A-kinase-interacting protein 1 overexpression correlates with deteriorative tumor features and worse survival profiles, and promotes cell proliferation but represses apoptosis in non-small-cell lung cancer.

BACKGROUND: This study aimed to explore the correlation of A-kinase-interacting protein 1 (AKIP1) expression with clinical characteristics as well as survival profiles in non-small-cell lung cancer (NSCLC) patients, and further investigate its underlying effect on regulating NSCLC cell functions.
METHODS: 319 NSCLC patients who underwent resection were consecutively reviewed, and AKIP1 expression (in 319 tumor tissues and 145 adjacent tissues) was determined by immunohistochemistry. Disease-free survival (DFS) and overall survival (OS) were calculated. In vitro, control overexpression, AKIP1 overexpression, control shRNA and AKIP1 shRNA plasmids were transfected into A549 cells to evaluate the effect of AKIP1 on cell proliferation and apoptosis.
RESULTS: A-kinase-interacting protein 1 expression was increased in tumor tissues compared to adjacent tissues, and it positively correlated with tumor size, lymph node metastasis and TNM stage in NSCLC patients. Kaplan-Meier curves displayed that AKIP1 high expression correlated with worse DFS and OS, and multivariate Cox's regression revealed that it was an independent predictive factor for poor survival profiles. In vitro experiments displayed that AKIP1 expression was elevated in PC9 and A549 cells compared to normal lung epithelial cells; moreover, cell proliferation was increased by AKIP1 upregulation but reduced by AKIP1 downregulation, and cell apoptosis was decreased by AKIP1 upregulation but increased by AKIP downregulation in A549 cells. Interestingly, AKIP1 promoted fibronectin and zinc finger E-box binding homeobox 1 expressions while reduced E-cadherin expression in A549 cells.
CONCLUSION: A-kinase-interacting protein 1 overexpression correlates with deteriorative tumor features and worse survival profiles and promotes cell proliferation but represses apoptosis in NSCLC.

INTRODUCTION

Lung cancer, one of the most frequently diagnosed cancers, has become the leading cause of cancer‐related death worldwide.1, 2 According to Global Cancer Statistics 2018, lung cancer is responsible for approximately 2.09 million newly diagnosed cancer cases and 1.76 million cancer‐related deaths worldwide.3 Non‐small‐cell lung cancer (NSCLC), which involves adenocarcinoma, squamous cell carcinoma, and large‐cell carcinoma, accounts for 85% of newly diagnosed lung cancers.1 During the past two decades, some advancements in the treatments of NSCLC have been achieved, such as the application of small molecule tyrosine kinase inhibitors and immunotherapy, which benefit to some selected patients.4 Nevertheless, the prognosis of NSCLC patients (especially those with metastasis disease) is still dismal, with a 5‐year survival rate of just 15%.2 Hence, continued research into more effective screening for predicting disease progression in NSCLC patients at an early stage is required, and with this aim, molecular markers may expand the screening approach to improve prognosis in NSCLC.

A‐kinase‐interacting protein 1 (AKIP1) is 23‐kDa protein that localizes in the cytoplasm, nucleus, and mitochondria, and it serves as an adaptor of intracellular structural protein.5, 6, 7 Limited studies have displayed that AKIP1 is upregulated in tumor cells or tumor tissues such as colorectal cancer, cervical cancer, and esophageal squamous cell carcinoma, and its overexpression associates with abnormal physiological and pathological changes in patients with these cancers, indicating that AKIP1 may behave as an oncogene in these cancers.8, 9 Moreover, AKIP1 has been reported to act as a molecular determinant of protein kinase (PKA) in nuclear factor‐kB (NF‐κB) signaling, which is an inducible transcriptional factor for genes involved in inflammatory responses and cell proliferation in many diseases, particular in cancers.10, 11 For NSCLC, Guo et al12 reveal that overexpression of AKIP1 in NSLCC tissues was positively correlated with tumor features and AKIP promoted epithelial‐mesenchymal transition (EMT) of NSCLC via transactivating zinc finger E‐box binding homeobox 1 (ZEB1), while the sample size of 139 was relatively low, which might result in low statistical efficacy, and no further study was found to verify their results. Thus, this study enrolled 319 NSCLC patients to explore the correlation of AKIP1 expression with clinical characteristics as well as survival profiles and further investigate its underlying effect on regulating NSCLC cell functions. We envisaged that our study would contribute to expand the clinical data and deepen the understanding of mechanisms in NSCLC.

