αV Integrin Induces Multicellular Radioresistance in Human Nasopharyngeal Carcinoma via Activating SAPK/JNK Pathway

Background Tumor cells acquire the capacity of resistance to chemotherapy or radiotherapy via cell–matrix and cell–cell crosstalk. Integrins are the most important cell adhesion molecules, in which αV integrin mainly mediating the tight contact between tumor cells. Methodology/Principal Findings To investigate the role of αV integrin in multi-cellular radioresistance (MCR) of human nasopharyngeal carcinoma (NPC), we performed immunohistochemistry and Western blotting to find that the expression of αV integrin in the tumor tissue of radioresistant patients is much higher than that in radiosensitive patients. In vitro, we cultured human NPC cell line CNE-2 cells as multi-cellular spheroids (MCSs) or as monolayer cells (MCs), and found that the expression of αV integrin in MCSs is significantly higher than that in MCs. MTT, flow cytometry and clonogenic suvival assays showed that MCSs are less sensitive to X-ray irradiation than MCs while blocking of αV integrin in MCSs dramatically reversed their radioresistance. Furthermore, as detected by Western blotting, MCSs displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway in presence of irradiation. Blocking of αV integrin in MCSs decreased the expression of phosphorylated JNK. Additionally, blocking of SAPK/JNK signaling pathway synergistically induced apoptosis of MCSs exposed to irradiation by increasing the expression of cleaved caspase-3. In vivo, we found that irradiation combined with αV integrin blocking treatment significantly enhanced the radiosensitivity of NPC xenografts. Conclusions Our results indicate a novel role of αV integrin in multi-cellular radioresistance of NPCs.


Introduction
Nasopharyngeal carcinoma is the most common malignancy of head and neck in Southeast Asia, and radiotherapy is the most effective treatment [1]. Nevertheless, radioresistance still occurs in a high proportion of NPC patients, which is the main risk factor contributing to poor prognosis [2]. Thus understanding of the molecular mechanisms underlying radioresistance may provide opportunity to develop more effective anti-cancer strategy. The previous researches about tumor radiosensitivity mainly focus on a single tumor cell, disregarding the fact that in tumor mass, tumor cells acquire some new characteristics by interacting with each other to become more resistant to chemo-or radiotherapy, termed multi-cellular resistance (MCR) [3].
Integrins are critical cell adhesion molecules mediating the crosstalk between tumor cells and participating in cell invasion, metastasis, angiogenesis, cell survival and some other important biological behaviors of tumor cells [4][5][6][7][8]. More specifically, aV integrin is expressed in most cancer cells playing an essential role mediating cell-matrix and cell-cell interactions. Meanwhile, aV integrin is a key molecule contributing to cell proliferation and apoptosis [9][10][11]. Given the correlations between apoptosis and radiosensitivity, We then hypothesized that aV integrin may act as a pivotal factor inducing radioresisitance in NPCs.
In this study, we tested the hypothesis that aV integrin may cause multi-cellular radioresistance of NPC in a three-dimensional culture condition mimicking a tumor microenvironment, and we found that aV integrin expression is required for sustaining multicellular radioresistance in human NPC cell line CNE-2. Furthermore, we demonstrated that SAPK/JNK signaling pathway was involved in aV integrin mediated radioresistance. Our finding for the first time shows the important role of aV integrin in multicellular radioresistance of nasopharyngeal carcinomas.

Results
aV Integrin is Highly Expressed in NPC Tissues of Radioresistant Patient, and is Correlated to the

Expressions of Apoptosis Related Genes
To determine whether the expressions of aV integrin of NPC tumors are different in patients with different radiosensitivity, immunohistochemical technique was performed to detect the expressions of av integrin in the 105 cases of tumor tissues and 20 cases of adjacent tissues. The positive expressions of av integrin in NPC tumor tissues were shown to be significantly higher than those in the adjacent tissues. The expression of av integrin are correlated to the differentiation degree of cancer cells and lymph node metastases (p,0.01), but not correlated to the patient's gender, age, tumor location or tumor size (p.0.05) ( Table 1). We also found that the expressions of aV integrin in radioresisitant patients are much higher than those of radiosensitive patients (Figure 1 A, B) and the levels of av integrin are highly correlated with the Objective Response Rate (ORR) of NPCs (Table 2). Given apoptosis is an unarguably common pathway to cell death initiating from irradiation. We speculate that aV integrin may affect the levels of apoptotic genes. We therefore measured the expressions of cleaved Caspase-3 and cleaved PARP in these 105 cases of NPC patients, and found that the expressions of aV integrin are negatively correlated with the levels of cleaved Caspase-3 and PARP (P,0.01) ( Table 3).

