Molecular Carcinogenesis

Correspondence Yu Deng, Department of Thoracic Surgery, Tongji Hospital, Huazhong University of Science and Technology, 430030 Wuhan, China. Email: dengyuyayayhu@163.com Abstract Lung cancer is one of the most common causes of cancer‐related mortality worldwide, which is partially due to its metastasis. However, the mechanism underlying its metastasis remains elusive. In this study, we showed that a low Krüppel‐like factor 3 (KLF3) expression level is correlated with a poor prognosis and TNM stages in clinical patients with lung cancer and further demonstrated that KLF3 expression is downregulated in lung cancer tissues compared with adjacent normal samples. In addition, bioinformatics analysis results showed that KLF3 expression is related to lung cancer epithelial‐mesenchymal transition (EMT). In vitro and in vivo experiments also showed that KLF3 silencing promotes lung cancer EMT and enhances lung cancer metastasis. More importantly, bioinformatics analysis and in vitro experiments indicated that the role of KLF3 in lung cancer metastasis is dependent on the STAT3 signaling pathway. Overall, our data indicated the crucial function of KLF3 in lung cancer metastasis and suggested opportunities to improve the therapy of patients with lung cancer.

different types of tumors. [12][13][14][15] KLF3, which is a member of the Krüppellike factor family, has also shown aberrant expression in many kinds of cancers. In human metastatic sarcomas, dysregulated expression of KLF3 has been observed, and the knockdown of KLF3 has been shown to promote cancer cell metastasis. 16 RNA sequencing analysis has demonstrated that KLF3 may play a protective role in colorectal cancer. 17 However, the precise mechanism of the regulation of cancer metastasis mediated by KLF3 remains incompletely explained, and the role of KLF3 in lung cancer remains unclear.
Metastasis is a multistep process that is dependent on the regulation of abnormal signal transduction through pathways, including the TGFβ, JAK2/STAT3, and Wnt signaling pathways. 18 Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family, which is composed of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. 19,20 Under normal conditions, STAT3 mainly localizes in the cytoplasm, and upon stimulation from external environmental factors, such as IL6, STAT3 translocates into the nucleus and binds to DNA to activate the expression of related genes.
In multiple cell types, increasing evidence has demonstrated that STAT3 functions as a tumor oncogene. Increasing studies have shown that the overexpression of STAT3 enhances cell proliferation and metastasis in a variety of cancers. For example, the overexpression of IL-22RA1 promotes stemness and tumorigenicity in pancreatic cancer by regulating the STAT3 signaling pathway. 21 In addition, it has recently been shown that the activation of the STAT3 signaling pathway promotes colon cancer metastasis by regulating epithelialmesenchymal transition (EMT). 22,23 The STAT3 signaling pathway seems to be necessary for cancer metastasis. Despite these findings, the reason for the abnormal activation of STAT3, especially in lung cancer, has yet to be determined.
Considering the urgent need to explore novel therapeutic targets for lung cancer metastasis, we determined the potential role of KLF3 in lung cancer metastasis and examined the expression levels of KLF3 in clinical lung cancer tissues in this study. More importantly, we found that the biological function of KLF3 in metastasis is dependent on transcriptional STAT3 expression. Our findings suggested that the KLF3/STAT3 signaling pathway is a potential therapeutic target for patients with lung cancer. Antibodies against STAT3, KLF3, E-cadherin, and N-cadherin were obtained from Cell Signaling Technology (Boston), and antibodies against vimentin, Zo-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam.

| Western blot analysis
Western blot analysis was performed using total cell lysates that were lysed in NP40 (150 mM NaCl, 0.1% SDS, 1% NaMoO 4 , 1% NP-40, 50 mM Tris-HCl [pH 7.5], and 0.02% NaN 3 ) buffer with protease and phosphatase inhibitors (Roche). The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The bands were incubated with primary antibodies at 4°C overnight. Next, the bands were incubated with HRPconjugated secondary antibodies. Then, signals were examined by ECL reagents (Thermo Fisher Scientific).

| Plasmid constructs and stable cell lines
The short hairpin RNA (shRNA) sequences for KLF3 were obtained from Sigma-Aldrich. shKLF3 that was used in all the experiments was constructed using the following sequence: CCGGCCCACTTGAAAG

| Luciferase reporter assay
For the luciferase reporter assay, cells were seeded into 24-well plates.

| Statistical analysis
The data are presented as the mean ± standard deviation (SD).
Pearson's χ 2 test was used to analyze associations between KLF3, E-cadherin, and vimentin to evaluate relationships between KLF3 expression and clinicopathological characteristics. P < .05 was considered statistically significant. All statistical analyses were performed using SPSS 21.0 statistical software (SPSS Inc, Chicago, IL) and GraphPad Prism 5.0 software (GraphPad Software, Inc, La Jolla, CA).

