Lung cancer is one of the most common cancers and the leading cause of cancer deaths in the world and China. Metastasis is an essential aspect of cancer progression and most lung cancer patients present with metastatic diseases. Chemotherapy, radiation, and surgery have generally not been satisfactory in the management of lung cancer metastasis and about 90% of lung cancer patients die from metastasis. Therefore, the treatment of metastasis is of great importance to the clinical management of patients with lung cancer. Metastasis is a complex process involving loss of cell adhesion, invasion, entry into the circulation, and finally colonization of distant sites. Many factors contribute to the metastasis of tumor cells such as, expression of metastasis-related genes, matrix metalloproteinases (MMPs), and molecular signaling pathways.
Integrins exist on the surface of cancer cells and promote the metastatic potential of cancer cells via mediating cell–cell adhesion and invasion. They mediate interactions between cancer cells and the extracellular matrix. The different integrin subfamilies are determined by the β subunit. The β8-integrin is known to bind to the αv subunit and encoded by the ITGB8 gene. ITGB8 has been found to be increased in many types of cancer, including breast cancer, lung cancer, laryngeal cancer, and gastric cancer (Ni et al., 2010). Furthermore, ITGB8 has shown increased expression in highly metastatic tumors. ITGB8 is considered as a critical metastasis-related gene and potential target for treating metastatic cancer. However, mechanisms underlying the relationship between lung cancer metastasis and ITGB8 are still not very clear.
In this study, we presented evidence that silencing of ITGB8 in lung cancer cell lines A549 and PC9 dramatically changed the cell cycle, invasion, and adhesion potential in vitro. Increased expressions of E-Cadherin and cystatin B, while decreased expressions of CXCL1, CXCL 2, CXCL 5, MMP-2, and MMP-9 were found. Furthermore, the changes in the cell cycle, expression of metastasis-related genes, and metastatic potential were accompanied by decreased tumor cell signal transduction molecular activity, for example, NF-κB, Snail, MEK, and Akt. Our studies indicate that silencing of ITGB8 may decrease the metastatic potential of the lung cancer cell lines A549 and PC9 by controlling the expression of metastasis-related genes and cell signal transduction molecular activity.
MATERIALS AND METHODS
Cell Lines and Cell Culture
Human lung cancer cell lines A549 and PC9 (American Type Culture Collection) were grown and maintained in DEME (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies) at 37°C with 5% CO2.
Cell Transfection and Stable Clones Establishment
An empty vector and ITGB8 shRNA plasmid (Santa Cruz biotechnology) were transfected into human lung cancer cell lines A549 and PC9 with Lipofectamine 2000 reagent. Cells were screened with 400 μg mL−1 of G418 (Invitrogen) to generate ITGB8 silencing and negative control sublines. ITGB8 expression was measured using western blotting and real time PCR.
Cell Invasion Assay In Vitro
The invasion assay in vitro was examined by Matrigel invasion chambers (Costar). The 20 μL fibronectin (0.2 μg μL−1) was added on the lower surface of the membrane and 50 μL Matrigel (0.2 μg μL−1) was added on the upper surface of the membrane. About 2 × 105 cells were planted and incubated in 0.1% BSA medium for 24 hr. The cells invading to the chamber membranes were counted by photographing the membrane in five microscopic fields (100× magnification) after H&E staining.
Cell Adhesion Assay
The 96-well plates were covered with 25 μL Matrigel (0.2 μg μL−1) overnight at 37°C and blocked by 2% BSA medium for 1 hr. A 4 × 104 cells suspended in 100 μL 0.1% BSA medium were added to wells and incubated for 1 hr at 37°C with 5% CO2, then washed by PBS. The 96-well plates were determined by MTS assay at 490 nm.
Cell Cycle Analysis Assay
Nearly 2 × 105 cells were seeded into six-well plates for 24 hr. Then cells were harvested and fixed in 70% ice-cold ethanol overnight. Cells were treated with RNase A, stained with propidium iodide, and then subjected to a FACSAria flow cytometer. The data were analyzed with the ModFit LT software.
Levels of CXCL1, CXCL2, and CXCL5 in supernatant were quantified using commercially available enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's protocol (R&D Systems).
Western Blot Assay
Cells were treated in RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, and 1 mg mL−1 Aprotinin] and protein concentration was detected by BCA protein assay kit. About 20 μg of total cell lysate was separated on 12% SDS-PAGE, transferred to nitrocellulose membrane. Membrane was blocked for 1 hr at room temperature with 5% milk protein, and then probed with appointed antibodies with 5% BSA overnight at 4°C. Membranes were incubated with HRP-conjugated goat anti-rabbit antibody with 3% milk protein for 1 hr. After washing, blots were visualized with the Phototope HRP Western blot detection system.