aV Integrin is Differently Expressed in MCSs and MCs
It has been demonstrated in our previous study that aV integrin is a critical factor mediating MCR to chemotherapy in MCSs [12]. We therefore hypothesized that the expression of aV integrin in NPC MCSs and MCs may be different. MCSs are cultured as previously described [12] (Fig. 2 A). As detected by flow cytometry assay and Western blot. The expression of aV integrin is much higher in MCSs than that in MCs ( Fig. 2 B, C).

Blocking the Function of aV Integrin Reversed Radioresistance of MCSs
To determine if aV integrin is critical in multi-cellular radioresistance, we compared the cell survival rate of different groups with or without aV integrin function blockade in the presence of irradiation. It showed that blocking the function of aV integrin dramatically increased the radiosensitivity of MCSs (Fig. 3A), and more intriguingly, no changes of radiosensitivity were detected in MCs even after aV integrin blockade (Fig. 3A). Clonogenic survival assay was also performed to measure the radiation response. As shown in Figure 3 B, in the presence of irradiation, blocking the function of aV integrin in MCSs resulted in a significantly increased radiosensitivity relative to the control groups, indicating that aV integrin critically contribute to the radioresistance of MCSs. Meanwhile, aV integrin blocked MCSs resulted in a substantially decreased cell survival (Fig. 3C) and increased apoptosis (Fig. 3D) when exposed to 2 Gy fractionated irradiation. Additionally, the expressions of apoptotic genes cleaved Caspase-3 and cleaved PARP were found to be increased significantly in aV integrin blocked MCSs (Fig. 3E).

SAPK/JNK Pathway is Involved in aV Integrin Mediated Multi-cellular Radioresistance of NPC MCSs
Irradiation is a stress inducing apoptosis in cancer cells, and it is well known that SAPK/JNK pathway is a critical signaling activated by stress. To determine the mechanism mediating aV integrin's inhibitory function on apoptosis, we investigated the effect of aV integrin on SAPK/JNK signaling pathways in MCSs. Western blotting showed that SAPK/JNK was substantially phosphorylated in MCSs of CNE-2 cells in response to irradiation (Fig. 4A). Blocking the function of aV integrin in MCSs substantially decreased the expression of phosphorylated JNK (Fig. 4B), and blocking of SAPK/JNK pathway increased the expression of cleaved casepase3 (Fig. 4C). Flow cytometry assay also showed that irradiation induced apoptosis of MCSs was increased by blocking SAPK/JNK pathway (Fig. 4D).

aV Integrin Blocking Enhances the Radiosensitivity of NPC Xenografts
To further confirm the effect of aV integrin on radiosensitivity of NPCs, we injected equal number (1.0610 6 per mouse) of CNE-2 cells subcutaneously into nude mice (3 mice for each group). The mice were exposed to 6 Gy fractionated irradiation, and a peritumoral injection of aV integrin blocking peptide or isotype blocking peptide (10 mg/kg, twice a week) were also administrated when the xenografts reached a mean diameter of 0.8-1.0 cm. The xenografts were excised and weighed 3 weeks after treatment. As shown in Fig. 5A and Fig. 5B, aV integrin blockade synergistically increased the effect of irradiation on xenografts. Xenografts were then fixed with 2% paraformaldehyde and dissected into sections at 8 mm. Immunochemistry staining of TUNEL was performed and found that the apoptosis of tumor in aV integrin blockade combined group is significantly higher than that in control groups (Fig. 5C). All of these indicate that aV integrin blockade may increase radiosensitivity of NPCs.