METHODS

Patients

From January 2010 to December 2013, 319 patients with NSCLC who underwent resection at the First Hospital of Qinhuangdao were consecutively reviewed in this retrospective study. The inclusion criteria were as follows: (a) histologically or cytologically confirmed NSCLC; (b) received resection, and tumor tissue was well preserved and eligible for immunohistochemistry (IHC) assay; (c) clinical and follow‐up records were complete and available; (d) no distant metastasis. The patients who underwent neoadjuvant therapy or complicated with other malignancies were excluded. Meanwhile, 145 adjacent noncancerous pulmonary tissues from NSCLC patients were collected as controls. The study protocol was approved by the Ethics Committee of First Hospital of Qinhuangdao. All patients or their guardians provided informed consents (paper version or electronic edition).

Data collection

All enrolled patients' clinical data were collected from electronic medical records, which included age, gender, tumor size, lymph node metastasis, distant metastasis, TNM stage, and pathological differentiation. Disease‐free survival (DFS) and overall survival (OS) were calculated according to survival data which were obtained from follow‐up records. The DFS was defined as the duration from resection to recurrence or progression of disease or death; the OS was defined as the time interval from resection to death.

Tissue preparation for IHC assay

The tumor tissues and adjacent noncancerous pulmonary tissues were freshly isolated from surgery, then fixed in 10% neutral‐buffered formalin, and then embedded in paraffin wax. The level of AKIP1 in the paraffin‐embedded specimens was assessed by IHC assay as follows: The specimens were cut into 4‐µm sections, deparaffinized with xylene, rehydrated with ethanol followed by antigen retrieval, and incubated with H2O2 for endogenous peroxidase blocking, then were blocked using 1.5% normal goat serum (Yeasen Biotechnology Co., Ltd). Subsequently, the tissue sections were incubated at 4℃ with rabbit‐anti‐C11orf17/AKIP1 antibody overnight (1:100 dilution) (Abcam). Next day, the tissue sections were incubated with horseradish peroxidase‐conjugated goat‐anti‐rabbit immunoglobulin G antibody (1:1000 dilution) (Abcam) for 30 minutes. Afterward, tissue sections were stained with diaminobenzidine (DAB) and hematoxylin and were sealed with neutral tree gum. Finally, the positive cells were observed and counted using a fluorescence microscope (Olympu0073).

Assessment of AKIP1 expression by IHC assay

A‐kinase‐interacting protein 1 expression in tumor tissues and adjacent noncancerous pulmonary tissues was assessed by a semi‐quantitative scoring method based on the average intensity and percentage of positively stained tumor cells according to previous studies.13 The intensity of staining was scored as follows: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (dark yellow/light brown); and 3, strong staining (dark brown). The proportion of positively stained tumor cells was scored as follows: 0, 0%; 1, ≤25%; 2, 26%‐50%; 3, 51%‐75%; 4, >75%. The total score was calculated by multiplying staining intensity score and the proportion score of positively stained tumor cells, and high expression of AKIP1 was defined as the total score >3, low expression of AKIP1 was defined as the total score ≤3.

Assessment of AKIP1, EMT markers, and transcriptional marker expressions by Western blot and quantitative polymerase chain reaction (qPCR)

Fresh tumor tissues and adjacent nontumor tissue were further obtained, and then, the AKIP1, E‐cadherin (EMT marker), fibronectin (EMT marker), and ZEB1 (transcriptional marker) mRNA expressions were detected by qPCR, and their protein expressions were assessed by Western blot.