RNA Reverse Transcription and Real-Time PCR Assay
Total cellular RNA was extracted via the Trizol reagent (Invitrogen) and quantified using UV spectrophotometer. RNA was reverse transcribed by M-MLV reverse transcriptase kit according to the manufacturer's instructions. The cDNA was mixed with ABI SYBR Green Master Mix and appointed genes primer, then amplified in ABI7500 Real-time PCR. The results were normalized by glyceraldehyde-3-phoshate dehydrogenase (GAPDH). Primer sequences were as follows: ITGB8 forward: 5′-CTGAAGAAATACCCCGTGGA-3′, reverse: 5′-ATGGGGAGGCATACAGTCT-3′. E-Cadherin forward: 5′-CTGAAGTGACTCGTAACGAC-3′, reverse: 5′-CATGTCAGCCAGCTTCTTGAAG-3′; MMP-2 forward: 5′-AGCTGCAACCTGTTTGTG-3′, reverse: 5′-CCAATGATCCTGTATGTGATC-3′; MMP-9 forward: 5′-ACGAGGACTCCCCTCTGCAT-3′; Reverse: 5′-AGGCCTTGGGTCAGGTTTAGA-3′;cystatin B forward 5′-GAGCGTGCACTTGTGATCCTAA-3′, reverse 5′-GCCCCTTCCACCCCAA-3′; CXCL1, forward: 5′-AACCGAAGTCATAGCCACAC-3′; reverse: 5′-GTTGGATTTGTCACTGTTCAGC-3′; CXCL2 forward 5′-CGCTGTCAATGCCTGAAG-3′; CXCL2 reverse, 3′-GGCGTCACACTCAAGCTCT-5; CXCL5 forward 5′ 5′-TTCATGAGAAGGCAATGCTG-3′, reverse 5′-CCCAGGCTCAGACGTAAGAA-3′; GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′, reverse 5′-TCCACCACCCTGTTGCTGTA-3′.
DNA Transfection Assay
The 1 × 105 cells were plated in 24-well plate and were cotransfected with 400 ng of psnail-luc, pNF-κB-luc, and 4 ng of pRL-SV40 as an internal control. Luciferase assays were performed using the Dual-luciferase Reporter Assay System, on a BERTHOLD TriStar LB 941 microplate reader after 24 hr.
Data are presented as means ± SD. One-way ANOVA test was used to analyze the differences between parent, negative control, and ITGB8 silencing groups. Differences were considered statistically significant at P < 0.05.
Total cellular protein and RNA were extracted to verify the expression change of ITGB8 in the human lung cancer cell lines A549 and PC9 by Western blot assay and real-time PCR. As shown in Fig. 1, ITGB8 siRNA-induced cells dramatically decreased at both mRNA and protein expression levels compared to the parent and negative control groups.
There was a significant change in cell metastasis behavior between the ITGB8 silencing group and the parent or negative control groups. In adhesion and invasion assays, the adhesion and invasion abilities of the human lung cancer cell lines with ITGB8 silencing were dramatically decreased compared to the parent and negative control groups (Figs. 2, 3).
The flow cytometric cell cycle analysis revealed that A549- and PC9-ITGB8 silencing cells decreased in G0/G1 phase and increased in S phase compared to the parent and negative control groups (Fig. 4). This indicated that decreased lung cancer cell metastatic ability may be due to the cell cycle arrest.
The protein expression of MMP-2/-9, E-Cadherin and cystatin B were verified by Western blot. The protein expression of E-Cadherin and cystatin B was clearly increased, while the expression of MMP-2 and -9 were dramatically decreased in the ITGB8 silencing cell lines compared to the parent and negative control groups. We also used real-time PCR to verify the mRNA expression levels of MMP-2/-9, E-Cadherin, and cystatin B. The mRNA expression of E-Cadherin and cystatin B were dramatically increased, while the expression of MMP-2 and -9 were clearly decreased in ITGB8 silencing cell lines compared to the parent and negative control groups (Figs. 5, 6). Meanwhile, we verified the expression of CXCL-1, -2, and -5 in tumor culture media supernatant. The results showed decreased expression of CXCL-1, -2, and -5 in ITGB8 silencing cell lines compared to the parent and negative control groups (Fig. 7).
Because ITGB8 modulates a series of molecules that play important roles in tumor metastasis, we investigated whether ITGB8 was able to inactivate MEK, Akt, Snail, and NF-κB. ITGB8-silenced cell lines showed a significant decrease in the levels of phosphorylated MEK/Akt proteins but no such finding was observed in the levels of total MEK/Akt proteins (Figs. 8–10). We then examined the effect of ITGB8 on snail and NF-κB transcriptional activity. Cells were transfected with pSnail-luc and pNF-κB-luc, and the transcriptional activity was detected by luciferase reporter assay. ITGB8 silencing suppressed Snail and NF-κB transcriptional activity in human lung cancer cell lines (Fig. 8).
