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Introduction

In many developed countries, cancer is one of the most common causes of death. The incidence of colorectal cancer (CRC) has recently increased in Japan, in concert with changing lifestyles (1). The major cause of death from cancer is distant metastases. Identification of genes responsible for development and progression of CRC and understanding their clinical significance are critical for the establishment of adequate treatments for this disease (2,3).

Inhibin β A (INHBA) is a member of the transforming growth factor β (TGF-β) superfamily (4). INHBA forms a disulfide-linked homodimer known as activin A, which was originally described in 1978 for its role in the hypothalamic-pituitary-gonadal axis (5,6). It is able to strongly induce embryonic stem cell differentiation (7). Its expression is increased in carcinoma tissues, as established by studies of activin A levels in esophageal (8), pancreatic (9), prostate (10), and ovarian (11,12) cancers, and patients with endometrial and cervical carcinomas have high serum levels of activin A (13).

The aim of this study was to analyze the correlation between INHBA expression in CRC tissues obtained from patients and clinicopathological factors. In addition, we performed an in vitro study in which gene knockdown techniques and the introduction of INHBA were used to investigate the relevance of INHBA expression and its relationship with clinicopathological characteristics.

Materials and methods

Cell culture

Human CRC cell lines (CaR1, CCK81 and DLD-1) were obtained and cultured in minimum essential medium (MEM; Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL, Carlsbad, CA, USA) and antibiotics at 37°C in a humidified atmosphere containing 5% CO2. Caco-2 cells were cultured in MEM containing 20% FBS. For the small interference RNA (siRNA) knockdown experiment, RNA duplexes that targeted human INHBA (5′ end) were synthesized (Hs_INHBA_4HPsiRNA; Qiagen, Valencia, CA, USA). AllStars Neg siRNA was used as a negative control (sense sequence, UUCUCCGAACGUGUCACGU; Qiagen). CRC cell lines were transfected with 15 μmol/l siRNA using HiPerFect transfection reagent (Qiagen). The growth rate of the cell culture was measured by counting cells using a CellTac kit (Nihon Koden, Tokyo, Japan). Triple transfection was performed using all the siRNA duplexes together. Plasmids containing human INHBA NM_002192 (OriGene Inc., Rockville, MA, USA) were transfected into CCK81, DLD-1 and Caco-2 cells using Lipofectamine™ 2000 (Invitrogen Life Technologies). An empty vector was used as a mock control. Values are presented as means ± standard deviation (SD) of 3 independent experiments.

Clinical tissue samples

From 1992 to 2002, 126 patients (75 men and 51 women) diagnosed with CRC underwent surgical resection at the Medical Institute of Bioregulation at Kyushu University. Primary CRC specimens and their adjacent normal colorectal mucosa were obtained from patients after obtaining their informed consent and in accordance with the institutional guidelines. Each patient was definitively diagnosed as having CRC on the basis of clinicopathological findings. The resected surgical specimens were equally divided into two halves; one half was frozen in liquid nitrogen and preserved at −80°C for RNA study, and the other half was fixed in formalin, processed through graded ethanol, and embedded in paraffin. The formalin-fixed sections were stained with hematoxylin and eosin and elastic van Gieson, and the degree of histological differentiation, lymphatic invasion, and venous invasion was microscopically examined. None of the patients received chemotherapy or radiotherapy before surgery. Clinicopathological factors were assessed according to the tumor-node-metastasis (TNM) classification criteria as defined by the International Union Against Cancer (14,15). The patients were followed up with blood examination, including for levels of tumor markers such as serum carcinoembryonic antigen and cancer antigen, and underwent imaging investigations such as abdominal ultrasonography and/or computer tomography as well as chest radiography every 3–6 months.

RNA study

Total RNA was extracted from the frozen tissues, and reverse transcription was performed (16,17). Two human INHBA oligonucleotide primers used for PCR were designed as 238-bp INHBA fragments [5′-CCTCGGAGATCATCACG TTT-3′ (forward) and 5′-CCCTTTAAGCCCACTTCCTC-3′ (reverse)]. The forward primer was located in exon 1, and the reverse primer was located in exon 2. As an internal control, a PCR assay was performed using primers specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These GAPDH primers, 5′-TTGGTATCGTGGAAGGACTCA-3′ (forward) and 5′-TGTCATCATATTGGCAGGTT-3′ (reverse), produced a 270-bp amplicon. Real-time PCR monitoring was performed using the LightCycler system (Roche Diagnostics, Tokyo, Japan) for complementary DNA (cDNA) amplification of INHBA and GAPDH. The amplification protocol consisted of 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec, and elongation at 72°C for 10 sec. The PCR products were then subjected to a temperature gradient from 55°C to 95°C at 0.1°C sec−1 with continuous fluorescence monitoring to produce product melting curves. The mRNA expression ratio of tumor to normal tissues was calculated and normalized against GAPDH mRNA expression.

