HBXIP suppression reduces cell proliferation and migration and its overexpression predicts poor prognosis in non-small-cell lung cancer

Abstract

Emerging evidence has demonstrated that the high expression of HBXIP has been correlated with many cancers. With evaluation of the functional role of HBXIP in non-small-cell lung cancer, the primary aim of this study is to investigate the correlation between HBXIP expression and the prognosis of non-small-cell lung cancer patients. The protein levels of HBXIP were detected using western blotting in non-small-cell lung cancer cells. Cell proliferation and migration assays were measured to evaluate the function of HBXIP in non-small-cell lung cancer cells. A total of 120 non-small-cell lung cancer patients with strict follow-up and 60 adjacent non-tumor lung tissues were selected for immunohistochemical staining of the HBXIP protein. The localization of the HBXIP protein was detected in A549 non-small-cell lung cancer cells using immunofluorescence staining. The correlation between HBXIP expression and the clinicopathological features of non-small-cell lung cancer patients was analyzed by a chi-squared and Fisher’s exact test. The overall survival rates of all of the non-small-cell lung cancer patients were calculated using the Kaplan–Meier method, and univariate and multivariate analyses were performed using the Cox proportional hazards regression model. In function, we showed that suppression of HBXIP decreased A549 cell proliferation and migration. HBXIP protein showed a mainly cytoplasmic staining pattern in non-small-cell lung cancer using immunohistochemical staining in paraffin-embedded non-small-cell lung cancer tissues and immunofluorescence staining in A549 cells. The HBXIP protein had strong positive staining in the non-small-cell lung cancer tissues, which was significantly higher than the percentage of adjacent non-tumor tissues. The overexpression of HBXIP was closely correlated with histological grade, clinical stage, lymph node metastasis, and lower overall survival rates of patients with non-small-cell lung cancer. Moreover, multivariate analysis suggested that HBXIP emerged as a significant independent prognostic factor along with clinical stage in patients with non-small-cell lung cancer. In conclusion, a high level of expression of HBXIP is associated with the progression of non-small-cell lung cancer and may be a useful biomarker for poor prognostic evaluation and a potential molecular therapy target for patients with non-small-cell lung cancer.

Introduction

Lung cancer is the leading cause of cancer-related disease worldwide and causes 1.8 million deaths per year.^1–3 An overall 5-year survival rate lower than 16%^4–6 demonstrates its high mortality rate. Based on histology, there are two major types of lung cancer: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Approximately 85% of primary lung cancer patients have NSCLC, which includes the main histological subtypes of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. ⁷ In the advanced stages of NSCLC, some therapies have led to improvements in treatment, such as surgical resection, chemotherapy, and radiotherapy. Despite advances in multimodal therapies, the prognosis of lung cancer remains disappointingly poor. Because most patients present with locally advanced or metastatic disease, ⁸ an efficient method of early detection is urgently needed in the clinic. Therefore, a better understanding of the molecular pathogenesis and mechanisms involved in NSCLC initiation and progression will likely contribute to providing useful prognostic biomarkers and therapeutic targets for NSCLC therapy.

Hepatitis B X–interacting protein (HBXIP) is a cellular 18 kDa protein among mammalian species that was originally discovered by identifying the binding partners of hepatitis B virus (HBV) X protein in hepatoma HepG2 cells. ⁹ The overexpression of HBXIP suppresses both hepatitis B viral replication in HepG2 cells and the transactivation phenotype of HBx. ⁹ Interestingly, emerging evidence has demonstrated that the high expression of HBXIP has been correlated with the development of many cancers, including liver, breast, and ovarian cancer. It has been reported that HBXIP functions as an interaction partner to survivin and suppresses the apoptosis involved in hepatocellular carcinogenesis. ¹⁰ Previously, investigators have shown that HBXIP is a regulator of centrosome duplication and causes excessive centrosome replication, resulting in tripolar and multipolar spindles and defective cytokinesis, which affects genetic stability. ¹¹ Furthermore, HBXIP plays a critical role in regulating cellular proliferation. ¹² HBXIP can upregulate S100A4 by activating S100A4 promoters such as signal transducer and activator of transcription 4 (STAT4) and by inducing phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/Akt signaling to promote the growth and migration of breast cancer cells. ¹³ Similarly, HBXIP is able to upregulate platelet-derived growth factor subunit B (PDGFB) and fibroblast growth factor (FGF)4 to activate the transcriptional factor Sp1, which promotes the proliferation and migration of breast cancer cells.^14,15 Although HBXIP is associated with a variety of cancers, the frequency of HBXIP overexpression and the clinical significance in NSCLC have not yet been determined.

