High expression of the ANKRD49 protein is associated with progression and poor prognosis of gastric cancer

Abstract

BACKGROUND:

Gastric cancer is one of the most common malignant tumours. Identifying novel genes that govern the development of gastric cancer will help to elucidate its molecular mechanisms and find novel biomarkers.

METHODS:

Expression of the ANKRD49 protein was assessed by immunohistochemical analysis of tissue microarrays containing 92 sets of human gastric cancer specimens with adjacent non-cancerous tissue. Associations between ANKRD49 levels and clinicopathological characteristics of the patient were investigated. The correlation between ANKRD49 expression and patient survival was analysed by the Kaplan-Meier method.

RESULTS:

The results revealed that the expression level of the ANKRD49 protein in gastric cancer was significantly upregulated and correlated with the tumour size, tumour-node-metastasis (TNM) stage, histological grade, depth of invasion, vessel invasion, lymph node metastasis and distant metastasis. The mean survival time of patients with low expression levels of ANKRD49 was significantly longer than that of patients with high expression levels of ANKRD49. Multivariate Cox regression analysis demonstrated that the ANKRD49 protein expression level was an independent prognostic indicator for the survival rate of patients with gastric cancer.

CONCLUSION:

The results of the present study highlighted an important role of the ANKRD49 protein in the progression of gastric cancer. The ANKRD49 protein could act as a potential biomarker for prognosis evaluation of gastric cancer and may be used as a molecular target for gastric cancer treatment.

Keywords

ANKRD49 gastric cancer tissue microarray immunohistochemistry prognosis

1. Introduction

Gastric cancer (GC) is one of the most common malignant tumours worldwide and is the third leading cause of cancer-associated mortality in China [1]. Every year, approximately 405,000 novel cases are diagnosed in China; approximately 70% of patients are diagnosed at an advanced stage, and the prognosis is poor [2, 3]. It is well known that environmental factors, such as Helicobacter pylori infection [4] and poor diet, and genetic factors, such as abnormal expression of cancer-related genes [5], contribute to gastric carcinogenesis. It is pivotal to investigate novel genes that govern the development of GC to elucidate the molecular mechanisms, identify novel biomarkers and develop personalized therapies for individual patients in the future.

Ankyrin repeat domain 49 (ANKRD49) contains four ankyrin repeat motifs, which include 30 to 34 amino acid residues [6]. Ankyrin repeat proteins mediate protein-protein interactions and have been proven to be involved in the development of diverse human cancers [7, 8]. ANKRD49 has been reported to contribute to the progression of human malignant gliomas and non-small cell lung cancer via promoting cell proliferation and cell invasion [9, 10]. However, the expression pattern of ANKRD49 in GC remains largely unclear. At present, molecular profiling of GC can be performed using gene/tissue expression microarray analysis or DNA sequencing [11, 12, 13], which facilitates the identification of novel biomarkers for subtype classification, prognosis and therapeutic targets. In the present study, we investigate the expression of ANKRD49 by immunohistochemistry (IHC) and identify its potential roles in tumour occurrence and development in and prognosis of patients with GC.

2. Materials and methods

2.1 Patients and follow-up

A total of 92 patients who underwent surgery for histologically proven gastric cancer from June 2007 to July 2008 at hospitals that cooperated with the National Engineering Center for Biochip at Shanghai were selected for this research. There were 62 men and 30 women, whose ages ranged from 37 to 84 years (median: 63 years). Patients had not received any previous treatment, and histological diagnoses were made from formalin-fixed, paraffin-embedded tissues by two pathologists. The clinicopathological characteristics, including gender, age, tumour size, tumour location, histological grade, differentiation grade and surgical record, were obtained from medical records. All patients were followed up, and the follow-up deadline was August 2013. The survival time was determined from the date of surgery to the follow-up deadline or date of mortality. Written informed consent was obtained from the patients for the use of their samples for research, and the research protocols were approved by the Ethics Committee of Shanxi Medical University. The clinicopathological characteristics of our study sample are presented in Table 1.