Statistical analysis

For continuous variables used in an in vitro analysis, data are expressed as means ± SD and were analyzed using the Wilcoxon rank test. The relationship between mRNA expression and clinicopathological factors was analyzed using the Chi-square and Student's t-tests. Kaplan-Meier survival curves were plotted and compared using a generalized log-rank test. Univariate and multivariate analyses for the identification of factors prognostic for overall survival were performed using the Cox proportional hazard regression model. All tests were analyzed using JMP software (SAS Institute Inc., Cary, NC, USA). P-values of <0.05 were considered statistically significant.

Results

INHBA mRNA expression in CRC clinical tissue specimens and clinicopathological characteristics

We performed quantitative real-time RT-PCR analysis using primary CRC and adjacent noncancerous CRC tissues. RT-PCR of 126 paired clinical samples showed that 116 (92.1%) of these samples exhibited higher INHBA mRNA levels in tumor tissues than in paired normal tissues (Fig. 1A). INHBA expression was calculated as INHBA/GAPDH expression. For the evaluation of clinicopathological factors, the tissue samples were divided into 2 groups according to INHBA expression. Patients with tumors that had a more than median INHBA expression were assigned to the high expression group (n=63); the others were assigned to the low expression group (n=63). Clinicopathological factors related to the INHBA expression status of the 126 patients are summarized in Table I. The data indicated that INHBA expression was correlated with the tumor stage (P<0.0001), lymph node metastasis (P<0.0001), lymphatic invasion (P=0.0013), venous invasion (P<0.0001) and liver metastasis (P=0.0024). Other factors were not significantly correlated with INHBA expression.

Figure 1 (A) INHBA mRNA expression in clinical tissue specimens. Quantitative RT-PCR of 126 clinical samples showed that 116 (92.1%) of these samples had higher levels of INHBA mRNA compared with the matched normal tissues. Mean INHBA mRNA expression in tumor tissues (normalized against GAPDH expression) was significantly higher than that of the corresponding normal tissues (*P<0.001; Student's t-test). (B) Overall survival curves of CRC patients, based on the INHBA mRNA expression status. The overall survival rate was better in the low expression group than that in the high expression group (*P=0.0016). INHBA, inhibin β A; CRC, colorectal cancer.

Table I Clinicopathological factors and INHBA mRNA expression in 126 CRC patients.

Table I

Clinicopathological factors and INHBA mRNA expression in 126 CRC patients.

	INHBA/GAPDH expression
Factors	High n=63, n (%)	Low n=63, n (%)	P-value
Age (years)
≤68	31 (49.2)	30 (47.6)	0.87
>68	32 (50.8)	33 (52.6)
Gender
Male	41 (65.1)	35 (55.6)	0.26
Female	22 (34.9)	28 (44.4)
Histological grade
Well, mod	58 (92.1)	61 (96.8)	0.22
Others	5 (7.9)	2 (3.2)
Tumor stage
T0–T2	6 (9.5)	29 (46.0)	<0.0001a
T3–T4	57 (90.5)	34 (54.0)
Lymph node metastasis
Absent	21 (33.3)	47 (74.6)	<0.0001a
Present	42 (66.7)	16 (25.4)
Lymphatic invasion
Absent	37 (58.7)	44 (69.8)	0.0013a
Present	26 (41.3)	19 (30.2)
Venous invasion
Absent	43 (68.3)	60 (95.2)	<0.0001a
Present	20 (31.7)	3 (4.8)
Liver metastasis
Absent	49 (77.8)	60 (95.2)	0.0024a
Present	14 (22.2)	3 (4.8)
Peritoneal dissemination
Absent	59 (93.7)	62 (98.4)	0.15
Present	4 (6.3)	1 (1.6)

a P<0.01.

{ label (or @symbol) needed for fn[@id='tfn2-or-30-06-2903'] } CRC, colorectal cancer; INHBA, inhibin β A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; well, well-differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; others, poorly differentiated adenocarcinoma and signet cell carcinoma.