This aim of this study is to investigate the association between HBXIP expression and the clinicopathological features of NSCLC patients and thereby determine whether the HBXIP protein may be correlated with poor prognosis in NSCLC patients. We performed immunohistochemistry (IHC) of HBXIP in NSCLC tissues and examined the association of HBXIP protein expression with the clinicopathological features of NSCLC. We also assessed the prognostic value of high HBXIP expression in patients with NSCLC. In addition, suppression of HBXIP by siRNA inhibited cell proliferation, increased cell apoptosis, and reduced cell migration.

Materials and methods

Ethics statement

This research complied with the Helsinki Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of Yanbian University Medical College. Patients were informed that the resected specimens were stored by the hospital and could potentially be used for scientific research and that their privacy would be maintained. Follow-up survival data were collected retrospectively through medical record analyses.

Cell culture and siRNA transfection

NSCLC cell lines, A549, H1975, H1944, and H1299, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Grand Island, NY, USA). H1299, H1975, and H1944 were grown in RPMI 1640. All cell lines were supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) with appropriate antibiotics at 37°C in 5% CO₂ humidified incubator. We purchased three different HBXIP small interfering RNA (siRNA) from RiboBio (Guangzhou, China). According to the KD effect, siRNA3 was used in this study. The sequence of siRNA3 was 5′-GTTGCACCGTGATCAATTT-3′. Additionally, control siRNA was also used in this study. Cells were seeded onto six-well plates. After 16 h, cells were transfected with HBXIP and control siRNA. In each well, 100 nM of HBXIP or negative control (NC) siRNA and Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) were added to Opti-MEM (Life Technologies, NY, USA) and mixed. After 20 min of incubation, the mixture was gently added to the cells. The plate was incubated for an indicated time until further assay.

Western blot analysis

Cells were lysed on ice for 30 min in radioimmunoprecipitation assay (RIPA) buffer and then centrifuged at 12,000 r/min for 15 min at 4°C. Protein samples were separated on 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 2 h. After blocking procedure, membranes were incubated overnight at 4°C with primary antibodies (1:1000) and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000) and visualized in Imager (Bio-Rad, USA) using enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Rochester,NY, USA).

EdU proliferation assay

Proliferating A549 cells were determined using a 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit (RiboBio) according to the manufacturer’s protocol. Briefly, 50 µM EdU labeling medium was added to the cell culture to allow incubation for 12 h at 37°C under 5% CO₂. Afterwards, cultured A549 was fixed with 4% paraformaldehyde (pH 7.4) for 30 min and incubated with glycine for 5 min. After washing with phosphate-buffered saline (PBS), staining with anti-EdU working solution was performed at room temperature (RT) for 30 min. Following washing with 0.5% Triton X-100 in PBS, the cells were incubated with 5 µg/mL Hoechst 33342 dye at RT for 30 min, followed by observation under a confocal laser scanning microscope.

Cell proliferation assays

Cell proliferation assays were performed by culturing 2 × 10⁴ cells in a six-well plate and culturing the cells at 37°C in 5% CO₂ humidified incubator; 24 h after seeding, cell proliferation was determined by cell numbers recorded at 24, 48, 72, and 96 h after being seeded and normalized to cell numbers at the starting time.

