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Introduction

Esophageal carcinoma is an age-related neoplasm with a 5-year overall survival rate of less than 35% (1,2). Males more than 40 years of age are at the highest risk of esophageal carcinoma (3). Results of studies have revealed that age-related changes affect the molecular crosstalk between the stromal and epithelial cells, generating a more permissive environment for tumor growth and metastasis (4). Senescence may be one of the mechanism which is responsible for this age-related change in the microenvironment. The definition of senescence is a process that keeps the stable form of cell cycle arrest at the G1 phase (5), as first reported by Hayflick (6). At the earlier stage of the process, senescence acts as an antiproliferative factor, which prevents tumorigenesis and leads to cell cycle arrest. However, if oncogenic mutations are present, these senescent cells may become immortal and initiate the development of additional genetic ‘hits’ in tumorigenesis (7–9). Cell cycle regulators within the retinoblastoma (Rb) and p53 pathways, including the cyclin-dependent kinase (CDK) and CDK inhibitor (CDKI) proteins, are the gatekeepers which maintain the senescence program. p14ARF, p15INK4b and p16INK4a have been identified as CDKIs and act as senescence maintenance factors.

The INK4a/ARF locus (9p21) is crucial in the pathogenesis of several types of malignant disorders and encodes two unrelated cell cycle regulators, p14ARF and p16INK4a, which have been regarded as significant senescence markers in clinical diagnosis (10). The p16INK4a gene encodes a p16 protein that binds competitively to CDK4 and, during G1 phase, inhibits the interaction of CDK4 and cyclin D1 to stimulate passage through the cell cycle (11–13). The p16 protein is often highly expressed in senescent cells in culture and is inactivated in a variety of human cancers. p15INK4b is located centromeric to the p16/p14 gene locus p14ARF, which is a major tumor suppressor and causes cell cycle arrest through transforming growth factor β (14).

Previous data revealed, through staining, that p14ARF, p15INK4b and p16INK4a are abundantly expressed in premalignant lung cancers and have essentially no expression in malignant lung cancers, which indicates that senescence is inactivated in the proliferated lung epithelial cells of lung cancers (15). The same results have been observed in pancreatic, hepatic and breast cancers. Tumorigenesis is defined as an outcome of the accumulation of abnormal stroma facilitated by peripheral senescent fibroblasts and the inactivation of the senescence of proliferating epithelial cells. However, more recent evidence has revealed the high expression of the senescence markers p14ARF, p15INK4b, p16INK4a and DCR2 in prostate cancer epithelial cells, indicating that senescence continued to be activated in these proliferating epithelial cells.

A balance between proliferation and senescence, instead of the inactivity of senescence only, appears to decide the fate of epithelial cells, converging on the probability of epithelial tumorigenesis.

Since the true mechanism involved in the process from senescence to tumor formation in epithelial cells remains elusive (16–19), in the present study we compared the expression of the senescence markers p14ARF, p15INK4b and p16INK4a and the proliferation markers bcl-2, ki-67 and skp2 in tissue blocks from normal esophageal epithelium, esophageal intraepithelial dysplasia (EID) and esophageal squamous cell carcinoma (ESCC). The purpose of the present study was to describe the predictors of biological activity in esophageal carcinoma and to provide evidence that may assist in clinical diagnosis.

Materials and methods

Sample selection

The preparation of the tissue slides was performed as previously described (20). Tissue blocks (n=91) were created from samples obtained from 80 patients from the People's Hospital of Sichuan (Sichuan, China). The samples included 20 cases of normal esophageal tissue, 11 cases of EID, 49 cases of low-grade ESCC and 11 cases of high-grade ESCC Specimens were obtained from patients (aged 42–74 years, mean age 57) who had not received either chemotherapy or irradiation prior to surgery. The basic information of the ESCC patients is summarized in Table I. All samples were diagnosed in duplicate by pathologists from Sichuan University (Sichuan, China) who observed the samples under the microscope and individually scored each slide. The use of human tissue in this study was approved by the Institutional Review Board.

Table I. Clinicopathological features of the ESCC patients.

Table I.

Clinicopathological features of the ESCC patients.

Feature	n (%)
Gender
Male	46 (77)
Female	14 (23)
Degree of differentiation
Well	17 (28)
Poor	43 (72)
Lymph node metastasis
Negative	28 (47)
Positive	32 (53)
TNM stage
I	6 (10)
IIA	22 (37)
IIB	14 (23)
III	18 (30)

[i] ESCC, esophageal squamous cell carcinoma.

