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SIRT1 overexpression protects non-small cell lung cancer cells against osteopontin-induced epithelial-mesenchymal transition by suppressing NF-κB signaling

Osteopontin (OPN) is a promoter for tumor progression. It has been reported to promote non-small cell lung cancer (NSCLC) progression via the activation of nuclear factor-κB (NF-κB) signaling. As the increased acetylation of NF-κB p65 is linked to NF-κB activation, the regulation of NF-κB p65 acetylation could be a potential treatment target for OPN-induced NSCLC progression. Sirtuin 1 (SIRT1) is a deacetylase, and the role of SIRT1 in tumor progression is still controversial. The effect and mechanism of SIRT1 on OPN-induced tumor progression remains unknown. The results presented in this research demonstrated that OPN inhibited SIRT1 expression and promoted NF-κB p65 acetylation in NSCLC cell lines (A549 and NCI-H358). In this article, overexpression of SIRT1 was induced by infection of SIRT1-overexpressing lentiviral vectors. The overexpression of SIRT1 protected NSCLC cells against OPN-induced NF-κB p65 acetylation and epithelial-mesenchymal transition (EMT), as indicated by the reduction of OPN-induced changes in the expression levels of EMT-related markers and cellular morphology. Furthermore, SIRT1 overexpression significantly attenuated OPN-induced cell proliferation, migration and invasion. Moreover, overexpression of SIRT1 inhibited OPN-induced NF-κB activation. As OPN induced NSCLC cell EMT through activation of NF-κB signaling, OPN-induced SIRT1 downregulation may play an important role in NSCLC cell EMT via NF-κB signaling. The results suggest that SIRT1 could be a tumor suppressor to attenuate OPN-induced NSCLC progression through the regulation of NF-κB signaling.


Introduction
Lung cancer is one of the main reasons for cancer-related deaths worldwide. 1 Tumor metastasis is considered as the primary cause of mortality. Non-small cell lung cancer (NSCLC) is the dominant form of lung cancer, accounting for nearly 85% of the cases. 2 Study has indicated that more than 65% of patients show regional lymph node or distant site metastases when they were initially diagnosed with NSCLC. 3 Therefore, it is necessary to explore the mechanisms regulating NSCLC metastasis for the development of potential new therapeutic targets. Epithelial-mesenchymal transition (EMT) is associated with multiple pathologies including lung cancer metastasis, during which epithelial cells acquire enhanced mobility and invasiveness by the loss of E-cadherin expression and the increase of mesenchymal marker (N-cadherin and Vimentin) expression. 4,5 Further studies are needed to explore the molecular mechanism that regulates EMT, in order to find therapeutic target for the treatment of tumor invasion and metastasis. Osteopontin (OPN) is an extracellular matrix protein that plays a key role in tumor progression through binding with avβ3-integrin and CD44 receptor. 6 The overexpression of OPN has been shown to correlate with poor prognosis in NSCLC. 7 It has been demonstrated that OPN promotes EMT of several types of cancer cells, including endometrial cancer, prostate cancer, breast cancer and liver cancer. [8][9][10][11] However, the mechanism underlying OPN-induced EMT remains poorly understood. Nuclear factor-κB (NF-κB) is a nuclear transcription factor that stimulates the expression of transcription factors that drive the EMT process. It has been shown to be involved in OPN-induced tumor progression. [12][13][14] It has been shown that the acetylation of RelA/p65, a subunit of NF-κB, can increase its specific transcriptional activity and the deacetylation will inhibit its transactivation. 15,16 Therefore, it can be inferred that deacetylation of NF-κB p65 could be a potential target to suppress OPN-induced NSCLC cell EMT. However, the acetylation level of NF-κB p65 in OPN-induced EMT remains unclear.
Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent lysine deacetylase. 17 The role of SIRT1 in tumor progression is still controversial. Initially SIRT1 was shown to suppress apoptosis by deacetylation of p53, a well-known tumor suppressor. 18 However, SIRT1 is regarded as a tumor suppressor that inhibits tumor progression by targeting HIF-1a, TGF-β/Smad4 or NF-κB/cyclin D1 signaling pathway. [19][20][21] In addition, resveratrol, the SIRT1 activator, has been shown to activate caspase-3 and reduce chemoresistance in breast tumor cells through the inhibition of NF-κB-specific transcriptional activation. 22 However, little is known regarding to the role of SIRT1 as regulator of NF-κB activation during EMT process. SIRT1 inhibits NF-κB activation by deacetylation of the RelA/p65 subunit at lysine 310. 16 Therefore, we examined the effect of OPN on SIRT1 expression and the role of SIRT1 in OPN-induced EMT process.
This study aimed to examine the mechanisms of OPNinduced EMT in NSCLC. It is found that OPN inhibits SIRT1 expression, and SIRT1 protects against OPN-induced EMT by inhibiting NF-κB signaling activation.