2.2 Tissue microarray construction

Paraffin-embedded tumour specimens and paired adjacent non-tumour specimens ( $\geqslant$ 1.5 cm away from the tumour) were carefully collected before any treatment. A tissue-arraying instrument (Beecher Instruments, Silver Spring, MD, USA) was used to create cylinder-shaped holes in a square recipient paraffin block and then to remove tissue cores from the donor block, such as clinical biopsies or tumour samples, by a hollow needle with an inner diameter of 1.5 mm held in an X-Y axis precision guide. The cylindrical samples were retrieved from the selected regions in the donors and extruded directly into the recipient blocks with defined array coordinates. After the construction of the array block, multiple 4- $\mu$ m-thick sections were cut with a microtome using an adhesive-coated tape sectioning system (Instrumedics, Hackensack, NJ, USA).

2.3 Immunohistochemistry

The immunohistochemical staining procedure was performed by using the two-step method. Slides were deparaffinized in xylene, rehydrated through graded concentrations of ethanol, and rinsed in distilled water. Endogenous peroxidase activity was blocked by incubating the slides with 30 mL/L H ${}_{2}$ O ${}_{2}$ in methanol for 10 min at room temperature. Then, the sections were submitted to antigen retrieval in a pressure cooker containing 0.01 mmol/L sodium citrate buffer for 10 min. The slides were subsequently incubated in 100 $\mu$ L normal goat serum for 20 min at room temperature and then incubated with rabbit anti-human ANKRD49 antibody (1:100 dilution with PBS-Triton; Abcam, Cambridge, UK) overnight at 4 ${}^{\circ}$ C. After washing with PBS-Triton, the slides were incubated with goat anti-rabbit immunoglobulin/HRP (1:10000; BOSTER, Wuhan, China), and the reaction product was visualized with diaminobenzidine as the chromogen and counterstained with haematoxylin.

The evaluation of the immunohistochemical staining was performed independently by two authors without knowledge of the clinicopathological information. The expression of ANKRD49 was examined by viewing the cells with light microscopy, calculating 1000 cells per 5 sites and evaluating the mean number. The percentage of positive cells was quantified under the microscope and classified into five groups as follows: 0% positive cells for 0; 1% to 25% positive cells for 1; 26% to 50% positive cells for 2; 51% to 75% positive cells for 3; and $\geqslant$ 76% positive cells for 4. Intensity was scored as 0 for absence of staining, 1 for weak, 2 for moderate, and 3 for strong staining. The score of the intensity plus the percentage of positive staining was used to define the following expression levels: 0–1: negative; 2–3: slightly positive ( $+$ 1); 4–5: moderately positive ( $+$ 2); and 6–7: strongly positive ( $+$ 3).

Table 1
Clinicopathological characteristics of gastric cancer cases

Characteristic	GC, $N=$ 92	%
Gender
Male	62	67.4
Female	30	32.6
Miss	0	0
Age (years)
$\leqslant$ 60 years	38	41.3
$>$ 60 years	53	57.6
Miss	1	1.1
Location of tumour
Cardia	10	10.9
Body	48	52.2
Antrum	34	36.9
Miss	0	0
Tumour size
$<$ 5 cm	45	48.9
$\geqslant$ 5 cm	46	50.0
Miss	1	1.1
Deep tumour invasion
T1-T2	7	7.6
T2	8	8.7
T3	52	56.5
T4	14	15.3
Miss	11	11.9
TNM stage
I–II	30	32.6
III–IV	61	66.3
Miss	1	1.1
Vessel invasion
Negative	22	23.9
Positive	15	16.3
Miss	55	59.8
Histological grade
I
II	23	25.0
II–III	33	35.9
III	21	22.8
III–IV	14	15.2
Miss	1	1.1
Lymph node metastasis
Absence	22	23.9
Presence	68	73.9
Miss	2	2.2
Distant metastasis
Absence	82	89.1
Presence	10	10.9
Miss	0	0
Follow-ups
Dead	56	60.9
Survival or lost	36	39.1
Miss	0	0

TNM, tumour-node-metastasis.