Immunohistochemical staining

The streptavidin-peroxidase immunohistostaining method was performed as described previously (20). Briefly, samples were fixed in 10% formalin buffer and embedded in paraffin. Tissue sections (4 μm thick) were steamed in universal decloaker (Biocare Medical, Walnut Creek, CA, USA) for antigen retrieval, followed by 19 min protein-blocking (Biocare Medical). All slides were first incubated against p14ARF, p15INK4b and p16INK4a (1:300, for 1 h at room temperature; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and then treated with anti-rabbit secondary antibody (Biocare Medical) and horseradish peroxidase for 15 min each. The tissues were stained for 3 min with high sensitivity 3,3′-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated and then mounted (21,22).

On the basis of the expression patterns in the esophageal epithelial cells, the expression of the p14ARF, p15INK4b, p16INK4a, bcl-2, ki-67 and skp2 proteins was considered positive when any positive staining was observed in the epithelial cells. Immunostained tissue slides were semi-quantitatively scored by two independent pathologists (Z.J. and Y.F.) blinded to the clinical information. Quantification was performed using a four-score grading system.

Statistical analysis

All statistical analyses were performed using the SPSS 10.0 statistical software program (SPSS, Chicago, IL, USA). The χ2 and Fisher's exact tests were used to assess the correlation between the expression of these biological markers and the clinical parameters of the patients. P<0.05 was considered to indicate a statistically significant result.

Results

As shown in Fig. 1, p14ARF, p15INK4b, p16INK4a and bcl-2 expression was observed in the cytoplasm of esophageal epithelial cells with no evidence of nuclear staining, while skp2 and ki-67 showed predominant nuclear staining at different portion of epithelial.

Figure 1. Expression of different markers in esophageal tissues (×40): Normal tissue, from top to bottom: (A) p16, negative; (B) p15, negative; (C) p14, negative; (D) skp2, negative; (E) ki-67, negative; (F) bcl-2, positive. EID, from top to bottom: (A) p16, positive; (B) p15, positive; (C) p14, negative; (D) skp2, positive, (E) ki-67, positive; (F) bcl-2, positive. Well-differentiated ESCC, from top to bottom: (A) p16, positive; (B) p15, positive; (C) p14, positive; (D) skp2, positive, (E) ki-67, positive; (F) bcl-2, positive. Poorly differentiated ESCC, from top to bottom: (A) p16, positive; (B) p15, positive; (C) p14, positive; (D) skp2, positive, (E) ki-67, positive; (F) bcl-2, positive. ESCC, esophageal squamous cell carcinoma; EID, esophageal intraepithelial dysplasia.

As shown in Table II, overall, p14ARF, p15INK4b and p16INK4a showed almost complete negative expression in the normal esophageal epithelium, while marked positive expression was observed in the EID tissues and different pathological lesions of the ESCC tissues. More specifically, the p14ARF expression was negative in most of the normal esophageal tissues (85%), but diffuse positive staining was observed in the EID (82%) and ESCC (100%) tissues, including 9 cases of EID and 60 cases of ESCC. The p16INK4a staining was similar to that of p15INK4b, with only 2 cases of normal esophageal tissue having weakly positive and most of the EID and ESCC tissues showing marked staining (73 and 88%, respectively). p15INK4b showed positive staining in 73% of the EID and 92% of the ESCC specimens, while no positive staining was observed in the normal esophageal tissue specimens.

Table II. Comparison of the immunohistochemical staining results for p14ARF, p15INK4b and p16INK4a in the ESCC tissue specimens and corresponding normal esophageal tissue specimens.

Table II.

Comparison of the immunohistochemical staining results for p14ARF, p15INK4b and p16INK4a in the ESCC tissue specimens and corresponding normal esophageal tissue specimens.

	p14ARF			p15INK4b			p16INK4a
	Negative	Positive	P-value	Negative	Positive	P-value	Negative	Positive	P-value
Normal (20)	17	3	<0.001	20	0	<0.001	18	2	<0.001
EID (11)	2	9		3	8		3	8
ESCC (60)	0	60		5	55		7	53

[i] ESCC, esophageal squamous cell carcinoma; EID, esophageal intraepithelial dysplasia.

A total of 46 male and 14 female ESCC patients participated in this study, 53% of whom had lymph node metastasis. The TNM stages of these patients are listed in Table I. As shown in Table III, a statistically significant correlation was observed between p16INK4a expression and the degree of differentiation. The poorly differentiated ESCC showed stronger p16INK4a staining compared with the well-differentiated ESCC (P<0.05). p16INK4a expression was observed in 95% of the poorly differentiated ESCC and only 71% of the well-differentiated ESCC tissues. The positive expression of p15INK4b was observed in 95% cases of poorly differentiated ESCC and 82% cases of well-differentiated ESCC. The positive staining of p14ARF was found in all the ESCC cases. Although there is no statistical evidence to verify that cases with lymph mode metastasis had a more marked immunostaining of p14ARF, p15INK4b and p16INK4a, the positive staining ratios are high in these specimens. There was no statistical correlation between the expression of p14ARF, p15INK4b and p16INK4a between the genders and among the TNM stages.