Cell experiments about the effect of AKIP1 on NSCLC cell viability and apoptosis

Non‐small‐cell lung cancer cell lines including PC9, A549, NCI‐H1299, NCI‐H460, and normal lung epithelial cell line BEAS‐2B were purchased from Shanghai Institutes for Biological Science or American Type Culture Collection (ATCC). PC9 cells were cultured in 90% Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), A549 cells were cultured in 90% Roswell Park Memorial Institute (RPMI) F12K Medium (Gibco) supplemented with 10% FBS (Gibco), NCI‐H1299 and NCI‐H460 cells were cultured in 90% RPMI‐1640 medium (Gibco) supplemented with 10% FBS (Gibco), and BEAS‐2B cells were cultured in 90% minimum Eagle's medium (MEM) (Sigma) supplemented with 10% FBS (Gibco). All cells were incubated at 37°C under 95% air and 5% CO2 condition. After cells thawing and culture, AKIP1 protein and mRNA expressions in NSCLC cell lines and normal lung epithelial cell line were detected using Western blot and qPCR. Then, control overexpression, AKIP1 overexpression, control shRNA, and AKIP1 shRNA plasmids were constructed using pEX‐2 and pGPU6 plasmids (GenePharma) and transfected into A549 cells by HilyMax (Dojindo) and were categorized as Control (+), AKIP1 (+), Control (−), and AKIP1 (−) groups. Subsequently, AKIP1 protein and mRNA expressions in each group were detected by Western blot and qPCR at 24 hours; cell proliferation ability in each group was detected by Cell Counting Kit‐8 (Sigma) at 0 hours, 24 hours, 48 hours, and 72 hours; cell apoptosis rate in each group was detected by FITC Annexin V Apoptosis Detection Kit II with propidium iodide (AV/PI) (BD) at 24 hours.

The effect of AKIP1 on EMT markers and transcriptional marker in NSCLC

At 24 hours after transfection, E‐cadherin (EMT marker), fibronectin (EMT marker), and ZEB1 (transcriptional marker) mRNA expressions were detected by qPCR, and their protein expressions were assessed by Western blot.

Western blot

After extraction of total protein using RIPA Buffer (Thermo), the concentration of total protein was measured by Pierce™ BCA Protein Assay Kit (Thermo). Then, thermal denaturation was conducted at 98℃ for 5 minutes, and 20 μg proteins was added to NuPAGE™ 4%‐12% Bis‐Tris Protein Gel (Invitrogen). After electrophoresis, proteins were transferred to Nitrocellulose filter membrane (Millipore), followed by blocking with 5% bovine serum albumin (BSA) (Sigma). Subsequently, membranes were incubated with primary antibodies (listed in Table S1) and secondary antibody horseradish peroxidase‐conjugated goat‐anti‐rabbit Immunoglobulin G antibody (1:5000 dilution) (Abcam). Finally, the bands were visualized by Pierce™ECL Plus Western Blotting Substrate (Thermo) and X‐ray film (Kodak). In addition, all the antibodies used in the Western blot assay were presented in Table S1.

qPCR

Total RNA was extracted from plasma with TRIzol Reagent (Thermo), and then, cDNA transcription was conducted using ReverTra Ace® qPCR RT Kit (Toyobo). qPCR was performed by SYBR® Green Realtime PCR Master Mix (Toyobo). Finally, qPCR results were read using ABI 7500 (ABI) and calculated by the 2−△△CT formula. GAPDH was applied as the internal reference for AKIP1, E‐cadherin, fibronectin, and ZEB1. Primers used for qPCR are listed in Table S2.

Statistical analysis

All statistical analyses were performed with the use of SPSS 22.0 software (SPSS Inc), and figures were made with the use of GraphPad Prism 7.00 (GraphPad Software). The continuous variable was presented as mean value ± standard deviation; the categorized variable was presented as count (percentage). The AKIP1 expression difference between tumor tissue and adjacent noncancerous pulmonary tissues was determined by chi‐square test, and the AKIP1 expression difference among patients with different clinical features was determined by chi‐square test or Wilcoxon rank‐sum test. The expressions of AKIP1, E‐cadherin, fibronectin, and ZEB1 among groups were compared by Kruskal‐Wallis H test. The DFS and OS were illustrated by Kaplan‐Meier curve. The differences of DFS and OS between AKIP1 low expression and high expression patients were determined by log‐rank test. The factors affecting DFS and OS were determined by univariate and multivariate Cox's proportional hazards regression model analyses. The difference of AKIP1 expression among cell lines was determined by one‐way ANOVA, and difference of AKIP1, cell proliferation, and cell apoptosis between two groups was determined by t test. P value < .05 was considered as significant.

RESULTS

Study flow

There were totally 559 NSCLC patients underwent resection who were screened in this study, while 205 patients were excluded (including 85 patients without well‐preserved tumor tissue, 52 patients with incomplete or missing clinical or follow‐up records, 47 patients underwent neoadjuvant therapy, 13 patients with distant metastasis, and 8 patients with other malignancies) (Figure 1). Among the remaining 354 eligible patients, 35 patients were excluded, among which 25 patients were unable to contact to obtain informed consents and 10 patients refused to sign informed consents. Finally, 319 patients were enrolled and analyzed in this study.