Relationship between INHBA expression and prognosis

The data showed that the overall survival rate was significantly lower in the high expression group than in the low expression group (P=0.0016) (Fig. 1B). The median follow-up period was 3.33±2.67 years. Table II shows the results of the univariate and multivariate analyses of factors related to overall survival. Univariate analysis showed that the following factors were significantly related to overall survival: histological grade (P=0.0139), tumor stage (P=0.0006), lymph node metastasis (P<0.0001), lymphatic invasion (P<0.0001), venous invasion (P=0.0011), liver metastasis (P<0.0001) and INHBA mRNA expression (P=0.0007). Multivariate analysis indicated that lymphatic invasion and liver metastasis were independent predictors of overall survival. INHBA mRNA high expression was not an independent predictor.

Table II Univariate and multivariate analysis of the clinicopathological factors affecting survival rate.

Table II

Univariate and multivariate analysis of the clinicopathological factors affecting survival rate.

		Univariate analysis		Multivariate analysis
Factors	No. of patients	5-year survival rate (%)	P-value	Relative risk (95% CI)	P-value
Age (years)
≤68	60	76.5	0.180
>68	66	61.8
Gender
Male	75	67.4	0.389
Female	51	73.1
Histological grade
Well, mod	119	71.8	0.0139a	1.75 (0.81–3.25)	0.139
Others	7	33.3
Tumor stage
T0–T2	36	93.1	0.0006b	1.26 (0.60–3.34)	0.566
T3–T4	90	58.9
Lymph node metastasis
Absent	69	85.9	<0.0001b	1.20 (0.75–2.03)	0.461
Present	57	50.0
Lymphatic invasion
Absent	71	84.1	<0.0001b	2.23 (1.40–3.73)	0.0006b
Present	55	51.8
Venous invasion
Absent	103	77.5	0.0011b	1.41 (0.91–2.11)	0.112
Present	23	36.2
Liver metastasis
Absent	17	79.5	<0.0001b	2.56 (1.67–3.97)	0.0000b
Present	109	20.3
INHBA expression
High	63	49.9	0.0007b	1.16 (0.73–1.92)	0.546
Low	63	86.5

a p<0.05;

b p<0.01.

{ label (or @symbol) needed for fn[@id='tfn5-or-30-06-2903'] } RR, relative risk; CI, confidence interval; well, well-differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; others, poorly differentiated adenocarcinoma and signet cell carcinoma; INHBA, inhibin β A.

Relationship between INHBA expression and lymph node metastasis

Table III shows the univariate and multivariate analyses of factors affecting lymph node metastasis. Univariate analysis showed that the following factors were significantly related to lymph node metastasis: histological grade (P=0.0341), tumor stage (P=0.0028), lymphatic invasion (P=0.0215) and INHBA mRNA high expression (P=0.0074). Multivariate analysis indicated that inclusion in the INHBA mRNA high expression group [relative risk (RR), 3.95; 95% confidence interval (CI), 1.71–9.35; P=0.0014] was an independent predictor of lymph node metastasis, as was lymphatic invasion (RR, 3.25; 95% CI, 1.39–7.72; P=0.0067).

Table III Results of the univariate and multivariate analysis of clinicopathological factors affecting lymph node metastasis.

Table III

Results of the univariate and multivariate analysis of clinicopathological factors affecting lymph node metastasis.

	Univariate analysis			Multivariate analysis
Factors	RR	95% CI	P-value	RR	95% CI	P-value
Age (years) (≤68/>68)	0.593	0.63–3.54	0.324	-	-	-
Gender (Male/female)	0.571	0.172–1.65	0.322	-	-	-
Histological grade (Well, mod/others)	5.62	1.02–28.2	0.0341a	4.91	0.73–98.0	0.16
Depth (T0–T2/T3–T4)	5.53	1.89–18.5	0.0028b	2.25	0.93–5.50	0.072
Lymphatic invasion (Absent/present)	3.68	1.27–12.3	0.0215a	3.25	1.39–7.72	0.0067b
Venous invasion (Absent/present)	2.95	0.914–8.91	0.0585	-	-	-
INHBA mRNA expression (Low/high)	5.93	1.81–26.8	0.0074b	3.95	1.71–9.35	0.0014b

a p<0.05;

b p<0.01.

{ label (or @symbol) needed for fn[@id='tfn8-or-30-06-2903'] } RR, relative risk; CI, confidence interval; Well, well differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; others, poorly differentiated adenocarcinoma and signet cell carcinoma; INHBA, inhibin β A.

