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Subcellular localization of Klf4 in non-small cell lung cancer and its clinical significance
Biomedicine & Pharmacotherapy 99 (2018) 480–485
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Subcellular localization of Klf4 in non-small cell lung cancer and its clinical significance
T
Mingyue Liua, Xiqing Lia, Ke-Zheng Pengb, Tianhui Gaoa, Yao Cuia, Ning Maa, Yun Zhoua, ⁎ Guiqin Houb, a b
Department of Oncology, People’s Hospital of Henan Province (Zhengzhou University), Zhengzhou 450003, China School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Klf4 non-small cell lung cancer Subcellular localization Cisplatin
Kruppel-like factor 4 (Klf4) was reported to have both tumor suppressive and oncogenic roles on tumorigenesis, which is depend on its subcellular localization. In this study, the expression and subcellular localization of Klf4 in non-small cell lung cancer (NSCLC) patients as well as its clinical significance were analyzed, and the expression and subcellular localization of Klf4 in A549 cells and A549/DDP cells were detected. The results showed that the expression of Klf4 in nucleus was related to the histological grade and clinical stage of NSCLC patients. Moreover, the subcellular localization of Klf4 is the independent risk factor for NSCLC, and the high expression of Klf4 in nucleus could lead to a poor prognosis, while the high expression of Klf4 in cytoplasm could lead to a prominent prognosis for NSCLC patients. In addition, the nuclear Klf4 expression in A549/DDP cells was higher than that in A549 cells, while the cytoplasmic Klf4 expression in A549/DDP cells was lower than that in A549 cells, indicating that the subcellular localization of Klf4 might be related to the resistance of A549 cells to cisplatin. The study indicates that Klf4 could be a potential therapeutic target in NSCLC.
1. Introduction Lung cancer is well-known because of its high incidence and mortality [1]. As a malignancy originated in epidermal cells, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer, which contains a variety of histologic subtypes including lung adenocarcinoma, lung squamous cell carcinoma and large cell lung cancer. In recent years, the 5-year survival rate and median survival time of NSCLC are still low with current therapeutic methods [2–4]. Therefore, it is imperative to explore the molecular mechanism of NSCLC and find new strategies for the diagnosis of NSCLC. Kruppel-like factor 4 (Klf4) is a nuclear transcription factor with a zinc-finger-like domain in eukaryotes, which contains some multiple functional areas, such as the N-terminal transcriptional activation domain, C-terminal transcription inhibitory region and nuclear localization sequences for regulating protein-protein interactions [5]. Klf4 could induce fibroblasts into pluripotent stem cells, and could be as the first started gene to regulate the transcription of Oct4, Sox2 and C-myc, thus inducing the transformation of non-neoplastic epithelial cells to cancer stem cells [6–8]. It has been reported that Klf4 could express both in cytoplasm and in nucleus, which has different functions on drug resistance by affecting the formation of tumor stem cells [5]. However,
⁎
the subcellular localization of Klf4 in NSCLC as well as its clinical significance have not been scientifically evaluated before. In this study, the subcellular localization and clinical significance of Klf4 in human NSCLC specimens were analyzed. In addition, we explored the potential role of Klf4 in the resistance of NSCLC cells to cisplatin by analyzed the expression and subcellular localization of Klf4 in A549 cells and A549/DDP cells. 2. Materials and methods 2.1. Ethical approval The studies have been approved by Ethics Committee of Life Science of Zhengzhou University and obtained the informed consent from participants. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Corresponding author at: School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China. E-mail address: [email protected] (G. Hou).
https://doi.org/10.1016/j.biopha.2018.01.090 Received 29 June 2017; Received in revised form 2 January 2018; Accepted 16 January 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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were extracted using CelLyticTM NuCLEARTM Extraction Kit (SigmaAldrich, USA). The protocols of the protein extraction of cytoplasm and nucleus were according to the instruction of the Kit. The proteins extracted from cytoplasm or nucleus was used for Western blot analysis as described before [11]. In brief, the protein extracts were separated by SDS-PAGE and electrotransferred to support nitrocellulose membranes (Amersham, Sweden) by a semidry transferor. After the membranes were blocked for 2 h in 5% skimmed milk in PBS-T at room temperature (RT), they were incubated with anti-Klf4 (Abcam Inc., USA, catalog #ab215036), anti-Histone H3 (Cell Signaling Technology, USA, catalog #D1H2) or anti-β-Actin (Cell Signaling Technology, USA, catalog #13E5) of 1:1000 diluted in 1% skimmed milk in PBS-T, respectively, at RT for 2 h, followed by being incubated with the appropriate HRPlinked secondary antibodies of 1:8000. Finally, the bands of specific proteins on the membranes were visualized with chemiluminescent substrate (Santa Cruz Biotechnology, USA) according to the manufacturer’s instructions. The membranes were rinsed three times with PBS-T between the incubations.