Discussion
Previously, our group have found that downregulation of aV integrin promoted drug sensitivity in colorectal carcinoma multicellular spheroids [12]. We therefore propose that loss of aV integrin function also enhances multi-cellular radiosensitivity. Our present study shows that aV integrin also contributes to multicellular radioresistance in NPCs by exacerbating irradiation induced apoptosis. More significantly, the expressions of aV integrin in human NPC tumors negatively correlate to the levels of apoptosis related genes, highlighting the potential role of aV integrin-mediated anti-apoptosis reprogramming in human NPCs. Taken together, our data provide a mechanism whereby aV integrin acting as a tumor protector by regulating multi-cellular radioresistance in NPCs.
Our findings are consistent with the previous work showing that anti-aV integrin can enhance the efficacy of radiation therapy and reduce metastasis of human cancer xenografts in nude mice [13,14]. More importantly and intriguingly, in our study, we present data to demonstrate that blocking the function of aV integrin in monolayers has little effect on their response to irradiation, indicating that aV integrin is only crucial for multi-cellular spheroids or biomass tumor in vivo. Furthermore, our studies have shed light on the mechanism through which aV integrin regulating apoptosis. Factors activating aV integrin are extensive, including intra-and extra-cellular factors, such as cytoskeleton, fibronectin, virus, force, shear stress, cell-cell adhesion, and cell-ECM adhesion [15][16][17][18][19]. In MCSs, cells adhere with each other and cell-cell junctions exist generally, leading to the hypothesis that aV integrin may be activated by cell-cell adhesion in MCSs and biomass tumor [20,21]. Otherwise, cell adhesion might provide a precondition for facilitators to activate aV integrin. aV integrin has been thought of as a cell adhesion receptor regulating signal transduction pathways of cell   proliferation, survival and apoptosis [22,23]. Given cell proliferation, survival, and apoptosis are three of the most critical factors impacting radiosensitivity. [24][25][26]. This may be in part of the mechanism of activation of aV integrin in MCR. Apoptosis is an unarguably common pathway to cell death initiating from irradiation [27], and NF-kB and JNK2 are two of the most important apoptotic factors, especially underlying stress [28]. It has already been demonstrated that aV integrin can activate NF-kB and inactivate JNK in some kinds of cells [29]. As a result in our study, we found that blocking SAPK/JNK pathway reversed radioresistance in MCSs, indicating that SAPK/JNK pathway is critical mediating MCR. It has been reported that SAPK/JNK pathway can be dramatically activated by endoplasmic reticulum stress (ER stress) [30,31] and endoplasmic reticulum is well known to be the compartment of protein synthesis, including apoptotic related proteins. This correlation may explain how aV integrin blocking results in an increased expression of caspase 3 and PARP. Although we can not draw a conclusion that SAPK/JNK pathway is the only pathway triggered by aV integrin mediated multicellular radioresisitance, the evidence we got has given us a hint that SAPK/ JNK pathway can be directly or indirectly activated by aV integrin. Our studies have revealed the profound impact of aV integrin on MCR to radiosensitivity, and it will be important for future work to examine the effect of aV integrin on each stage of NPC tumorigenesis in mechanistic detail.
The combination of molecular-targeted agents with irradiation is a highly promising avenue for cancer research and patient care. Given the role of aV integrin in mediating NPC radioresistance, aV integrin should be a potential target to improve the efficiency of radiosensitivity in NPCs.

Samples Collection
A tissue chip consisting of 105 human nasopharyngeal carcinoma (NPC) specimens was purchased from Shanghai Outdo Biotech Co.,Itd. A separated set of tissue specimens used for immunohistochemistry and Western blotting studies were collected from NPC patients who had undergone biopsies at Southwest Hospital under a protocol approved by Southwest Hospital. The Objective Response Rate (ORR) and histological subtypes were defined by an oncologist (pathologist) in the Southwest Cancer Center, Southwest Hospital. Complete Response (CR) means all detectable tumor has disappeared; Partial Response (PR) corresponds to at least a 50% decrease in the total tumor volume but with evidence of some residual disease still remaining; Stable Disease (SD) means the tumors stay the same size, to account for  measurement errors on scans and to discount ''insignificant'' changes, stable disease includes either a small amount of growth (typically less than 20 or 25%) or a small amount of shrinkage (anything less than a PR unless minor responses are broken out). Radiosensitive patients are clarified as those reached CR 2 to 4 weeks after irradiation therapy (GTV.70 Gy), and radioresistant patients are clarified as those of PR or SD or even with disease progression 2 to 4 weeks after irradiation therapy (GTV.70 Gy).