Figure 1

Study flow

Baseline characteristics

Mean age of 319 NSCLC patients in our study was 62.3 ± 10.5 years, and 145 (45.5%) patients were with age ≤60 years and 174 (54.5%) patients were with age ≥60 years (Table 1). There were 259 (81.2%) males and 60 (18.8%) females. As to the tumor characteristics, 122 (38.2%) patients suffered lymph node metastasis, while the others (N = 197 (61.8%)) were without lymph node metastasis. And the mean value of tumor size was 5.4 ± 1.9 cm, and there were 182 (57.1%) patients with tumors ≤5 cm and 137 (42.9%) patients with tumors >5 cm. Besides, the numbers of patients with TNM stage I, II, and III were 107 (33.5%), 100 (31.3%), and 112 (35.2%), respectively. With respect to the pathological differentiation, the numbers of patients with well differentiation, moderate differentiation, and poor differentiation were 49 (15.4%), 201 (63.0%), and 69 (21.6%), respectively.

Table 1

Baseline characteristics of NSCLC patients

Characteristics	NSCLC patients (N = 319)
Age (y)	62.3 ± 10.5
≤60 y (n/%)	145 (45.5)
>60 y (n/%)	174 (54.5)
Gender (n/%)
Male	259 (81.2)
Female	60 (18.8)
Tumor size (cm)	5.4 ± 1.9
≤5 cm (n/%)	182 (57.1)
>5 cm (n/%)	137 (42.9)
Lymph node metastasis (n/%)
No	197 (61.8)
Yes	122 (38.2)
TNM stage (n/%)
I	107 (33.5)
II	100 (31.3)
III	112 (35.2)
Pathological differentiation (n/%)
Well	49 (15.4)
Moderate	201 (63.0)
Poor	69 (21.6)

Data were presented as mean value ± standard deviation or count (percentage).

Abbreviations: NSCLC, non‐small‐cell lung cancer; TNM, tumor node metastasis.

Comparison of AKIP1 expression between tumor tissue and adjacent noncancerous pulmonary tissue in NSCLC patients

A‐kinase‐interacting protein 1 expression was detected in totally 319 tumor tissues and 145 adjacent tissues, and the examples of AKIP1 high or low expression are shown in Figure 2A. The comparison of AKIP1 expression in tumor tissue and adjacent noncancerous pulmonary tissue was determined by chi‐square test (Table 2), which revealed that AKIP1 expression was elevated in tumor tissues compared to adjacent tissues (P < .001).

Figure 2

DFS and OS in AKIP1 high expression group and AKIP1 low expression group. AKIP1 high expression and low expression tissue examples (A). Compared to AKIP1 low expression group, DFS was shorter in AKIP1 high expression group (B). Compared to AKIP1 low expression group, OS was worse in AKIP1 high expression group (C). K‐M curves were displayed to exhibit DFS and OS. Comparison of DFS or OS between two groups was determined by log‐rank test. P value < .05 was considered significant. AKIP1, A‐kinase‐interacting protein 1; DFS, disease‐free survival; K‐M curves, Kaplan‐Meier curves; OS, overall survival

Table 2

AKIP1 expression in tumor tissue and adjacent noncancerous pulmonary tissue

Tissues	AKIP1 expression		P value
	High (n = 259)	Low (n = 205)
Tumor tissue (n = 319)	201 (63.0)	118 (37.0)	<.001
Adjacent tissue (n = 145)	58 (40.0)	87 (60.0)

Data were presented as count (percentage). Comparison was determined by chi‐square test. P value < .05 was considered significant.

Abbreviation: AKIP1, A‐kinase‐interacting protein 1.

Correlation of AKIP1 expression with clinical characteristics in NSCLC patients

A‐kinase‐interacting protein 1 high expression was correlated with increased tumor size (P = .023), lymph node metastasis (P = .029), and elevated TNM stage (P = .001) in NSCLC patients (Table 3). Besides, AKIP1 high expression was numerically associated with poorer pathological differentiation in NSCLC patients (P = .074), whereas no correlation of AKIP1 high expression with age (P = .703) or gender (P = .723) was found.