Table III. Immunohistochemical expression of p14ARF, p15INK4b and p16INK4a and their correlation with the clinicopathological parameters of the ESCC patients.

Table III.

Immunohistochemical expression of p14ARF, p15INK4b and p16INK4a and their correlation with the clinicopathological parameters of the ESCC patients.

	p14ARF			p15INK4b			p16INK4a
	Negative	Positive	P-value	Negative	Positive	P-value	Negative	Positive	P-value
Gender
Male	0	46	-	2	44	0.141	4	42	0.410
Female	0	14		3	11		3	11
Differentiation
Well	0	17	-	3	14	0.261	5	12	0.025
Poor	0	43		2	41		2	41
Lymph node metastasis
No	0	28	-	4	24	0.275	4	24	0.851
Yes	0	32		1	31		3	29
TNM stage
I	0	6	-	2	4	0.088	0	6	1.000
IIA	0	22		2	20		3	19
IIB	0	14		1	13		2	12
III	0	18		0	18		2	16

As shown in Table IV, there was positive staining for the proliferation marker skp2 in 64% of the EID and 72% of the ESCC cases but only 10% of the normal esophageal tissues. The analysis of ki-67 revealed a markedly high level of expression in the EID (91%) and ESCC (100%) cases, while there was a low incidence of positive staining in the basal epithelial cells of the normal tissue (20%). The bcl-2 immunostaining revealed that 36 ESCC cases (60%) and only 2 (10%) of the normal esophageal tissues had positive bcl-2 expression. As shown in Table V, cells that showed a positive expression of bcl-2 were found in 15 (88%) of the well-differentiated and 21 (49%) of the poorly differentiated ESCC cases. The variant expression of bcl-2 may be due to the heterogeneity of the cells.

Table IV. Comparison of the immunohistochemical staining results for bcl-2, skp2 and ki-67 in the ESCC tissue specimens and corresponding normal esophageal tissue specimens.

Table IV.

Comparison of the immunohistochemical staining results for bcl-2, skp2 and ki-67 in the ESCC tissue specimens and corresponding normal esophageal tissue specimens.

	bcl-2			skp2			ki-67
	Negative	Positive	P-value	Negative	Positive	P-value	Negative	Positive	P-value
Normal (20)	18	2	<0.001	18	2	<0.001	16	4	<0.001
EID (11)	0	11		4	7		1	10
ESCC (60)	24	36		17	43		0	60

[i] ESCC, esophageal squamous cell carcinoma; EID, esophageal intraepithelial dysplasia.

Table V. Immunohistochemical expression of bcl-2, ki-67 and skp2 and their correlation with the clinicopathological parameters of patients.

Table V.

Immunohistochemical expression of bcl-2, ki-67 and skp2 and their correlation with the clinicopathological parameters of patients.

	bcl-2			ki-67			skp2
	Negative	Positive	P-value	Negative	Positive	P-value	Negative	Positive	P-value
Gender
Male	19	27	0.709	0	46	-	13	33	1.000
Female	5	9		0	14		4	10
Differentiation
Well	2	15	0.005	0	17	-	9	8	0.028
Poor	22	21		0	43		8	35
Lymph node metastasis
No	16	12	0.011	0	28	-	10	18	0.235
Yes	8	24		0	32		7	25
TNM stage
I	3	3	0.472	0	6	-	4	2	0.223
IIA	6	16		0	22		6	16
IIB	7	7		0	14		3	11
III	8	10		0	18		4	14

Discussion

The incidence of esophageal carcinoma rises exponentially with age, beginning from the fourth decade of life and peaking at the age of 75 years. Evidence suggests that cellular senescence is involved in causing organismal aging. Senescence, the process by which cells permanently withdraw from the cell cycle in response to diverse stress, may also contribute to this age-related disease (23). The senescence marker p16 was found to be upregulated with age in the progenitor cells of the mouse brain, bone marrow and pancreas. However, cellular senescence has two roles in cells. On one hand, senescence is associated with aging, which significantly increases the transformation of epithelial tumors. On the other hand, it is acknowledged that senescence is crucial in the prevention of cancer, which is achieved by inducing reversible proliferative arrest mediated by ARF/p53 and irreversible proliferative arrest mediated by the concomitant actions of INK4a/Rb and the p53 pathway (4,24). In this process, the main barrier to the overgrowth of cancer is the derepression of the INK4a/ARF locus (25,26). Overall, cellular senescence has two roles: cancer protection in the young and age promotion in the old. To the best of our knowledge, epithelial cells are the origin of most carcinomas, resulting from either the mutation of an oncogene in an epithelial cell or, in a paracrine manner, by an adjacent senescent fibroblast cell, converging on the promotion of epithelial tumorigenesis. However, whether senescence occurs in epithelial cells during transformation is unknown.

