IFN-gamma withdrawal after immunotherapy potentiates B16 melanoma invasion and metastasis by intensifying tumor integrin alphavbeta3 signaling

Immunotherapy can effectively suppress tumor, yet complete tumor eradication occurs infrequently. The metastatic potential of remnant tumor cells after immunotherapy and the underlying mechanisms have not been fully elucidated. Here, we report that the termination of immunotherapy strikingly increases the metastatic potential of remnant melanoma. This is mainly due to the withdrawal of IFN-gamma after immunotherapy. The relief of IFN-gamma stress led to the increase of alphavbeta3 integrin expression in B16 cells, which increased the adhesion of B16 cells to fibrinogen, fibronectin and laminin. Through alphavbeta3 signaling, the activation of FAK, upregulation of cdc2, production of active MMP-2 and MMP-9 and actin polymerization were intensified in B16 cells stimulated with ECM molecules 24 h after the withdrawal of IFN-gamma. The i.v. injection of such tumor cells into mice resulted in more metastatic tumor nodes in lung and shortened the survival of mice. The pitfall of immunotherapy termination can be remedied by the administration of recombinant CBD-HepII polypeptide of fibronectin, which effectively inhibits alphavbeta3 signaling. These findings suggest that the risk of tumor metastasis can be increased after the termination of immunotherapy, due to the withdrawal of IFN-gamma and that targeting alphavbeta3 signaling pathway can improve the therapeutic effect of immunotherapeutic approaches by reducing such metastatic risk.     

Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation

We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.     

RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward alphavbeta3-expressing endothelial cells

To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both alphavbeta3 and CD3. Monocyclic RGD peptides (cRGDfK) were covalently coupled to an anti-CD3 monoclonal antibody at different peptide:protein ratios. The resulting RGDpep-anti-CD3 conjugates bound specifically to alphavbeta3-expressing endothelial cells. Displacement studies with radiolabeled alphavbeta3 ligand demonstrated that the alphavbeta3 binding affinity of RGDpep-anti-CD3 conjugates was elevated as compared to the non-conjugated RGDpep. IC50 values ranged from 150-1.1 nM, depending on the amount of coupled RGDpep molecules per antibody molecule. RGD modification did not affect the ability of anti-CD3 antibodies to bind to CTL. Furthermore, RGDpep-anti-CD3 was fully capable of activating T cells upon CD3 binding as was shown in a Jurkat/NFAT reporter-gene activation assay. All RGDpep-anti-CD3 conjugates were able to induce RGDpep, CD3-dependent lysis of human primary endothelial cells by anti-CD3/IL-2 activated human peripheral blood mononuclear cells (PBMC), with a significant induction of cytotoxicity observed at an E/T ratio as low as 10. Redirecting cytolytic activity reached up to 50% cytotoxicity using the conjugate with the highest RGD peptide load. Combining the good accessibility of tumor blood vessel endothelium for CTL with the efficiency of target cell killing warrants further investigations on anti-tumor effects of this type of conjugates in vivo.     

Diagnostic value of integrin alpha3, beta4, and beta5 gene expression levels for the clinical outcome of tongue squamous cell carcinoma

BACKGROUND: The objective of the current study was to identify biomarkers that reflect the clinical course of squamous cell carcinoma of the tongue (TSCC). METHODS: TSCC tissue samples from 66 patients were subjected to gene expression analysis by real-time polymerase chain reaction. Eleven integrin family genes and 14 genes used for normalization, including housekeeping genes and genes that encode desmosomal, cytoskeletal, and extracellular matrix molecules, were considered. Multivariate statistical analysis was performed on 154 expression ratios of integrin genes with clinical parameters. RESULTS: In principal-component analysis, the first principal component was related to the outcome of death, and the second principal component mainly reflected the tendency for cervical lymph node (LN) metastasis. The former axis consisted of the variance of the integrin beta4 gene (ITGB4) and ITGB5 expression levels, and the latter axis agreed with the expression level of the integrin alpha3 gene (ITGA3). Multivariate logistic regression analysis with cervical LN metastasis as the response variable concordantly identified ITGA3/junction plakoglobin gene (JUP) expression (P=.02) and ITGB5/paxillin gene (PXN) expression (P=.04) as significant factors. Only ITGB4/JUP expression was identified as a significant factor in terms of the outcome of death (P<.00049) by a Cox proportional hazards model. The group with high ITGB4/JUP levels exhibited a significantly high death rate on a Kaplan-Meier curve (P<.0001; Wilcoxon and log-rank tests). CONCLUSIONS: The expression levels of ITGA3, ITGB4, and ITGB5 with functional normalization by desmosomal or cytoskeletal molecule genes were selected as candidate biomarkers for cervical LN metastasis or for the outcome of death in TSCC.     