In vitro assessment of knockdown and transfection of INHBA

The INHBA expression study indicated that CaR1 cells had higher levels of expression than other CRC cell lines such as DLD1, Caco-2 and CCK81 (Fig. 2A). We performed a knockdown experiment of INHBA expression using the CaR1 cell line. After 48 h of siRNA transfection, quantitative real-time RT-PCR was used to confirm the reduction in INHBA expression due to siRNA treatment (Fig. 2B). A proliferation assay indicated that the knockdown resulted in a reduction in the number of CaR1 cells at 72 h (P<0.05; Fig. 2C). We induced INHBA expression in the cell lines (DLD1, Caco-2 and CCK81) using a plasmid technique, and quantitative real-time RT-PCR confirmed successful induction in them (Fig. 3A–C). Proliferation assays indicated that high INHBA expression increased the cell numbers of CRC cells, which have low INHBA expression by default (P<0.05; Fig. 3D–F) at 48 h.

Figure 2 (A) INHBA mRNA expression in 4 CRC cell lines. INHBA expression status was evaluated using 4 cell lines derived from human CRC (DLD1, Caco-2, CCK81 and CaR1) using quantitative real-time RT-PCR analysis. CaR1 had significantly higher INHBA mRNA expression than the other cell lines. (B) Knockdown assessment of INHBA by siRNA in the CaR1 cell line. Quantitative real-time RT-PCR confirmed reduced INHBA mRNA levels (*P<0.05). (C) A proliferation assay was performed using the CaR1 cell line. Significant differences were observed between NC and INHBA siRNA (n=3, *P<0.05). An in vitro assay showed differences in the ratio when compared to the control (with no treatment) cells. Values are presented as means and SEM. INHBA, inhibin β A; CRC, colorectal cancer; NC, negative control.

Figure 3 INHBA transfection assessment by plasmids in 3 CRC cell lines. Quantitative real-time RT-PCR was used to confirm INHBA mRNA levels in the cell lines: (A) DLD1, (B) Caco-2 and (C) CCK81. Proliferation assays were performed using (D) DLD1, (E) Caco-2 and (F) CCK81 cells. Significant differences were observed between mock- and INHBA-transfected cells (n=3). INHBA, inhibin β A; CRC, colorectal cancer. (*P<0.05)

Discussion

INHBA is a subunit of both activin and inhibin, two closely related glycoproteins with opposing biological effects, which belong to the TGF-β superfamily (18–20). The TGF-β superfamily comprises a structurally similar, although functionally diverse group of proteins that play important roles in embryonic development as well as in the functions of terminally differentiated tissues. Activins play fundamental roles in cell differentiation and development and are known to induce cellular responses via activin receptors and the SMAD2/3 pathway, while inhibits function to antagonize activins through either competition of receptor binding or β-glycan.

In the present study, we determined that INHBA is highly expressed in CRC tissues when compared with that in the corresponding normal tissues. In addition, high INHBA expression in CRC tissues was a predictor of poor prognosis when compared with low INHBA expression. Clinicopathological factors related to the INHBA expression status indicated that the high expression group displayed a worse histological grade, higher tumor stage, more lymph node metastasis, poorer lymphatic invasion, greater vascular invasion and more extensive liver metastasis. Therefore, INHBA expression was not an independent prognostic factor but may be strongly related to one of the other prognostic factors of CRC. Thus, this study of the association of high INHBA expression with other prognostic factors indicated that high INHBA expression is an independent prognostic factor for lymph node metastasis. Inclusion in the INHBA mRNA high expression group was an independent predictor of lymph node metastasis, as was lymphatic invasion. The overexpression and knockdown experiments were performed in vitro. These experiments showed that high INHBA expression induced cell growth, whereas low INHBA expression induced an opposite effect.

It is useful to determine the necessity for intensive follow-up and adjuvant therapy for CRC by predicting recurrence and metastases after curative surgical resection (21–23). In the present study, clinicopathological analysis revealed that patients who had CRCs with high INHBA expression had a poor prognosis for overall survival than those with low expression. The data indicated that INHBA expression is presumably a novel predictor of CRC prognosis. Several adjuvant chemotherapies are helpful at certain disease stages, particularly in CRC (23–27). For such cases, an informative prognostic marker, which is independent of the traditional TNM classification, is extremely important and can contribute to diagnosis and treatment. Adjuvant chemotherapy for CRC is necessary for highly suspicious recurrent cases. In such cases, analysis of INHBA expression may be useful for predicting CRC prognosis, and INHBA is also proposed to be a therapeutic target in treatment for patients with poor prognosis.

References