Cell migration assay and wound-healing assay

The migration assay used 24-well plates with 8-µm polyethylene terephthalate (PET). Cells were seeded into the upper insert in serum-free media, while media containing 10% FBS was added to the lower chambers as a chemoattractant for 24 h. The cells were removed from the upper surface of the filter by scraping with a cotton swab. Cells that infiltrated through the filter were fixed and stained with Giemsa. The images were taken with OLYMPUS BX53. The number of migration n cells in three to five fields was counted. Cells were seeded in six-well plates and cultured with DMEM containing 10% FBS until cells reached sub-confluence. After removal of the culture medium, a monolayer of the sub-confluent cells was scratched with a 200 µL pipette tip to create a wound area. The wounded monolayers were washed with PBS twice and cultured in FBS-free medium for 48 h. Cell migration into the wound area was monitored by inverted microscopy, photographed at the indicated time points, and analyzed using ImageJ software. The migration distance (distance of cell migration, millimeter) was calculated by subtracting the distance measured between the edges of the lesion at 48 h from the distance measured at 0 h.

Immunofluorescence staining analysis

An immunofluorescence (IF) staining method was used to detect the subcellular localization of the HBXIP protein in A549 NSCLC cells. All steps were performed at RT. A549 cells were grown on coverslips to reach 70%–80% confluence and fixed with 4% paraformaldehyde for 10 min; after 24 h, the cells were permeabilized with 0.5% Triton X-100 for 10 min. After blocking with 3% Albumin Bovine V (A8020; Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the HBXIP antibody (1:1000) at 4°C overnight, followed by incubation with Alexa Fluor 488 Goat Anti-Rabbit IgG (H + C; A11008, 1:1000; Invitrogen) for 1 h. After washing with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; C1006; Beyotime, Shanghai, China), and the coverslips were mounted with Antifade Mounting Medium (P0126; Beyotime). Finally, the IF signals were visualized and recorded using a BX53 Olympus microscope.

Clinical samples

A total of 180 human lung tissue samples were used for this study, including 120 NSCLC lung cancer tissues and 60 adjacent non-tumor tissues. All of the tissues were collected from the Tissue Bank of Yanbian University Medical College. All tissues were routinely fixed in 10% buffered formalin and embedded in paraffin blocks. The study protocol was approved by the Institutional Review Board of Yanbian University Medical College. The pathological parameters, including gender, age, tumor size, clinical stage, differentiation, nodal metastasis, and survival data, were carefully reviewed in all 120 NSCLC cases.

The patients with NSCLC include 64 males and 56 females, ranging from 44 to 77 years of age, with a mean age of 62 years. Of the 120 patients, 70 were 62 years old or older, and 50 were younger than 62 years old. All NSCLC diagnoses were confirmed by pathological examination. For the tumor sizes, 63 cases were less than or equal to 4 cm and 57 cases were greater than 4 cm. Tumor–node–metastasis (TNM) staging was assessed according to the staging system established by the American Joint Committee on Cancer (AJCC). Of the 120 NSCLC cases, 64 were stages I–II and 56 cases were stages III–IV. In all, 34 cases were defined as histological Grade 1, 35 cases were Grade 2, and 51 cases were Grade 3. Additionally, 63 cases had lymph node (LN) metastases, and 57 cases had no LN metastases. None of the patients received radiochemotherapy before surgery. The 120 patients with NSCLC were followed for 10 years or until death. There were 60 samples of adjacent non-tumor lung tissues collected from the cancer resection margin for this study

