Decreased expression of telomerase-associated RNAs in the proliferation of stem cells in comparison with continuous expression in malignant tumors

The p16INK4A tumor suppressor gene is frequently inactivated in non-small cell lung carcinoma (NSCLC) and is less frequently inactivated in small cell lung carcinoma (SCLC) by mutation, deletion or DNA methylation. There are several reports that the reintroduction of the p16INK4A gene into p16(-) NSCLC cells results in significant growth suppression. However, there have been no reports of reintroduction of the p16INK4A gene into SCLC cells. To assess the biological significance of p16INK4A inactivation in the development of SCLC, full-length p16INK4A cDNA was introduced into an SCLC cell line, Ms-13, in which the p16INK4A protein was not detected. SCLC cells stably transfected with the p16INK4A expression vector formed only 2-16% of the number of neomycin-resistant colonies formed by cells transfected with a control vector, and no expression of exogenous p16INK4A protein was detected in any of 16 expanded clones. Transient transfection of the p16INK4A gene into SCLC cells resulted in exogeneous p16INK4A protein expression and dephosphorylation of endogenous retinoblastoma (RB) protein. These results suggest that the restoration of the p16INK4A function suppresses the growth of SCLC cells by dephosphorylation of the RB protein. Therefore, inactivation of p16INK4A may play an important role in the enhancement of growth of p16INK4A(-) RB(+) SCLC tumors in vivo.     

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.     

Demethylation by 5-aza-2'-deoxycytidine (5-azadC) of p16INK4A gene results in downregulation of vascular endothelial growth factor expression in human lung cancer cell lines

Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor progression via angiogenesis. Recently, gene transduction of wild-type p16INK4A, tumor suppressor gene, has been shown to result in downregulation of VEGF expression in p16INK4A-deleted glioma cells. Because expression of p16INK4A is regulated by methylation of the p16INK4A gene, we examined whether demethylation of the p16INK4A gene by 5-aza-2'-deoxycytidine (5-azadC) could cause the protein expression of VEGF as well as of p16INK4A in human lung cancer cells. For this, five different lung cancer cell lines with or without loss of p16 activity were used. H841 and Ma-10 cells had the methylated p16INK4A gene without expression of p16INK4A protein, whereas Ma-1 and H209 cells had the unmethylated p16INK4A gene with constitutive expression of p16INK4A protein. Neither the p16INK4A gene nor p16INK4A protein was detected in A549 cells. Treatment with 5-azadC caused demethylation of the p16INK4A gene with reexpression of p16INK4A protein in H841 and Ma-10 (methylated p16INK4A gene dominant) cell, but not in other cell lines such as Ma-1, H209 (unmethylated p16INK4A gene dominant), or A549 (p16INK4A gene deleted). In a parallel experiment, 5-azadC inhibited production of VEGF protein by H841 and Ma-10 cells, especially in the later hypermethylated cells, but not Ma-1, H209, or A549 cells. RT-PCR analysis showed that Ma-10 cells expressed VEGF isoforms 121, 165, and 189, all of which were inhibited by 5-azadC. These findings indicate that the methylation status of the p16INK4A gene plays an important role in the regulation of angiogenesis associated with progression of lung cancer, through regulation of VEGF expression.     

肺癌p14ARF和p16INK4a基因协同表达缺失及其意义

目的：研究抑癌基因位点ＩＮＫ４ａ－ＡＲＦ在肺肿瘤细胞中的表达状况,揭示ｐ１４ＡＲＦ和ｐ１６ＩＮＫ４ａ协同表达缺失与肺癌发生发展的相关性。方法：用ＲＴ－ＰＣＲ和Ｗｅｓｔｅｒｎ ｂｌｏｔ对６株肺癌细胞（ＳＰＣ－Ａ－１,Ｃａｌｕ－１,Ｈ４４６,ＳＨ７７,Ａ５４９,Ｈ４６０）的ＩＮＫ－４ａ－ＡＲＦ基因位点在ｍＲＮＡ、蛋白水平上进行检测,对ＰＣＲ产物进行纯化和测序分析。结果：６株肺癌细胞中,有３株细胞（Ｈ４     

外源性p16基因对wtp53型人肺腺癌细胞生长的影响

目的 探讨Ｐ１６对人肺腺癌的作用及Ｐ１６基因治疗肺癌的可能性。方法 运用分子克隆技术构建重组人Ｐ１６基因表达载,以电穿孔法将Ｐ１６基因导入到该基因缺失的ｗｔｐ５３型的人肺腺癌Ａ５４９和Ｈ４６０细胞中,得到稳定表达Ｐ１６的人肺腺癌细胞。详细研究Ｐ１６基因对ｗｔｐ５３型人肺腺癌细胞周期的影响和细胞增殖的作用。结果 导入Ｐ１６基因能使人Ａ５４９和Ｈ４６０细胞生长速度较对照细胞明显减慢,细胞周期阻滞在Ｇ１     

Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells

A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells.     

