consist of noncovalently associated α and β subunits (6). These receptors influence cell functions, including adhesion, differentiation, proliferation, migration, and cell survival. Alteration of integrin expression in cancer cells correlates with tumor growth, progression, invasiveness, and metastatic potential. In particular, αvβ3 integrin remains one of the most actively investigated members of the integrin family because it has been shown to promote angiogenesis, tumor growth, and metastasis (7, 8). Its expression correlates strongly with malignancy in many tumor types including prostate cancer. Expression of αvβ3 integrin has been shown in prostate adenocarcinoma, as well as in the invasive prostate cancer PC-3 cell line, whereas it is absent in normal prostate epithelial cells and the less aggressive LNCaP cell line (9). Overexpression of αvβ3 integrin in LNCaP prostate cancer cells upregulates cdc2 level and increases cell migration (10).

In the past decade, there has been emerging evidence to suggest that αvβ3 integrin may promote radioresistance of a tumor. In 2005, Gruber and colleagues reported that patients with cervical cancer with αvβ3 expression had significantly worse local control, metastasis, and survival after curative radiotherapy (11). Also in 2005, Abdollahi and colleagues demonstrated that S247 (an αvβ3 petidomimetic antagonist) potentiates antiangiogenic effect of ionizing radiation (IR) on endothelial cells and xenograft tumors (12). In 2006, Albert and colleagues demonstrated that cilengitide (αvβ3 cyclic peptideantagonist) increased sensitivity of human endothelial cells and non–small cell lung cancer (NSCLC) cells in vitro (13). In an orthotopic rat glioma xenograft model, application of a single dose of cilengitide (4 mg/kg), 4–12 hours prior to radiation potentiates radiation efficacy (14). Although phase II clinical trial of cilengitide in patients with nonmetastatic castration-resistant prostate cancer shows no detectable clinical activity (15), application of cyclic RGD peptide with liposomal drug delivery system enhances therapeutic efficacy in treating prostate cancer bone metastasis, implying a complex prostate cancer response to the integrin antagonist (16).

Survivin belongs to a family of inhibitors of apoptosis (IAP; ref. 17). It plays an important role in mitosis, inhibition of apoptosis and autophagy, repair of DNA breaks, and resistance to chemo- (18) or radiotherapy (19, 20). Notably, survivin is overexpressed in many types of cancer cells including prostate cancer while absent in normal differentiated tissues (21). Thus, survivin expression level is found to be positively correlated with tumor progression and inversely correlated with the overall survival in patients after treatment (22, 23). The purpose of this study is thus to investigate whether αvβ3 integrin can promote intrinsic radioresistance of prostate cancer cells and to determine whether the survivin is involved in the regulation of cell survival controlled by αvβ3 integrin.

Antibodies and reagents

The following antibodies were used for immunoblotting analysis: anti-ERK1 (Santa Cruz Biotechnology, Inc.), anti-AKT (Cell Signaling Technology, Inc.), anti-Bcl-xL and anti-XIAP (BD Biosciences), anti-survivin (Novus Biologicals Inc.), anti-β3 integrin AP-3 (ATCC), anti-αv integrin (NKI-M9) and anti-β1 integrin (TS2/16; Thermo Fisher Scientific), Cyclo (-Arg-Gly-Asp-D-Phe-Val; cRGD), and the control Cyclo (-Arg-Ala-Asp-D-Phe-Val; cRAD) peptides were from Bachem. Survivin-derived (S4) double-stranded RNA oligonucleotide and control siRNA (VIII) were from GE Healthcare Dharmacon Inc. PowerPrep HP plasmid purification system was from Origene. BSA, Lipofectamine 2000, and Opti-MEM were purchased from Invitrogen.

Cell line and transfectants

Human prostate cancer cells PC-3, LNCaP, and LNCaP stable cell lines expressing pRc/CMV vector alone (mock-LNCaP) or vector containing full-length β3 integrin (αvβ3-LNCaP) were described previously (9). Cells were cultured in RPMI1640 medium supplemented with 5% heat-inactivated FBS (from Gemini Bio-Products, Inc.), 2 mmol/L l-glutamine, 10 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Invitrogen). PC-3- or LNCaP-stable cell lines infected with lentivirus carrying shRNA were maintained in the presence of 0.5 or 1 μg/mL of puromycin, respectively. LNCaP-β3 integrin transfectants were maintained in the presence of 100 μg/mL of geneticin. Integrin surface expression was analyzed by FACS analysis as described previously (9). Cells were maintained in the culture medium at 37°C with 5% CO2.

FACS analysis

One-color FACS analysis was performed using nonpermeabilized cell suspensions with one of the following mAbs to human integrins: TS2/16 to β1, NKI-M9 to αv, and AP-3 to β3 while 12CA5 (ATCC) to hemagglutinin (HA) or mouse IgG as negative controls. The cells were incubated with goat anti-mouse FITC-conjugated secondary antibody (40 μg/mL; Cappel) or goat anti-mouse PE-conjugated secondary antibody (The Jackson Laboratory) at 4°C for 30 minutes for FACS staining. FACS analysis and sorting were performed using FACSort (Becton Dickinson).

Immunoblot analysis

Cells were collected using trypsin-EDTA and lysed with the lysis buffer containing: 50 mmol/L Tris (pH 7.5; American Bioanalytical), 1% NP40 (Calbiochem), 150 mmol/L NaCl, 1 mmol/L sodium pyrophosphate, 50 mmol/L NaF, and 2 mmol/L EDTA (pH 8), 1 mmol/L PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin, and 1 mmol/L sodium orthovanadate (all from Sigma Chemical Co.). Protein concentrations were determined using the BCA protein assay reagent (Pierce Chemical Co.), and proteins in lysates were resolved by 10% or 15% SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes (Schleicher and Schuell). Quantitative analysis was conducted using a computing densitometer (Molecular Dynamics) or Imagelab (Bio-Rad).

Radiation exposure

Cells were irradiated at room temperature using a 6 MeV Varian 2300CD linear accelerator (Varian Medical Systems). Single doses of 2–10 Gy were delivered at 3 Gy/minute with a 1-cm surface bolus application to ensure dose uniformity at a depth of 100-cm source to tray distance. Nonirradiated cells were given a sham irradiation as controls.

Cell survival assay

We used clonogenic assay and colorimetric sulforhodamine B (SRB) assay to evaluate survival of cells in response to radiation. For SRB assay, cells were harvested, resuspended, counted, and irradiated at room temperature over the dose range of 2–10 Gy. Appropriate cells with optimal seeding density were then immediately plated into a 96-well plate and incubated for 6–7 days before determination of the survival by SRB assay. SRB assay was performed as described previously (24, 25). Optical density was measured at 562 nm (Bio-Rad 550 microplate reader). For survival curve, surviving fractions were calculated as: OD of irradiated cells/OD of nonirradiated cells. For inhibition assays, cRGD or cRAD (control) were added into cell culture 1 hour before irradiation, and fresh peptides with media were exchanged every 3 days. To determine synergistic effect of peptide in combination with IR, survival curves were corrected for the drug effect of peptide itself: surviving fraction = OD of irradiated cells with the peptide/OD of nonirradiated cells with the peptide. For clonogenic assay, cells were exposed to IR at doses from 2 to 10 Gy and seeded in either 6-cm dish or 6-well plates. Cells were fixed with formalin and stained with crystal violet 10–12 days post IR. The surviving fraction was scored with the colony containing more than 50 cells.