Table 3

Correlation of AKIP1 expression with clinical characteristics

Items	AKIP1 expression		P value
	High (n = 201)	Low (n = 118)
Age (n/%)
≤60 y	93 (64.1)	52 (35.9)	.703
>60 y	108 (62.1)	66 (37.9)
Gender (n/%)
Male	162 (62.5)	97 (37.5)	.723
Female	39 (65.0)	21 (35.0)
Tumor size (n/%)
≤5 cm	105 (57.7)	77 (42.3)	.023
>5 cm	96 (70.1)	41 (29.9)
Lymph node metastasis (n/%)
No	115 (58.4)	82 (41.6)	.029
Yes	86 (70.5)	36 (29.5)
TNM stage (n/%)
I	54 (50.5)	53 (49.5)	.001
II	65 (65.0)	35 (35.0)
III	82 (73.2)	30 (26.8)
Pathological differentiation (n/%)
Well	26 (53.1)	23 (46.9)	.074
Moderate	127 (63.2)	74 (36.8)
Poor	48 (69.6)	21 (30.4)

Data were presented as count (percentage). Comparison was determined by chi‐square test or Wilcoxon rank‐sum test. P value < .05 was considered significant.

Abbreviations: AKIP1, A‐kinase‐interacting protein 1; TNM, tumor node metastasis.

Correlation of AKIP1, EMT, and transcriptional factor mRNA/protein expressions with TNM stage

Furthermore, considering EMT and transcriptional factor play a key role in the TNM stage of tumors, and AKIP1 is reported to regulate EMT in cancers, we compared the AKIP1, EMT, and transcriptional markers (qPCR and Western blot) between tumor tissues with different TNM stages and adjacent tissues. We observed that AKIP1, fibronectin, and ZEB1 were increased in tumor tissues compared with adjacent tissues (P < .001) and correlated with advanced TNM stage (P < .001) (Figure S1A,C,D,E), while E‐cadherin was decreased in tumor tissues compared with adjacent tissues (P < .001) and correlated with reduced TNM stage (P < .001) (Figure S1B,E).

Correlation of AKIP1 expression with survival profiles in NSCLC patients

Disease‐free survival in AKIP1 high expression patients (median: 29.0 months, 95% CI: 25.3‐32.7 months) was shorter than that in AKIP1 low expression patients (median: 47.0 months, 95% CI: 34.5‐59.5 months) (P < .001) (Figure 2B); moreover, OS in AKIP1 high expression patients (median: 41.0 months, 95% CI: 35.2‐46.8 months) was decreased than that in AKIP1 low expression patients (median: 64.0 months, 95% CI: 42.5‐85.5 months) (P < .001) (Figure 2C). These results indicated that AKIP1 high expression was associated with worse DFS and OS in NSCLC patients.

Analysis of factors affecting DFS and OS in NSCLC patients

Univariate Cox's regression disclosed that AKIP1 high expression (P < .001) was associated with decreased DFS in NSCLC patients, and lymph node metastasis (P < .001), increased TNM stage (P = .004), as well as worse pathological differentiation (P = .001) were also correlated with reduced DFS in NSCLC patients (Table 4). And further multivariate Cox's regression revealed that AKIP1 high expression (P = .001) was an independent factor predicting shorter DFS, as well as lymph node metastasis (P < .001) and poor pathological differentiation (P < .001). As to factors affecting OS in NSCLC patients, AKIP1 high expression (P < .001), larger tumor size (P = .002), presence of lymph node metastasis (P < .001), raised TNM stage (P = .002), as well as poor pathological differentiation (P = .016) were associated with worse OS in NSCLC patients, and AKIP1 high expression (P < .001), presence of lymph node metastasis (P < .001), as well as worse pathological differentiation (P = .009) were verified as independent predictive factors for poor OS in NSCLC patients (Table 5).

Table 4

Cox's regression model analysis of factors effecting DFS

Items	Univariate Cox's regression				Multivariate Cox's regression
	P value	HR	95% CI		P value	HR	95% CI
			Lower	Higher			Lower	Higher
AKIP1 expression (high vs low)	<.001	1.663	1.288	2.147	.001	1.569	1.210	2.035
Age (>60 y vs ≤60 y)	.191	1.178	0.922	1.506	.110	1.235	0.953	1.599
Gender (male vs female)	.896	0.979	0.712	1.346	.988	1.003	0.719	1.397
Tumor size (>5 cm vs ≤5 cm)	.099	1.231	0.962	1.575	.102	0.740	0.516	1.061
Lymph node metastasis (yes vs no)	<.001	1.752	1.357	2.262	<.001	2.050	1.397	3.007
TNM stage (III vs II/I)	.004	1.462	1.133	1.888	.899	1.027	0.681	1.548
Pathological differentiation (poor vs moderate/well)	.001	1.611	1.211	2.142	<.001	1.732	1.281	2.341

Factors effecting DFS were determined by univariate and multivariate Cox's proportional hazards regression model analyses. P value < .05 was considered significant.