Targeting of RGD-modified proteins to tumor vasculature: a pharmacokinetic and cellular distribution study

Angiogenesis-associated integrin alpha(v)beta(3) represents an attractive target for therapeutic intervention because it becomes highly upregulated on angiogenic endothelium and plays an important role in the survival of endothelial cells. Cyclic RGD peptides were prior shown to have a high affinity for alpha(v)beta(3) and can induce apoptosis of endothelial cells. In our laboratory, monocyclic RGD peptides (cRGDfK) were chemically coupled to a protein backbone. Previous results demonstrated that the resulting RGDpep-HuMab conjugate bound with increased avidity to alpha(v)beta(3)/alpha(v)beta(5) on endothelial cells. In our present study, RGDpep-HuMab was injected intravenously and intraperitoneally in B16.F10 tumor-bearing mice to determine its pharmacokinetics and organ distribution. In the tumor, the RGDpep-HuMab conjugate specifically localized at the endothelium as was demonstrated by immunohistochemistry. The control RADpep-HuMab conjugate was not detected in the tumor. Besides tumor localization RGDpep-HuMab was found in liver and spleen associated with macrophages. This uptake by macrophages is probably responsible for the more rapid clearance of RGDpep-HuMab from the circulation than HuMab and RADpep-HuMab. The half-life of RGDpep-HuMab (90 min) was still considerably longer than that of free RGD peptides (<10 min). This prolonged circulation time may be favorable for drug targeting strategies because the target cells are exposed to the conjugate for a longer time period. Taken together these results indicate that RGD-modified proteins are suitable carriers to deliver therapeutic agents into tumor or inflammation induced angiogenic endothelial cells.     

Role for beta3 integrins in human melanoma growth and survival

The role of alphaIIbbeta3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of alphaIIbbeta3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that alphaIIbbeta3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of alphaIIbbeta3 was 8% (+/-1.8), 33% (+/-10.4) and 62% (+/-5) in melanomas ranging in thickness from 0-1.5 mm, 1.5-4.0 mm and >4 mm, respectively; alphavbeta3 was equally high all categories. To determine biological function, we stably transfected alphaIIbbeta3 into human melanoma cells that express alphavbeta3, but not alphaIIbbeta3. Surface expression of alphavbeta3 remained unaltered between alphaIIbbeta3 (+) and mock transfected counterparts. The alphaIIbbeta3 (+) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of alphaIIbbeta3 displaced alphavbeta3 from focal contact points. When implanted subcutaneously into SCID mice, the alphaIIbbeta3 (+) cells developed approximately 4-fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co-expression of the 2 beta3 integrins, alphavbeta3 and alphaIIbbeta3, in human melanoma cells enhanced cell survival and promoted growth in vivo.     

Osteoblasts-derived TGF-beta1 enhance motility and integrin upregulation through Akt, ERK, and NF-kappaB-dependent pathway in human breast cancer cells

Bone metastases are common complications of breast cancer. Integrins are the major adhesive molecules in mammalian cells. Here we found that osteoblast conditioned medium (OBCM) increased the migration and cell surface expression of beta1 or beta3 integrin in human breast cancer cells (MDA-MB-231 cells). beta1 or beta3 integrin monoclonal antibodies (mAbs) or small interference RNA (siRNA) against beta1 or beta3 integrin inhibited the OBCM-induced increase in the migration of breast cancer cells. Transforming growth factor-beta1 (TGF-beta1) siRNA could remarkably blocked OBCM-induced chemomigration and beta1 and beta3 integrin expression in breast cancer cells. Stimulation of cells with OBCM caused an increase in Akt and extracellular signal-regulated kinase (ERK) phosphorylation in a time-dependent manner. In addition, treatment of MDA-MB-231 cells with phosphatidylinositol 3-kinase inhibitor (LY294002), ERK inhibitor (PD98059), NF-kappaB inhibitor (PDTC), or IkappaB protease inhibitor (TPCK) inhibited OBCM-induced cells migration and integrins expression. Treatment of MDA-MB-231 cells with OBCM induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OBCM-mediated increases in IKK alpha/beta phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity were inhibited by LY294002 and PD98059. In addition, TGF-beta1 siRNA also reduced the OBCM-induced ERK, Akt, IKKalpha/beta, IkappaBalpha, and p65 phosphorylation. Taken together, these results suggest that the osteoblast-derived TGF-beta1 acts through Akt and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 and beta3 integrins and contributing the migration of breast cancer cell.     

