Qianru Zhuang,Guolong Liu,Wenqiang Huang,Zhijiang He
Knockdown of LAMB3 suppressed radioresistance in nasopharyngeal carcinoma via deactivating NRF2 signaling pathway
nasopharyngeal carcinoma,LAMB3,reactive oxygen species,apoptosis,radioresistance,NRF2 signaling pathway
Abstract Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and Southern China. Laminin subunit beta-3 (LAMB3) has been validated to participate in diverse cancers. Nevertheless, the role and mechanism of LAMB3 in NPC remain unclear. In this study, LAMB3 expression is upregulated in NPC cells and tissues. Interestingly, knockdown of LAMB3 promoted apoptosis and reduced the radioresistance of NPC cells. Besides, shLAMB3 enhanced X-ray-induced reactive oxygen species (ROS) accumulation. Mechanically, knockdown of LAMB3 deactivated nuclear factor erythroid-2-related factor 2 (NRF2) signaling pathway via enhancing forkhead box 3 (FOXO3) expression. In rescue experiments, suppression of NRF2 signaling pathway abrogated shLAMB3-induced NPC cell apoptosis and ROS accumulation under X-ray treatment. Similarly, LAMB3 knockdown restrains NPC tumor growth and reduces radioresistance in vivo. Thus, these findings concluded that knockdown of LAMB3 enhanced apoptosis and ROS accumulation, and suppressed radioresistance in NPC via enhancing FOXO3 expression and deactivating NRF2 signaling pathway, facilitating the development of novel strategies for NPC radioresistance.
Knockdown of LAMB3 suppressed radioresistance in nasopharyngeal carcinoma via deactivating NRF2 signaling pathway

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and Southern China. Laminin subunit beta-3 (LAMB3) has been validated to participate in diverse cancers. Nevertheless, the role and mechanism of LAMB3 in NPC remain unclear. In this study, LAMB3 expression is upregulated in NPC cells and tissues. Interestingly, knockdown of LAMB3 promoted apoptosis and reduced the radioresistance of NPC cells. Besides, shLAMB3 enhanced X-ray-induced reactive oxygen species (ROS) accumulation. Mechanically, knockdown of LAMB3 deactivated nuclear factor erythroid-2-related factor 2 (NRF2) signaling pathway via enhancing forkhead box 3 (FOXO3) expression. In rescue experiments, suppression of NRF2 signaling pathway abrogated shLAMB3-induced NPC cell apoptosis and ROS accumulation under X-ray treatment. Similarly, LAMB3 knockdown restrains NPC tumor growth and reduces radioresistance in vivo. Thus, these findings concluded that knockdown of LAMB3 enhanced apoptosis and ROS accumulation, and suppressed radioresistance in NPC via enhancing FOXO3 expression and deactivating NRF2 signaling pathway, facilitating the development of novel strategies for NPC radioresistance.

Nasopharyngeal carcinoma (NPC) is an endemic malignancy in Southeast Asia and Southern China [1]. Owing to the sensitivity of NPC to ionizing radiation (IR) and the difficulty of surgery implementation, radiotherapy is administered as a primary option for NPC treatment [2]. Nevertheless, the efficacy of radiotherapy is impaired by inherent or acquired radioresistance, resulting in tumor recurrence or distant metastasis of NPC patients [3]. Thus, to improve the outcomes of NPC patients, the mechanism of radioresistance is employed, which is essential for the treatment of NPC patients. Notably, accumulating reactive oxygen species (ROS) plays an important role in mitigating the damaging effects of IR on normal tissues [4]. ROS elimination promotes the radiosensitivity of NPC cells, indicating that the reduction of ROS levels is one of the ways to address the resistance of cancer cells to radiotherapy [5]. Laminins are important bioactive components of basal lamina, which contribute to cell proliferation and survival in multiple cancers [6, 7]. Laminin subunit beta-3 (LAMB3), which encodes one of the three subunits of LM-332, expresses in diverse cancers and is involved in tumorigenesis. LAMB3 can inhibit pancreatic cancer cells apoptosis, promote proliferation, invasion and metastasis by regulating PI3K/Akt signaling pathway [8]. In thyroid cancer, LAMB3 predicts poor prognosis, which is upregulated and associated with lymph node metastasis [9]. High expression of LAMB3 is closely related to the resistance of tumor cells to chemotherapy drugs. Furthermore, LAMB3 silencing induces the sensitivity of anti-cancer drugs such as cisplatin in head and neck squamous cell carcinoma [10]. However, the role and mechanism of LAMB3 have been rarely reported in NPC. Mechanically, LAMB3 suppresses the expression of FOXO3, which is a member of forkhead box (FOXO) protein family and serves as a tumor suppressor gene [11]. On the one hand, a recent study has reported that knockdown of FOXO3 enhances radioresistance in NPC by inducing epithelial–mesenchymal transition and activating Wnt/β-catenin signaling pathway [12]. On the other hand, FOXO3 deactivate snuclear factor erythroid-2-related factor 2 (NRF2) signaling pathway and reverses the resistance of human colorectal cancer cells to 5-fluorouracil [13]. Besides, activation of NRF2 pathway can enhance ROS elimination and increase the radioresistance of NPC cells [5]. Based on these findings, we hypothesized that LAMB3 may regulate the resistance of NPC to radiotherapy by regulating the FOXO3/NRF2 axis.