Abbreviations: AKIP1, A‐kinase‐interacting protein 1; DFS, disease‐free survival; TNM, tumor node metastasis.

Table 5

Cox's regression model analysis of factors effecting OS

Items	Univariate Cox's regression				Multivariate Cox's regression
	P value	HR	95% CI		P value	HR	95% CI
			Lower	Higher			Lower	Higher
AKIP1 expression (high vs low)	<.001	1.933	1.435	2.604	<.001	1.830	1.353	2.476
Age (>60 y vs ≤60 y)	.637	1.068	0.813	1.402	.781	1.042	0.779	1.395
Gender (male vs female)	.682	0.930	0.657	1.317	.796	0.954	0.665	1.367
Tumor size (>5 cm vs ≤5 cm)	.002	1.521	1.160	1.994	.865	0.969	0.673	1.394
Lymph node metastasis (yes vs no)	<.001	2.300	1.744	3.034	<.001	2.873	1.941	4.253
TNM stage (III vs II/I)	.002	1.557	1.172	2.069	.156	0.736	0.481	1.125
Pathological differentiation (poor vs moderate/well)	.016	1.469	1.075	2.007	.009	1.571	1.121	2.202

Factors effecting OS were determined by univariate and multivariate Cox's proportional hazards regression model analyses. P value < .05 was considered significant.

Abbreviations: AKIP1, A‐kinase‐interacting protein 1; OS, overall survival; TNM, tumor node metastasis.

Effect of AKIP1 on cell proliferation and cell apoptosis in A549 cells

For the purpose of assessing the expression of AKIP1 in NSCLC cells and normal lung epithelial cells, we detected AKIP1 mRNA expression by qPCR assay and AKIP1 protein expression by Western blot assay, which disclosed that AKIP1 expression was elevated in NSCLC cell lines including PC9 as well as A549 compared to normal lung epithelial cell line BEAS‐2B, while it was nondifferentiated in NCI‐H1299 cells or NCI‐H460 cells compared to normal lung epithelial cell line BEAS‐2B cells, and A549 cell line was selected for the subsequent assays (Figure 3A,B). Moreover, control overexpression, AKIP1 overexpression, control shRNA, and AKIP1 shRNA plasmids were transfected into A549 cells, and AKIP1 mRNA expression as well as protein expression was increased in AKIP1 (+) group compared to Control (+) group (P < .001) but decreased in AKIP1 (−) group compared to Control (−) group (P < .001) (Figure 3C,D). Besides, CCK‐8 assay showed that cell proliferation was raised in AKIP1 (+) group compared to Control (+) group at 72 hours (P < .05) in A549 cells (Figure 3E), while it was reduced in AKIP1 (−) group compared to Control (−) group at 48 hours (P < .05) and 72 hours (P < .05). Furthermore, AV/PI assay disclosed that cell apoptosis rate was decreased in AKIP1 (+) group compared to Control (+) group (P < .01) but increased in AKIP1 (−) group compared to Control (−) group in A549 cells (P < .01) (Figure 3F,G). These data suggested that AKIP1 enhanced cell proliferation but repressed cell apoptosis in A549 cells.

Figure 3

Effect of AKIP1 on cell proliferation and apoptosis. qPCR assay displayed that AKIP1 mRNA expression was elevated in PC9 cells and A549 cells compared to BEAS‐2B cells, but nondifferentiated in NCI‐H1299 cells and NCI‐H460 cells (A). In A549 cells, Western blot assay showed that AKIP1 expression was higher in PC9 and A549 cells than that in BEAS‐2B cells (B). AKIP1 mRNA expression was elevated in AKIP1 (+) group than that in Control (+) group, while it was lower in AKIP1 (−) group than that in Control (−) group (C). Western blot assay showed that AKIP1 expression was higher in AKIP1 (+) group than that in Control (+) group but reduced in AKIP1 (−) group than that in Control (−) group (D). CCK‐8 assay disclosed that cell proliferation was promoted in AKIP1 (+) group at 72 h (vs Control (+) group) but suppressed in AKIP1 (−) group (vs Control (−) group) at 48 h and 72 h (E). AV/PI assay revealed that cell apoptosis rate was decreased in AKIP1 (+) group (vs Control (+) group) but enhanced in AKIP1 (−) group (vs Control (−) group) (F, G). Comparison between two groups was determined by one‐way ANOVA and t test. P value < .05 was considered significant. *P < .05; **P < .01; ***P < .001. AKIP1, A‐kinase‐interacting protein 1; AV/PI, Annexin V Apoptosis Detection Kit II with propidium iodide; CCK‐8, Cell Counting Kit‐8; qPCR, quantitative polymerase chain reaction

