α_vβ₃ Integrin Mediates Radioresistance of Prostate Cancer Cells through Regulation of Survivin Free

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, α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ integrin may promote radioresistance of a tumor. In 2005, Gruber and colleagues reported that patients with cervical cancer with α_vβ₃ expression had significantly worse local control, metastasis, and survival after curative radiotherapy (11). Also in 2005, Abdollahi and colleagues demonstrated that S247 (an α_vβ₃ petidomimetic antagonist) potentiates antiangiogenic effect of ionizing radiation (IR) on endothelial cells and xenograft tumors (12). In 2006, Albert and colleagues demonstrated that cilengitide (α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ integrin.

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-α_v 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.

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 (α_vβ₃-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.

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 α_v, 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).

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).

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.

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.

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.

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).

Silencing survivin using siRNA transfection was performed as described previously (29). Briefly, mock- and α_vβ₃-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.

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.

To determine whether α_vβ₃ 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 α_v and β₁ integrin and determined whether there is a difference between mock- and α_vβ₃-LNCaP cells. Our data showed that no difference of β₁ integrin between mock- and α_vβ₃-LNCaP cells. However, there is a significant increase of α_v integrin level in α_vβ₃-LNCaP than in mock-LNCaP (Supplementary Fig. S1). Because radiation has been shown to upregulate α_vβ₃ in endothelial cells, NSCLC, and glioblastoma cells (31, 32), we examined this integrin expression in the transfectants after radiation. No change of α_vβ₃ expression level was observed in cells upon IR as shown by immunoblotting (Fig. 1B).

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 α_vβ₃-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 α_vβ₃-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 α_vβ₃-LNCaP were evaluated and show similar results.

PC-3, another prostate cancer cell line, contains endogenous β₃ integrin. To see whether enhanced survival in α_vβ₃-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 α_v- or β₁-integrin level among PC-3 β₃ integrin shRNA treated and the control cells (Supplementary Fig. S2)

To further validate the impact of α_vβ₃ 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 α_vβ₃ 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 α_vβ₃-LNCaP cells were still able to form colonies in the agar (Fig. 2A). The difference of colony formation between irradiated mock- and α_vβ₃-LNCaP is statistically significant (P < 0.01).

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 is the first-generation α_vβ₃-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 α_vβ₃-specific relationship for radioresistance. We confirmed the specificity of the peptide to α_vβ₃ by blocking adhesion of α_vβ₃-LNCaP cells to VN but not to FN, which is generally α_vβ₁ mediated. On the other hand, control peptide, cRAD, does not affect α_vβ₃-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 α_vβ₃-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 α_vβ₃-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.

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 α_vβ₃-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 α_vβ₃-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 α_vβ₃-LNCAP cells (Fig. 4B and C). Taken together, expression of α_vβ₃ integrin in LNCaP cells prevents downregulation of survivin of cells in response to IR.

We further investigated the effect of IR on survivin level using PC-3 cells with downregulated β₃ integrin (Sh1 and Sh2). Unlike α_vβ₃-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).

To investigate the role of survivin in α_vβ₃-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 α_vβ₃-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).

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 α_vβ₃-LNCaP cells. The combination of IR and survivin siRNA/shRNA completely inhibits anchorage-independent growth in α_vβ₃-LNCaP cells (Fig. 5A and B, left).

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-α_vβ₃-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 α_vβ₃ knockdown cells promotes cell survival in response to IR.

The α_vβ₃ 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 α_vβ₃ integrin significantly increases the radioresistance of prostate cancer cells. Inhibition of α_vβ₃ integrin by its antagonist cRGD reduces the radioprotective effect of the integrin. Furthermore, we showed that α_vβ₃ integrin regulates survivin level upon IR. Finally, disruption of survivin expression using siRNA significantly abolishes α_vβ₃ integrin–mediated radioresistance of prostate cancer cells. Thus, our study proposes a novel approach to target α_vβ₃ integrin prior to and/or during radiotherapy for patients with prostate cancer with integrin expression. Cilengitide, the cyclic peptide antagonist of α_vβ₃ 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 α_vβ₃ 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, α_vβ₃ integrin maintained survivin expression after IR, which would be otherwise downregulated in the absence of the α_vβ₃ integrin. In contrast, α_vβ₃ 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 α_vβ₃ integrin. By maintaining survivin expression after irradiation (as shown in Fig. 4), α_vβ₃ 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 α_vβ₃ shRNA enhances cell survival in response to radiation. To our knowledge, such observation linking α_vβ₃ 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) α_vβ₃-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) α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ 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.