Transcription Factor KLF4: A Potential Biomarker of Non-small Cell Lung Cancertranscription Factor KLF4: A Potential Biomarker of Non-small Cell Lung Cancer

Background Krüppel-like factor 4 (KLF4) is a member of the zinc nger transcription factor family and plays an important role in cell proliferation, differentiation, apoptosis, embryonic development, organ formation, and tumor invasion. There is little research on the clinicopathological characteristics and KLF4 expression in non-small cell lung cancer (NSCLC). Methods We conducted a retrospective study to further explore the correlation between KLF4 expression and clinicopathological features of NSCLC by immunohistochemical staining. Results This study included 81 males (52.3%), 74 females (47.7%), and 101 (65.2%) ages <65, 54 (34.8%) ages ≥ 65 years. A total number of 127 cases (81.9%) were adenocarcinoma, 26 cases (16.8%) were squamous cell carcinoma, and 2 cases (1.3%) were large cell carcinoma. There were 140 patients (89.7%) in T1-T2 phase and 15 patients in T3-T4 phase; 130 patients (83.9%) in N0 phase and 25 patients (16.1%) in N1-N3 phase. There were no distant metastases in 154 patients (99.4%), and only 1 patient (0.6%) was in M1 phase. In terms of clinical stage, 142 patients (91.6%) in stage I-II and 13 patients (8.4%) in stage III-IV. The number of patients with smoking history was 40 (25.8%). KLF4 expression in NSCLC tissues was signicantly decreased compared with normal lung tissues (P<0.001); KLF4 expression in adenocarcinoma was lower than that in normal lung tissues (P<0.001), while there was no signicant difference in quamous cell carcinoma (P=0.314). Higher KLF4 expression was signicantly associated with clinicopathological features of NSCLC such as squamous cell carcinoma (P<0.001), later T stage (T3-T4 stage) (P=0.013), lymph node metastasis (P=0.011), later clinical stage (III-IV stage) (P=0.001). KLF4 expression was not associated with age (P=0.082); there was no signicant difference in KLF4 expression between male and female patients with adenocarcinoma (P=0.709). It was also found that KLF4 was positively correlated with Ki-67 expression in patients with adenocarcinoma (r=0.272, P=0.002). Conclusions Aboval all, the tumor suppressor gene KLF4 become a potential biomarker and a new therapeutic target for lung cancer patients.


Introduction
After entering the 21st century, despite the progress in the diagnosis and treatment of lung cancer, the prognosis of lung cancer is still poor due to the lack of early diagnosis, and the 5-year survival rate is still very low, about only 18% [1] . There were 18.1 million new cancer cases worldwide in 2018, of which 11.6% were lung cancer patients, while 18.4% of 9.6 million cancer deaths died from lung cancer so lung cancer is the most common type of cancer diagnosed and the leading cause of cancer death [2] . With the development of our economy, the accumulation of factors such as air pollution, tobacco and occupational exposure, the incidence and mortality of lung cancer are on the rise, which brings huge social and economic burden [3] . Non-small cell lung cancer (NSCLC) accounted for about 80% of the total lung cancer [4] . Patients with non-small cell lung cancer are often diagnosed later in clinical stage, resulting in poor prognosis. Therefore, the related mechanism of lung cancer development needs to be further studied in order to provide more support for early diagnosis and early treatment in clinical work.
Recently, some biomarkers have also provided new perspectives for targeted therapy in lung cancer patients, such as human epidermal growth factor receptor 2 (Her2), phosphatase and tensin-like protein (PTEN) and Krüppel like transcription factor 4 (KLF4), etc. Of which KLF4 was rst identi ed by Shields et al. in 1996 [5,6] , also known as gastrointestinal enrichment Krüppel like transcription factor, is a member of the KLF family and belongs to the larger Sp1 like transcription factor family [7][8][9] . KLF4 is a eukaryotic zinc nger protein transcript with binding site speci city, which is involved in the regulation of cell proliferation, differentiation, apoptosis and somatic reprogramming. KLF4 has gradually received widespread attention due to the diversity of its biological functions, and many studies have explored the role of KLF4 in lung cancer. Fadous-Khalife et al. reported that KLF4 expression is related to the clinicopathological characteristics of lung cancer patients and is a potential biomarker of lung cancer [10] , but its research has some shortcomings such as the small number of included tissue samples. Our preliminary basic research found that KLF4 can inhibit the metastasis of NSCLC [11] .