Effect of AKIP1 on EMT markers and transcriptional marker

E‐cadherin mRNA expression was decreased in AKIP1 (+) group compared to Control (+) group (P < .01) but increased in AKIP1 (−) group compared to Control (−) group (P < .01) (Figure S2A). Meanwhile, fibronectin mRNA expression (Figure S2B) and ZEB1 miRNA expression (Figure S2C) were elevated in AKIP1 (+) group compared to Control (+) group but reduced in AKIP1 (−) group compared to Control (−) group (all P < .05). For their protein expressions, they showed the same trends as mRNA expressions in each group (Figure S2D).

(P < .01).

DISCUSSION

In this study, we discovered that (a) AKIP1 was overexpressed in tumor tissues compared to adjacent tissues, and it positively correlated with tumor size, lymph node metastasis, as well as TNM stage. A‐kinase‐interacting protein 1 higher expression independently predicted worse DFS and OS in NSCLC patients; (b) AKIP1 was upregulated in some NSCLC cell lines compared to normal lung epithelial cell line, and it promoted cell proliferation and reduced cell apoptosis in A549 cells.

A‐kinase‐interacting protein 1, is a nuclear protein whose transcripts are differentially expressed in different cells and tissues, and it is initially found highly expressed in the cell lines of breast cancer and prostate cancer.10, 14, 15 Despite the biochemical or biological functions of AKIP1 is not well illuminated, the influence of AKIP1 on cancer cells have been illuminated by several studies.8, 9, 16 For example, a study reveals that AKIP1 transcriptionally upregulates vascular endothelial growth factor‐C (VEGF‐C) by cooperating with several transcriptional factors such as specificity protein 1 (Sp1), activator protein 2 (AP2), and NF‐kB, thereby enhances angiogenesis and lymphangiogenesis in human esophageal squamous cell carcinoma.9 Also, a study displays that AKIP1 upregulates the expression of chemokine (C‐X‐C motif) ligand 1 (CXCL1), CXCL2 and CXCL8 via activating IKKβ/NF‐κB signaling pathway, and thus contributes to cell proliferation in cervical cancer cells, which further facilitates angiogenesis as well as enhances tumor growth in BALB/c nude mouse xenograft model.8 Another study shows that AKIP1 suppression inhibits cell motility and invasion via repressing Akt/GSK‐3β/Snail signal pathway in breast cancer cells.16 And these data suggest that AKIP1 might participate in the initiation and progression of some cancers through affecting biological processes such as proliferation, mobility or invasion ability of cancer cells via regulating multiple genes such as VEGF‐C, CXCL1, and NF‐κB pathway‐related genes.

As to the clinical practices investigating the effect of AKIP1 in cancers, the dysregulation and correlation of AKIP1 with clinical characteristics in cancer patients have been displayed in some previous data.9, 16 For instance, a previous study displays that AKIP1 is overexpressed in clinical esophageal squamous cell carcinoma samples, and its expression is positively correlated with TNM stage in ESCC patients.9 Another study discloses that AKIP1 is overexpressed in breast cancer tissues compared to normal breast tissues, and its high expression correlates with advanced tumor stage as well as lymph node metastasis in breast cancer patients.16 These studies reveal that AKIP1 is upregulated in tumor tissues of several cancers, and its high expression is associated with increased disease progression in these cancer patients, whereas evidence about the correlation of AKIP1 expression with clinical characteristics in NSCLC patients is limited, only a study with small sample size displayed a positive correlation of AKIP1 expression with some tumor features, which needs to be further validated.12 Thus, 319 NSCLC patients were enrolled and the correlation of AKIP1 expression with disease risk as well as clinical characteristics in NSCLC patients was investigated. The results in our study indicated that AKIP1 was overexpressed in tumor tissues compared to adjacent tissues, and its expression positively correlated with tumor size, lymph node metastasis, and TNM stage in NSCLC patients, which might result from the following reasons: (a) AKIP1 upregulated NF‐κB‐dependent chemokines including CXCL1, CXCL2, and CXCL8 to promote NSCLC angiogenesis and tumor growth, thereby caused larger tumor size in NSCLC patients with AKIP1 high expression8; (b) AKIP1 enhanced the ability of cellular motility, cell metastasis, as well as cell invasion via activating Akt/GSK‐3β/Snail signal pathway, thus it contributed to epithelial‐to‐mesenchymal transition and further led to lymph node metastasis in NSCLC patients16; (c) considering tumor size and lymph node metastasis were important contents that were involved in the classification of TNM stage, and AKIP1 was able to increase tumor growth and enhance lymph node metastasis as we mentioned above, thus AKIP1 overexpression resulted in more advanced TNM stage in NSCLC patients.17