2.2. Patients tissue samples and cell lines 78 NSCLC tissue samples (33 men and 45 women with the mean age of 61 ± 7 years old) from Chinese patients were collected from People's Hospital of Zhengzhou University (Henan Province), Zhengzhou, China. No patients had undergone chemotherapy or radiotherapy prior to surgery. Among them, histologic subtype was 38 (squamous carcinoma) and 40 (adenocarcinoma), and according to the histologic grade, the number of grade 1 was 29, grade 2 was 28 and grade 3 was 21 tissues, respectively. Furthermore, T classification was T1-T2 of 30 and T3-T4 of 48, respectively, and the clinical stage was I-II of 48 and III-IV of 30, respectively. NSCLC cell line A549 and A549/ DDP were purchased from Cell Centre of XiangYa School of Medicine, Central South University, Changsha, China, and were cultured in RPMI1640 medium (Gibco-BRI, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in the presence of 5% CO2. The resistance index of A549/DDP is 2.524, and for keeping the resistance of cisplatin in A549/DDP, 2 μg/mL cisplatin was added in the culture medium when A549/DDP cells were cultured.
2.6. Statistical analysis SPSS19.0 software was used for statistical analysis. χ2-test was used to analyze the clinical significance of Klf4 protein expression, the correlation between the expression level of Klf4 and survival time or prognosis of patients with NSCLC were analyzed by Kaplan-Meier method, and the compare of protein expression level of Klf4 in cytoplasm and nucleus by Western blot was performed by one-way ANOVO. p < 0.05 was considered as statistically significant.
2.3. Immunohistochemical and clinical significance analysis After the NSCLC tissues above were fixed in 10% formalin and embedded in paraffin wax, 4 μm thick sections were cut, and then, the expressions of Klf4 in them were measured immunohistochemically and the relationships between the expression levels and site of Klf4 protein and gender, Age, histologic subtype, histological grade, T classification and clinical stage were analyzed, respectively. The protocols of immunohistochemistry were described as our previous paper [9] and immunohistochemical evaluation was performed by a pathologist without knowledge of the clinical and pathologic characteristics of these patients. The tumor cells were further scored according to the intensity (I), distribution (D), and pattern (P) reported by Dong et al. [10]: I score: 0, negative; 1, weak; 2, moderate; and 3, strong. D score (%): 0, negative; 1, 10–50%; 2, 51–90%; and 3, > 90%. P score: 0, no staining; 1, sporadic positive staining; 2, focal positive staining; and 3, diffuse positive staining. The total scores of each tissue = I × D × P; the 0 score was negative, and ≥1 score was positive. In addition, the information of patients above suffered conventional chemotherapy after surgery operation were tracked and the relationships between the expression level and site of Klf4 in tissues and survival time and prognosis of NSCLC patients were analyzed.
3. Results 3.1. Expression and subcellular localization of Klf4 in NSCLC patients and its clinical significance To examine the potential role of the Klf4 in NSCLC, the expression of Klf4 was examined immunohistochemically in NSCLC tissues. The results showed that Klf4 was expressed both in cytoplasm and in nucleus (Fig. 1). Then the correlation between clinicopathological features and subcellular localization of Klf4 expression in NSCLC patients was analyzed and showed in Table 1. The expression level of Klf4 in cytoplasm had no relation to the gender, age, histologic subtype and T classification (p > 0.05). However, when Klf4 was expressed in nucleus, its expression level was related to the histological grade and clinical stage (p < 0.05).
2.4. Immunofluorescence 3.2. Correlation between NSCLC patients’ survival time and the subcellular localization of Klf4
After the sterile coverslips were placed previously into 6-well plates, A549 cells and A549/DDP cells were seeded into 6-well plates and cultured for 24 h. Then the coverslips were picked out, the cells were fixed with 4% formaldehyde for 10 min and then incubated with 1% BSA in PBS-T for 1 h to block non-specific protein-protein interactions. The cells were then incubated with anti-Klf4 antibody (Abcam Inc., USA, catalog #ab215036) at a 1:2000 dilution overnight at 4 °C. The secondary antibody marked by green fluorescence was added at a 1:250 dilution and incubated for 1 h, and then 4′6-Diamidino-2-phenylindole dihydrochloride (DAPI) was added and incubated for 15 min. Finally, mounting medium preventing fluorescence attenuation was added and the fluorescence of cells was observed using fluorescence confocal microscope (Olympus, FV10i, Olympus Corporation, Japan).