Effect of p16INK4a on chemosensitivity in nasopharyngeal carcinoma cells

The p16INK4a tumor suppressor gene is frequently inactivated in nasopharyngeal carcinoma (NPC) and hence it may play an important role in the suppression of this tumor. In order to study the effect of p16INK4a restoration in NPC cells, full-length human p16INK4a gene was transfected into a NPC cell line, CNE1. Four individual clones with differential levels of p16INK4a protein expression, were selected for further studies. The introduction of p16INK4a into CNE1 cells induced growth suppression through G0/G1 cell cycle arrest; however, the cell growth rate was not correlated to the levels of p16INK4a protein expression. To study whether transfection of p16INK4a could protect NPC cells from radiation, cisplatin and 5-fluorouracil (5FU), the cellular sensitivity of p16INK4a transfectants and vector control were investigated. An increase in sensitivity to 5FU was observed (2-fold compared to IC50) in all 4 clones compared to vector-transfected control. P16INK4a transfection also resulted in increased sensitivity to cisplatin (1.5-1.8-fold) in 3 out of 4 cell lines. However, no difference in radiosensitivity was found between the p16INK4a transfectants and the control. These findings indicate that p16INK4a suppresses NPC cell growth through G0/G1 arrest and modulating cellular response to chemotherapeutic drugs in NPC cells. Therefore, restoration of p16INK4a may have a therapeutic purpose in the treatment of NPC.     

Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21(WAF1/CIP1) and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity

Cell cycle regulatory proteins are important candidates for therapeutic tumour suppressors. Adenovirus vectors were constructed to overexpress cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21(WAF1/CIP1) and p27KIP1 under the control of the murine cytomegalovirus immediate early gene promoter. These vectors directed the efficient expression of each of the cyclin kinase inhibitors and induced growth arrest, inhibited DNA synthesis, and prevented phosphorylation of the retinoblastoma protein (pRb) in cell lines expressing functional pRb. In pRb-deficient cells, expression of the cyclin kinase inhibitors was not effective in inhibiting DNA replication or growth arrest. Interestingly, three of the cyclin kinase inhibitors, p16, p18 and p27 were found to induce apoptotic death in transduced HeLa and A549 cells. When the vectors were tested for their ability to inhibit tumorigenicity in a polyomavirus middle T antigen model of murine breast carcinoma, expression of the cyclin kinase inhibitors resulted in a delay in tumour formation that varied from several weeks for the p19 expressing vector to greater than 25 weeks for the p27 expressing vector. When tumours were injected directly with the adenovirus vectors expressing the cyclin kinase inhibitors, only treatment with the vector expressing p16 resulted in a delay in tumour growth.     

Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells

We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0 - G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (-p16, -p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2.     

Hypermethylation of E-cadherin gene is frequent and independent of p16INK4A methylation in non-small cell lung cancer: potential prognostic implication

E-cadherin gene is often termed a 'metastasis suppressor' gene and inactivation of this gene through promoter methylation occurs in various epithelial cancer. This study assessed the methylation status of p16INK4a and E-cadherin genes, correlated with clinical characteristics in lung cancer patients. Forty-five patients with non-small cell lung cancer (NSCLC) were evaluated for methylation status of p16INK4a and E-cadherin genes by using the methylation-specific PCR. E-cadherin expression in tumor samples was examined by immunohistochemistry. Overall duration of survival in different subsets of NSCLC with or without p16INK4a or E-cad methylation at diagnosis was compared by using the Kaplan-Meier method and log-rank test. We found the hypermethylation of p16INK4a gene in 38% (17/45) of our subjects. While the E-cadherin gene was hypermethylated in 62% (28/45) related with reduced E-cadherin expression, and methylation status of both p16INK4a and E-cadherin genes seemed to be independent. Seventy-six percent (34/45) of NSCLC patients had an abnormal methylation pattern in at least one gene. Although there was no difference in overall survival of patients between methylated p16INK4a and unmethylated p16INK4a, NSCLS patients with hypermethylation of both genes (concordant pattern) had a significantly good prognosis. In contrast, NSCLC patients with hypermethylated p16INK4a but un-methylated E-cadherin gene (discordant pattern) had a significantly poor prognosis. E-cadherin and p16INK4a are commonly methylated in NSCLC and the methylation pattern of p16INK4a and E-cadherin genes may have prognostic value for the outcome of NSCLC patients.     

Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4

The function of p16^INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16^INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16^INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16^INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16^INK4 independent of Rb. The transfected p16^INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1), p16^INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16^INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16^INK4 or expressed reduced amounts of p16^INK4 protein. Therefore, p16^INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16^INK4 is a tumor suppressor gene. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Exogenous Expression of p16INK4a is Associated with Decrease in Telomerase Activity

In this study, gene transfection was used to determine whether the exogenous expression of p16INK4a modulated the biological characteristics of glioblastoma cells. The human glioblastoma cell line U87MG was doubly transfected with the plasmids pVgRXR and pIND harboring the wild-type p16 gene. The expression of p16INK4a in the resulting transfectants was regulated by the addition of the ecdysone homologue, muristerone A. When the cells expressed p16INK4a, their growth capacity was reduced and morphological changes such as an increase in cell size and cellular flattening were observed. The analysis of cell cycle regulation provided evidence that cells expressing p16INK4a were inhibited from entry into the cell cycle, as assessed by Ki-67 antigen expression. In addition, it was observed that the exogenous expression of p16INK4a was associated with decrease in telomerase activity.     

Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p16(INK4a)/pRB pathway

It has been suggested that the helix-loop-helix protein Id-1 plays an important role in tumourigenesis in certain types of human cancer. Previously, we reported that Id-1 was up-regulated during sex hormone-induced prostate carcinogenesis in a Noble rat model (Ouyang et al. (2001) Carcinogenesis, 22, 965-973). In the present study, we investigated the direct effect of Id-1 expression on human prostate cancer cell proliferation by transfecting an Id-1 expression vector into a prostate cancer cell line LNCaP. Ten stable transfectant clones were isolated and the ectopic Id-1 expression resulted in both increased DNA synthesis rate and the percentage of S phase cells. To study the possible mechanisms involved in the Id-1 induced prostate cancer cell growth, we examined the expression of several factors responsible for G(1) to S phase progression. We found that Id-1 expression induced phosphorylation of RB and down-regulation of p16(INK4a) but not p21(Waf1)or p27(Kip1). Our results indicate that the Id-1 induced inactivation of p16(INK4a)/pRB pathway may be responsible for the increased cell proliferation in prostate cancer cells. Given the fact that both Id-1 over-expression and inactivation of p16(INK4a)/pRB are common events in prostate cancer, our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.     

Association of p16(INK4a) and pRb inactivation with immortalization of human cells

To examine the association of cell cycle regulatory gene inactivation with human cell immortalization, we determined the expression status of INK4a, Rb, and WAF1/ CIP1, in eleven in vitro immortalized human cell lines, including fibroblasts and keratinocytes. Two human papillomavirus type 16 E6 expressing cell lines with telomerase activity, including a fibroblast cell line and a keratinocyte cell line, expressed no detectable p16(INK4a). These cell lines had a hyperphosphorylated pRb and reduced expression of p21(WAF1/CIP1). All of seven fibroblast cell lines immortalized either spontaneously or by (60)Co, X-rays, 4-nitroquinoline 1-oxide or aflatoxin B(1), maintaining their telomeres by the ALT (alternative lengthening of telomeres) pathway, displayed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. Levels of p21(WAF1/CIP1) expression varied among the cell lines. Two fibroblast cell lines that became immortalized following infection with a retrovirus vector encoding human telomerase catalytic subunit (hTERT) cDNA were also accompanied by inactivation of p16(INK4a) and pRb pathways. Acquisition of telomerase activity alone was not sufficient for immortalization of these cell lines. Taken together, all the cell lines including fibroblasts and keratinocytes, with either telomerase activity or the ALT pathway for telomere maintenance showed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. These demonstrate the association of inactivation of both p16(INK4a) and pRb with immortalization of human cells including fibroblasts and epithelial cells and telomerase-positive cells and ALT-positive cells.     

The tumor-suppressive functions of the human INK4A locus

The INK4A locus is often inactivated in human cancer. INK4A encodes for p14ARF and p16INK4A that inhibit growth through p53 and pRb, respectively. We used RNA interference vectors in transformation assays of human primary cells to analyze tumor-suppressive functions. We first show that a concerted inactivation of pRb and p53 is required for transformation. We then demonstrate that loss of p14ARF enhances growth in a p53-dependent manner but has little tumorigenic effect. In contrast, suppression of p16INK4A expression does not affect cellular proliferation but synergizes with p53 loss to accelerate growth and cause transformation. Our results delineate the functions of the human INK4A genes in normal and tumorigenic growth.     