IHC staining analysis

IHC analysis was performed using the Dako LSAB kit (Dako A/S, Glostrup, Denmark). Briefly, to eliminate endogenous peroxidase activity, 4-µm-thick tissue sections were de-waxed with xylene and rehydrated through a graded series of ethanol. The antigen was retrieved by placing the slides in 0.01 M sodium citrate buffer (pH 6.0) at 95°C for 20 min, and the slides were cooled for 30 min at RT. The slides were incubated with 3% H₂O₂ for 15 min at RT to inactivate the endogenous peroxidase. The slides were then incubated with the HBXIP antibody (1:500, #14492-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4°C in a humidified chamber. After washing in PBS, the sections were incubated with the biotinylated secondary antibody at RT for 30 min, followed by another 30-min incubation with the streptavidin–peroxidase complex at RT. IHC staining was developed using 3,3′-diaminobenzidine, and Mayer’s hematoxylin was used for counterstaining. We used a rabbit IgG isotype control, which showed negative staining. The positive tissue sections were processed without the primary antibody to serve as another NC.

Two pathologists (Z.L. and S.C.) who did not possess knowledge of the clinical data examined and scored all tissues specimens. In the case of a discrepancy, a final score was established by reassessment by both pathologists using a double-headed microscope. Briefly, the IHC staining for HBXIP was semi-quantitatively scored as “−” (negative; no or less than 5% positive cells), “+” (5%–25% positive cells), “++” (26%–50% positive cells), and “+++” (more than 50% positive cells). A cytoplasmic/nuclear expression pattern was considered positive staining. Tissue sections that were scored as “++” and “+++” were considered strong positives (high-level expression) for HBXIP protein.

Statistical methods

All statistical analyses were analyzed using the SPSS 17.0 statistical software (SPSS Inc, Chicago, IL, USA). Statistical significances between HBXIP expression and clinicopathological parameters were analyzed by a chi-squared (χ²) or Fisher’s test. The survival rates were calculated using the Kaplan–Meier method, and differences in the survival curves were analyzed using a log-rank test. Multivariate survival analysis was performed on all of the significant characteristics measured by univariate survival analysis through the Cox proportional hazard regression model; p < 0.05 was considered statistically significant.

Results

High expression of the HBXIP protein in A549 non-small lung cell and suppression of HBXIP reduce cell proliferation and migration in vitro

First, we measured the expression levels of HBXIP in NSCLC cell lines, H1975, A549, H1299, and H1944, using western blotting. We found that in the A549 cells, the protein expression of HBXIP was the highest among these four cell lines (Figure 1(a) and (b)). Thus, A549 cell was selected for subsequent analysis of the functional role of HBXIP in NSCLC. To further investigate the underlying mechanism of HBXIP in NSCLC, the effect of HBXIP on A549 cell proliferation and migration was investigated using NC siRNA and three different HBXIP-siRNAs (siRNA1, siRNA2, and siRNA3). Then, western blot analysis showed that HBXIP expression was significantly downregulated by siRNA3 as compared to the NC siRNA (Figure 2(a) and (b)). Among the analyzed siRNAs, siRNA3 appeared to have the greatest mean knockdown efficiency and was therefore selected for subsequent in vitro experiments. Cell proliferation was determined by cell counting and the EdU assay. The migration ability of cells was measured by wound-healing assay and Transwell migration assay. Compared with the control group, HBXIP-knockdown significantly repressed A549 cell proliferation and migration (Figure 3(a)–(g)).

Figure 1.

Expression of HBXIP in NSCLC cell lines. (a and b) Western blot analysis of HBXIP expression in NSCLC cell lines, equal amount of protein loading was ensured by β-actin level. Data represent the mean ± SD of three independent experiments.

Figure 2.

HBXIP-knockdown in A549 cells. (a and b) The expression of HBXIP in A549 cells transfected with control siRNA and HBXIP-siRNA was analyzed by western blotting, equal amount of protein loading was ensured by β-actin level. Data represent the mean ± SD of three independent experiments (**p < 0.01 as compared with negative control).

Figure 3.