Next, the Kaplan-Meier assay were performed and the results were showed in Fig. 2. The survival time of patients with high expression of Klf4 in cytoplasm was significantly longer than that with low expression (p < 0.05), whereas the survival time was significantly decreased in patients with high expression of Klf4 in nucleus than that with low expression (p < 0.05). The results above indicated that the subcellular localization of Klf4 had obvious relevance to patient’s survival time, and the expression of Klf4 in cytoplasm helped to prolong patients’ survival time. 3.3. Correlation between NSCLC patients’ prognosis and subcellular localization of Klf4
2.5. Western blot Moreover, Cox regression was used to have univariate analysis with all of the statistical two-tailed parameters. As shown in Table 2, the
The nuclear and cytoplasmic proteins of A549 and A549/DDP cells
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Fig. 1. Expression of Klf4 in NSCLC tissues by immunohistochemical analysis (400×). (a) Klf4 was positive expressed in cytoplasm. (b) Klf4 was moderate positive expressed in cytoplasm. (c) Klf4 was negative expressed in cytoplasm. (d) Klf4 was positive expressed in nucleus. (e) Klf4 was moderate positive expressed in nucleus. (f) Klf4 was negative expressed in nucleus.
(p > 0.05), which indicated that the subcellular localization of Klf4 had obvious relevance to patients’ prognosis.
differentiation, cytoplasmic expression of Klf4 and nuclear expression of Klf4 are prognostic risk factors for NSCLC (p < 0.05). Subsequently, multivariate Cox proportional hazards regression was used to analyze the cytoplasmic and nuclear expression of Klf4, and we found the cytoplasmic and nuclear expression of Klf4 are independent risk factors for NSCLC (p < 0.05; Table 2). Then we analyzed the correlation between the patients’ prognosis and subcellular localization of Klf4 expression in NSCLC patients and showed in Table 3. The results demonstrated that the patients with low expression of Klf4 in nucleus had better prognosis than that of high expression (p < 0.05), while the prognosis of patients with different expression level of Klf4 in cytoplasm had no obvious difference
3.4. Expression and subcellular localization of Klf4 in A549 and A549/ DDP cells To explored the role of Klf4 in the resistance of NSCLC cells to cisplatin, the expression and subcellular localizations of Klf4 in A549 cells and A549/DDP cells were analyzed by immunofluorescence and Western blot. As shown in Fig. 3, Klf4 was expressed in both cytoplasm and nucleus of A549 cells, while there was strong expression of Klf4 in nucleus of A549/DDP cells but weak expression in cytoplasm. As shown
Table 1 Correlation between clinicopathological features and subcellular localization of Klf4 expression in NSCLC patients. Variables
Gender Male Female Age < 65 ≥65 Histologic subtype Squamous carcinoma Adenocarcinoma Histological grade Grade 1 Grade 2 Grade 3 T classification T1-T2 T3-T4 Clinical stage I–II III–IV
n
Cytoplasm n (%)
Nucleus n (%)
Low
High
P
Low
High
P
33 45
18(54.5) 23(51.1)
15(45.5) 22(48.9)
0.764
22(66.7) 38(84.4)
11(33.3) 7(15.6)
0.066
45 33
23(51.1) 18(54.5)
22(48.9) 15(45.5)
0.764
33(73.3) 27(81.8)
12(26.7) 6(18.2)
0.380
38 40
21(55.3) 20(50.0)
17(44.7) 20(50.0)
0.642
31(81.6) 29(72.5)
7(18.4) 11(27.5)
0.341
29 28 21
12(41.4) 17(60.7) 12(57.1)
17(58.6) 11(39.3) 9(42.9)
0.305
26(89.7) 22(78.6) 12(57.1)
3(10.3) 6(21.4) 9(42.9)
0.026
30 48
18(60.0) 23(47.9)
12(40.0) 25(52.1)
0.298
23(76.7) 37(77.1)
7(23.3) 11(22.9)
0.966
48 30
22(45.8) 19(63.3)
26(54.2) 11(36.7)
0.132
33(68.8) 27(90.0)
15(31.2) 3(10.0)
0.030
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Fig. 2. Effect of subcellular localization of Klf4 to patients’ survival time. The relationship of the expression site of Klf4 in tissues and survival time of patients with NSCLC was analyzed by Kaplan-Meier method. (a) The effect of cytoplasmic Klf4 on patient’s survival time. (b) The effect of nuclear Klf4 on patient’s survival time.