The patient tissues were in accordance with the Declaration of Helsinki and the animal experiments obeyed the Guide for the Care and Use of Laboratory Animals, which was approved by Maoming People’s Hospital.

Human NPC cell lines CNE1, CNE2 and HNE1 were obtained from the Cellbank of Chinese Academy of Sciences (Shanghai, China), maintained at 37°C with 5% CO2 in Dulbecco's Modified Eagle Medium (Invitrogen, California, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco, USA). The human nasal epithelial cell line RPMI2650 was obtained from ATCC (USA) and cultured in minimum essential medium (Gibco, USA) supplemented with 10% FBS at 37°C with 5% CO2. The CNE1 and HNE1 cells were transfected by pcDNA3.0 plasmid (NC), pcDNA3.0-LAMB3 (LAMB3), shNC, shLAMB-1# or shLAMB-2# (Ribobio, China) following the manufacturer’s standard protocol. For co-transfection, the CNE1 and HNE1 cells were co-transfected with shLAMB-2# or shNC and shFOXO3 following the manufacturer’s standard protocol. To activate NRF2 signaling pathway, the cells were pretreated with 10 nM bardoxolone methyl (BM; Sigma-Aldrich, USA).

The mRNA expression profile of gene chip GSE61218 and GSE64634 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61218 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64634) [14, 15]. The data of LAMB3 mRNA level in GSE61218 were obtained from 10 NPC and 6 normal healthy nasopharyngeal tissue specimens. The data of LAMB3 mRNA level in GSE64634 were obtained from 12 NPC and 4 normal healthy nasopharyngeal tissue specimens.

Human NPC tissues and paired adjacent normal tissues were collected from Maoming People’s Hospital. The patients without other cancers, autoimmune diseases or infectious diseases who also never received chemotherapy or radiotherapy provided informed consent to participate in the study. All collected samples were immediately frozen and stored for subsequent analysis.

The X-ray irradiation was performed by an RS2000 X-ray Biological Research Irradiator (3 mm copper filter, 160 kV, 25 mA; Rad Source Technologies, USA). Single irradiation doses were 0, 2, 4, 6, 8 Gy with a 1.151 Gy/min dose rate.

The tissues from patients and mice were embedded in paraffin, sliced into 5 μm-thick sections. All the sections were stained for LAMB3 antibody (1:200 dilution; Abcam, UK) or Ki67 antibody (1:200 dilution; Abcam, UK) following the standard protocols.

The CNE1 and HNE1 cells after transfection were seeded in the 6-well plates and exposed to different doses of X-ray beam. After being cultured for 10–14 days, the colonies were then stained with 0.5% crystal violet (Sigma-Aldrich, USA) solution for 10 min and counted under a light microscopy. A colony that has at least 50 cells was regarded as a viable colony. The survival fraction was calculated by the method: the colonies (Gy)./the colonies (0Gy).

The cells after transfection were seeded in 24-well plates (3 × 104 cells/well) and received radiation exposure (0 or 4 Gy). After 24 h, the cells were harvested and subsequently stained with Annexin V-FITC/PI apoptosis detection kit (Invitrogen, USA), which was further detected by a flow cytometry.

The ROS accumulation was detected by the use of a Reactive Oxygen Species Assay kit (Beyotime, China). Briefly, the cells were incubated with DCFH-DA probe in serum-free medium at 37°C for 30 min, washed with phosphate buffer saline and then visualized under a fluorescent microscope (Leica, Germany).