To date, evidence suggests that p14ARF, p15INK4b and p16INK4a are widely downregulated in several solid tumors (27), including hepatic (28), breast, urinary bladder, pancreatic and esophageal carcinomas and gliomas (29). The INK4a/ARF locus encodes two significant tumor suppressors, p16INK4 and ARF, which share the same exons but encode different reading frames. p16INK4a is an inhibitor of CDK4 and CDK6 and acts by imposing a G1 cell cycle arrest. The INK4a/ARF locus has been regarded as a controller of cancer evolution which is expressed at low levels in most tissues in young organisms but becomes derepressed with age. The inactivation of senescence markers, due to homozygous deletion or hypermethylation of the genes which encode them, may be a significant mechanism in the dysfunction of the Rb and p53 growth regulation pathways during ESCC development (30). However, it has been reported that the senescence pathway remains intact in a large number of prostate cancer and cervical squamous carcinoma cases (31) even if the ki-67 index indicates increased proliferation in these cancers. Zhang et al found that the senescence markers p14ARF, p15INK4b, p16INK4a and DCR2 were expressed more frequently in prostate carcinomas than in benign tissues (32) and Meng et al identified activated senescence markers in colon cancer cells (33). Moreover, Schwarze identified an alteration in the p16/pRb pathway in the majority of primary prostate cancers in vitro (34). In our ESCC samples, the expression of p14ARF, p15INK4b and p16INK4a increased during ESCC progression and was also associated with greater age, poor differentiation and, to some degree, a high tumor stage. That is, although cancer cells generally lose their ability to undergo senescence and apoptosis, certain tumor cells trigger senescence in response to severe DNA damage or other stimuli (16,35). Therefore, senescence may play a role in cancer development.

skp2 is a member of the Skp1-Cullin-F-box protein (SCF) complex and considered to be a proto-oncogene, as its overexpression causes increased proliferation and metastasis, at least in part through increased p27 proteolysis (24). Wang et al found that phosphorylation at Ser72 is crucial for the ability of the skp2 protein to promote cell proliferation and tumorigenesis, through several complementary mechanisms (36). skp2 also induces cells to undergo a mitotic division cycle by degrading p27 in early G1 (37). The results of our immunostaining assays revealed that skp2 is activated at the dysplasia stage of esophageal tumorigenesis and maintains a high level of expression in most ESCCs (38). The uninhibited proliferation eventually leads to the development of ESCC.

The nuclear antigen ki-67, which is markedly expressed in the S and M phases of the cell cycle (39–41), was used to estimating cell growth. bcl-2 is a proto-oncogene which has been identified as a biologically significant inhibitor of apoptosis, whose overexpression leads to cell proliferation (42–44). In our data, a high expression of ki-67 was associated with the high expression of skp2 and the low expression of bcl-2, which acts as a marker of proliferation (27,45). We detected the expression of bcl-2 in EID and ESCC tissues and half of the ESCC cases showed a paradoxical loss of bcl-2, which indicated a poor prognosis. The high expression of bcl-2 in EID showed its anti-apoptotic function by blocking p53-mediated G1 arrest (46). The paradoxical loss of bcl-2 in ESCC may not be explained as a result of chemo-radiotherapy, since none of the cases in our database were treated prior to surgery. However, the same results were obtained in cervical and endothelial carcinoma, indicating that other mechanisms may be involved.

In general, the role of senescence in tumorigenesis attracts a great deal of attention, particularly concerning the functions of p14ARF, p15INK4b and p16INK4a. According to previous studies, tumorigenesis in old age not only reflects the accumulation of oncogenic mutations but also stromal alteration (47). Campisi (48) addresses these issues as good citizen and bad neighbors. The epithelial cells in squamous carcinomas always show senescence and apoptosis. Senescence serves as a powerful barrier for tumorigenesis in epithelial cells. However, in ESCC, the high expression of p14ARF, p15INK4b and p16INK4a provides evidence for the activation of senescence in the epithelial rather than stromal cells. Further studies are required to explain this phenomenon. However, the expression of markers of senescence and proliferation, including p14ARF, p15INK4b, p16INK4a, bcl-2, skp2 and ki-67 in ESCC and promalignancies and a loss of their expression in normal tissue and neoplasm may augment routine histological diagnostic methods for difficult cases.

References