alpha(v)beta(3) Integrin-targeting radionuclide therapy and imaging with monomeric RGD peptide

The alpha(v)beta(3) integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress alpha(v)beta(3) integrin, as in tumor neovascularization, and alpha(v)beta(3) integrin expression in other microvascular beds and organs is limited. Therefore, alpha(v)beta(3) integrin is a suitable receptor for tumor-targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to alpha(v)beta(3) integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor-targeting properties and the therapeutic potential of 111In- and 90Y-labeled monomeric RGD peptides in BALB/c nude mice with SKOV-3 human ovarian carcinoma tumors. DOTA-c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to alpha(v)beta(3) integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In-DOTA-c(RGDfK) and 90Y-DOTA-c(RGDfK) showed high uptake in tumors (7.3 +/- 0.6% ID/g and 4.6 +/- 0.8% ID/g, respectively) and gradually decreased over time (2.3 +/- 0.4% ID/g and 1.5 +/- 0.5% ID/g at 24 hr, respectively). High tumor-to-blood and -muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple-dose administration of 90Y-DOTA-c(RGDfK) (3 x 11.1 MBq) suppressed tumor growth in comparison to the control group and a single-dose administration (11.1 MBq). Monomeric RGD peptides, 111In-DOTA-c(RGDfK) and (90)Y-DOTA-c(RGDfK), could be promising tracers for alpha(v)beta(3) integrin-targeting imaging and radiotherapy.     

Expression level of integrin alpha 5 on tumour cells affects the rate of metastasis to the kidney

Tumour metastasis is known clinically to have organ specificity. We hypothesised that integrins might be involved in determining the organ specificity of tumour metastasis. Here, we report the results of spontaneous metastasis tested in nude mice that were inoculated with Chinese hamster ovary (CHO) cells expressing integrin alpha 5 beta 1 at various levels. The growth of the primary tumour inversely correlated with the alpha 5 expression level on CHO cells, which is consistent with a previous report (Schreiner et al, 1991). The rates of pulmonary, lymph node, and adrenal metastases that developed in nude mice were not related to changes of the alpha 5 expression level on CHO cells. Kidney metastasis developed in 40% of nude mice inoculated with alpha 5B2 cells (CHO cells overexpressing alpha 5) and in 20% of mice with CHO-K1 cells (CHO cells expressing native alpha 5), whereas inoculation with CHO-B2 cells (alpha 5-defective mutants) and alpha 5CHO cells with the highest expression of alpha 5 did not lead to development of kidney metastasis. Furthermore, alpha 5CHO, which shows the slowest growth of these cell types, did not lead to primary tumours in nude mice. These findings suggest that there is an appropriate level of alpha 5 expression on tumour cells that leads to metastasis. Microscopic observations revealed that micrometastasis in the kidney was formed in glomeruli. An adhesion assay using frozen sections of the kidney demonstrated that alpha 5B2 cells, but not CHO-B2 cells, effectively adhered to glomeruli. Kidney metastasis in vivo and the adhesion of alpha 5B2 to glomeruli shown ex vivo were significantly suppressed by the administration of GRGDS peptide. Finally, we conclude that the interaction of alpha 5 beta 1 on tumour cells with fibronectin in kidney glomeruli is involved in kidney metastasis and that the tumour has appropriate levels of integrins crucial for metastasis.     

Suppression of integrin alphaupsilonbeta6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway

Integrin alphaupsilonbeta6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of alphaupsilonbeta6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down-regulate alphaupsilonbeta6 expression in protein and mRNA levels. Supression of integrin alphaupsilonbeta6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro-MMP-9 and pro-MMP-2 in tumor conditioned medium, and more important, inhibits MAPK-dependent [(3)H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin alphaupsilonbeta6 inhibits extracellular matrix degradation through the MAPK pathway.     

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Core 3‐derived glycans, a major type of O‐glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional β3‐N‐acetylglucosaminyltransferase‐6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re‐expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re‐expressed core 3 synthase in human pancreatic cancer cells (Capan‐2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O‐glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor‐associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3‐derived O‐glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.     