4. Discussion
Table 2 Univariate and multivariate prognostic analysis of influencing factors in NSCLC patients. Variables
Category
Univariate analysis Gender Age (years old) Histologic subtype T classification C classification Differentiation Cytoplasmic Klf4 Nuclear Klf4 Multivariate analysis Cytoplasmic Klf4 Nuclear Klf4
OS (overall survival) HR
95% CI
In recent years, although the strategies of early diagnosis, treatment, radiotherapy and chemotherapy of lung cancer have been greatly improved, the recurrence, metastasis, drug resistance and poor prognosis of lung cancer were still serious [12]. Klf4 was reported to have different functions on the proliferation and apoptosis of tumor cells by changing their subcellular localization, which has both tumor suppressive and oncogenic roles [13–16]. In gastric cancer, cervical cancer, bladder carcinoma and liver cancer, Klf4 plays the role of a tumor suppressor [17–22]. However, Klf4 plays the oncogenic role in breast cancer and laryngeal squamous cell carcinoma [21–23]. In addition, it is reported that the effects of Klf4 on tumorigenesis depend on its cell context [24]. Our previous study had showed that the influence of stem cell-related factors in different sub-position on prognosis of lung cancer, and Klf4 could increase malignancy degree of lung tumor through increase its stemness [25]. However, the function and mechanism of Klf4 in NSCLC are still unclear. In the present study, we analyzed the expression and clinical significance of Klf4 in patient’s tissues of NSCLC, and the correlation between the subcellular localization of Klf4 and survival time/prognosis of NSCLC patients. These results indicated that the subcellular localization of Klf4 in NSCLC cells has significant different effects on survival time and prognosis of NSCLC patients, and the expression of Klf4 in cytoplasm may help to prolong survival time and improve prognosis of NSCLC patients. It is explicit that the nuclear transcription factor Exportin-1/ Chromosome Region Maintenance 1/CRM1 (XPO1) could transfer some proteins from nucleus to cytoplasm of cells through binding with nuclear localization sequences [26,27]. And some drugs were reported to reverse the transport of proteins between nucleus and cytoplasm to suppress tumor [28,29]. In our present study, we confirmed that the nuclear Klf4 expression of A549/DDP cells was higher than that in A549 cells, while the cytoplasmic Klf4 expression of A549/DDP cells was lower than that in A549 cells, which indicated that nuclear Klf4 may participated in the resistance of A549 cells to cisplatin. Therefore, finding a novel pharmacological strategies that targets the subcellular localization of Klf4 and promotes the transfer of Klf4 from nucleus to
p
Male vs. female < 65 vs. ≥65 Squamous carcinoma vs. Adenocarcinoma T1–T2 vs. T3–T4 N0–N1 vs. N2–N3 G1,G2,G3 positive and negative positive and negative
1.156 0.897 1.201
0.698–1.455 0.875–1.145 0.987–1.541
0.785 0.100 0.514
0.785 0.741 1.654 1.846 2.059
0.562–1.121 0.548–0.975 1.101–2.009 1.452–2.401 1.549–2.737
0.098 0.697 0.000 0.000 0.000
positive and negative positive and negative
2.157 1.727
1.487–2.824 1.270–2.348
0.000 0.000
Table 3 Correlation between prognosis and subcellular localization of Klf4 expression in NSCLC patients. Variables
CR + PR SD + PD
Cases
40 38
Cytoplasm n (%)
Nucleus n (%)
Low
High
P
Low
High
P
22(55.0) 19(50.0)
18(45.0) 19(50.0)
0.223
37(92.5) 23(60.5)
3(7.5) 15(39.5)
0.001
Note: CR, completely remission; PR: partly remission; SD, stable disease; PD, disease in progress.
in Fig. 4, compared to the A549 cells, the nuclear Klf4 expression of A549/DDP cells increased significantly (p < 0.05), while the cytoplasmic Klf4 expression of A549/DDP cells decreased significantly (p < 0.05). The results above indicated that the subcellular localization of Klf4 in cells might be related to the resistance of A549 cells to cisplatin.
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Fig. 3. The expression and subcellular localization of Klf4 in A549 and A549/DDP cells were detected by immunofluorescence. After A549 and A549/DDP cells were fixed with 4% formaldehyde, anti-Klf4 antibody and secondary antibody marked by green fluorescence as well as DAPI were used. Then the expression and subcellular localization of Klf4 in A549 and A549/DDP cells were observed by fluorescence confocal microscope (200×).
Fig. 4. The expression and subcellular localization of Klf4 in A549 and A549/DDP cells were detected by Western blot. (a) the cytoplasmic and nuclear proteins of A549 and A549/DDP cells were extracted, and the expressions of Klf4 in cytoplasm and nucleus were detected by Western blot. Histone H3 and β-Actin was used as control. (b) Semiquantitative values of the bands in Fig. 4a from three independently repeated experiments, which were statistically analyzed by densitometry using Image J software, were expressed as means ± SD. *p < 0.05 compared to control group.
cytoplasm will be of significance to NSCLC treatment.