ITGB8 activates metastasis and some studies suggest that it may act as an upstream regulator that modulates downstream metastasis-related genes, inhibiting tumor metastasis. Preliminary data suggested that ITGB8 silencing could reduce tumor cell adhesion, invasion, and cell cycle arrest in human lung cancer cells in vitro. However, the mechanism by which ITGB8 participates in tumor metastasis is not fully understood.
Our results showed that down regulation of ITGB8 expression induced decreased tumor cell adhesion and invasion abilities, and cell cycle arrest in vitro. We further explored the mechanism by which ITGB8 silencing decreased cancer cells metastasis using the Western blot, real-time PCR, ELISA, and luciferase reporter assays. ITGB8 was found to regulate metastasis-related genes by increasing the expression of E-cadherin and cystatin B, and decreasing the expression of MMP-2, MMP-9, CXCL1, CXCL 2, and CXCL 5.
E-cadherin is a calcium-dependent glycoprotein which can promote cell adhesion. The decreased expression of E-cadherin was observed in many tumors (Kato et al., 2011; Ucvet et al., 2011). Some studies indicate that tumor cells can secrete CXCL-1,-2, and -5 to attract neutrophils and activate MMP-2 and -9 to degrade the extracellular matrix (ECM), leading to tumor metastasis (Kulbe et al., 2004; Põld et al., 2004). Silencing of ITGB8 resulted in decreased expression of CXCL-1, -2, -5 and reduced metastatic potential of the cancer cell lines A549 and PC9. Abundant evidence shows that the tumor microenvironment could regulate cell cycle progression (Li et al., 2009). We inferred that the effect of ITGB8 on the cell cycle occurs at the G1–S transition, a critical point in cell cycle progression. This may be one of the mechanisms by which ITGB8 affect the tumor cell cycle.
ECM degradation, which is one of the first steps in tumor invasion and metastasis, relies mainly on relative gene expression of proteinases, including MMPs and their inhibitors. MMPs, a Zn2+-independent protease, may regulate tumor cell adhesion, proliferation, and migration directly or through the release of growth factors. MMP-2 and -9 have been suggested to play an important role in the early metastatic invasion of tumor (Ertan et al., 2011; Gingis-Velitski et al., 2011; Kim et al., 2012) Cystatin B is the inhibitor of MMPs, and MMPs/cystatin B imbalance could cause degradation of ECM and vascular basement membrane. The expression of cystatin B has been shown to significantly reduce in lung cancer (Rivenbark et al., 2009). Our study showed that MMP-2 and -9 were significantly decreased and cystatin B was dramatically increased at mRNA levels and protein levels both in A549- and PC9-ITGB8 silencing cells. So we inferred that cystatin B was first activated and then resulted in the subsequent dramatic changes. The molecular mechanism on how ITGB8 upregulate cystatin B and downregulate MMP-2 and -9 is not very clear.
To further explore the molecular mechanisms involved in inhibitory effect of ITGB8 silencing on tumor metastasis, we studied the Akt pathway. The activation of Akt is one of the most frequent alterations observed in human cancer cells. Accumulating evidence shows that Akt activation plays an important role in tumor metastasis through regulation of metastasis-related genes expression (Wang et al., 2012). Akt phosphorylation promotes tumor cell metastasis by activating cell substrates such as NF-κB. NF-κB is a transcription factor which is activated by various intra- and extracellular stimuli. It controls the expression of numerous genes involved in tumor metastasis, including MMP-2/-9, E-Cadherin, and cystatin B. Inhibition of NF-κB transcription activation effectively suppressed tumor cell metastasis through upregulation of E-Cadherin and cystatin B and downregulation of MMP-2 and -9 (Kim et al., 2009). MEK is another important cell signaling pathway. It also regulates various cellular activities, such as gene expression, differentiation, proliferation, survival, and metastasis. CXCL families can be regulated by Ras gene. Our findings indicated that ITGB8 silencing may regulate the expression of CXCL -1, -2, and -5 through inhibition of the MEK-related pathway (Hong et al., 2011). The zinc-finger transcription factor Snail has been confirmed to inhibit the E-cadherin expression by interacting with the E-box proximal promoter (Dong et al., 2012). In this study, ITGB8 silencing significantly decreased the transcriptional activity of Snail and increased the expression of E-cadherin, resulting in reduced metastatic potential of human lung cancer cell lines A549 and PC9. We also inferred that ITGB8 silencing may directly regulate the activity of Snail transcription by combining with the Zn2+structural domain.
Our results clearly demonstrate that ITGB8 silencing can suppress the metastatic potential of human lung cancer cell lines A549 and PC9. The modulation of metastasis-related genes, the inhibition of Akt and MEK phosphorylation, and the inactivation of NF-κB and Snail transcription could be part of the mechanism by which ITGB8 silencing suppressed lung cancer cells metastatic potential. Although further in vivo studies will be required, these results raise the possibility that ITGB8 might be a potential therapeutic strategy for the management of lung cancer, and possibly other types of cancer.