Anchorage-independent growth assay

Cell growth in soft agar was evaluated as described previously (26). Cells were harvested with trypsin/EDTA. Cells were exposed to IR with dose ranges of 2–10 Gy at room temperature. After radiation, cells were seeded onto soft agar. A total of 5 × 104 cells were suspended in the complete medium containing 0.3% low-melt agarose (Invitrogen). The mixture was then plated into 60-mm tissue culture plates containing 0.5% agar in complete medium. Cells were allowed to grow for 11–14 days, and colonies with the size equal to or greater than 100 μm were counted. Duplicate plates were prepared, and 20 fields per plate were counted. For inhibition assays, the cells were incubated in the presence of cRGD or control cRAD 1 hour prior to irradiation, and fresh peptides were added every 3 days.

Cell adhesion assay

Adhesion assays were performed as described previously (9). Vitronectin (VN) and fibronectin (FN) were purified as described previously (27, 28). Cell adhesion was scored after measuring the absorbance at 630 nm. Inhibition assay was performed with incubating cells in the presence of either cRGD or cRAD (as control).

Transfection, gene silencing, and plasmid construction

Silencing survivin using siRNA transfection was performed as described previously (29). Briefly, mock- and αvβ3-LNCaP cells were seeded in 6-well plates at a density of 2 × 105 cells per well. Cells were transfected with either control siRNA (VIII) or survivin siRNA (S4) at a concentration of 10 nmol/L using Lipofectamine 2000. Survivin expression was analyzed using immunoblot analysis.

Meanwhile, we also silenced 1) surivin expression in αvβ3-LNCaP cells using pLKO.1 lentivirus carrying shRNA; 2) β3-integrin expression in PC-3 cells using pGIPZ lentivirus carrying shRNA (UMASS Medical School RNAi Core, Worcester, MA). The mature antisense sequences of survivin shRNA were 5′-TTCTTGAATGTAGAGATGCGG-3′ (SV-1) or 5′- AACTGCTTCTTGACAGAAAGG-3′ (SV-2). The mature antisense sequences of β3 integrin shRNA were 5′-ACAGTTGCAGTAGTAGCCG-3′ (β3-Sh1) or 5′-TCACTTCCTCATTGAAGCG-3′ (β3-Sh2). The lentivirus containing vectors without the target shRNA were used as a control. The cells were infected with lentivirus in the presence of 10 μg/mL polybrene, selected with the puromycin, and maintained in the puromycin-containing culture medium.

The wild-type full-length survivin cDNA was inserted in the pcDAN3.1 carrying a HA tag as described previously (30). Plasmid DNA was prepared using PowerPrep HP plasmid purification system (Origene) for cell transfection. For transient transfection experiments, cells were mixed with plasmids and Lipofectamine (Invitrogen) using the manufacturer's protocol.

Statistical analysis

For the SRB assay, the survival curves were calculated as: mean OD of treated cells/mean OD of untreated control cells. The results are expressed as mean ± SEM. All experiments in this study were repeated at least two times, confirmed with at least two clones of each transfectant cell line. Significance of differences was analyzed using the Student t test. Statistical significance is considered to be P < 0.01.

For anchorage-independent cell growth, significance of differences was analyzed using the Student t test. P value smaller than 0.01 or 0.05 are considered statistically significant.

αvβ3 expression in LNCaP transfectants remains unchanged after IR

To determine whether αvβ3 can promote intrinsic radioresistance of prostate cancer cells, we used LNCaP transfectants stably transfected with β3 integrin (Fig. 1A) as described previously (9). To exclude confounding factors of alteration of other integrin subtypes in cells, we also surveyed αv and β1 integrin and determined whether there is a difference between mock- and αvβ3-LNCaP cells. Our data showed that no difference of β1 integrin between mock- and αvβ3-LNCaP cells. However, there is a significant increase of αv integrin level in αvβ3-LNCaP than in mock-LNCaP (Supplementary Fig. S1). Because radiation has been shown to upregulate αvβ3 in endothelial cells, NSCLC, and glioblastoma cells (31, 32), we examined this integrin expression in the transfectants after radiation. No change of αvβ3 expression level was observed in cells upon IR as shown by immunoblotting (Fig. 1B).

Figure 1.

αvβ3 integrin expression increases survival of LNCaP cells in response to IR. A, Surface expression of ectopic αvβ3 integrin in αvβ3-LNCaP, mock-LNCaP, and parental LNCaP cells were analyzed using FACS. Cells were incubated with mAb specific to αvβ3 integrin (AP3) and mAb specific to HA epitope (12CA5) as a negative control. B, Cells were treated with 0 or 5 Gy and were lysed 48 hours after radiation. Proteins in cell lysates were separated in 10% polyacrylamide gels, immunoblotted, and probed with Ab to carboxyl end of β3 integrin (41). ERK1/2 is used as a loading control. C, Survival curves of mock-LNCaP and αvβ3-LNCaP were determined using colorimetric SRB assay. Cells (1,000/well) were plated in 96-well plates at each radiation dose. Cells were incubated for 6–7 days before the SRB assay. Surviving fractions were calculated as: OD of irradiated cells/OD of nonirradiated cells. Each dose was carried out with 6 replicates, and the results are expressed as mean ± SEM. Two different clones for either mock-LNCaP or αvβ3-LNCaP were also tested to yield similar results. Each experiment was repeated at least two times. D, Survival curves of PC-3-NS, PC-3-β3-sh1, and PC-3-β3-sh2 were determined using clonogenic assay. Cells were plated in 6-cm plates at each radiation dose. Cells were incubated for 10–12 days in the culture. Survival of cells was scored with colonies with more than 50 cells. Each experiment was repeated three times. E, Downregulation of β3 integrin using shRNA containing lentivirus was confirmed by using immunoblot analysis with antibody specific to β3 integrin. β-Actin was used as a control.

Figure 1.

αvβ3 integrin expression increases survival of LNCaP cells in response to IR. A, Surface expression of ectopic αvβ3 integrin in αvβ3-LNCaP, mock-LNCaP, and parental LNCaP cells were analyzed using FACS. Cells were incubated with mAb specific to αvβ3 integrin (AP3) and mAb specific to HA epitope (12CA5) as a negative control. B, Cells were treated with 0 or 5 Gy and were lysed 48 hours after radiation. Proteins in cell lysates were separated in 10% polyacrylamide gels, immunoblotted, and probed with Ab to carboxyl end of β3 integrin (41). ERK1/2 is used as a loading control. C, Survival curves of mock-LNCaP and αvβ3-LNCaP were determined using colorimetric SRB assay. Cells (1,000/well) were plated in 96-well plates at each radiation dose. Cells were incubated for 6–7 days before the SRB assay. Surviving fractions were calculated as: OD of irradiated cells/OD of nonirradiated cells. Each dose was carried out with 6 replicates, and the results are expressed as mean ± SEM. Two different clones for either mock-LNCaP or αvβ3-LNCaP were also tested to yield similar results. Each experiment was repeated at least two times. D, Survival curves of PC-3-NS, PC-3-β3-sh1, and PC-3-β3-sh2 were determined using clonogenic assay. Cells were plated in 6-cm plates at each radiation dose. Cells were incubated for 10–12 days in the culture. Survival of cells was scored with colonies with more than 50 cells. Each experiment was repeated three times. E, Downregulation of β3 integrin using shRNA containing lentivirus was confirmed by using immunoblot analysis with antibody specific to β3 integrin. β-Actin was used as a control.