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Subcellular localization of Klf4 in non-small cell lung cancer and its clinical significance
T
Mingyue Liua, Xiqing Lia, Ke-Zheng Pengb, Tianhui Gaoa, Yao Cuia, Ning Maa, Yun Zhoua, ⁎ Guiqin Houb, a b
Department of Oncology, People’s Hospital of Henan Province (Zhengzhou University), Zhengzhou 450003, China School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Klf4 non-small cell lung cancer Subcellular localization Cisplatin
Kruppel-like factor 4 (Klf4) was reported to have both tumor suppressive and oncogenic roles on tumorigenesis, which is depend on its subcellular localization. In this study, the expression and subcellular localization of Klf4 in non-small cell lung cancer (NSCLC) patients as well as its clinical significance were analyzed, and the expression and subcellular localization of Klf4 in A549 cells and A549/DDP cells were detected. The results showed that the expression of Klf4 in nucleus was related to the histological grade and clinical stage of NSCLC patients. Moreover, the subcellular localization of Klf4 is the independent risk factor for NSCLC, and the high expression of Klf4 in nucleus could lead to a poor prognosis, while the high expression of Klf4 in cytoplasm could lead to a prominent prognosis for NSCLC patients. In addition, the nuclear Klf4 expression in A549/DDP cells was higher than that in A549 cells, while the cytoplasmic Klf4 expression in A549/DDP cells was lower than that in A549 cells, indicating that the subcellular localization of Klf4 might be related to the resistance of A549 cells to cisplatin. The study indicates that Klf4 could be a potential therapeutic target in NSCLC.
1. Introduction Lung cancer is well-known because of its high incidence and mortality [1]. As a malignancy originated in epidermal cells, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer, which contains a variety of histologic subtypes including lung adenocarcinoma, lung squamous cell carcinoma and large cell lung cancer. In recent years, the 5-year survival rate and median survival time of NSCLC are still low with current therapeutic methods [2–4]. Therefore, it is imperative to explore the molecular mechanism of NSCLC and find new strategies for the diagnosis of NSCLC. Kruppel-like factor 4 (Klf4) is a nuclear transcription factor with a zinc-finger-like domain in eukaryotes, which contains some multiple functional areas, such as the N-terminal transcriptional activation domain, C-terminal transcription inhibitory region and nuclear localization sequences for regulating protein-protein interactions [5]. Klf4 could induce fibroblasts into pluripotent stem cells, and could be as the first started gene to regulate the transcription of Oct4, Sox2 and C-myc, thus inducing the transformation of non-neoplastic epithelial cells to cancer stem cells [6–8]. It has been reported that Klf4 could express both in cytoplasm and in nucleus, which has different functions on drug resistance by affecting the formation of tumor stem cells [5]. However,
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the subcellular localization of Klf4 in NSCLC as well as its clinical significance have not been scientifically evaluated before. In this study, the subcellular localization and clinical significance of Klf4 in human NSCLC specimens were analyzed. In addition, we explored the potential role of Klf4 in the resistance of NSCLC cells to cisplatin by analyzed the expression and subcellular localization of Klf4 in A549 cells and A549/DDP cells. 2. Materials and methods 2.1. Ethical approval The studies have been approved by Ethics Committee of Life Science of Zhengzhou University and obtained the informed consent from participants. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Corresponding author at: School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China. E-mail address: [email protected] (G. Hou).
https://doi.org/10.1016/j.biopha.2018.01.090 Received 29 June 2017; Received in revised form 2 January 2018; Accepted 16 January 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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were extracted using CelLyticTM NuCLEARTM Extraction Kit (SigmaAldrich, USA). The protocols of the protein extraction of cytoplasm and nucleus were according to the instruction of the Kit. The proteins extracted from cytoplasm or nucleus was used for Western blot analysis as described before [11]. In brief, the protein extracts were separated by SDS-PAGE and electrotransferred to support nitrocellulose membranes (Amersham, Sweden) by a semidry transferor. After the membranes were blocked for 2 h in 5% skimmed milk in PBS-T at room temperature (RT), they were incubated with anti-Klf4 (Abcam Inc., USA, catalog #ab215036), anti-Histone H3 (Cell Signaling Technology, USA, catalog #D1H2) or anti-β-Actin (Cell Signaling Technology, USA, catalog #13E5) of 1:1000 diluted in 1% skimmed milk in PBS-T, respectively, at RT for 2 h, followed by being incubated with the appropriate HRPlinked secondary antibodies of 1:8000. Finally, the bands of specific proteins on the membranes were visualized with chemiluminescent substrate (Santa Cruz Biotechnology, USA) according to the manufacturer’s instructions. The membranes were rinsed three times with PBS-T between the incubations.