Effect of HBXIP-knockdown on A549 cells. (a) Proliferating A549 cells were labeled with EdU. The click-it reaction revealed EdU staining (red). Cell nuclei were stained with Hoechst 33342 (blue). The images are representative of the results obtained. (b) The percentage of EdU-positive A549 cells was quantified. (c and d) Representative images showing the migration ability of A549 cells transfected with control siRNA and HBXIP-siRNA, as assessed by wound-healing assays (magnification 200×). (e) A549 cell proliferation rates were determined by cell counting. (f and g) A549 cells were transfected with control or HBXIP siRNA3. After 24 h, cells were seeded onto 24-well Transwell plates for migration assay. After 48 h, the number of cells that crossed the Transwell membrane was counted. The average of three independent experiments is shown. Data represent the mean ± SD of three independent experiments (**p < 0.01 as compared with negative control).

High expression of the HBXIP protein in NSCLC tissues

Next, our data confirmed that HBXIP staining in A549 NSCLC cells was observed in the cytoplasm by IF staining. IF staining indicated that the HBXIP protein was mainly located in the cytoplasm of A549 lung cancer cells (Figure 4). Then, we test the expression of the HBXIP in NSCLC patients’ tissues. IHC staining also demonstrated that the expression of HBXIP was positive in 98 (81.7%) of the 120 NSCLC patients, and 71 (59.2%) of the 120 NSCLC samples were considered strong positives. Further analysis revealed that HBXIP staining was found primarily in NSCLC tissues and not in the adjacent normal lung tissues (p < 0.01; Table 1). As shown in Figure 5, HBXIP was detected in the cytoplasm of cancer cells.

Figure 4.

Immunofluorescence staining for HBXIP protein in A549 and non-small-cell lung cancer cells. A549 non-small-cell lung cancer cells were immunostained for HBXIP (red). Nuclei were visualized by DAPI staining (blue). HBXIP protein is mainly located in the cytoplasm of A549 non-small-cell lung cancer cells.

Table 1.

Expression of HBXIP protein in NSCLC.

Diagnosis	No. of cases	HBXIP				Positive case rate (%)	Strongly positive case rate (%)
		−	+	++	+++
Non-small-cell lung cancer	120	22	27	36	35	81.7**	59.2**
Adjacent non-tumor lung	60	46	13	1	0	23.3	1.67

Positive rate: percentage of positive cases with +, ++, and +++ staining scores.

Strongly positive rate (high-level expression): percentage of positive cases with ++ and +++ staining scores.

**

p < 0.01 compared with adjacent non-tumor lung.

Figure 5.

IHC staining of HBXIP protein in lung tissues. (a) HBXIP protein was negative in adjacent non-tumor tissues. (b) Diffuse and strong positive HBXIP protein signal in papillary growth patterns of lung adenocarcinoma. (c) HBXIP protein shows positive staining in cytoplasm of lung squamous cell carcinoma cells. (d) Diffuse and strong positive HBXIP protein signal in bronchioloalveolar carcinoma. (e and f) Diffuse and strong positive HBXIP protein signal in lung adenocarcinoma (original magnification: 100× in a and b; 200× in c–f).

Correlation between HBXIP expression status and clinicopathological features of NSCLC

The relationship between HBXIP protein and clinicopathological features of NSCLC was summarized in Table 2. Overexpression of the HBXIP protein was significantly correlated with histological grade, LN metastasis, and clinical stage, but not with patient’s age, gender, or tumor size. We found that tissues with strong positive HBXIP staining were found more often in Grade-3 NSCLC (76.5%, 39/51) than in Grade-2 (57.1%, 20/35) or Grade-1 (35.3%, 12/34) and were more common in cases of NSCLC with LN metastasis (71.4%, 45/63) than in cases without metastasis (45.6%, 26/57). Similarly, overexpression of HBXIP showed a correlation with the clinical stage of lung cancers. We found that 71.4% (40/56) of the advanced clinical stage (stages III and IV) NSCLC samples showed strong positive HBXIP staining, which was significantly higher than in cases with early clinical stage (stages I and II; 48.4%, 31/64). Overall, the expression of HBXIP protein was positively correlated with LN metastasis, histological grade, and clinical stage (Table 2).