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αvβ3 increases survival of prostate cancer cells upon IR

The standard for radiosensitivity testing in vitro is the clonogenic assay by scoring colonies upon IR. However, LNCaP cells cannot be assessed with this traditional clonogenic assay because of cell density and poor plating efficiency (33). We performed clonogenic soft-agar assay and the colorimetric assay to analyze the radiosensitivity of cells transfected with αvβ3-LNCaP as compared with their mock counterparts. The colorimetric assay has been adopted in situations where colony counting cannot be performed, and Pauwels and colleagues have previously demonstrated that the colorimetric SRB assay generates similar radiation–dose response curve and radiosensitivity parameters as the clonogenic assay (25). Significantly improved survival of αvβ3-LNCaP was observed with radiation dose from 2 Gy to 10 Gy (P < 0.01; Fig. 1C left). To eliminate the possibility of transfectant artifact, two different clones of either mock-LNCaP or αvβ3-LNCaP were evaluated and show similar results.

PC-3, another prostate cancer cell line, contains endogenous β3 integrin. To see whether enhanced survival in αvβ3-LNCaP cells was due to the expression of β3 integrin, we downregulated β3 integrin in PC-3 cells using lentivirus-containing β3 integrin shRNA (Sh1 and Sh2 cells). Cells with β3 integrin downregulated up to 70% (Sh2; Fig. 1E) showed a significant sensitivity to IR starting at a dose as low as 4 Gy (Fig. 1D). Cells with β3 integrin downregulated for −52% showed a significant sensitivity to IR at higher doses (Sh1; Fig. 1D). The result suggested the role of β3 integrin in prevention of cell death from IR damage. There is no significant difference of αv- or β1-integrin level among PC-3 β3 integrin shRNA treated and the control cells (Supplementary Fig. S2)

To further validate the impact of αvβ3 on radioresistance and to eliminate the effect of adhesion, cell growth in soft agar was used to determine the anchorage-independent growth as a response to IR. Overexpression of αvβ3 integrin in LNCaP cells significantly increased the survival of cells in response to IR when cells were exposed to IR up to 10 Gy (P < 0.01; Fig. 2A). Radiation significantly decreased colony formation in both mock-LNCaP and αvβ3-LNCaP cells (P < 0.01). However, mock-LNCaP irradiated with 5 Gy completely failed to generate colonies in the soft agar, whereas irradiated αvβ3-LNCaP cells were still able to form colonies in the agar (Fig. 2A). The difference of colony formation between irradiated mock- and αvβ3-LNCaP is statistically significant (P < 0.01).

Figure 2.

αvβ3 integrin expression increases anchorage-independent growth of LNCaP or PC-3 cells and radioresistance to IR. A, Soft agar assay was used to determine anchorage-independent cell growth (left). Both mock- and αvβ3-LNCaP cells were irradiated up to 10 Gy. The experiment was repeated two times with two clones of αvβ3-LNCaP with similar results. The differences between irradiated and nonirradiated cells for both mock- and αvβ3-LNCaP are significant (P < 0.01). Representatives of anchorage-independent growth of LNCaP transfectants were presented (right). B, Soft agar assay was used to determine anchorage-independent cell growth. PC-3-NS, PC-3-β3-sh1, and PC-3-β3-sh2 were irradiated up to 8 Gy. The experiment was repeated two times. The differences between irradiated and nonirradiated cells for PC-3-NS and PC-3-β3-shRNA cells and PC-3-β3-sh2 are significant (P < 0.01) at high dose of IR (left). Representative images of anchorage-independent growth of LNCaP transfectants are presented (right).

Figure 2.

αvβ3 integrin expression increases anchorage-independent growth of LNCaP or PC-3 cells and radioresistance to IR. A, Soft agar assay was used to determine anchorage-independent cell growth (left). Both mock- and αvβ3-LNCaP cells were irradiated up to 10 Gy. The experiment was repeated two times with two clones of αvβ3-LNCaP with similar results. The differences between irradiated and nonirradiated cells for both mock- and αvβ3-LNCaP are significant (P < 0.01). Representatives of anchorage-independent growth of LNCaP transfectants were presented (right). B, Soft agar assay was used to determine anchorage-independent cell growth. PC-3-NS, PC-3-β3-sh1, and PC-3-β3-sh2 were irradiated up to 8 Gy. The experiment was repeated two times. The differences between irradiated and nonirradiated cells for PC-3-NS and PC-3-β3-shRNA cells and PC-3-β3-sh2 are significant (P < 0.01) at high dose of IR (left). Representative images of anchorage-independent growth of LNCaP transfectants are presented (right).

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Meanwhile, we also performed on clonogenic soft-agar assay using PC-3 cells infected with lentivirus carrying β3 integrin shRNA. Downregulation of β3 integrin in PC-3 cells leads to significantly enhanced radiosensitivity in PC-3 cells at high dose (Fig. 2B), which is again replicated in two different clones of PC-3 cells.

cRGD increases radiosensitivity of prostate cancer cells

cRGD is the first-generation αvβ3-selective cyclic peptide antagonist. Cilengitide was derived with similar purpose (with N-methylation modification) for increased antitumor activity (34). We used cRGD peptide for our in vitro testing and to illustrate αvβ3-specific relationship for radioresistance. We confirmed the specificity of the peptide to αvβ3 by blocking adhesion of αvβ3-LNCaP cells to VN but not to FN, which is generally αvβ1 mediated. On the other hand, control peptide, cRAD, does not affect αvβ3-mediated adhesion to VN (Fig. 3A). When the cells were treated with the peptides before irradiation and incubated afterward, the SRB assay revealed that cRGD has a synergistic effect with IR independent of the drug effect, with statistical significance at 2 and 4 Gy (P < 0.01; Fig. 3C). However, the peptide alone also had profound effect on survival, which might be at least partially due to its effect on adhesion (Fig. 3B). To exclude the confounding effect of adhesion, we performed soft-agar assay using cRGD to assess for its effect on anchorage-independent growth of αvβ3-LNCaP upon 5 Gy of IR. cRGD alone or IR with control cRAD did not completely inhibit colony formation, but combination of cRGD and IR produced dramatic inhibition of colony formation, which is statistically significant (P < 0.01) as compared with the control cRAD combined with IR (Fig. 3D and E). Similarly, we also performed the soft-agar assay on β3 integrin-expressing PC-3 cells treated with 2 μmol/L cRGD or cRAD in combination with IR. cRGD significantly enhanced sensitivity of cells upon IR at a high-dose of 8 Gy as a dose-dependent manner (Fig. 3F and G). Unlike αvβ3-LNCaP cells, 2 μmol/L cRGD has little effect on PC-3 cell survival at the lower doses of radiation (2–6 Gy) and the increased dose of cRGD (3 μmol/L) can sensitize PC-3 cells to 6 Gy (Supplementary Fig. S4) suggesting the heterogeneous response of cRGD treatment in different cell types in response to IR.