The methylation status and protein expression of CDH1, p16(INK4A), and fragile histidine triad in nonsmall cell lung carcinoma: epigenetic silencing, clinical features, and prognostic significance

BACKGROUND: Aberrant methylation of the promoter CpG island (methylation) is known as a major inactivation mechanism of tumor suppressor and tumor-related genes. In this study, the authors studied the presence of methylation by investigated the inactivation of genes and prognostic factors in patients with nonsmall cell lung carcinoma (NSCLC) by examining resection samples for the presence of methylation. METHODS: Samples were obtained from 224 patients who underwent pulmonary resection for NSCLC. The authors used those samples to study methylation status with methylation-specific polymerase chain reaction analysis and to study protein expression with immunohistochemistry for 3 different genes: CDH1, p16INK4A, and fragile histidine triad (FHIT). RESULTS: The frequency of methylation in NSCLC was determined as 58.0% for CDH1, 21.9% for p16INK4A, and 52.2% for FHIT. The methylation of p16INK4A was observed significantly in heavy smokers compared either with nonsmokers or with patients who had smoked for <20 pack-years (P = .0420); it also was more significant in squamous cell carcinomas than in adenocarcinomas (P = .0343). FHIT methylation also was correlated significantly with lymph node metastasis (P = .0361). Patients who had tumors with both methylation and reduced expression of CDH1 had a significantly poorer prognosis compared with patients who had tumors both without methylation and with positive expression of CDH1 (P = .0259 and P = .0369, respectively; multivariate Cox analysis). For p16INK4A methylation, 63.3% of tumors showed reduced expression; whereas, in p16INK4A-unmethylated tumors, 33.7% showed reduced expression (P = .0002). However, for CDH1 and FHIT, no significant correlation was found for either methylation or reduced expression. CONCLUSIONS: Although protein expression was not inactivated by methylation alone, p16 expression was inactivated strongly by methylation. In addition, the analysis of methylation and expression of CDH1 played a clinically important role in treatment strategies for patients with NSCLC.     

Over-expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines

p27kip1 is a cyclin-dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. p27 has been associated with cell-cycle arrest and apoptosis. In this study, we transferred the full-length human p27 cDNA using a replication-deficient recombinant adenoviral vector (Ax-p27) into lung cancer cell lines and evaluated the potential of this strategy for anti-cancer gene therapy. After infection with Ax-p27, the growth of H322, A549 and SQ-5 cells, which express pRb, was almost completely suppressed, though no such effect was found in H69 and Lu-135 cells, which do not express pRb. In addition, cell death from day 4 after infection with Ax-p27 was observed only in H322, A549 and SQ-5 cells but not in H69 and Lu-135 cells. The cell cycle of H322 cells treated with Ax-p27 became arrested at the G1 phase from day 1 to day 3 despite continued over-expression of p27. When we examined the changes in expression level of pRb and E2F-1, which play important roles in cell-cycle progression from G1 to S phase, down-regulation of pRb expression was detected in H322 cells 3 days after infection with Ax-p27. These data suggest that (i) the growth-inhibitory effect and induction of apoptosis by over-expression of p27 require expression of pRb and (ii) adenovirus-mediated p27 gene transfer may have promise as a novel strategy in cancer gene therapy.     

Loss of p16INK4a expression is associated with vascular endothelial growth factor expression in squamous cell carcinoma of the esophagus

Vascular endothelial growth factor (VEGF) expression has been suggested to correlate with intratumoral microvessel density, tumor advancement and prognosis in esophageal squamous cell carcinoma (ESCC). Previous studies have showed that disruption of cell cycle regulator p16 is related to oncogenesis and tumor progression in ESCC. We hypothesized that VEGF expression in ESCC is reflected by abnormalities in the p16(INK4a) gene. To clarify the regulatory role of p16(INK4a) in VEGF expression in vitro, we transferred the p16(INK4a) gene into a p16(INK4a)-deleted ESCC cell line and observed changes in VEGF expression. Furthermore, we immunohistochemically assessed the expression of the cell cycle regulators (p16, p53 and RB) and VEGF in 90 surgically resected specimens of ESCC. Introduction of p16(INK4a) cDNA by the p16 expression vector significantly suppressed cell proliferation in the p16(INK4a)-deleted cell line TE8 (p < 0.0001). VEGF secretion by TE8 cells transfected with the p16(INK4a) vector was significantly suppressed as compared to non-transfected TE8 cells (p < 0.0001) and TE8 cells transfected with a control vector (p = 0.0015). The immunohistochemical studies of ESCC primary tumor specimens showed that loss of p16 expression was significantly correlated with VEGF-positive expression (p = 0.0004). The cumulative postoperative survival rate in the group with p16-positive and VEGF-negative expression was significantly higher than in the other groups. Neither p53 nor RB expression had any impact on outcome. Aberrant p53 expression tended to be associated with VEGF expression, but the trend did not reach statistical significance. Our study demonstrated that VEGF expression was correlated with p16 expression in ESCC. Our results suggest that p16 may have a regulatory role in VEGF expression in ESCC.