The supernatant of cell culture was collected, and the malondialdehyde (MDA) and glutathione (GSH) levels in supernatant were measured by Lipid Peroxidation MDA Assay kit (Beyotime, China) and GSSG Assay kit (Beyotime, China) according to the instruction, respectively.

The proteins from collected tissues or cells were lysed by RIPA buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking in 5% skim milk, the membranes were incubated with specific antibodies: LAMB3 (1:600 dilution; Abcam, UK), FOXO3 (1:600 dilution; Abcam, UK), NRF2 (1:600 dilution; Abcam, UK), KEAP1 (1:600 dilution; Abcam, UK), HO-1 (1:600 dilution; Abcam, UK) or β-actin (1:600 dilution; Abcam, UK). Subsequently, the membranes were incubated with proper horseradish peroxidase–labeled second antibodies (1:5000 dilution; Abcam, UK) and visualized by ECL luminescence reagent (Absin, China).

A total of 24 female BALB/c nude mice (6 weeks old, Vitalriver, China) were received adaptive feeding for a week. In brief, 1.5 × 107 CNE1 cells were injected subcutaneously into the right armpit of mice. The animals were randomly divided into four groups: shNC+0Gy, shLAMB3–2# + 0Gy, shNC+4Gy, shLAMB3–2# + 4Gy groups. Tumors were measured every 7 days. The experiments were then terminated with 21 days of tracking.

Data are shown as mean ± standard deviation (SD) and analyzed by Student’s t-test and analysis of variance using SPSS 21.0 (USA). P < 0.05 was considered significant.

Interestingly, we found the abnormal mRNA levels of LAMB3 in two GEO data sets: GSE61218 and GSE64634, indicating that LAMB3 was upregulated in NPC tumor tissues (Fig. 1A). Similarly, we observed that the tumor tissues displayed a relatively high level of LAMB3 compared with the normal tissues in NPC patients (Fig. 1B and C). The immunohistochemistry was employed to assess the LAMB3 expression in NPC samples, implying that the high expression of LAMB3 was detected in tumor tissues compared with the normal tissues in NPC patients (Fig. 1D). Furthermore, LAMB3 expression was also enhanced in NPC cell lines (CNE1, HNE1 and CNE2) compared with human nasal epithelial cell line RPMI2650 (Fig. 1E). Consequently, these results indicated that LAMB3 expression is upregulated in NPC cells and tissues.

To estimate whether LAMB3 levels contribute to NPC radioresistance, we generated the LAMB3-knockdown CNE1 and HNE1 cells, as well as the CNE1 and HNE1 cells overexpressing LAMB3. The transfection efficiency was confirmed by western blot (Fig. 2A). Besides, knockdown of LAMB3 reduced cell growth, whereas overexpression of LAMB3 promoted CNE1 and HNE1 cell growth without irradiation (Supplementary Fig. S1A). Subsequently, the cells suffered from the irradiation doses of 0, 2, 4, 6 and 8 Gy, and a colony assay was performed to investigate the impact of LAMB3 on cell proliferation after irradiation. As shown in Figure 2B and C, the LAMB3-overexpressing CNE1 and HNE1 cells had relatively higher colony survival after exposure to irradiation at the dose of 2, 4, 6 or 8 Gy compared with NC cells. Conversely, the colony survival was markedly reduced in LAMB3-knockdown cells after exposure to irradiation at the dose of 2, 4, 6, or 8 Gy compared with that of in shLAMB3 cells. Interestingly, the CNE1 and HNE1 cells suffered from 4 Gy showed an increase apoptosis rate compared with the cells with 0 Gy. Notably, overexpression of LAMB3 suppressed cell apoptosis and knockdown of LAMB3 promoted apoptosis after exposure to irradiation at the doses of 0 and 4 Gy (Fig. 2D). Thus, these findings concluded that knockdown of LAMB3 reduced the radioresistance of NPC cells.

Subsequently, the ROS levels in LAMB3-depleted or LAMB3-overexpressing NPC cells were examined by DCFH-DA fluorescence staining. As shown in Figure 3A, X-ray induced ROS accumulation in CNE1 and HNE1 cells. More importantly, overexpression of LAMB3 suppressed ROS accumulation, whereas knockdown of LAMB3 promoted ROS accumulation. Besides, X-ray increased MDA level, and decreased GSH level and GSH/GSSG ratio. Moreover, overexpression of LAMB3 reduced MDA level, but enhanced GSH level and GSH/GSSG ratio, whereas knockdown of LAMB3 had opposite effect (Fig. 3B). Thus, these findings concluded that knockdown of LAMB3 enhanced X-ray-induced ROS accumulation.