Figure 3.

cRGD increases radiosensitivity of αvβ3-LNCaP and PC-3 cells. A, cRGD peptide blocks αvβ3 integrin–mediated adhesion to VN, but not to FN (P < 0.01). Adhesion of αvβ3-LNCaP cells to FN or VN was performed in the presence or absence of cRGD peptide (1.74 μmol/L) or control peptide cRAD (1.74 μmol/L). Triplicate experiments were performed. B, SRB assay also showed that cRGD alone caused reduction of survival of αvβ3-LNCaP cells. Surviving fraction because of different treatments was calculated as OD of treated cells/OD of cells treated with control cRAD. cRGD (2 μmol/L) alone caused significant reduction of cell survival (P < 0.01). For comparison, the absolute surviving fraction of cells treated with IR (4 Gy) or combination of cRGD and IR (4 Gy) were shown. C, cRGD produced synergistic inhibition of αvβ3-LNCaP cell survival in combination with IR at dose-dependent manner. αvβ3-LNCaP cells were collected, counted, and incubated with either cRGD or control cRAD for 1 hour prior to IR. Immediately after IR, cells were plated in 96-well plates and incubated at 37 °C in 5% CO2 in the presence of the peptide. A fresh peptide–containing medium was exchanged every 3 days during the incubation. To determine the synergistic effect of peptide in combination with IR, survival curves were corrected for the drug effect of the peptide itself: surviving fraction = OD of irradiated cells with the peptide/OD of nonirradiated cells with the peptide. Results were achieved in two experiments using two clones of αvβ3-LNCaP. D, cRGD in combination with IR completely inhibits anchorage-independent growth of αvβ3-LNCaP cells. αvβ3-LNCaP or mock-LNCaP cells were incubated in the presence or absence of 2 μmol/L cRGD 1 hour prior to irradiation. After either 0 or 5 Gy, 5 × 104 cells were seeded in a 6-cm soft-agar dishes with duplicates. The experiment was repeated twice with two different clones of αvβ3-LNCaP with similar results. E, Representative images of anchorage-independent growth of LNCaP transfectants (0 Gy vs. 5 Gy) are presented. The scale bar (equivalent to 400 μm) was added using a software Freehand. F, Soft-agar assay was used to determine anchorage-independent cell growth. PC-3 cells were incubated in the presence or absence of 2 μmol/L cRGD 1 hour prior to irradiation up to 8 Gy. The experiment was repeated twice. The differences between cRGD and cRAD (control) treatments are significant at high dose of IR (P < 0.01). G, Representative images of anchorage-independent growth of PC-3 cells (0 Gy vs. 8 Gy) are presented. The scale bar equals to 400 μm as measured using a microscope.

Figure 3.

cRGD increases radiosensitivity of αvβ3-LNCaP and PC-3 cells. A, cRGD peptide blocks αvβ3 integrin–mediated adhesion to VN, but not to FN (P < 0.01). Adhesion of αvβ3-LNCaP cells to FN or VN was performed in the presence or absence of cRGD peptide (1.74 μmol/L) or control peptide cRAD (1.74 μmol/L). Triplicate experiments were performed. B, SRB assay also showed that cRGD alone caused reduction of survival of αvβ3-LNCaP cells. Surviving fraction because of different treatments was calculated as OD of treated cells/OD of cells treated with control cRAD. cRGD (2 μmol/L) alone caused significant reduction of cell survival (P < 0.01). For comparison, the absolute surviving fraction of cells treated with IR (4 Gy) or combination of cRGD and IR (4 Gy) were shown. C, cRGD produced synergistic inhibition of αvβ3-LNCaP cell survival in combination with IR at dose-dependent manner. αvβ3-LNCaP cells were collected, counted, and incubated with either cRGD or control cRAD for 1 hour prior to IR. Immediately after IR, cells were plated in 96-well plates and incubated at 37 °C in 5% CO2 in the presence of the peptide. A fresh peptide–containing medium was exchanged every 3 days during the incubation. To determine the synergistic effect of peptide in combination with IR, survival curves were corrected for the drug effect of the peptide itself: surviving fraction = OD of irradiated cells with the peptide/OD of nonirradiated cells with the peptide. Results were achieved in two experiments using two clones of αvβ3-LNCaP. D, cRGD in combination with IR completely inhibits anchorage-independent growth of αvβ3-LNCaP cells. αvβ3-LNCaP or mock-LNCaP cells were incubated in the presence or absence of 2 μmol/L cRGD 1 hour prior to irradiation. After either 0 or 5 Gy, 5 × 104 cells were seeded in a 6-cm soft-agar dishes with duplicates. The experiment was repeated twice with two different clones of αvβ3-LNCaP with similar results. E, Representative images of anchorage-independent growth of LNCaP transfectants (0 Gy vs. 5 Gy) are presented. The scale bar (equivalent to 400 μm) was added using a software Freehand. F, Soft-agar assay was used to determine anchorage-independent cell growth. PC-3 cells were incubated in the presence or absence of 2 μmol/L cRGD 1 hour prior to irradiation up to 8 Gy. The experiment was repeated twice. The differences between cRGD and cRAD (control) treatments are significant at high dose of IR (P < 0.01). G, Representative images of anchorage-independent growth of PC-3 cells (0 Gy vs. 8 Gy) are presented. The scale bar equals to 400 μm as measured using a microscope.

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αvβ3 prevents radiation-induced downregulation of survivin

Apoptosis is an active process characterized by programmed cell death in which a cascade of events is triggered in response to IR. In head and neck squamous cancer cells, cilengitide has been found to enhance radiation sensitivity through downregulation of Bcl-2 expression (35). Survivin is a member of the IAP gene family that plays an important role in antiapoptosis and cell-cycle division (17). Thus, we further investigated whether survivin is involved in the αvβ3-mediated survival of cells upon IR by looking at the expression levels of IAP members including survivin and XIAP as well as Bcl-XL. When mock-LNCaP cells were exposed to 5 Gy of IR, survivin expression was significantly downregulated (∼4-fold) 48 hours postirradiation while maintained in irradiated αvβ3-LNCaP cells (Fig. 4A). In contrast, the expression of XIAP (another important member of IAP) and Bcl-XL (a member of Bcl-2 family) were either stable or increased after radiation in both mock- and αvβ3-LNCAP cells (Fig. 4B and C). Taken together, expression of αvβ3 integrin in LNCaP cells prevents downregulation of survivin of cells in response to IR.

Figure 4.

αvβ3 integrin prevents downregulation of survivin in αvβ3-LNCaP cells upon IR. Overexpression of αvβ3 integrin prevents radiation-induced downregulation of survivin. Cells were exposed to 0 Gy or 5 Gy and harvested 48 hours postradiation. Proteins in cell lysates were separated in 15% polyacrylamide gels, immunoblotted, and probed with pAb specific to survivin (A), XIAP (B), or Bcl-XL (C). Experiment was confirmed at least two times with two different clones of the transfectants. ERK 1/2 was used as a loading control. D, Downregulation of β3-integrin does not change the survivin level at 5 Gy 48 hours postradiation as shown in immunoblots. α-Tubulin was used as a loading control. E, Downregulation of β3 integrin in PC-3 cells significantly reduces the survivin level and XIAP levels at 10 Gy 24–48 hours postradiation as shown in immunoblots. α-Tubulin was used as a loading control. Bcl-XL does not show changes.