Immunohistochemistry staining
Surgical specimens were xed in 10% formalin and embedded in para n. Four-micrometer sections from NSCLC tumors were depara nized and antigen retrieval was performed by microwaving. Immunohistochemistry staining was performed using KLF4 monoclonal antibody (Abcam, Cat. No. Ab215036). The adjacent normal tissues were used as positive controls and negative controls were used PBS as primary antibody.

Evaluation of immunohhistochemistry staining
For all the sections only nuclear positive staining was assessed. A combined score based on the intensity of nuclear positive staining and the percentage of positive labeled cells was de ned. The intensity of nuclear staining was assessed as follows: 0, no positive staining; 1, mild ; 2, moderate; 3, strong. The

Statistical analysis
Statistical analysis was performed using SPSS 25.0. The median and quartile were used to describe the grade data. The expression of KLF4 between tumor tissues and adjacent normal tissues was compared by nonparametric test of paired data. Correlation between KLF4 expression and clinicopathological characteristics was analyzed by nonparametric test of two independent samples. Chi square test or Fisher exact test was performed to analyze the correlation beetween Ki67 exprssion and clinicopathological characteristics. The correlation between KLF4 expression and Ki67 expression was analyzed by Spearman rank correlation test. P <0.05 was considered signi cant.

Patient characteristics
This study selected 155 patients who met the study criteria between September 2018 and February 2019. Patient characteristics were shown in Table 1. The median (Q25, Q75) of KLF4 expression was 0 (0, 3) in adenocarcinoma tissues, of which in control tissues was 5 (4, 5). KLF4 expression was signi cantly different between lung adenocarcinoma tumor samples and squamous tumors samples (P<0.001), shown in Table 3. Immunohistochemical staining was shown in Figure 1.  Table 2. The median of KLF4 expression (Q25,Q75) was 0 (0,3) and 4 (0,4) in tissue samples of lung cancer patients during the T1-T2 and T3-T4 stage, respectively. Further strati cation analysis revealed that there was no signi cant difference of KLF4 expression in adenocarcinoma or squamous cell carcinoma.
The median (Q25,Q75) expression of KLF4 in N0 and N1-N3 patients was 0(0,3.25) and 3(1,4), respectively. The expression was signi cantly higher in patients with lymph node metastasis than in those without lymph node metastasis (P=0.011). Further strati cation analysis revealed that there was no signi cant difference of KLF4 expression in adenocarcinoma or squamous cell carcinoma.
The median KLF4 expression (Q25,Q75) was 0(0,3) and 4(2.5,4) in patients with stage I-II and stage III-IV, respectively. KLF4 expression was signi cantly higher in patients with clinical stage III-IV than in patients with stage I-II (P=0.001). The further analysis showed that 7 adenocarcinoma patients with III-IV stage had higher expression than 120 patients with I-II stage (P=0.013); there was no signi cant difference in squamous cell carcinoma patients (P=0.744).
We next detect the relationship between KLF4 expression and clinicopathologic variables. No signi cant correlation was found between KLF4 expression and age, gender. There was a positive correlation between tumor tissue KLF4 expression and Ki-67 proliferation index in non-small cell lung cancer (r=0.512, P<0.001), shown in Figure 2A.