Role of alpha4 integrin and its ligand VCAM-1 in the specific extravasation of a tumor-specific TH2 clone into tumor tissue that initiates its rejection

The effectiveness of anticancer immunotherapeutic strategies involving the transfer of tumor-specific T cells depends on appropriate lymphocyte-endothelial cell interactions that facilitate the migration of lymphocytes into tumor. Here, we investigated the molecular mechanisms underlying the migration of the antigen-specific Th2 CD4(+) T-cell clone YS1093 into S1509a tumor tissue. YS1093 is specific for the S1509a tumor but does not recognize the S713a tumor. Transfer of YS1093 cells into mice bearing both S1509a and S713a tumors caused only the S1509a tumor to regress. This regression was markedly inhibited by pretreating YS1093 cells with an anti-alpha4 integrin MAb and administering an anti-VCAM-1 MAb at T-cell transfer. Since vascular endothelial cells in S1509a tumor tissues express VCAM-1 and the MHC class II (I-E(k)) molecule restricting YS1093 activity, labeled YS1093 cells migrated specifically into the S1509a tumor, and this migration was also blocked by the anti-TCRbeta F(ab')(2) and anti-I-E(k) MAbs. Furthermore, in vitro assays revealed that anti-CD3 MAb-mediated TCR cross-linkage initiated the binding of alpha4 integrin on YS1093 cells to VCAM-1. This adhesive activity was completely blocked by the anti-alpha4 integrin MAb. These results strongly suggest that i.v.-transferred YS1093 cells act in tumor regression by specifically recognizing their tumor antigen peptide in the context of I-E(k) on vascular endothelial cells in the S1509a tumor, which activates the binding of alpha4 integrin to VCAM-1 on the endothelial cells, facilitating YS1093 extravasation into the tumor. It is likely that this initial migration of specific CD4(+) T cells into tumor tissues promotes the subsequent infiltration into the tumor of other immunocytes that effect tumor destruction.     

Signaling and regulatory mechanisms of integrin alphavbeta6 on the apoptosis of colon cancer cells

Considerable researches have been done about integrin alphanubeta6 and carcinomas, but little information has been shown about the relationship between integrin alphanubeta6 and apoptosis. In this study, we investigated the apoptosis and its related signal pathways to integrin alphavbeta6 in colon cancer cells. After we blocked the function of integrin alphavbeta6 in HT29 cells used the monoclonal antibody, the apoptotic cells increased markedly. Meanwhile, cytochrome C released from mitochondria into cytosol, Bcl-2 decreased while Bax increased significantly, and Fas and Fas-ligand had no change. The activities of caspase-3 and caspase-9 increased, while caspase-8 remained no change. Moreover, the expression of phosphorylated extracellular signal-related kinase (P-ERK) decreased. We confirmed that integrin alphavbeta6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involved the mitochondrial pathway.     

Enhancement of integrins by interleukin-1alpha,and their relationship with metastatic and invasive behavior of human pancreatic ductal adenocarcinoma cells

BACKGROUND AND OBJECTIVES: Adhesion and invasion of tumor cells to extracellular matrix (ECM) proteins play an important role in tumor metastasis formation. We investigated the enhancement of adhesive and invasive behavior to ECM proteins of human pancreatic cancer cells by interleukin-1alpha (IL-1alpha) to examine the mechanism of adhesion and invasion of metastatic human pancreatic cancer cells to ECM proteins. METHODS: The enhancement of integrin subunits by IL-1alpha was examined by cellular enzyme-linked immunosorbent assay (CELISA) in two metastatic human pancreatic cancer cell lines (BxPC-3 and SW1990) and two nonmetastatic pancreatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion and invasion to ECM proteins were performed to investigate whether increased integrin expression affected the invasive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: Expression of the alpha6 subunit by metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also exhibited preferential adherence and invasion to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSIONS: The enhancement of alpha6beta1-integrin by Il-1alpha acting through IL-1RI, as well as the expression of alpha6beta1-integrin, plays an important role in metastasis formation in pancreatic cancer     

Bone sialoprotein‐αvβ3 integrin axis promotes breast cancer metastasis to the bone

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvβ3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvβ3 integrin. Interestingly, we found the αvβ3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvβ3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvβ3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvβ3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvβ3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT‐PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvβ3 integrin is one of the downstream factors regulated by BSP in the breast cancer‐bone metastatic cascade.     

CpG island hypermethylation of E-cadherin (CDH1) and integrin alpha4 is associated with recurrence of early stage esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence-associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif-1, sFRP1, integrin alpha4, CDH1, DAP kinase and RARbeta2 genes in 251 ESCCs. The methylation status was determined by methylation-specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARbeta2, 43% for CDH1, 21% for integrin alpha4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif-1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48-18.67; p = 0.01) and a poor recurrence-free survival after surgery (HR = 3.13, 95% CI = 1.21-8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46-70.41; p = 0.003) times higher in patients with Wif-1 methylation than in those without. For stage II cancers, integrin alpha4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09-8.37; p = 0.03) and a poor recurrence-free survival (HR = 2.12, 95% CI = 1.13-3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin alpha4 genes may be used as recurrence-associated prognostic indicators in stage I and stage II ESCCs, respectively.