Table 2.

Relationship between HBXIP protein overexpression and the clinicopathological features of NSCLC.

Variables	No. of cases (n)	HBXIP strong positive rate (%)	χ2	p value
Gender
Male	64	41 (64.1%)	1.361	0.243
Female	56	30 (53.6%)
Age (years)
>62	70	42 (60.0%)	0.048	0.826
≤62	50	29 (58.0%)
Tumor size (cm)
≤4	63	35 (55.6%)	0.716	0.398
>4	57	36 (63.2%)
Histological grade
Grade 1	34	12 (35.3%)	14.400	0.001**
Grade 2	35	20 (57.1%)
Grade 3	51	39 (76.5%)
Clinical stage
I + II	64	31 (48.4%)	6.535	0.011*
III + IV	56	40 (71.4%)
Lymph node status
N0	57	26 (45.6%)	8.254	0.004**
N+	63	45 (71.4%)

*

p < 0.05; **p < 0.01.

High HBXIP expression is an independent biomarker of poor prognosis in patients with NSCLC

To further substantiate the importance of high HBXIP expression in NSCLC progression, 120 NSCLC cases were analyzed using the Kaplan–Meier method. We found that patients with high HBXIP expression exhibited a lower rate of overall survival (OS) than did those with low HBXIP expression (log rank = 26.459, p = 0.000; Figure 6(a)). Similarly, survival of patients with Grade-1 (log rank = 5.579, p = 0.018), Grade-2 (log rank = 4.104, p = 0.043), and Grade-3 (log rank = 8.059, p = 0.005) NSCLC were significantly lower in patients with tumors exhibiting high versus low HBXIP expression (Figure 6(b)–(d)). Moreover, the patients with high HBXIP expression had decreased OS compared to those with low HBXIP expression in either LN metastasis (−) cases (log rank = 7.900, p = 0.005) or LN metastasis (+) cases (log rank = 13.600, p = 0.000; Figure 6(e) and (f)). Additionally, NSCLC patients with high HBXIP expression had decreased OS compared to those with low HBXIP expression in either early-stage (log rank = 6.122, p = 0.013) or late-stage cases (log rank = 16.690, p = 0.000; Figure 6(g) and (h)).

Figure 6.

Kaplan–Meier survival curves illustrating the significance of HBXIP expression in NSCLC. (a) OS rates of patients with high (n = 71) and low (n = 49) HBXIP expression. (b–d) High HBXIP expression was strongly associated with poor OS in G1 (n = 12), G2 (n = 20), and G3 (n = 39). (e and f) High HBXIP expression was strongly associated with poor OS in patients without lymph node metastasis (n = 26) and with lymph node metastasis (n = 45). (g and h) High HBXIP expression was strongly associated with poor OS in early stage (n = 31) and late stage (n = 40). ((a) log rank = 26.459, p = 0.000; (b) log rank = 5.579, p = 0.018; (c) log rank = 4.104, p = 0.043; (d) log rank = 8.059, p = 0.005; (e) log rank = 7.900, p = 0.005; (f) log rank = 13.600, p = 0.000; (g) log rank = 6.122, p = 0.013; and (h) log rank = 16.690, p = 0.000.)

Additionally, univariate analysis demonstrated that LN metastasis (p = 0.006), histological grade (p = 0.001), clinical stage (p = 0.000), and HBXIP expression status (p = 0.000) were all significantly associated with OS in patients with NSCLC. These data suggested that HBXIP might be a valuable prognostic factor in NSCLC. Moreover, multivariate analysis was performed using the Cox proportional hazards model for all of the significant variables that were examined in the univariate survival analysis. We found that the clinical stage (p = 0.003) proved to be an independent poor prognostic factor for survival in NSCLC. Importantly, high HBXIP expression also emerged as a significant independent poor prognostic factor in NSCLC (p = 0.005; Table 3).