Figure 4.

αvβ3 integrin prevents downregulation of survivin in αvβ3-LNCaP cells upon IR. Overexpression of αvβ3 integrin prevents radiation-induced downregulation of survivin. Cells were exposed to 0 Gy or 5 Gy and harvested 48 hours postradiation. Proteins in cell lysates were separated in 15% polyacrylamide gels, immunoblotted, and probed with pAb specific to survivin (A), XIAP (B), or Bcl-XL (C). Experiment was confirmed at least two times with two different clones of the transfectants. ERK 1/2 was used as a loading control. D, Downregulation of β3-integrin does not change the survivin level at 5 Gy 48 hours postradiation as shown in immunoblots. α-Tubulin was used as a loading control. E, Downregulation of β3 integrin in PC-3 cells significantly reduces the survivin level and XIAP levels at 10 Gy 24–48 hours postradiation as shown in immunoblots. α-Tubulin was used as a loading control. Bcl-XL does not show changes.

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We further investigated the effect of IR on survivin level using PC-3 cells with downregulated β3 integrin (Sh1 and Sh2). Unlike αvβ3-LNCaP cells, PC-3 cells did not show a change of survivin level when cells were exposed to 5 Gy of radiation (Fig. 4D). However, when the IR dose was increased to 10 Gy, PC-3 cells showed a significantly reduced level (30%–80%) of survivin (Fig. 4E).

RNA interference of survivin enhances the radiation-induced inhibition of anchorage-independent growth of αvβ3-LNCaP Cells

To investigate the role of survivin in αvβ3-mediated survival of LNCaP cells in response to IR, we disrupted survivin expression using siRNA or shRNA. For survivin siRNA, a titration experiment was performed to determine the appropriate siRNA dosages (ranging from 5 to 20 nmol/L) for radiation experiment. Survivin siRNA inhibited survivin levels in αvβ3-LNCaP at a dose-dependent manner (Supplementary Fig. S5A). We chose 10 nmol/L siRNA as the optimal concentration for further experiment because 10 nmol/L siRNA significantly reduced survivin level with minimal toxicity and partially inhibited cell growth (Supplementary Fig. S5B). The cells stably transfected with survivin shRNA also showed a significant downregulation of survivin level (Fig. 5B, right).

Figure 5.

Survivin siRNA or shRNA significantly enhances radiosensitivity of cells in anchorage-independent growth. A, Left, IR or survivin siRNA alone partially decreases colony formation in αvβ3-LNCaP cells. The combination of IR and survivin siRNA inhibits anchorage-independent growth (colony formation) of αvβ3-LNCaP cells. Results are displayed as mean ± SEM. Two independent experiments were performed, and similar results were achieved. The differences of colony size and number between irradiated and nonirradiated survivin siRNA transfected αvβ3-LNCaP cells are statistically significant (*, P < 0.01). Right, survivin siRNA (10 nmol/L) downregulates survivin expression in αvβ3-LNCaP cells upon IR. The control siRNA (NS) was used to compare the effect of survivin siRNA. All survivin siRNA–transfected cells were exposed to 0 or 5 Gy 48 hours after transfection. Akt was used as a loading control. B, Left, IR or survivin shRNA alone partially decreases colony formation in αvβ3-LNCaP cells. The combination of IR and survivin shRNA inhibits anchorage-independent growth (colony formation) of αvβ3-LNCaP cells. Results are displayed as mean ± SEM. Two independent experiments were performed, and similar results were achieved. The differences of colony number between irradiated and nonirradiated survivin shRNA–transfected αvβ3-LNCaP cells are statistically significant (*, P < 0.01). Right, Survivin shRNA downregulates survivin expression in αvβ3-LNCaP cells upon IR. The control shRNA (NS) was used to compare the effect of survivin shRNA. C, Survival curves of PC-3-β3-sh cells overexpressed with the plasmids containing either SV-WT or empty vector PCDNA3-HA (control) were determined using clonogenic assay. Cells were plated in 6-cm plates at each radiation dose. Cells were incubated for 10–12 days in the culture postradiation. Survival of cells was scored with the colonies containing more than 50 cells. D, Overexpression of SV-WT in PC-3-β3-sh cells significantly rescues the survivin level 10 Gy 24–48 hours postradiation as shown in immunoblots. α-Tubulin is used as a loading control.

Figure 5.

Survivin siRNA or shRNA significantly enhances radiosensitivity of cells in anchorage-independent growth. A, Left, IR or survivin siRNA alone partially decreases colony formation in αvβ3-LNCaP cells. The combination of IR and survivin siRNA inhibits anchorage-independent growth (colony formation) of αvβ3-LNCaP cells. Results are displayed as mean ± SEM. Two independent experiments were performed, and similar results were achieved. The differences of colony size and number between irradiated and nonirradiated survivin siRNA transfected αvβ3-LNCaP cells are statistically significant (*, P < 0.01). Right, survivin siRNA (10 nmol/L) downregulates survivin expression in αvβ3-LNCaP cells upon IR. The control siRNA (NS) was used to compare the effect of survivin siRNA. All survivin siRNA–transfected cells were exposed to 0 or 5 Gy 48 hours after transfection. Akt was used as a loading control. B, Left, IR or survivin shRNA alone partially decreases colony formation in αvβ3-LNCaP cells. The combination of IR and survivin shRNA inhibits anchorage-independent growth (colony formation) of αvβ3-LNCaP cells. Results are displayed as mean ± SEM. Two independent experiments were performed, and similar results were achieved. The differences of colony number between irradiated and nonirradiated survivin shRNA–transfected αvβ3-LNCaP cells are statistically significant (*, P < 0.01). Right, Survivin shRNA downregulates survivin expression in αvβ3-LNCaP cells upon IR. The control shRNA (NS) was used to compare the effect of survivin shRNA. C, Survival curves of PC-3-β3-sh cells overexpressed with the plasmids containing either SV-WT or empty vector PCDNA3-HA (control) were determined using clonogenic assay. Cells were plated in 6-cm plates at each radiation dose. Cells were incubated for 10–12 days in the culture postradiation. Survival of cells was scored with the colonies containing more than 50 cells. D, Overexpression of SV-WT in PC-3-β3-sh cells significantly rescues the survivin level 10 Gy 24–48 hours postradiation as shown in immunoblots. α-Tubulin is used as a loading control.

Close modal

We further studied whether downregulation of survivin can enhance sensitivity of cells upon IR. Soft-agar assay was used to determine anchorage-independent cell growth. After 10–12 days of incubation of cells treated by either survivin siRNA or shRNA ± IR, colonies larger than 100 μm were scored under a microscope. Although IR had a minor effect on the survivin level in either control (NS) or siRNA-infected cells (Fig. 5A, right), IR or survivin siRNA/shRNA alone partially reduced colony formation in αvβ3-LNCaP cells. The combination of IR and survivin siRNA/shRNA completely inhibits anchorage-independent growth in αvβ3-LNCaP cells (Fig. 5A and B, left).