| Reduced KLF3 expression in human lung cancer is associated with tumor progression
To investigate the role of KLF3 in human lung cancer progression, we first examined KLF3 expression in normal and cancerous human lung tissues by bioinformatics analysis (data were obtained from the Oncomine database). As shown in Figure 1A and 1B, we found that the SUN ET AL. messenger RNA (mRNA) expression of KLF3 was markedly downregulated in lung cancer tissues compared to normal tissues. Next, as shown in Figure 1C and 1D, we found that the protein expression of KLF3 was markedly downregulated in eight matched lung cancer and normal tissues. Furthermore, we examined the mRNA expression levels of KLF3 in the abovementioned tissues, and reduced KLF3 mRNA expression was observed in the lung cancer specimens ( Figure   1E). In addition, the levels of KLF3 expression were further evaluated by IHC. Similar to the abovementioned results, we observed that the levels of KLF3 expression were reduced in lung cancer specimens compared with normal tissues ( Figure 1F and 1G).
To further assess the clinical value of KLF3, we first detected the KLF3 protein expression levels in 56 pairs of human lung cancer tissues and matched normal lung tissues. As shown in Table 1, we found that the levels of KLF3 expression were negatively correlated with the tumor, node, metastasis (TNM) stage and lymph node metastasis. Importantly, the bioinformatics analysis results also showed that KLF3 expression was positively correlated with the TNM stage ( Figure 1H). Collectively, the abovementioned data indicated that KLF3 expression is downregulated in lung cancer tissues and closely related to tumor progression and might act as a molecular marker for predicting lung cancer metastasis.

| Knockdown of KLF3 promotes lung cancer cell migration and invasion
Considering that KLF3 is related to lung cancer metastasis, we established stable cell lines expressing shRNA-KLF3 via lentiviral infection. As shown in Figure 2A and 2B, the silencing efficiency was analyzed by Western blot and qPCR assays in H1299 and A549 cells.
Then, we examined the migratory ability of control and shRNA-KLF3 cells. As shown in Figure 2C and 2D, the wound-healing assay surprisingly showed that the knockdown of KLF3 markedly promoted cell migration. Consistent with these results, the Transwell assay using Transwells coated with and without Matrigel showed that the knockdown of KLF3 substantially promoted the migration ( Figure 3E and 3F) and invasion ( Figure 3G and 3H) of lung cancer cells.
To assess the role of KLF3 in lung cancer cell proliferation, we compared changes in cell proliferation between the three groups. As shown in Figure S1, no significant changes were observed between the three groups, indicating that the promotion of migration and invasion by KLF3 knockdown was not dependent on cell proliferation. Overall, the abovementioned data further implicated that the knockdown of KLF3 enhances lung cancer migration and invasion and that KLF3 might suppress the metastasis of lung cancer cells.

| KLF3 silencing promotes EMT in lung cancer
Metastasis is a multistep process. EMT is considered an important step in the metastasis cascade and enables cell migration and invasion.
Indeed, the enrichment of genes that mediate EMT in lung cancer tissues with low KLF3 expression levels was observed ( Figure 3A). This finding led us to hypothesize that KLF3 regulates the EMT of cancer cells that affects metastasis. To evaluate the effect of KLF3 on EMT, we first examined the protein expression of mesenchymal and epithelial markers in control and shRNA-KLF3 cells. Interestingly, the expression levels of the mesenchymal markers were high in the shRNA-KLF3 cells compared to the control cells ( Figure 3B and 3C).
More importantly, shKLF3 cells exhibited an obvious mesenchymal transition phenotype ( Figure 3D). The immunofluorescence (IF) assay results also showed that the shRNA-KLF3 cells expressed substantially more mesenchymal markers than the control cells ( Figure 3E).
Consistent with the phenomena that are affected by KLF3 knockdown, compared with the control cells, the shRNA-KLF3 cells obviously exhibited a mesenchymal phenotype throughout the general observation period.
Collectively, these data suggest that KLF3 silencing mediates EMT progression and that KLF3 is involved in lung cancer development.