Table 3.

Cox regression model analysis of the clinicopathological features in 120 patients with NSCLC.

Characteristics	B	SE	Wald	HR	95% CI		p value
					Lower	Upper
Univariate
Age	0.026	0.188	0.020	1.027	0.710	1.484	0.889
Gender	0.038	0.185	0.043	1.039	0.723	1.492	0.836
Tumor size	0.164	0.184	0.796	1.178	0.822	1.689	0.372
LN	0.512	0.187	7.528	1.668	1.157	2.405	0.006*
Clinical stage	0.724	0.193	14.071	2.062	1.413	3.009	0.000*
Histological grade	0.410	0.118	12.005	1.507	1.195	1.900	0.001**
HBXIP	0.679	0.193	12.298	1.971	1.349	2.880	0.000**
Multivariate
LN	0.329	0.191	2.970	1.390	0.956	2.021	0.085
Histological grade	0.224	0.129	3.006	1.252	0.971	1.613	0.083
Clinical stage	0.591	0.202	8.545	1.806	1.215	2.683	0.003**
HBXIP	0.571	0.201	8.063	1.770	1.193	2.625	0.005**

B: coefficient; SE: standard error; Wald: Wald statistic; HR: hazard ratio; CI: confidence interval.

*

p < 0.05; **p < 0.01.

Discussion

Lung cancer is the leading cause of cancer-related death worldwide. It is a disease that occurs as a result of the dysregulation of oncogenes or tumor-suppressor genes. Multiple genetic alterations change the biological characteristics of cancer cells, including cell growth, apoptosis, migration, invasion, and metabolism. ¹⁶ The majority of patients are often diagnosed with lung cancer at an advanced stage, accompanied by extensive invasion and metastasis. ¹⁷ Of the types of lung cancer, NSCLC accounts for 85% of all lung cancers. ⁷ Despite recent advances in the diagnosis and chemotherapeutic and targeted treatment of NSCLC, including immunotherapies such as epidermal growth factor receptor (EGFR)-targeted treatment, insulin-like growth factor 1 receptor, or EML4-ALK fusion protein interference, ¹⁸ 80% of all NSCLC cases are in the advanced stage, where the prognosis is poor, ¹⁹ the OS rate is low (5-year survival rate of 15%), and the recurrence rate of NSCLC is high, even with early diagnosis. ²⁰ Therefore, investigations of a molecular predictive protein underlying the progression of lung cancer are necessary and may help develop novel prognostic biomarkers and therapeutic targets for this malignancy.

The HBXIP, encoding a 9.6-kDa protein, was originally identified by its interaction with the C-terminus of the HBV X protein (HBX) and is located at human chromosome 1p13.3. ⁹ The HBXIP sequence is well conserved among mammalian species. ¹¹ Marusawa et al. ¹⁰ reported that HBXIP acts as a bridge between HBX and survivin. Moreover, Zhang et al. ²¹ found that HBXIP inhibited the apoptosis induced by HBX in hepatoma cells. Previously, some studies reported that HBXIP functioned as an oncoprotein in breast cancer and served as a coactivator of transcriptional factors, such as STAT4, SP1, E2F1, and TFIID, to transactivate S100A4, LMO4, Skp2, and Lin28B and to promote cellular proliferation and migration in breast cancer.^13,22–24 Cui et al. ²⁵ found that HBXIP upregulates CD46, CD55, and CD59 via extracellular signal–regulated protein kinase 1/2 (ERK1/2)/nuclear factor-κB (NF-κB) signaling to protect breast cancer cells from complement attack. Moreover, Xu et al. ²⁶ reported that HBXIP was highly expressed in ovarian cancer and could promote the migration of ovarian cancer cells by stimulating the activity of Skp2 promoter via the transcription factor Sp1. The overexpression of HBXIP can also enhance the tumor-induced angiogenesis of HepG2 human hepatocellular carcinoma cells. ²⁷ Similarly, HBXIP enhanced angiogenesis and growth of breast cancer by modulating FGF8 and vascular endothelial growth factor (VEGF). ²⁸ Until now, there have been no reports that explored the status of HBXIP and its potential impacts on lung tumorigenesis.