Overexpression of survivin rescues cell survival in response to IR

Downregulation of β3 integrin enhanced loss of survivin in PC-3 cells (Fig. 4E) and subsequently survival of cells (Fig. 1D) in response to IR. We further asked whether overexpression of survivin in PC-3-αvβ3-shRNA cells could rescue the survival of cells upon radiation. Cells were transiently transfected with either the plasmid containing wild-type survivin (SV-WT) or empty plasmid vector as a control. As shown in Fig. 5C, cells transfected with SV-WT showed a significantly higher survival at the doses higher than 6 Gy. The survivin levels in cells postradiation were significantly lower (70% lower) in control cells in comparison with the cells that overexpressed SV-WT (30% lower; Fig. 5D). This suggests that the expression of survivin in αvβ3 knockdown cells promotes cell survival in response to IR.

The αvβ3 integrin is attracting increased attention as a target for cancer therapy; however, this is often related to its role in angiogenesis (12, 13). The effect of integrins on tumor response with conventional treatment modalities including radiotherapy is not understood, and the molecular signaling mechanism affecting cellular response to IR has not been defined. Our study demonstrates a novel finding that introduction of αvβ3 integrin significantly increases the radioresistance of prostate cancer cells. Inhibition of αvβ3 integrin by its antagonist cRGD reduces the radioprotective effect of the integrin. Furthermore, we showed that αvβ3 integrin regulates survivin level upon IR. Finally, disruption of survivin expression using siRNA significantly abolishes αvβ3 integrin–mediated radioresistance of prostate cancer cells. Thus, our study proposes a novel approach to target αvβ3 integrin prior to and/or during radiotherapy for patients with prostate cancer with integrin expression. Cilengitide, the cyclic peptide antagonist of αvβ3 integrin, has demonstrated a very tolerable toxicity profile in phase I trial, although the drug produced minimal tumor response in the limited number of patients tested (36). In our previous study, our laboratory has demonstrated that IR exhibits profound inhibitory effect on β1 integrin expression of prostate cancer cells and has impact on the adhesion of androgen-dependent prostate cancer cells treated with bicalutamide (37, 38). Inhibition of β1 integrin enhances the cytotoxicity in both prostate cancer and breast cancer cells exposed to IR (39, 40). Therefore, combining integrin-targeted drug with radiotherapy can potentially produce a synergistic effect. However, simply inhibiting integrin with antagonist may not be sufficient. Selectively targeting molecules of downstream signaling pathways may further enhance radiation kill of tumors and prevent cancer cell repopulation.

The relationship between αvβ3 integrin and survivin is novel from a mechanistic perspective. Survivin is a member of the IAP gene family. It plays an important role in the regulation of cellular apoptosis and cell-cycle division (17). It is highly expressed in most malignant tissues, and its expression has been linked to resistance to radiotherapy and chemotherapy. Chakravarti and colleagues have previously shown that radioresistant glioblastoma cell lines have higher level of survivin with additional upregulation after irradiation. The inhibition of survivin function with dominant-negative survivin increases the radiation response of the more radioresistant cell lines. They reported that the mechanism of survivin-mediated radioresistance is caspase-independent and related to enhanced double-strand DNA repair (20). In our data, αvβ3 integrin maintained survivin expression after IR, which would be otherwise downregulated in the absence of the αvβ3 integrin. In contrast, αvβ3 integrin itself does not increase survivin levels in the absence of radiation (41). Radiation-induced increase of survivin levels is found in several tumor cells which also endogenously express αvβ3 integrin. By maintaining survivin expression after irradiation (as shown in Fig. 4), αvβ3 integrin increases the likelihood of cells to survive posttreatment and leads to a more radioresistant phenotype (as shown in Figs. 1 and 2). This is further supported by our rescue experiment whereby reintroduction of survivin into PC-3 cells that were treated with αvβ3 shRNA enhances cell survival in response to radiation. To our knowledge, such observation linking αvβ3 integrin with survivin has not been reported, and the pronounced effect seen only after irradiation may open a new area of translational research. For example, upregulation of survivin may work in synchrony with G2-checkpoint regulation to enhance cellular survival because (i) αvβ3-LNCaP cells show significant increase in G2–M population after IR; (ii) G2 delay of a variety of cell lines is associated with increasing radioresistance (42); and (iii) αvβ3 integrin upregulates CDC2, which is the key regulator for maintaining G2 arrest (10).

The link between integrin-mediated signaling pathway and survivin expression has been found in a variety of cell types. In keratinocyte stem cells, inhibition of β1 integrin signaling using anti-β1 antibody downregulates SV-WT expression and induces anoikis (43). Treatment of Keratin 8(-/-) mice with the anti-β1 integrin antibody upregulates survivin expression with decreased activation of caspases (44). In pancreatic cancer cells chemoresistant to gemcitabine, the constitutive FAK activation induced by laminin (a β1 integrin ligand) has been shown to be required for AKT activation that mediates an increased expression of survivin (45). In osteosarcoma MG64 cells, knockdown of survivin expression inhibits the invasion and migration of osteosarcoma in vitro and the expression of α5 integrin on cell surface (46). In melanoma, knockdown of α5 integrin with siRNA abrogates the enhanced tumor growth of melanoma cells overexpressed with survivin. Xenograft of survivin-overexpressing cells in mice demonstrated increased α5 integrin level in both the primary tumor and metastatic colonies in the pulmonary parenchyma (47). However, how integrin regulates survivin expression is still not understood. In prostate cancer, adhesion of PC-3 cells to fibronectin via β1 integrin upregulates survivin expression. This prevents cells from apoptosis induced by TNFα in which the regulation of survivin level is mediated by protein kinase B/AKT mechanism (48). Moreover, overexpression of β6 integrin leads to enhancement of survivin level through JNK pathway while inhibition of JNK activity with JBD significantly abolished the expression of survivin (41). In glioma cells, α5β1 integrin cross-talks with p53 pathway in regulation of survivin and PEA-15 (49). Although similar mechanisms may be predicted for this pathway, the regulation of β3 integrin on survivin level in prostate cancer cells in response to IR remains to be explored.

In conclusion, the data observed in androgen-dependent prostate cancer cell lines with endogenous β1 integrin expression provide evidence for αvβ3 integrin as a promoter of radioresistance through regulation of survivin expression that subsequently may contribute to the radioresistance of prostate cancer. Our results and other articles suggest the complexity of prostate cancers in which the differential expression of molecular profile, that is, integrins and antiapoptotic molecules, plays an important role in regulation of various survival pathways in the heterogeneous subpopulations of prostate cancer cells. Future investigation on the signaling pathways downstream of integrins and regulation of antiapoptotic molecules should be continued to guide the design of novel therapeutic treatments with different combination of radiotherapy, including αvβ3 peptide antagonist and survivin antagonist.

No potential conflicts of interest were disclosed.