2.4 Statistical analysis

Values are expressed as the mean $\pm$ standard error of the means. The differential expression of ANKRD49 proteins between tumourous tissue and non-tumourous tissue was determined by the Mann-Whitney U-test. Spearman’s rank correlation coefficient was used to analyse the relationships between clinicopathological and molecular parameters. Survival curves were estimated based on Kaplan-Meier curve analysis. Overall survival (OS) was defined as the time from the date of histological diagnosis to the date of last contact or death from any cause. Multivariate analysis was performed by using the Cox regression model to assess whether a factor was an independent predictor of prognosis of gastric cancer. In all statistical analyses, a two-tailed $P$ value $\leqslant$ 0.05 was considered statistically significant. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) 17.0 software (SPSS Inc, Chicago, IL, USA).

3. Results

3.1 Expression levels of the ANKRD49 protein in gastric cancer and non-cancerous gastric mucosae

We investigated the protein expression of ANKRD 49 in gastric cancer specimens and adjacent non-tumourous tissue using a large tissue microarray (92 cores). The tumourous or non-tumourous mucosa-specific staining was semi-quantitatively scored by the intensity and the percentage of positive staining. ANKRD49 protein expression was detected in 92/92 (100%) patients with gastric cancer, including high expression levels in 68 (73.9%) cases and low expression levels in 24 (26.1%) patients, whereas the ANKRD49 protein was weakly (13/92 cases) or not expressed in non-cancerous gastric mucosa tissues. The percentage of tissues with a high ANKRD49 protein expression level was significantly higher ( $P<$ 0.0001) in gastric cancer tissues compared with adjacent non-cancerous gastric mucosa tissues. Furthermore, immunohistochemistry analysis showed that the ANKRD49 protein was mainly expressed in the cytoplasm of malignant cells, although some expression was detected in the nucleus. Images of representative immunostaining are presented in Fig. 1, and the results are shown in Table 2.

Figure 1.

Immunohistochemical expression of ANKRD49 in gastric cancer and adjacent non-cancer tissues. The black arrow indicates ANKRD49 expression in the cytoplasm; the red arrow indicates ANKRD49 expression in the nucleus. Magnification: $\times$ 200.

3.2 Relationship between the expression of the ANKRD49 protein and clinicopathological parameters

The correlation between ANKRD49 protein expression and the clinicopathological parameters of patients with gastric cancer was evaluated. The expression level of the ANKRD49 protein in gastric cancer was associated with tumour size, deep tumour invasion, TNM stage, vessel invasion, lymph node metastasis and distant metastasis of the tumour, whereas it was not associated with gender, age, location or the histological grade of the tumour. Gastric cancer tissues from patients with large tumour size ( $\geqslant$ 5 cm), deep tumour invasion (T3 and T4), vessel invasion, lymph node metastasis and distant metastasis had significantly higher expression levels of ANKRD49 than those with small tumour size ( $<$ 5 cm), superficial tumour invasion (T1 and T2) and without vessel invasion or lymph node and distant metastasis (Table 3). The Spearman correlation coefficients of ANKRD49 expression with tumour size, depth of invasion, TNM stage, vessel invasion, lymph node metastasis and distant metastasis of tumour were 0.465, 0.387, 0.517, 0.331, 0.538 and 0.279, respectively.

3.3 Correlation between the ANKRD49 protein expression level and prognosis of patients with gastric cancer

Table 2
ANKRD49 expression in tumourous tissue and adjacent non-tumourous tissue

Protein	Histotype	Expression levels (Number)				$P$
		–	$+$ 1	$+$ 2	$+$ 3
ANKRD49	N	76	13	0	0	0.0001
	T	0	24	38	30

T: Tumourous tissue; N: Non-tumourous tissue. Mann-Whitney U-test.