To explore the mechanism of LAMB3 in NPC cells under X-ray, the western blot assay was performed. As shown in Figure 4A, knockdown of LAMB3 promoted FOXO3 expression, and reduced NRF2 and HO-1 expression, whereas overexpression of LAMB3 had opposite effect, indicating that knockdown of LAMB3 promoted FOXO3 expression and deactivated NRF2 signaling pathway in NPC cells under X-ray treatment. Of note, knockdown of LAMB3 increased KEAP1 expression, whereas overexpression of LAMB3 decreased KEAP1 expression (Supplementary Fig. S1B). Interestingly, knockdown of FOXO3 abolished the effect of knockdown of LAMB3 on deactivation of NRF2 signaling pathway in CNE1 and HNE1 cells under X-ray treatment (Fig. 4B and Supplementary Fig. S1C). Therefore, these results revealed that knockdown of LAMB3 deactivated NRF2 signaling pathway via enhancing FOXO3 expression.

To verify the effect of NRF2 signaling pathway on LAMB3 in NPC cells, the BM, a kind of NRF2 signaling pathway activator, was added. In Figure 5A, the NRF2 and HO-1 expression was markedly upregulated by BM treatment in CNE1 cells. Moreover, the shLAMB3-induced apoptosis was reduced by BM in CNE1 cells under X-ray treatment (Fig. 5B). Besides, BM abrogated shLAMB3-induced ROS accumulation in CNE1 cells under X-ray treatment (Fig. 5C). Thus, suppression of NRF2 signaling pathway abrogated shLAMB3-induced apoptosis and ROS accumulation in NPC cells under X-ray treatment.

Subsequently, we validated the effects of depletion of LAMB3 on tumor growth and radiosensitivity in vivo. As shown in Figure 6A, radiation significantly reduced the tumor growth. More importantly, depletion of LAMB3 further suppressed the tumor growth in mice. These findings implied that LAMB3 might suppress tumor growth via decreasing resistance to radiation exposure. Immunohistochemical staining showed a reduction of Ki-67-positive proliferative cells in LAMB3-depleted tumors after radiation treatment (Fig. 6B). Besides, an increase of ROS accumulation was observed in LAMB3-depleted tumors after radiation treatment (Fig. 6C). At the same time, LAMB3-depleted mice received radiation treatment that exhibited higher expression of FOXO3 and lower expression of NRF2 and HO-1 than shNC mice under 4 Gay radiation exposure (Fig. 6D). Taken together, LAMB3 knockdown restrained NPC tumor growth and reduced radioresistance in vivo.