Materials and methods cell culture and reagents
NSCLC cell lines A549, NCI-H358 (H358), NCI-H1299 (H1299) and NCI-H460 (H460) and normal lung epithelium cell line BEAS-2B were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, People's Republic of China) and were cultured in RPMI 1640 (HyClone, Logan, UT, USA) supplemented with a mixture of 10% fetal bovine serum (FBS) and antibiotics at 37°C in an incubator filled with 5% CO 2 . Recombinant human OPN protein (rhOPN) was purchased from R&D Systems (Minneapolis, MN, USA). enzyme linked immunosorbent assay A549, NCI-H358, NCI-H1299, NCI-H460 and BEAS-2B cell lines were seeded in T25 culture bottle at a density of 3×10 6 /bottle; cell culture supernatants were collected after cells were cultured for 24 hours. The secretory levels of OPN in these cells were detected using a human OPN enzymelinked immune detection kit according to the manufacturer's instructions (R&D Systems).

cell infection
Lentiviral vectors overexpressing SIRT1 (LV-SIRT1) and their negative control (LV-NC) were constructed by GeneChem Co., Ltd (Shanghai, People's Republic of China) and used to infect A549 and NCI-H358 cell lines. For infection, cells were plated in 6-well plates at a density of 1×10 5 cells/well. After 24 hours, the cells were infected with 5 μL of the lentiviral vector by using polybrene (8 μg/mL) according to the manufacturer's instructions. At 96 hours after infection, the effect of the infection was evaluated by real-time quantitative polymerase chain reaction (qPCR) and Western blot analysis.

ccK-8 assay
Cell suspension (5×10 4 /mL) was seeded in 96-well plate (100 μL per well). Cells were cultured overnight at 37°C in an incubator filled with 5% CO 2 . The cells infected with LV-SIRT1 and LV-NC were treated with different concentrations of OPN (0, 100, 200 and 400 ng/mL) for 24 hours or treated with 400 ng/mL OPN for various times (0, 12, 24 and 48 hours). The concentration of FBS in culture medium was reduced to 5%. To detect cell proliferation, cells were incubated with 10 μL Cell Counting Kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) for 2 hours in an incubator at 37°C. The absorbance was measured at a wavelength of 450 nm.

real-time Pcr (quantitative Pcr)
Total RNA was extracted from NSCLC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using the PrimeScript™ RT reagent Kit (Takara, Dalian, People's Republic of China). Aliquots of the resulting cDNA were used for PCR amplification in Roche light cycler 480 (Roche Diagnostics GmbH, Basel, Switzerland) using the SYBR Green Supermix (Takara). The specific primer sequences

G A G G A G T T G A A T G G T G C A T A C -3 ′ , R : 5 ′ -T T T C A G C A C T C T G G T C A T C C A -3 ′ ;
Vimentin F: 5′-ACCGCTTTGCCAACTACAT-3′, R: 5′ -TTGTCCCGCTCCACCTC-3 ′ ; E -c a d h e r i n F : GAPDH F: 5′-GAGTCAACGGATTTGGTCGT-3′, R: 5′-GACAAGCTTCCCGTTCTCAG-3′. GAPDH was used as an internal control. The relative expressions of the genes were quantified using the 2 −ΔΔCT method.

Wound healing migration assay
The cells (infected with either LV-SIRT1 or LV-NC) were plated onto 6-well plates and then cultured to 90% confluence. Cell monolayers were carefully wounded by scratching with the 200 μL pipette tip. The cells were then washed twice with PBS to remove dislodged cells and cultured in serumfree RPMI 1640 culture medium with either control buffer (CB) or 400 ng/mL OPN for 48 hours. Wound closure rate = (wound width at 0 hour − wound width at 48 hours)/wound width at 0 hour ×100%.

Transwell invasion assay
Cells (infected with either LV-SIRT1 or LV-NC) were treated with either CB or 400 ng/mL OPN for 48 hours. The cells in serum-free medium were added to the upper chambers (8×10 4 cells/well) of a 24-well transwell chamber (8 μm, Corning Life Sciences, Corning, NY, USA) coated with 40 μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:8 in serum-free medium. The chambers were incubated for 48 hours with 600 μL complete culture medium, which contained 15% FBS added in the lower chamber. Non-invading cells were removed from the upper chamber with cotton swabs. Cells that had invaded through membrane on the lower surface of the membrane were fixed and stained with crystal violet (Beyotime). The membranes were captured with digital images, and the number of invading cells on the filters were counted in random fields.