Table 3

Association of ANKRD49 protein levels with clinicopathological parameters of patients with gastric cancer

Clinicopathological parameters	Total no. patients	ANKRD49 protein expression level		$\chi^{2}$	$P$ -value	r
		Low ( $n$ , %)	High ( $n$ , %)
Gender
Male	62	16 (25.8)	46 (74.2)	0.178	0.842	0.020
Female	30	8 (26.7)	22 (73.3)
Age (years)
$\leqslant$ 60 years	38	10 (26.3)	28 (73.7)	0.182	0.746	0.040
$>$ 60 years	53	14 (26.4)	39 (73.6)
Location of tumour
Cardia	10	3 (30.0)	7 (70.0)	0.120	0.349	0.067
Body	48	13 (27.1)	35 (72.9)
Antrum	34	8 (23.5)	26 (76.5)
Tumour size
$<$ 5 cm	45	14 (31.1)	31 (68.9)	11.283	0.028	0.465
$\geqslant$ 5 cm	46	10 (21.7)	36 (78.3)
Deep tumour invasion
T1–T2	7	6 (85.7)	1 (14.3)	30.948	$<$ 0.0001	0.387
T2	8	6 (75.0)	2 (25.0)
T3	52	11 (21.2)	41 (78.2)
T4	14	1 (7.1)	13 (92.9)
TNM stage
I–II	30	10 (33.3)	20 (66.7)	37.285	$<$ 0.0001	0.517
III–IV	61	13 (21.3)	48 (78.7)
Vessel invasion
Negative	22	18 (81.8)	4 (18.2)	41.285	$<$ 0.0001	0.331
Positive	15	5 (33.3)	9 (66.6)
Histological grade
I
II	23	6 (26.1)	17 (73.9)	63.472	0.904	0.751
II–III	33	8 (24.2)	25 (75.8)
III	21	6 (28.6)	15 (71.4)
III–IV	14	4 (28.6)	8 (71.4)
Lymph node metastasis
Absence	22	10 (45.5)	12 (54.5)	20.741	$<$ 0.0001	0.538
Presence	68	14 (20.6)	54 (79.4)
Distant metastasis
Absence	82	23 (28.0)	59 (72.0)	38.378	$<$ 0.0001	0.279
Presence	10	1 (10.0)	9 (90.0)

TNM, tumour-node-metastasis.

Table 4

Multivariate analysis of the correlation between clinicopathological parameters and the survival rate of 92 patients with gastric cancer

Covariates	CI	SE	HR (95% CI)	$P$ -value
ANKRD49 protein expression (high vs. low)	0.819	0.139	2.164 (1.723–2.615)	$<$ 0.0001
Tumour location (cardia vs. other locations)	$-$ 0.149	0.153	0.795 (0.603–1.102)	0.371
Tumour size ( $\geqslant$ 5 cm vs. $<$ 5 cm)	0.081	0.149	1.267 (0.963–1.573)	0.574
Lymph node metastasis (positive vs. negative)	0.227	0.314	1.371 (0.604–2.135)	0.413
Vessel invasion (positive vs. negative)	$-$ 0.108	0.141	0.817 (0.526–1.107)	0.649
Distant metastasis (positive vs. negative)	0.687	0.175	2.471 (1.046–3.896)	0.014
Age range ( $>$ 60 vs. $\leqslant$ 60)	0.183	0.107	1.316 (0.872–1.767)	0.143
Depth of invasion				0.013
T2 vs. T1	0.537	0.616	1.716 (0.427–4.519)	0.461
T3 vs. T1	0.843	0.639	2.539 (1.204–7.071)	0.058
T4 vs. T1	0.405	0.603	1.628 (0.617–4.327)	0.374
TNM stage				$<$ 0.001
Stages III–IV vs. stages I–II	1.935	0.647	8.382 (3.173–24.163)	$<$ 0.001

CI, confidence interval; HR, hazard ratio; SE, standard error; TNM, tumour-node-metastasis.