Assessment of Immunohistochemical Staining
Immunohistochemical staining was scored as 0-4. No staining or weak staining were scored was 0 and 1, respectively. Strong staining of 25% tumor cells or moderate staining of ,80% scored 2. Strong staining of 25-50% or moderate staining of .80%, and strong staining of .50% tumor cells, scored 3 and 4, respectively. Ten representative areas were counted in each case from high power fields. Slides were examined and scored independently by 2 researchers blinded to other pathological information.
Cell Culture CNE-2 cells were routinely grown and passaged as monolayers in RPMI1640 medium (HyClone, UK) supplemented with 5% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL) under a humidified atmosphere of 5% CO 2 at 37uC. MCSs were obtained by using the liquid overlay technique. Exponentially-growing CNE-2 cells were added in culture medium in plates which were previously coated with 2% agarose. The plates were gently horizontally swirled 10 min every 3 h in the first 24 h, then 10 min every 4 h. Appropriate medium was refreshed every other day. For antibody treatment, cells were incubated with purified endotoxin-free mAbs (175 mg/ml) for 24 h.

Cell Survival Assay
Cell survival was evaluated by using the cell counting kit 8 (Dojindo Laboratories, Japan). In contrast to monolayers, MCSs were digested by Non-enzyme Cell Detach Solution (Applygen Technologies, China) for 10 min before using the cell counting kit 8 to detect cell survival. Colony Survival Assay Cells were seeded into 24-well culture dishes in triplicates (1000 cells to each well). The cells were allowed to form colonies during 1 week, and then cells were treated with different doses of 6MV X-ray radiation [IBL 637; Cis-Bio International, Gif-sur-Yvette, France]. The radiation doses were 0, 2, 4, 6 and 8 Gy, respectively; the dose efficiency was 300 cGy/min. After an incubation period of 12-15 days, the colonies were fixed with methanol and stained with crystal violet. Colonies of .50 cells were counted and analyzed.

Flow Cytometry Analysis of Apoptosis
Flow cytometry was performed to detect apoptosis of trypsindissociated cells with AnnixinV-PE apoptosis Detection Kit (Beyotime). Cells were washed and resuspended in 0.5 ml PBS buffer, and fixed for 24 hr in 70% alcohol. Annixin V-PE (50 mg/ ml) was added and incubated for 30 min on ice, and then analyzed by FCM (FACScan, Becton Dickinson, San Jose, CA).

In vivo Study
Female BALB/c (nu/nu) nude mice, 4-5 weeks old, weighing 17-22 g, were housed in filter-capped cages kept in a sterile facility and maintained in a specific pathogen-free barrier system. After 3 weeks, xenografts established by subcutaneous injection CNE-2 MCSs in mouse hips reached a mean diameter of 0.8-1.0 cm, and then 6 Gy fractionated irradiation combined with or without daily peritumoral injection of aV integrin blocking peptide or isotype blocking peptide (Santa Cruz Biotechnology, Santa Cruz CA, USA) were administrated (10 mg/Kg, twice a week). Mice were sacrificed 3 weeks later and the xenografts were excised and weighed.

Statistical Analysis
Statistical analysis was performed with SPSS (SPSS, Chicago, IL) using Students t-test or one-way ANOVA. Differences were considered statistically significant when P-values were less than 0.05. Error bars represent standard error of the mean.  . Effect of aV integrin blocking on radioresponse of NPC xenografts in mice with irradiation treatment. Same volume of MCSs (100 ml per mouse) were injected subcutaneously, Mice were treated with local radiation at a fractionated dose of 6 Gy combined with aV integrin blocking peptide or isotype blocking peptide peritumoral treatment (10 mg/Kg, twice a week) when the xenografts reached a mean size of 0.8-1 cm. Tumor weight was measured when the mice were sacrificed after 3 weeks. Each data point represents the mean weight of 3 tumors; A: Xenograft weight was measured when the mice were sacrificed 3 weeks after radiotherapy. A photograph of a representative xenograft in each group is also shown, *P,0.01. B: The decrease of xenograft size was measured every 2 days after radiotherapy, *p,0.01. C: Apoptosis detected by TUNEL assay was shown to compare the effect of aV integrin blocking peptide on radiosensitivity of in irradiated xenografts (6100). doi:10.1371/journal.pone.0038737.g005