Immunofluorescence
Cells (infected with either LV-SIRT1 or LV-NC) were cultured in 12-well plates containing coverslips, treated with CB or OPN (400 ng/mL) for 2 hours, and then washed with PBS. The cells were fixed in 4% paraformaldehyde for 30 minutes and permeabilized in 0.1% Triton X-100 solution. Cells were blocked with goat serum for 1 hour and incubated overnight at 4°C with primary antibody against NF-κB p65 (Abcam). Subsequently, cells were washed three times with PBS, followed by incubation with FITC-conjugated antirabbit antibody (Proteintech) at room temperature for 3 hours. The cell nuclei were stained with DAPI and the cells were observed by fluorescence microscopy.

statistical analysis
All of the experiments were repeated at least three times for statistical analysis. All data would be presented as mean ± SD. Student's t-test was used to compare data between two groups. P,0.05 was considered statistically significant.

OPn is highly expressed by nsclc cells and promotes nsclc cells eMT
Real-time qPCR revealed that the mRNA expression levels of OPN in human NSCLC cell lines (A549, NCI-H358, NCI-H1299, NCI-H460) were higher than that in normal lung epithelium cell line BEAS-2B ( Figure 1A). Consistent with this, enzyme linked immunosorbent assay revealed that the protein secretion levels of OPN in these NSCLC cell lines were higher than that in normal lung epithelium cell line ( Figure 1B). In these cells, the protein secretion levels OPn inhibits sirT1 expression and promotes acetylation of nF-κB p65 subunit that incubation with OPN (0, 100, 200 and 400 ng/mL) for 24 hours resulted in a decrease of SIRT1 expression in A549 and NCI-H358 cells (Figure 2A-D). As SIRT1 can inhibit the activation of NF-κB by deacetylation of the RelA/p65 subunit at lysine 310, 16 the acetylation of RelA/p65 was evaluated after treatment with OPN for 24 hours. Figure 2B, C and E showed that OPN significantly increased the protein levels of acetyl NF-κB p65; this is consistent with the decrease of β β However, neither SIRT1 overexpression nor OPN treatment could influence NF-κB p65 protein expression levels ( Figure 3D and E).

sirT1 overexpression inhibits OPninduced nsclc cell proliferation
The effect of SIRT1 on OPN-induced NSCLC cell proliferation was assessed. A549 and NCI-H358 cells were infected with LV-SIRT1 or LV-NC followed by treatment with different concentrations of OPN (0, 100, 200, 400 ng/mL) for 24 hours. CCK-8 assays were, then, performed on A549 and NCI-H358 cells. As shown in Figure 4A and B, compared with untreated cells, OPN promoted A549 and NCI-H358 cell proliferation. However, in SIRT1 overexpression cells, OPN could not significantly promote cells proliferation. A549 and NCI-H358 cells infected with LV-SIRT1 or LV-NC were treated with CB or 400 ng/mL OPN for different times (0, 12, 24, 48 hours). CCK-8 assays showed that SIRT1 overexpression inhibited OPN-induced A549 and NCI-H358 cell proliferation at 12, 24 and 48 hours after OPN treatment ( Figure 4C and D).

sirT1 overexpression inhibits OPninduced nsclc cell migration and invasion
The roles of SIRT1 in OPN-induced NSCLC cell migration and invasion were explored using wound healing assay and transwell assay, respectively. Wound healing assay showed that OPN significantly increased the migration rate of the NSCLC cell lines A549 and NCI-H358 ( Figure 5A and B). The overexpression of SIRT1 in A549 and NCI-H358 cells significantly inhibited OPN-induced cell migration ( Figure 5A and B). Similarly, OPN promoted cell invasion, and overexpression of SIRT1 significantly depressed OPNinduced cell invasion ( Figure 5C). This phenomenon indicated that SIRT1 is involved in inhibition of OPN-induced NSCLC cell migration and invasion.