Figure 2.

Clinicopathological parameters associated with overall survival in gastric cancer based on Kaplan-Meier survival curves. The horizontal axis represents overall survival time (years); the vertical axis represents survival function.

Kaplan-Meier curve analysis showed that high expression levels of the ANKRD49 protein were associated with poor prognosis of patients with gastric cancer ( $P<$ 0.0001). The 1-, 3- and 5-year cumulative survival rates were 96.1, 80.3 and 40.7% for patients with low ANKRD49 expression level and 79.4, 24.7 and 5.68% for patients with high ANKRD49 expression levels, respectively. The mean survival time of patients with low expression levels of ANKRD49 was 50.4 months, which was significantly higher ( $P<$ 0.0001) than that of patients with high expression levels of ANKRD49, at 27.1 months. It was also revealed that age, tumour location, size, depth of invasion, TNM stage, vessel invasion, lymph node metastasis and distant metastasis were significantly associated with the survival of patients with gastric cancer (Fig. 2), whereas gender and histological grade were not significantly associated with survival (data not shown).

To avoid the influence caused by univariate analysis, the expression of ANKRD49 and other parameters were examined by multivariate Cox analysis. It was revealed that the ANKRD49 protein expression level, distant metastasis, TNM stage and depth of invasion were independent prognostic indicators for the survival of patients with gastric cancer, which indicated that the ANKRD49 protein could act as a potential biomarker for prognosis evaluation of gastric cancer. Of the other parameters, age, tumour location, size, lymph node metastasis and vessel invasion were not found to be independent prognostic factors for patient survival (Table 4).

4. Discussion

Ankyrin repeat proteins usually include a varying number of the ankyrin repeat domain, which is an $\sim$ 33-residue-long motif with a canonical helix-loop-helix- $\beta$ -hairpin/loop fold and which is known to function as a specific protein-protein interactor [6, 14]. These ankyrin repeat domain proteins are involved in many cellular processes, including tumour progression [8]. Some proteins, such as Notch, P16 and I $\kappa$ B, which contain an ankyrin repeat domain, have been associated with many types of cancer [15, 16, 17]. The ankyrin repeat domain 49 (ANKRD49) protein contains four ankyrin repeat motifs. It has been reported that ANKRD49 participates in the progression of human malignant gliomas and non-small cell lung cancer [9, 10], whereas the role and expression pattern of ANKRD49 in other cancers are still elusive.

Approximately 990,000 people are diagnosed with GC worldwide every year, and this disease is multifactorial [18]. Both environmental and genetic factors have a role in its aetiology [19]. In the present study, an elaborate experiment was conducted, and a rigorous analysis of the human ANKRD49 protein was performed on a cohort of 92 gastric cancer specimens to address the potential roles of the ANKRD49 protein in the occurrence and development of GC. Our results revealed that the expression levels of the ANKRD49 protein in gastric cancer tissues were significantly higher than those in adjacent non-cancerous gastric mucosae ( $P<$ 0.01).These findings are similar to those reported for other overexpressed proteins in GC, such as Musashi-1 and MDM2 binding protein, which are always associated with tumour location, size, depth of invasion, vessel invasion, histological grade, TNM stage, lymph node and distant metastasis of tumours [1, 20]. Our immunohistochemical assay showed that the expression level of ANKRD49 in gastric cancer tissues was significantly associated with these clinicopathological parameters. In particular, a high ANKRD49 expression level was more frequently observed in tumours at high TNM stages (stages III–IV), with deep invasion (T3 and T4), and the presence of vessel invasion, lymph node metastasis and distant metastasis compared with tumours at low TNM stages (stages I–II), with superficial invasion (T1 and T2), and the absence of vessel invasion, lymph node metastasis and distant metastasis. The results indicated that ANKRD49 may be involved in the invasion and metastasis of gastric cancer. Our present findings are in accord with a previous study that identified ANKRD49 as an invasion-associated gene in non-small cell lung cancer [9].