2.2. Patients tissue samples and cell lines 78 NSCLC tissue samples (33 men and 45 women with the mean age of 61 ± 7 years old) from Chinese patients were collected from People's Hospital of Zhengzhou University (Henan Province), Zhengzhou, China. No patients had undergone chemotherapy or radiotherapy prior to surgery. Among them, histologic subtype was 38 (squamous carcinoma) and 40 (adenocarcinoma), and according to the histologic grade, the number of grade 1 was 29, grade 2 was 28 and grade 3 was 21 tissues, respectively. Furthermore, T classification was T1-T2 of 30 and T3-T4 of 48, respectively, and the clinical stage was I-II of 48 and III-IV of 30, respectively. NSCLC cell line A549 and A549/ DDP were purchased from Cell Centre of XiangYa School of Medicine, Central South University, Changsha, China, and were cultured in RPMI1640 medium (Gibco-BRI, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in the presence of 5% CO2. The resistance index of A549/DDP is 2.524, and for keeping the resistance of cisplatin in A549/DDP, 2 μg/mL cisplatin was added in the culture medium when A549/DDP cells were cultured.
2.6. Statistical analysis SPSS19.0 software was used for statistical analysis. χ2-test was used to analyze the clinical significance of Klf4 protein expression, the correlation between the expression level of Klf4 and survival time or prognosis of patients with NSCLC were analyzed by Kaplan-Meier method, and the compare of protein expression level of Klf4 in cytoplasm and nucleus by Western blot was performed by one-way ANOVO. p < 0.05 was considered as statistically significant.
2.3. Immunohistochemical and clinical significance analysis After the NSCLC tissues above were fixed in 10% formalin and embedded in paraffin wax, 4 μm thick sections were cut, and then, the expressions of Klf4 in them were measured immunohistochemically and the relationships between the expression levels and site of Klf4 protein and gender, Age, histologic subtype, histological grade, T classification and clinical stage were analyzed, respectively. The protocols of immunohistochemistry were described as our previous paper [9] and immunohistochemical evaluation was performed by a pathologist without knowledge of the clinical and pathologic characteristics of these patients. The tumor cells were further scored according to the intensity (I), distribution (D), and pattern (P) reported by Dong et al. [10]: I score: 0, negative; 1, weak; 2, moderate; and 3, strong. D score (%): 0, negative; 1, 10–50%; 2, 51–90%; and 3, > 90%. P score: 0, no staining; 1, sporadic positive staining; 2, focal positive staining; and 3, diffuse positive staining. The total scores of each tissue = I × D × P; the 0 score was negative, and ≥1 score was positive. In addition, the information of patients above suffered conventional chemotherapy after surgery operation were tracked and the relationships between the expression level and site of Klf4 in tissues and survival time and prognosis of NSCLC patients were analyzed.
3. Results 3.1. Expression and subcellular localization of Klf4 in NSCLC patients and its clinical significance To examine the potential role of the Klf4 in NSCLC, the expression of Klf4 was examined immunohistochemically in NSCLC tissues. The results showed that Klf4 was expressed both in cytoplasm and in nucleus (Fig. 1). Then the correlation between clinicopathological features and subcellular localization of Klf4 expression in NSCLC patients was analyzed and showed in Table 1. The expression level of Klf4 in cytoplasm had no relation to the gender, age, histologic subtype and T classification (p > 0.05). However, when Klf4 was expressed in nucleus, its expression level was related to the histological grade and clinical stage (p < 0.05).
2.4. Immunofluorescence 3.2. Correlation between NSCLC patients’ survival time and the subcellular localization of Klf4
After the sterile coverslips were placed previously into 6-well plates, A549 cells and A549/DDP cells were seeded into 6-well plates and cultured for 24 h. Then the coverslips were picked out, the cells were fixed with 4% formaldehyde for 10 min and then incubated with 1% BSA in PBS-T for 1 h to block non-specific protein-protein interactions. The cells were then incubated with anti-Klf4 antibody (Abcam Inc., USA, catalog #ab215036) at a 1:2000 dilution overnight at 4 °C. The secondary antibody marked by green fluorescence was added at a 1:250 dilution and incubated for 1 h, and then 4′6-Diamidino-2-phenylindole dihydrochloride (DAPI) was added and incubated for 15 min. Finally, mounting medium preventing fluorescence attenuation was added and the fluorescence of cells was observed using fluorescence confocal microscope (Olympus, FV10i, Olympus Corporation, Japan).
Next, the Kaplan-Meier assay were performed and the results were showed in Fig. 2. The survival time of patients with high expression of Klf4 in cytoplasm was significantly longer than that with low expression (p < 0.05), whereas the survival time was significantly decreased in patients with high expression of Klf4 in nucleus than that with low expression (p < 0.05). The results above indicated that the subcellular localization of Klf4 had obvious relevance to patient’s survival time, and the expression of Klf4 in cytoplasm helped to prolong patients’ survival time. 3.3. Correlation between NSCLC patients’ prognosis and subcellular localization of Klf4
2.5. Western blot Moreover, Cox regression was used to have univariate analysis with all of the statistical two-tailed parameters. As shown in Table 2, the
The nuclear and cytoplasmic proteins of A549 and A549/DDP cells
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Fig. 1. Expression of Klf4 in NSCLC tissues by immunohistochemical analysis (400×). (a) Klf4 was positive expressed in cytoplasm. (b) Klf4 was moderate positive expressed in cytoplasm. (c) Klf4 was negative expressed in cytoplasm. (d) Klf4 was positive expressed in nucleus. (e) Klf4 was moderate positive expressed in nucleus. (f) Klf4 was negative expressed in nucleus.