sirT1 overexpression inhibits OPninduced nF-κB signaling activation
NF-κB is an important signaling pathway that mediates OPN-induced tumor progression. So, acetyl NF-κB p65 protein expression level was explored in cells treated with OPN, revealing that OPN promoted acetyl NF-κB p65 protein expression ( Figure 2B, C and E). Furthermore, SIRT1 overexpression inhibited OPN-induced NF-κB p65 acetylation ( Figure 3D, E and I). Therefore, the assumption, where OPN promotes NF-κB p65 subunit activation through inhibiting SIRT1 expression was explored. The role of SIRT1 in OPNinduced NF-κB activation was explored using Western blot and immunofluorescence assay in A549 and NCI-H358 cells. The analysis from the Western blot clearly showed that OPN increased the nuclear translocation of the NF-κB p65 subunit and hence OPN-induced NF-κB activation in comparison to untreated cells ( Figure 6A). However, OPN-induced NF-κB p65 nuclear translocation was greatly inhibited by the overexpression of SIRT1 ( Figure 6A). Similarly, immunofluorescence assay also showed that in both untreated cells and SIRT1-overexpressing cells treated with OPN, NF-κB p65 subunit was mainly located in the cytoplasm. However, in cells treated with OPN alone, NF-κB p65 was translocated to the nucleus ( Figure 6B). Furthermore, the effect of OPN on the upstream molecules involved in NF-κB activation was examined. Treatment of A549 and NCI-H358 cells with OPN caused a degradation of IκB-α and an increase in the expression of IKK-α ( Figure 7A). SIRT1 overexpression inhibited OPN-induced downregulation of IκB-α and upregulation of IKK-α ( Figure 7A). These results indicated that OPN induces NF-κB signaling activation through the inhibition of SIRT1 expression. In order to examine the role of NF-κB signaling in OPN-induced NSCLC cell EMT, A549 and NCI-H358 cells were pretreated with NF-κB inhibitor (PDTC, 10 μM) for 30 minutes followed by treatment with OPN (400 ng/mL) for 24 hours. Western blot showed that PDTC significantly inhibited OPN-induced decrease of E-cadherin expression and increase of N-cadherin expression ( Figure 7B). In conclusion, OPN promoted NSCLC cell EMT by activating NF-κB signaling, and SIRT1 overexpression inhibited OPN-induced EMT by inhibiting NF-κB signaling activation.
including gastric cancer, 23 liver cancer, 24 bladder urothelial cancer, 25 prostate cancer, 26 breast cancer 27 and NSCLC. 28 OPN is involved in multiple tumor-associated biological processes, including the induction of tumor cell immune evasion, survival, invasion, metastasis and angiogenesis. 29 The EMT process appears to be important for tumor cells to acquire multiple drug resistance, an increased migration and invasion ability, and cancer stem cells characteristics. 5 In our study, NSCLC cell lines were shown to be with a higher OPN expression than normal lung epithelium cell line, which is consistent with previous studies.
In this study, we found that OPN-induced EMT in NSCLC cells may depend, at least partially, on the downregulation of SIRT1. SIRT1 overexpression attenuated OPN-induced NSCLC cell migration and invasion via inhibition of EMT. Previously, it has been shown that SIRT1 regulates various physiological processes by the inhibition of NF-κB signaling pathway as a deacetylase. 22,30,31 Here, OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com Dovepress Dovepress 1165 sirT1 protects nsclc against OPn-induced eMT via nF-κB the results showed that treatment of A549 and NCI-H358 cells with OPN increased the expression of acetyl NF-κB p65, while SIRT1 overexpression attenuated OPN-induced NF-κB p65 acetylation. Furthermore, it is demonstrated that SIRT1 overexpression inhibited OPN-induced NF-κB p65 signaling activation. The results showed that SIRT1 may protect NSCLC cells against OPN-induced EMT by inhibiting NF-κB activity. It is well known that the EMT process plays an important role in the transition of lung cancer from early stage to invasive carcinoma. 4 Taken together, these data suggested that SIRT1 might be a new therapeutic modality in OPN-mediated NSCLC progression.