Regarding the predictive value of AKIP1 in cancers, a study shows that colorectal cancer patients with AKIP1 high expression present with worse OS compared to those with AKIP1 low expression. And another study discloses that AKIP1 high expression is associated with poorer OS and reduced recurrence‐free survival, and it is an independent prognostic factor for these unfavorable survival profiles in breast cancer patients.16 Most importantly, these data indicate that AKIP1 has good predictive value for worse prognosis in patients with different cancers, while limited studies disclose the effect of AKIP1 on prognosis in NSCLC. In our study, we found that AKIP1 high expression was an independent predictive factor for unfavorable DFS and OS in NSCLC patients. And there were several possible explanations for these results: (a) AKIP1 promoted cell proliferation (via regulating NF‐κB‐dependent chemokines) and enhanced cell invasion as well as cell metastasis (by affecting Akt/GSK‐3β/Snail signal pathway), thereby led to an aggravated disease progression and further reduced the survival time of NSCLC patients8, 16; (b) AKIP1 might induce the drug resistance, thereby led to worse treatment efficacy and further resulted in shorter survivals, while the detailed mechanism was not clear, and further investigation was need. Besides, there were some limitations existed in our study: (a) this was a retrospective study, which used IHC assay and assessed the AKIP1 expression restricted to the paraffin‐embedded specimens; thus, further studies with more detection methods to verify the results are needed; (b) the sample size was relatively small in our study (N = 319), thus the statistical power might be relatively low; (c) this was a single‐center study, which might lack wide representativeness.

Better understanding on the effects of AKIP1 in tumor cells might provide more supports for exploring novel treatment targets, and some previous studies have conducted some in vivo or in vitro experiments to investigate the underlying mechanism of AKIP1 in cancer cells.8, 10, 16 For instance, a former study displays that AKIP1 is overexpressed in breast cancer cells (MDA‐MB231 and MCF7).10 Additionally, another study shows that AKIP1 suppression inhibits cell proliferation in cervical cancer cell lines (C33A and Hela) and restrains growth as well as angiogenesis of xenografts in BALB/c nude mice.8 Moreover, other two studies disclose that AKIP1 downregulation suppresses motility and invasion of breast cancer cells and migration of colorectal cancer cells, while the effect of AKIP1 on cell proliferation and apoptosis was not investigated.16 Although these data reveal the function of AKIP1 as an oncogene in some cancers, the precise mechanism of AKIP1 in NSCLC remains largely unknown. In order to explore the role of AKIP1 in NSCLC cells, we conducted qPCR, Western blot, CCK‐8, and AV/PI assays and found that AKIP1 was overexpressed in several NSCLC cells compared to normal lung epithelial cells, and it enhanced cell proliferation but repressed cell apoptosis in A549 cells; meanwhile, AKIP1 enhanced the EMT process and transcriptional factor expression in A549 cells. These data might facilitate understanding the mechanisms of AKIP1 in NSCLC cells and provide support to the further exploration of applying AKIP1 as a treatment target in this cancer. As for possible reasons for these results, one probable explanation was that AKIP1 may affect the cancer cell survival (cell proliferation and apoptosis) via regulating the mitochondria permeability in NSCLC cells, which has been revealed in a previous study that illuminated that AKIP1 could stabilize the mitochondria permeability pore.18 Besides, the effect of AKIP1 on cell apoptosis might be relatively low to some extent (about 2‐fold change compared to control), this might be explained by: (a) cell apoptosis was naturally influenced by multiple factors, while AKIP1 was only one of the multiple factors; thus, its single effect was limited; (b) the transfection efficiency might also be an issue affecting this results.

In conclusion, AKIP1 is overexpressed and correlates with increased tumor size, lymph node metastasis, raised TNM stage, as well as worse survivals in NSCLC. Furthermore, AKIP1 promotes NSCLC cell proliferation while represses cell apoptosis.

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