(p > 0.05), which indicated that the subcellular localization of Klf4 had obvious relevance to patients’ prognosis.
differentiation, cytoplasmic expression of Klf4 and nuclear expression of Klf4 are prognostic risk factors for NSCLC (p < 0.05). Subsequently, multivariate Cox proportional hazards regression was used to analyze the cytoplasmic and nuclear expression of Klf4, and we found the cytoplasmic and nuclear expression of Klf4 are independent risk factors for NSCLC (p < 0.05; Table 2). Then we analyzed the correlation between the patients’ prognosis and subcellular localization of Klf4 expression in NSCLC patients and showed in Table 3. The results demonstrated that the patients with low expression of Klf4 in nucleus had better prognosis than that of high expression (p < 0.05), while the prognosis of patients with different expression level of Klf4 in cytoplasm had no obvious difference
3.4. Expression and subcellular localization of Klf4 in A549 and A549/ DDP cells To explored the role of Klf4 in the resistance of NSCLC cells to cisplatin, the expression and subcellular localizations of Klf4 in A549 cells and A549/DDP cells were analyzed by immunofluorescence and Western blot. As shown in Fig. 3, Klf4 was expressed in both cytoplasm and nucleus of A549 cells, while there was strong expression of Klf4 in nucleus of A549/DDP cells but weak expression in cytoplasm. As shown
Table 1 Correlation between clinicopathological features and subcellular localization of Klf4 expression in NSCLC patients. Variables
Gender Male Female Age < 65 ≥65 Histologic subtype Squamous carcinoma Adenocarcinoma Histological grade Grade 1 Grade 2 Grade 3 T classification T1-T2 T3-T4 Clinical stage I–II III–IV
n
Cytoplasm n (%)
Nucleus n (%)
Low
High
P
Low
High
P
33 45
18(54.5) 23(51.1)
15(45.5) 22(48.9)
0.764
22(66.7) 38(84.4)
11(33.3) 7(15.6)
0.066
45 33
23(51.1) 18(54.5)
22(48.9) 15(45.5)
0.764
33(73.3) 27(81.8)
12(26.7) 6(18.2)
0.380
38 40
21(55.3) 20(50.0)
17(44.7) 20(50.0)
0.642
31(81.6) 29(72.5)
7(18.4) 11(27.5)
0.341
29 28 21
12(41.4) 17(60.7) 12(57.1)
17(58.6) 11(39.3) 9(42.9)
0.305
26(89.7) 22(78.6) 12(57.1)
3(10.3) 6(21.4) 9(42.9)
0.026
30 48
18(60.0) 23(47.9)
12(40.0) 25(52.1)
0.298
23(76.7) 37(77.1)
7(23.3) 11(22.9)
0.966
48 30
22(45.8) 19(63.3)
26(54.2) 11(36.7)
0.132
33(68.8) 27(90.0)
15(31.2) 3(10.0)
0.030
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Fig. 2. Effect of subcellular localization of Klf4 to patients’ survival time. The relationship of the expression site of Klf4 in tissues and survival time of patients with NSCLC was analyzed by Kaplan-Meier method. (a) The effect of cytoplasmic Klf4 on patient’s survival time. (b) The effect of nuclear Klf4 on patient’s survival time.