It is worth noting that previous studies have indicated that SIRT1 exerted both stimulative [32][33][34] and inhibitory effects 20,35,36 on tumor cell EMT. In prostate cancer, SIRT1 has been reported to induce EMT by cooperating with EMT-related transcription factor ZEB1. 32 Moreover, in colorectal cancer, Cheng et al 33 found that SIRT1 promotes EMT and metastasis by regulating Fra-1 expression. Recently, non-steroidal anti-inflammatory drugs were found to inhibit TGF-β1-induced EMT via the downregulation of SIRT1. 34 However, SIRT1 has been documented to suppress EMT. In lung cancer, hypoxia promoted EMT and cancer metastasis by the repression of SIRT1 expression in a SUMOylation-dependent manner. 35 Consistent with this, in ovarian cancer, hypoxic stress induced EMT by the downregulation of SIRT1 transcription level. 36 In addition, SIRT1 has been shown to reduce EMT in breast cancer by  A and B) a549 and nci-h358 cells were infected with lV-sirT1 or lV-nc followed by stimulation with OPn (400 ng/ml). cell migration was examined by wound healing assay. representative images at 48 hours after scratching are presented (×100). results presented are mean ± sD analyzed with student's t-test from three independent experiments. **P,0.01 versus cB + lV-nc. ## P,0.01, ### P,0.001 versus OPn + lV-nc. (C) a549 and nci-h358 cells were infected with lV-sirT1 or lV-nc followed by stimulation with OPn (400 ng/ml) for 48 hours. cell invasion was examined by transwell assay. after incubation for 48 hours, the cells that had invaded to the lower chamber were fixed, stained and counted. Representative images are presented (×100). scale bars represent 100 μm. Abbreviations: sirT1, sirtuin 1; OPn, osteopontin; lV-sirT1, lentiviral vectors overexpressing sirT1; nc, negative control; sD, standard deviation.  Notes: (A) a549 and nci-h358 cells infected with lV-sirT1 or lV-nc were treated with OPn (400 ng/ml) for 2 hours. The levels of iκB-α and iKK-α were determined by Western blot analysis. β-actin was used as an internal control. The group treated with cB + lV-nc serving as control is presented as 1. results presented are mean ± sD analyzed with student's t-test from three independent experiments. *P,0.05, **P,0.01, ***P,0.001 versus cB + lV-nc. # P,0.05, ## P,0.01, ### P,0.001 versus OPn + lV-nc. (B) a549 and nci-h358 cells were pretreated with nF-κB inhibitor (PDTc, 10 μM) for 30 minutes followed by treatment with OPn (400 ng/ml) for 24 hours. Western blot was used to detect e-cadherin and n-cadherin expression. β-actin was used as an internal control. The group treated with cB serving as control is presented as 1. results presented are mean ± sD analyzed with student's t-test from three independent experiments. **P,0.01 versus control. # P,0.05, ### P,0.001 versus cB + OPn. Abbreviations: OPn, osteopontin; eMT, epithelial-mesenchymal transition; nF-κB, nuclear factor-κB; lV-sirT1, lentiviral vectors overexpressing sirtuin 1; nc, negative control; cB, control buffer; sD, standard deviation. deacetylating Smad4 in the TGF-β pathway and repressing TGF-β signaling. 20 Therefore, it is likely that the effect of SIRT1 on EMT depends on the tumor types, specific downstream targets and tumor microenvironment. However, the underlying mechanisms of these contradictory data in different cell types and culture conditions would require further investigation. OPN, as a secreted tumor regulatory protein in tumor microenvironment derived from both tumor cells and tumor associated stromal cells, mediates cell-matrix interactions and plays an important role in EMT initiation. 37 It is necessary to develop effective approaches to decrease OPN level or block the signaling cascades elicited by OPN. The SIRT1 activator, resveratrol, takes part in anti-nephrolithic activity mediated partially by the inhibition of OPN. 38 However, the relationship between OPN and SIRT1 in tumor is still unclear. Recent studies have showed that SIRT1 expression was regulated by various cytokines, such as TGF-β, TNF-α and IL-1β. 30,39,40 This is the first study to demonstrate that SIRT1 expression was decreased in NSCLC cells treated with OPN. Since NF-κB is a direct target of SIRT1, 16 the effect of SIRT1 overexpression on NF-κB signaling pathway activated by OPN was explored. It was discovered that SIRT1 overexpression inhibits OPN-induced NF-κB activation. Previous studies have demonstrated that SIRT1 suppresses tumor cell proliferation and reduces chemoresistance by inhibiting NF-κB signaling. 21,22 In our study, the results exhibited that SIRT1 overexpression could inhibit OPN-induced NSCLC cell EMT by the inhibition of NF-κB activation.

Conclusion
In conclusion, SIRT1 expression was inhibited and NF-κB acetylation was promoted by OPN treatment. Our study suggests that SIRT1 is a potential tumor suppressor that inhibits OPN-induced NF-κB activation by deacetylation of NF-κB p65 to protect NSCLC cells from OPN-induced EMT. Therefore, SIRT1 may be a novel therapeutic target for treating OPN-induced NSCLC progression. OPn promotes nsclc cell eMT. Notes: (A-C) The protein expression of target genes in control group is presented as 1. results presented are mean ± sD analyzed with student's t-test from three independent experiments. *P,0.05, **P,0.01, ***P,0.001 versus control. Abbreviations: OPn, osteopontin; nsclc, non-small cell lung cancer; eMT, epithelial-mesenchymal transition; sD, standard deviation.

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