Discussion
Lung cancer has caused a heavy economic and health burden in China. Recently, the diagnosis and treatment of lung cancer has been greatly improved in the early stage, and there are also problems of metastasis, recurrence, drug resistance and so on. Therefore, it is particularly important to nd better biomarkers and new therapeutic targets for lung cancer. KLF4 is a nuclear transcription factor and plays a tumor inhibitory role in lung cancer according to multiple clinical and experimental evidence. A total of 155 NSCLC patients were included in this study to explore KLF4 expression and its correlation with clinicopathological features.
The results showed that the expression of KLF4 in tumor tissues of NSCLC was signi cantly lower than that of normal lung tissues; the down-regulation of KLF4 expression may be associated with promoter hypermethylation, loss of heterozygosity in KLF4 loci or point mutations in the coding region [12] . Meanwhile, KLF4 expression in T3-T4 stage was higher than that in T1-T2 stage patients. The further analysis showed that there was no signi cant difference in adenocarcinoma patients and squamous cell carcinoma patients, separately. However, a study from Shaoli Li et al. included 60 patients found that the proportion of KLF4 low expression increased signi cantly in the T3, T4 group [13] . A recently published study [14] showed that there was no signi cant difference in KLF4 expression in tumor tissues of patients with stage T1-T2 and stage T3-T4 NSCLC. Our results showed that KLF4 expression was signi cantly higher in patients with N1-N3 stage. The further analysis showed that there was no signi cant difference in adenocarcinoma patients and squamous cell carcinoma patients, separately. Previous studies have shown [15][16][17] lower KLF4 expression in patients with lymph node metastasis. The expression of KLF4 in this study was signi cantly higher in patients with III-IV stage than in patients with I-II stage. KLF4 expression of patients with III-IV adenocarcinoma was higher than that of 120 patients with I-II adenocarcinoma; there was no signi cant difference in KLF4 expression between 20 patients with I-II and 6 patients with III-IV squamous cell carcinoma. Studies by Shaoli Li, Wenxian Hu, Zhang Zhiping, and others suggested that decreased KLF4 expression in patients with NSCLC following staging progression [13][14][15] . Marie Claude Fadous-Khalife's study found that KLF4 expression was absent in the stage I, and the expression of KLF4 was signi cantly higher in patients with II, III, IV stages [12] . The potential gene silencing due to hypermethylation might contribute to the lower KLF4 expression. However, the speci c mechanism needs to be elucidated by further studies in the future. Increased KLF4 expression in II, III and IV stages may be associated with reduced tumor differentiation and increased invasiveness [12] . As for correlation between KLF4 expression and distant metastasis of NSCLC, no statistical analysis was carried out. Therec was only one of 155 patients in this study was M1 because patients with distant metastasis generally did not choose surgical treatment. While a study incorporating 50 NSCLC patients with stage M0 and 10 patients with stage M1 showed that KLF4 expression decreased with distant metastasis [13] . The preliminary statistical analysis of this study showed signi cant differences in KLF4 expression between men and women, but further strati ed analysis revealed no correlation between KLF4 expression and sex in adenocarcinoma. This study does not include the female squamous cell carcinoma patient, failing to further analyze whether there is a difference in KLF4 expression between male and female lung squamous cell carcinoma patients.
KLF4 can inhibit cell proliferation, resulting in cell cycle arrest at G1/S and G2/M stages. Proliferation index Ki-67 re ects cell proliferation ability. To explore the correlation between KLF4 and Ki-67 expression of NSCLC, immunohistochemistry were performed. Results showed that there was a positive correlation in NSCLC and adenocarcinoma, the correlation in squamous cell carcinoma needs to be con rmed by larger sample size and cytology and animal experiments , because only 26 cases were included in this study.
Above all, there is still a lack of large-scale, multicenter research data on the correlation between KLF4 and NSCLC clinicopathological features such as T stage, N stage, M stage, clinical stage, etc. The number of samples retrieved in the literature is less than 100, and this study included 155 patients and could obtain more objective and accurate results. The KLF4 expression levels of previous studies were classi ed as positive, negative or high expression, low expression. The KLF4 expression of this study was evaluated as different grades between 0-6 points according to the positive area and intensity of KLF4 positive staining, which could re ect the difference of KLF4 expression more speci cally. At the same time, this study adopts the 8th edition of the TNM staging criteria, which has greatly changed compared with 7th edition that used in earlier, to determine the stage of NSCLC.
Our study revealed that KLF4 expression signi cantly higher in squamous cell carcinoma than in adenocarcinoma. There was no signi cant difference between 38 squamous cell carcinoma and 40 adenocarcinoma tissues in Mingyue Liu's and Zhiping Zhang's studies [14,16] . The difference of KLF4 expression in different pathological tissues might attribute to KLF4 biological function dependent on cell microenvironment and tissue speci city [15,18] , and might also associated with DNA mutations, molecular alterations, hypermethylation or miRNAs.

Conclusion
The role and mechanism of KLF4 in NSCLC has not been fully de ned at present, and the conclusions of the correlation between KLF4 and the clinicopathological features of NSCLC are not fully uni ed, which need to be con rmed by larger sample or prospective experiments. Our study showed that overall KLF4 expression in NSCLC decreased compared with normal lung tissue, while KLF4 expression in squamous cell carcinoma was signi cantly higher than that in adenocarcinoma, and increased with the progression of T stage, N stage, clinical stage, which could be expected to become a new biomarker for the diagnosis of NSCLC.