Conception and design: T. Wang, J. Huang, M. Vue, H.L. Goel, L.R. Languino, T.J. FitzGerald

Development of methodology: T. Wang, J. Huang, M. Vue, H.L. Goel, L.R. Languino, T.J. FitzGerald

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Wang, J. Huang, M.R. Alavian, L.R. Languino

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Wang, J. Huang, M. Vue, M.R. Alavian, L.R. Languino, T.J. FitzGerald

Writing, review, and/or revision of the manuscript: T. Wang, J. Huang, M. Vue, M.R. Alavian, H.L. Goel, D.C. Altieri, L.R. Languino, T.J. FitzGerald

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Wang, L.R. Languino, T.J. FitzGerald

Study supervision: T. Wang, L.R. Languino

The authors would like to thank Diane Safer, Thomas Lozeau, Marie Marley, Ginette Bailey, and Julie Trifone at the Department of Radiation Oncology for performing radiation and for their effort and patience, which made this study possible. We appreciate Huimin Lu for reading the manuscript and giving helpful suggestions. This work was supported by grants from NIH PO1 CA140043 (to L.R. Languino and D.C. Altieri), by a grant from Our Danny Cancer Fund P00010003300000 (to T. Wang) and ACS-IRG 93-033 (to H.L. Goel). M. Vue was supported by the NIH Summer Research Fellowship program at UMass Medical School. This project was also funded, in part, by a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.).

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.