In this study, we demonstrated that LAMB3 expression is upregulated in NPC cells and tissues. Interestingly, knockdown of LAMB3 promoted apoptosis and reduced the radioresistance of NPC cells. Besides, shLAMB3 enhanced X-ray-induced ROS accumulation. Mechanically, knockdown of LAMB3 deactivated NRF2 signaling pathway via enhancing FOXO3 expression. In rescue experiments, suppression of NRF2 signaling pathway abrogated shLAMB3-induced NPC cell apoptosis and ROS accumulation under X-ray treatment. Similarly, LAMB3 knockdown restrains NPC tumor growth and reduced radioresistance in vivo. Thus, these findings concluded that knockdown of LAMB3 enhanced apoptosis and ROS accumulation, and suppressed radioresistance in NPC via enhancing FOXO3 expression and deactivating NRF2 signaling pathway, facilitating the development of novel strategies for NPC radioresistance. Radiotherapy is a common therapeutic strategy for tumors, which has been applied for the treatment of NPC patients [16]. Although the increased radioresistance might be the major reason responsible for the recurrence of cancer after radiotherapy [17]. Thus, investigation on the mechanisms underlying LAMB3-induced radioresistance is of importance for the optimization of cancer radiotherapy [18]. LAMB3 is upregulated in diverse cancers and regulates tumorigenesis, including pancreatic cancer, colorectal cancer and papillary thyroid cancer [6, 8, 11]. Especially, at earlier, LAMB3 expression was enhanced in NPC and identified as a cancer biomarker [19]. Similarly, it was observed that LAMB3 expression is upregulated in NPC cells and tissues in our study. Nevertheless, the potential mechanism of LAMB3 in NPC remains unclear. In the current study, we demonstrated that knockdown of LAMB3 reduced radioresistance of NPC cells, whereas overexpression of LAMB3 promoted radioresistance of NPC cells, implying the vital role of LAMB3 in radioresistance of NPC cells. Majority of cancers start out being sensitive to radiotherapy at first, and can acquire radioresistance to radiotherapy during the evolution of cancers, ultimately leading to treatment failure. Accumulating evidences have demonstrated that inhibition of apoptosis serves as one of the main mechanisms underlying cancer radioresistance [20]. Notably, LAMB3 mediates apoptosis and metastatic behaviors in pancreatic cancer, pancreatic ductal adenocarcinoma [8, 21]. Similarly, we demonstrated that overexpression of LAMB3 promoted cell proliferation and inhibited cell apoptosis under X-ray treatment, revealing that upregulation of LAMB3 plays an essential role in radiosensitivity. Notably, the increases may influence the level of apoptosis induction in knockdown cells with irradiation. To some extent, high-expression of LAMB3 can aggravated apoptosis under irradiation. Although LAMB3 induced cell apoptosis, the cells overexpression of LAMB3 under irradiation presented suppressing cell apoptosis, indicating that the effect of LAMB3­-induced cell apoptosis can be ignored. The p53 is a tumor suppressor protein and promotes depolarization of Δψm by forming complexes with the proapoptotic Bcl-2 family proteins [22]. Besides, increased production of intracellular ROS is one of the signals that triggers the p53-induced apoptosis, whereas ROS-induced apoptosis has been identified in multiple cell systems [23]. Thus, ROS accumulation is closely associated with the radioresistance in cancers [24]. During apoptosis, a decreased intracellular ROS amount can reduce lipid peroxidation, ultimately protecting the cell death [25]. Unfortunately, the impact of LAMB3 on ROS accumulation remains unknown. In this study, we demonstrated that knockdown of LAMB3 promoted the ROS accumulation under X-ray treatment, whereas overexpression of LAMB3 inhibited the ROS accumulation, which might be the first time exploring the reductive effect of LAMB3 on ROS accumulation. It has been reported that overexpression of LAMB3 promoted cell proliferation and cell migration via decreasing FOXO3 protein expression and in colorectal cancer [11]. Consistently, our results also validated that overexpression of LAMB3 decreased FOXO3 but increased NRF2 expression, indicating that knockdown of LAMB3 deactivated NRF2 signaling pathway via enhancing FOXO3 expression. NRF2, a master regulator of antioxidant response, combines with the antioxidant response element and regulates the expression of antioxidant genes, thereby neutralizing ROS to restore cellular redox balance [26]. More importantly, NRF2-HO-1 signaling pathway can be activated by ROS elevation and conferred radioresistance in human lung adenocarcinoma [17]. As we expected, suppression of NRF2 signaling pathway rescued shLAMB3-induced NPC cell apoptosis and ROS accumulation under X-ray treatment. Therefore, we hypothesized that upregulation of LAMB3 decreased FOXO3 expression, activated NRF2 signaling pathway and then reduced ROS accumulation, subsequently suppressed apoptosis in NPC cells under X-ray treatment, thereby leading to the promotion of radioresistance. Nevertheless, there are many limitations in our research. We have yet to show whether LAMB3-manipulated ROS/FOXO3/NRF2-dependent mechanism is the only mechanism of LAMB3 in radioresistance in clinic. Besides, there might be other cell death pathways in sh-LAMB3-induced cell death, which can be a novel sight for further studies. Other therapeutic targets of LAMB3 are not known, which might be validated in the future. In summary, we demonstrated that knockdown of LAMB3 induces high ROS accumulation and apoptosis via enhancing FOXO3 expression and deactivating NRF2 signaling pathway, ultimately leading to the decline of therapeutic radioresistance in NPC, which might facilitate to the development of a novel predictive and therapeutic strategy against radiation-resistant NPC.

The authors state that there are no conflicts of interest to disclose.

Not applicable.

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of Maoming People’s Hospital and with those of the 1964 Helsinki Declaration and its later amendments for ethical research involving human subjects. All animal experiments were approved by the Ethics Committee of Maoming People’s Hospital for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.

Q.Z. designed the study, completed the experiment and supervised the data collection, G.L. analyzed the data, interpreted the data, W.H. and Z.H. prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

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