Recent studies have demonstrated that HBXIP plays a crucial role in the development of cancer through glucose metabolism reprogramming and abnormal lipid metabolism. In breast cancer, HBXIP enhances glucose metabolism reprogramming by suppressing SCO2 and PDHA1. It also contributes to abnormal lipid metabolism via LXRs/SREBP-1c/FAS signaling.^29,30 A new study has revealed that HBXIP was able to depress gluconeogenesis by suppressing PCK1 via the miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway to promote hepatocarcinogenesis. ³¹ These results indicate that HBXIP can serve as a coactivator of transcription factors to upregulate many of the genes involved in cancer metabolism and signaling and suggest that it is important to measure HBXIP expression in other cancer tissues.

In this study, the expression levels of HBXIP in several NSCLC cell lines were initially detected, and it was demonstrated that the expression levels of HBXIP in A549 cells were significantly higher compared with other NSCLC cells. For better elucidating the role and underlying mechanisms of HBXIP in A549 cells, the effects of HBXIP-knockdown on A549 cells were investigated. Furthermore, the expression levels of HBXIP was associated with the biological characteristics of A549 cells, since it was demonstrated that knockdown of HBXIP expression repressed cell proliferation and migration. Similar to Hu et al.’s ³² and Li’s ³³ researches, downregulating the expression of HBXIP can inhibit cell proliferation and promote cell apoptosis in hepatocellular carcinoma and in urothelial carcinoma of the bladder. Because its effect on the invasive, proliferative, and metastatic behaviors of cancer cells, we speculated whether overexpression of the HBXIP is associated with clinical features and prognosis of NSCLC. Thus, we discussed the relationship between HBXIP expression and long-term prognosis in these patients. This is the first study to show that HBXIP plays an important role in NSCLC. Then, we used IF staining to locate the HBXIP protein in A549 NSCLC. We found that the staining of HBXIP was mainly localized in the cytoplasm in A549 cells. Xu ²⁶ and Cheng ³⁴ found that the expression of HBXIP could be observed in both the cytoplasm and the nucleus in ovarian and breast cancer tissues. However, in this study, IHC staining showed that the expression of HBXIP was mainly localized in the cytoplasm in NSCLC tissues. The results indicated that the localization of HBXIP overexpression might differ across a range of tumor types. We then observed the staining intensity and percentage of positive tumor cells to score HBXIP expression. We determined that HBXIP expression was significantly higher in cancerous tissues than in adjacent non-tumor tissues. We found that the abnormal expression of HBXIP might play a crucial role in NSCLC. Cheng et al. ³⁴ reported that HBXIP expression was higher in breast cancer patients with LN metastasis than it was in those without metastasis. Moreover, our research analyzed the correlation between HBXIP expression and the clinicopathological features of NSCLC. We found that HBXIP protein was not only associated with LN metastasis but also related to both histological grade and clinical stage in the 120 NSCLC patients.

We also discovered that patients with high HBXIP expression had lower OS rates than did those with low HBXIP expression, as determined using the Kaplan–Meier method. These results suggested that HBXIP overexpression might be a crucial molecular marker in the prognosis of NSCLC patients. The Cox regression analysis showed that HBXIP protein is an independent prognostic factor in NSCLC, along with tumor clinical stage. Taken together, these statistics indicated that HBXIP may become a potential novel therapeutic target for the treatment of NSCLC.

Conclusion

In conclusion, HBXIP expression is frequently upregulated in NSCLC, and HBXIP overexpression plays an important role in NSCLC progression. HBXIP may act as an attractive new biomarker for prognostic evaluation and a molecular therapeutic target in patients with NSCLC.