Thus far, the general prognosis of gastric cancer is poor. The specificity and sensitivity of biomarkers are important for choosing the correct treatment options for and evaluating the prognosis of individual patients [21]. As a result of ANKRD49 involvement in the invasion and metastasis of tumours, ANKRD49 is proposed to be a biomarker associated with poor prognosis of GC. The present study demonstrated that the cumulative survival rates and mean survival time of patients with low ANKRD49 expression levels were significantly higher than those of patients with high ANKRD49 expression levels. Moreover, it was revealed that a high expression level of ANKRD49 was an independent prognostic factor for patients with gastric cancer. Other factors that were correlated with the survival rate of patients included age, tumour location, size, depth of invasion, TNM stage, vessel invasion, lymph node metastasis and distant metastasis. In addition, distant metastasis, TNM stage and depth of invasion were independent prognostic indicators for the survival rate of patients with gastric cancer. Thus far, only CEA or CA 19–9 markers are measured in clinical practice for detection of recurrence [22]. Therefore, the present study is of considerable importance since the results from univariate and multivariate survival analyses highlighted the prognostic relevance of the ANKRD49 protein in patients with gastric cancer.

Up to now, the function of ANKRD49 remains largely unclear. It has been reported that ANKRD49 is overexpressed in human malignant glioma and promotes the proliferation of human malignant glioma cells by increasing the cell cycle and inhibiting cell apoptosis [10]. In addition, it has been reported that ANKRD49 augments autophagy in male germ GC-1 cells by enhancing the nuclear factor kappa B (NF- $\kappa$ B) pathway and plays an important role in spermatogenesis [23]. It is well known that NF- $\kappa$ B signalling is involved in the occurrence and development of GC [24, 25]; in particular, it participates in the invasion and metastasis of gastric cancer cells [26, 27]. Herein, the ANKRD49 protein was primarily expressed in the cytoplasm of GC cells. Thus, we presume that overexpressed ANKRD49 in gastric cancer cells may activate NF- $\kappa$ B via ankyrin repeat motifs interacting with the NF- $\kappa$ B subunits in the cytoplasm and then contribute to the invasion and metastasis of gastric cancer cells. However, this hypothesis needs further investigation.

Although preliminary, our present results suggest that ANKRD49 may be involved in the progression of gastric cancer and that its expression level alone or in combination with other factors (such as TNM stage) is useful for predicting the prognosis of patients with gastric cancer. A limitation of the present study is the smaller size of tissue samples used in the tissue microarray analysis. In addition, the gastric cancer samples were collected from a single institution, which could introduce selection bias. Therefore, more gastric cancer specimens collected from different hospitals are needed to confirm the present analysis, and additional studies are required to elucidate the precise role of ANKRD49 in the development of GC and to examine its potential role as a therapeutic target.

Footnotes

Acknowledgments

This study was supported by the Youth Innovation Fund of the First Hospital of Shanxi Medical University (YC1410) and the Project of International Science and Technology Cooperation of Shanxi Province (2015081039).

Conflict of interest

The authors declare no conflict of interest.

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High expression of the ANKRD49 protein is associated with progression and poor prognosis of gastric cancer

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and follow-up

2.2 Tissue microarray construction

2.3 Immunohistochemistry

Table 1 Clinicopathological characteristics of gastric cancer cases

3. Results

3.1 Expression levels of the ANKRD49 protein in gastric cancer and non-cancerous gastric mucosae

3.3 Correlation between the ANKRD49 protein expression level and prognosis of patients with gastric cancer

Table 2 ANKRD49 expression in tumourous tissue and adjacent non-tumourous tissue

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Clinicopathological characteristics of gastric cancer cases

Table 2
ANKRD49 expression in tumourous tissue and adjacent non-tumourous tissue