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2018.
CA Cancer J Clin
2018
;
68
:
7
30
.
2.
Parker
CC
,
Dearnaley
DP
. 
Radical radiotherapy for prostate cancer.
Cancer Treat Rev
2003
;
29
:
161
9
.
3.
Catton
C
,
Milosevic
M
,
Warde
P
,
Bayley
A
,
Crook
J
,
Bristow
R
, et al
Recurrent prostate cancer following external beam radiotherapy: follow-up strategies and management.
Urol Clin North Am
2003
;
30
:
751
63
.
4.
Bromfield
GP
,
Meng
A
,
Warde
P
,
Bristow
RG
. 
Cell death in irradiated prostate epithelial cells: role of apoptotic and clonogenic cell kill.
Prostate Cancer Prostatic Dis
2003
;
6
:
73
85
.
5.
Wang
T
,
Languino
LR
,
Lian
J
,
Stein
G
,
Blute
M
,
Fitzgerald
TJ
. 
Molecular targets for radiation oncology in prostate cancer.
Front Oncol
2011
;
1
:
17
.
6.
Hynes
RO
. 
Integrins: bidirectional, allosteric signaling machines.
Cell
2002
;
110
:
673
87
.
7.
Pampori
N
,
Hato
T
,
Stupack
DG
,
Aidoudi
S
,
Cheresh
DA
,
Nemerow
GR
, et al
Mechanisms and consequences of affinity modulation of integrin αvβ3 detected with a novel patch-engineered monovalent ligand.
J Biol Chem
1999
;
274
:
21609
16
.
8.
Felding-Habermann
B
,
Fransvea
E
,
O'Toole
TE
,
Manzuk
L
,
Faha
B
,
Hensler
M
. 
Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells.
Clin Exp Metastasis
2002
;
19
:
427
36
.
9.
Zheng
DQ
,
Woodard
AS
,
Fornaro
M
,
Tallini
G
,
Languino
LR
. 
Prostatic carcinoma cell migration via αvβ3 integrin is modulated by a focal adhesion kinase pathway.
Cancer Res
1999
;
59
:
1655
64
.
10.
Manes
T
,
Zheng
DQ
,
Tognin
S
,
Woodard
AS
,
Marchisio
PC
,
Languino
LR
. 
αvβ3 integrin expression up-regulates cdc2, which modulates cell migration.
J Cell Biol
2003
;
161
:
817
26
.
11.
Gruber
G
,
Hess
J
,
Stiefel
C
,
Aebersold
DM
,
Zimmer
Y
,
Greiner
RH
, et al
Correlation between the tumoral expression of β3-integrin and outcome in cervical cancer patients who had undergone radiotherapy.
Br J Cancer
2005
;
92
:
41
6
.
12.
Abdollahi
A
,
Griggs
DW
,
Zieher
H
,
Roth
A
,
Lipson
KE
,
Saffrich
R
, et al
Inhibition of αvβ3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy.
Clin Cancer Res
2005
;
11
:
6270
9
.
13.
Albert
JM
,
Cao
C
,
Geng
L
,
Leavitt
L
,
Hallahan
DE
,
Lu
B
. 
Integrin αvβ3 antagonist cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models.
Int J Radiat Oncol Biol Phys
2006
;
65
:
1536
43
.
14.
Mikkelsen
T
,
Brodie
C
,
Finniss
S
,
Berens
ME
,
Rennert
JL
,
Nelson
K
, et al
Radiation sensitization of glioblastoma by cilengitide has unanticipated schedule dependency.
Int J Cancer
2009
;
124
:
2719
27
.
15.
Alva
A
,
Slovin
S
,
Daignault
S
,
Carducci
M
,
Dipaola
R
,
Pienta
K
, et al
Phase II study of cilengitide (EMD 121974, NSC 707544) in patients with nonmetastatic castration-resistant prostate cancer, NCI-6735. A study by the DOD/PCF prostate cancer clinical trials consortium.
Invest New Drugs
2012
;
30
:
749
57
.
16.
Wang
F
,
Chen
L
,
Zhang
R
,
Chen
Z
,
Zhu
L
. 
RGD peptide conjugated liposomal drug delivery system for enhance therapeutic efficacy in treating bone metastasis from prostate cancer.
J Control Release
2014
;
196
:
222
33
.
17.
Altieri
DC
. 
Survivin, versatile modulation of cell division and apoptosis in cancer.
Oncogene
2003
;
22
:
8581
9
.
18.
Zhang
M
,
Mukherjee
N
,
Bermudez
RS
,
Latham
DE
,
Delaney
MA
,
Zietman
AL
, et al
Adenovirus-mediated inhibition of survivin expression sensitizes human prostate cancer cells to paclitaxel in vitro and in vivo.
Prostate
2005
;
64
:
293
302
.
19.
Rodel
C
,
Haas
J
,
Groth
A
,
Grabenbauer
GG
,
Sauer
R
,
Rodel
F
. 
Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: survivin as a radioresistance factor.
Int J Radiat Oncol Biol Phys
2003
;
55
:
1341
7
.
20.
Chakravarti
A
,
Zhai
GG
,
Zhang
M
,
Malhotra
R
,
Latham
DE
,
Delaney
MA
, et al
Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms.
Oncogene
2004
;
23
:
7494
506
.
21.
Adida
C
,
Crotty
PL
,
McGrath
J
,
Berrebi
D
,
Diebold
J
,
Altieri
DC
. 
Developmentally regulated expression of the novel cancer antiapoptosis gene survivin in human and mouse differentiation.
Am J Pathol
1998
;
152
:
43
9
.
22.
Sarela
AI
,
Macadam
RC
,
Farmery
SM
,
Markham
AF
,
Guillou
PJ
. 
Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma.
Gut
2000
;
46
:
645
50
.
23.
Yamashita
S
,
Masuda
Y
,
Kurizaki
T
,
Haga
Y
,
Murayama
T
,
Ikei
S
, et al
Survivin expression predicts early recurrence in early-stage breast cancer.
Anticancer Res
2007
;
27
:
2803
8
.
24.
Goel
HL
,
Fornaro
M
,
Moro
L
,
Teider
N
,
Rhim
JS
,
King
M
, et al
Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by β1 integrins.
J Cell Biol
2004
;
166
:
407
18
.
25.
Pauwels
B
,
Korst
AE
,
de Pooter
CM
,
Pattyn
GG
,
Lambrechts
HA
,
Baay
MF
, et al
Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.
Cancer Chemother Pharmacol
2003
;
51
:
221
6
.
26.
Goel
HL
,
Breen
M
,
Zhang
J
,
Das
I
,
Aznavoorian-Cheshire
S
,
Greenberg
NM
, et al
β1A integrin expression is required for type 1 insulin-like growth factor receptor mitogenic and transforming activities and localization to focal contacts.
Cancer Res
2005
;
65
:
6692
700
.
27.
Yatohgo
T
,
Izumi
M
,
Kashiwagi
H
,
Hayashi
M
. 
Novel purification of vitronectin from human plasma by heparin affinity chromatography.
Cell Struct Funct
1988
;
13
:
281
92
.
28.
Engvall
E
,
Ruoslahti
E
. 
Binding of soluble form of fibroblast surface protein, fibronectin, to collagen.
Int J Cancer
1977
;
20
:
1
5
.
29.
Beltrami
E
,
Plescia
J
,
Wilkinson
JC
,
Duckett
CS
,
Altieri
DC
. 
Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis.
J Biol Chem
2004
;
279
:
2077
84
.
30.
Xia
F
,
Canovas
PM
,
Guadagno
TM
,
Altieri
DC
. 
A survivin–ran complex regulates spindle formation in tumor cells.
Mol Cell Biol
2008
;
28
:
5299
311
.
31.
Albert
JM
,
Cao
C
,
Geng
L
,
Leavitt
L
,
Hallahan
DE
,
Lu
B
. 
Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non–small cell lung cancer models.
Int J Radiat Oncol Biol Phys
2006
;
65
:
1536
43
.
32.
Wild-Bode
C
,
Weller
M
,
Rimner
A
,
Dichgans
J
,
Wick
W
. 
Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma.
Cancer Res
2001
;
61
:
2744
50
.
33.
Leith
JT
. 
In vitro radiation sensitivity of the LNCaP prostatic tumor cell line.
Prostate
1994
;
24
:
119
24
.
34.
Mas-Moruno
C
,
Rechenmacher
F
,
Kessler
H
. 
Cilengitide: the first anti-angiogenic small-molecule drug candidate design, synthesis, and clinical evaluation.
Anticancer Agents Med Chem
2010
;
10
:
753
68
.
35.
Heiduschka
G
,
Lill
C
,
Schneider
S
,
Seemann
R
,
Kornek
G
,
Schmid
R
, et al
The effect of cilengitide in combination with irradiation and chemotherapy in head and neck squamous cell carcinoma cell lines.
Strahlenther Onkol
2014
;
190
:
472
9
.
36.
Eskens
FA
,
Dumez
H
,
Hoekstra
R
,
Perschl
A
,
Brindley
C
,
Bottcher
S
, et al
Phase I and pharmacokinetic study of continuous twice weekly intravenous administration of cilengitide (EMD 121974), a novel inhibitor of the integrins alphavbeta3 and alphavbeta5 in patients with advanced solid tumours.
Eur J Cancer
2003
;
39
:
917
26
.
37.
Simon
EL
,
Goel
HL
,
Teider
N
,
Wang
T
,
Languino
LR
,
Fitzgerald
TJ
. 
High dose fractionated ionizing radiation inhibits prostate cancer cell adhesion and β1 integrin expression.
Prostate
2005
;
64
:
83
91
.
38.
Wang
T
,
Alavian
MR
,
Goel
HL
,
Languino
LR
,
Fitzgerald
TJ
. 
Bicalutamide inhibits androgen-mediated adhesion of prostate cancer cells exposed to ionizing radiation.
Prostate
2008
;
68
:
1734
42
.
39.
Goel
HL
,
Sayeed
A
,
Breen
M
,
Zarif
MJ
,
Garlick
DS
,
Leav
I
, et al
Beta1 integrins mediate resistance to ionizing radiation in vivo by inhibiting c-Jun amino terminal kinase 1.
J Cell Physiol
2013
;
228
:
1601
9
.
40.
Park
CC
,
Zhang
HJ
,
Yao
ES
,
Park
CJ
,
Bissell
MJ
. 
β1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts.
Cancer Res
2008
;
68
:
4398
405
.
41.
Lu
H
,
Wang
T
,
Li
J
,
Fedele
C
,
Liu
Q
,
Zhang
J
, et al
Alphavbeta6 integrin promotes castrate-resistant prostate cancer through JNK1-mediated activation of androgen receptor.
Cancer Res
2016
;
76
:
5163
74
.
42.
McKenna
WG
,
Iliakis
G
,
Weiss
MC
,
Bernhard
EJ
,
Muschel
RJ
. 
Increased G2 delay in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc.
Radiat Res
1991
;
125
:
283
7
.
43.
Marconi
A
,
Dallaglio
K
,
Lotti
R
,
Vaschieri
C
,
Truzzi
F
,
Fantini
F
, et al
Survivin identifies keratinocyte stem cells and is downregulated by anti-beta1 integrin during anoikis.
Stem Cells
2007
;
25
:
149
55
.
44.
Habtezion
A
,
Toivola
DM
,
Asghar
MN
,
Kronmal
GS
,
Brooks
JD
,
Butcher
EC
, et al
Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon.
Proc Natl Acad Sci U S A
2011
;
108
:
1445
50
.
45.
Huanwen
W
,
Zhiyong
L
,
Xiaohua
S
,
Xinyu
R
,
Kai
W
,
Tonghua
L
. 
Intrinsic chemoresistance to gemcitabine is associated with constitutive and laminin-induced phosphorylation of FAK in pancreatic cancer cell lines.
Mol Cancer
2009
;
8
:
125
.
46.
Liu
XZ
,
Li
CJ
,
Wu
SJ
,
Shi
X
,
Zhao
JN
. 
Involvement of alpha5 integrin in survivin-mediated osteosarcoma metastasis.
Asian Pac J Trop Med
2016
;
9
:
478
83
.
47.
McKenzie
JA
,
Liu
T
,
Jung
JY
,
Jones
BB
,
Ekiz
HA
,
Welm
AL
, et al
Survivin promotion of melanoma metastasis requires upregulation of alpha5 integrin.
Carcinogenesis
2013
;
34
:
2137
44
.
48.
Fornaro
M
,
Plescia
J
,
Chheang
S
,
Tallini
G
,
Zhu
YM
,
King
M
, et al
Fibronectin protects prostate cancer cells from tumor necrosis factor α-induced apoptosis via the AKT/Survivin pathway.
J Biol Chem
2003
;
278
:
50402
11
.
49.
Renner
G
,
Janouskova
H
,
Noulet
F
,
Koenig
V
,
Guerin
E
,
Bar
S
, et al
Integrin alpha5beta1 and p53 convergent pathways in the control of antiapoptotic proteins PEA-15 and survivin in high-grade glioma.
Cell Death Differ
2016
;
23
:
640
53
.