4. Discussion
Table 2 Univariate and multivariate prognostic analysis of influencing factors in NSCLC patients. Variables
Category
Univariate analysis Gender Age (years old) Histologic subtype T classification C classification Differentiation Cytoplasmic Klf4 Nuclear Klf4 Multivariate analysis Cytoplasmic Klf4 Nuclear Klf4
OS (overall survival) HR
95% CI
In recent years, although the strategies of early diagnosis, treatment, radiotherapy and chemotherapy of lung cancer have been greatly improved, the recurrence, metastasis, drug resistance and poor prognosis of lung cancer were still serious [12]. Klf4 was reported to have different functions on the proliferation and apoptosis of tumor cells by changing their subcellular localization, which has both tumor suppressive and oncogenic roles [13–16]. In gastric cancer, cervical cancer, bladder carcinoma and liver cancer, Klf4 plays the role of a tumor suppressor [17–22]. However, Klf4 plays the oncogenic role in breast cancer and laryngeal squamous cell carcinoma [21–23]. In addition, it is reported that the effects of Klf4 on tumorigenesis depend on its cell context [24]. Our previous study had showed that the influence of stem cell-related factors in different sub-position on prognosis of lung cancer, and Klf4 could increase malignancy degree of lung tumor through increase its stemness [25]. However, the function and mechanism of Klf4 in NSCLC are still unclear. In the present study, we analyzed the expression and clinical significance of Klf4 in patient’s tissues of NSCLC, and the correlation between the subcellular localization of Klf4 and survival time/prognosis of NSCLC patients. These results indicated that the subcellular localization of Klf4 in NSCLC cells has significant different effects on survival time and prognosis of NSCLC patients, and the expression of Klf4 in cytoplasm may help to prolong survival time and improve prognosis of NSCLC patients. It is explicit that the nuclear transcription factor Exportin-1/ Chromosome Region Maintenance 1/CRM1 (XPO1) could transfer some proteins from nucleus to cytoplasm of cells through binding with nuclear localization sequences [26,27]. And some drugs were reported to reverse the transport of proteins between nucleus and cytoplasm to suppress tumor [28,29]. In our present study, we confirmed that the nuclear Klf4 expression of A549/DDP cells was higher than that in A549 cells, while the cytoplasmic Klf4 expression of A549/DDP cells was lower than that in A549 cells, which indicated that nuclear Klf4 may participated in the resistance of A549 cells to cisplatin. Therefore, finding a novel pharmacological strategies that targets the subcellular localization of Klf4 and promotes the transfer of Klf4 from nucleus to
p
Male vs. female < 65 vs. ≥65 Squamous carcinoma vs. Adenocarcinoma T1–T2 vs. T3–T4 N0–N1 vs. N2–N3 G1,G2,G3 positive and negative positive and negative
1.156 0.897 1.201
0.698–1.455 0.875–1.145 0.987–1.541
0.785 0.100 0.514
0.785 0.741 1.654 1.846 2.059
0.562–1.121 0.548–0.975 1.101–2.009 1.452–2.401 1.549–2.737
0.098 0.697 0.000 0.000 0.000
positive and negative positive and negative
2.157 1.727
1.487–2.824 1.270–2.348
0.000 0.000
Table 3 Correlation between prognosis and subcellular localization of Klf4 expression in NSCLC patients. Variables
CR + PR SD + PD
Cases
40 38
Cytoplasm n (%)
Nucleus n (%)
Low
High
P
Low
High
P
22(55.0) 19(50.0)
18(45.0) 19(50.0)
0.223
37(92.5) 23(60.5)
3(7.5) 15(39.5)
0.001
Note: CR, completely remission; PR: partly remission; SD, stable disease; PD, disease in progress.
in Fig. 4, compared to the A549 cells, the nuclear Klf4 expression of A549/DDP cells increased significantly (p < 0.05), while the cytoplasmic Klf4 expression of A549/DDP cells decreased significantly (p < 0.05). The results above indicated that the subcellular localization of Klf4 in cells might be related to the resistance of A549 cells to cisplatin.
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Fig. 3. The expression and subcellular localization of Klf4 in A549 and A549/DDP cells were detected by immunofluorescence. After A549 and A549/DDP cells were fixed with 4% formaldehyde, anti-Klf4 antibody and secondary antibody marked by green fluorescence as well as DAPI were used. Then the expression and subcellular localization of Klf4 in A549 and A549/DDP cells were observed by fluorescence confocal microscope (200×).
Fig. 4. The expression and subcellular localization of Klf4 in A549 and A549/DDP cells were detected by Western blot. (a) the cytoplasmic and nuclear proteins of A549 and A549/DDP cells were extracted, and the expressions of Klf4 in cytoplasm and nucleus were detected by Western blot. Histone H3 and β-Actin was used as control. (b) Semiquantitative values of the bands in Fig. 4a from three independently repeated experiments, which were statistically analyzed by densitometry using Image J software, were expressed as means ± SD. *p < 0.05 compared to control group.
cytoplasm will be of significance to NSCLC treatment.
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5. Conclusion In conclusion, our present study showed that the subcellular localization of Klf4 have significant relevance to the survival time and prognosis of NSCLC patients, and may related to chemotherapeutic resistance of NSCLC cells to cisplatin, which indicated that Klf4 could be a potential therapeutic target in future treatment of NSCLC. Conflict of Interest There is no conflict of interest in this study. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant numbers U1504820], the Basic and Frontier Research Foundation of Henan Science and Technology Agency, China [grant numbers 122300410066 and 162300410122] and Medical Science and Technology PR Project of Henan Health Agency, China [grant number 201702182]. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (2016) 7–30. [2] E. Brambilla, W.D. Travis, T.V. Colby, B. Corrin, Y. Shimosato, The new World Health Organization classification of lung tumours, Eur. Respir. J. 18 (2001) 1059–1068.
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