| KLF3 silencing upregulates STAT3 expression in a transcriptional manner
To explore the molecular mechanism underlying how KLF3 modulates metastasis in lung cancer cells, we further analyzed an online database by gene set enrichment analysis (GSEA). Surprisingly, we found that the enrichment of STAT3 target gene sets is associated with reduced KLF3 expression in lung cancer ( Figure 4A and 4B), suggesting that KLF3 may be involved in the STAT3 signaling pathway. We determined whether KLF3 regulates STAT3 expression in lung cancer cells. As shown in Figure 4C, the Western blot results showed that the STAT3 expression level was reduced in control cells compared with shKLF3 cells. It has been reported that KLF3 mainly  controls gene expression by epigenetically regulating gene transcription. Therefore, the mRNA expression levels of STAT3 were examined in control cells and shKLF3 cells. Using qPCR assays, we observed that the knockdown of KLF3 markedly increased the STAT3 mRNA expression level ( Figure 4D). Moreover, to confirm the notion that KLF3 regulates STAT3 expression in a transcriptional manner, we constructed a luciferase plasmid containing the promoter of the STAT3 gene. Next, we cotransfected luciferase plasmids into cells. The dual-luciferase assay results demonstrated that compared with control cells, the knockdown of KLF3 strongly increased the luciferase activity of theSTAT3 promoter ( Figure 4E).
Considering the role of KLF3 in regulating gene expression, it is dependent on the CACCC box-binding transcription element. Thus, we speculated that KLF3 directly interacts with the promoter of STAT3 and subsequently decreases its expression level. As expected, we predicted KLF3 binding to the STAT3 promoter region ( Figure   4F). To confirm this speculation, Chip assays were performed using an anti-KLF3 antibody to pull-down KLF3-bound chromatin and PCR using primers for the STAT3 promoter. The ChIP results clearly suggested that KLF3 binds to the STAT3 gene promoter ( Figure 4G), indicating that KLF3 might suppress STAT3 expression by binding to the promoter of the STAT3 gene. In addition, we further mutated the binding site of the STAT3 promoter that is specifically recognized by KLF3 ( Figure 4F). Importantly, the dual-luciferase assays showed that the knockdown of KLF3 had no effect on the luciferase activity of the mutated STAT3 promoter ( Figure 4H). Overall, our data suggested that KLF3 regulates STAT3 expression in a transcriptional manner.

| KLF3 silencing promotes metastasis through the upregulation of STAT3 expression
Considering the crucial role of STAT3 in tumor progression and the contribution of KLF3 to the effects of STAT3, we examined whether STAT3 is essential for metastasis-mediated knockdown of KLF3 in lung cancer cells. First, we transfected shRNAs to knockdown STAT3 expression in shKLF3 cells. As shown in Figure 5A and 5B, the wound healing assay showed that STAT3 silencing markedly abrogated the knockdown of KLF3-induced cell migration. Furthermore, the Transwell assays showed that the knockdown of STAT3 abolished the migration-and invasion-promoting effects induced by the knockdown of KLF3 ( Figure 5C-F). These data indicated that STAT3 expression is at least partially essential for the prometastatic function of reducing the KLF3 expression level in lung cancer cells.

| Knockdown of KLF3 promotes lung cancer metastasis in vivo
To examine the role of KLF3 in lung cancer metastasis in vivo, 1 × 10 5 A549 cells were intravenously injected into Balb/c mice. After 6 weeks, all mice were killed, and the lung tissues were histologically examined. As shown in Figure 6A and 6B, compared with mice injected with the control cells, mice injected with the shKLF3 cells had more metastatic nodules and larger metastatic nodules on the lung surface. In addition, shKLF3 A549 cells shortened the survival time of the injected mice compared with the shNC group ( Figure 6C).
Furthermore, the number of metastatic nodules, wet lung weight, and percentage of tumor tissues to total lung tissues in the shKLF3 group mice were much greater than those in the shNC group mice ( Figure 6D-F). To further evaluate the role of the KLF3/STAT3 signaling pathway in lung cancer metastasis in vivo, we performed a rescue experiment in a mouse model. As shown in Figure 6G-I, compared with mice injected with the control cells, mice injected with the shKLF3 cells had more metastatic nodules and larger metastatic nodules on the lung surface; however, silencing STAT3 significantly abrogated the number and size of metastatic nodules induced by shKLF3. Overall, these results indicated that the KLF3/ STAT3 signaling axis plays a crucial role in lung cancer metastasis.

| Low KLF3 expression levels are associated with STAT3 expression in clinical lung cancer specimens
To understand whether KLF3 expression is related to EMT and the STAT3 signaling pathway in patients with lung cancer, we conducted an IHC assay to examine the correlation between KLF3, EMT marker, and STAT3 expression in 56 lung cancer specimens. As shown in Figure 7A and 7B, KLF3 expression was negatively correlated with STAT3 and vimentin expression in the clinical lung cancer specimens.
Furthermore, there was a strong positive correlation between KLF3 and E-cadherin expression. Overall, these findings strongly indicated that KLF3 acts as a crucial suppressor of lung cancer by regulating metastasis via STAT3 expression.

| DISCUSSION
Metastasis is the major reason for a poor prognosis in patients with lung cancer. EMT is the key factor that causes epithelial cancer cells to lose cellular junctions and vessels to infiltrate distant areas. 24,25 To elucidate abnormal signaling pathways involved in metastasis could provide a potential therapeutic target for treating lung cancer.
In this study, we demonstrated the molecular mechanism of metastasis regulated by KLF3 in vitro and in mouse models.  More importantly, we provided new insights into the KLF3/STAT3 signaling pathway in lung cancer. It has been reported that low expression levels of KLF3 in tumors are caused by DNA methylation. 16 More importantly, DNA methylation inhibitors have been applied in clinical practice, suggesting that the expression of KLF3 might be promoted by DNA methylation inhibitors in clinical patients with lung cancer to improve the prognosis. Thus, we propose that KLF3 may be a poor diagnostic marker and a potential therapeutic approach for patients with metastatic lung cancer.