Aberrant promoter 2 methylation‑mediated downregulation of protein tyrosine phosphatase, non‑receptor type 6, is associated with progression of esophageal squamous cell carcinoma
- Authors:
- Published online on: February 19, 2019 https://doi.org/10.3892/mmr.2019.9971
- Pages: 3273-3282
Abstract
Introduction
Esophageal cancer is a malignant tumor associated with poor prognosis and high mortality rates (1). The incidence of esophageal cancer exhibits marked geographic variation, appearing to be low in Western Africa, and high in Japan, Southeastern Africa and Northern China (2). Esophageal squamous cell carcinoma (ESCC) is the dominant histological type of esophageal cancer in China and it is associated with a particularly high morbidity in certain areas, including the south of Hebei in Northern China (3). The 5-year survival rate of patients with ESCC with advanced-stage or metastatic disease is <20% (4). Tobacco and alcohol use have been demonstrated to be independent risk factors for ESCC, but the exact pathogenetic mechanism remains to be elucidated. An improved understanding of the mechanisms underlying ESCC pathogenesis may identify promising molecular biomarkers for the early diagnosis and prevention of this malignancy.
Aberrant protein phosphorylation is a prerequisite for the occurrence and progression of several tumors, and it is one of the hallmarks of cancer cells (5). Protein kinases and protein phosphatases serve key roles in regulating cellular signal transduction. Protein phosphatases include protein serine/threonine phosphatases, protein tyrosine phosphatases (PTPs), tumor-suppressive metal-dependent protein phosphatases, tumor-suppressive phosphoprotein phosphatases, tumor-suppressive PTPs, receptor type and tumor-suppressive PTPs, non-receptor type (PTPN). Protein phosphatases were initially considered to be tumor suppressors, but several have been demonstrated to serve purely as oncogenes, whereas others may serve as tumor suppressors and oncogenes, according to the cellular environment or other unidentified factors (6). PTPNs are represented by 17 members and are absolutely specific to phospho-tyrosine. They consist of a highly conserved catalytic domain and variable regulatory domain arrays, acting by subcellular targeting or directly regulating phosphatase activity (7). A number of PTPNs serve as tumor suppressors by inhibiting Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, receptor tyrosine kinase-mediated growth factor signaling, or cell motility/invasion. Furthermore, certain PTPNs promote tumor-suppressive Hippo signaling and inhibit tyrosine-protein kinase-ABL1-mediated transformation through Yes-associated protein 1 (8).
Protein tyrosine phosphatase, non-receptor type 6 (PTPN6) (also known as HCP, HCPH or SHP1), is encoded by 17 exons and has 2 Src homology 2 domains, is primarily expressed in hematopoietic cells, and serves as a key regulator of multiple signaling pathways in hematopoietic cells. PTPN6 has been demonstrated to interact with and dephosphorylate a variety of phospho-proteins involved in hematopoietic cell signaling pathways. Plutzky et al (9) first identified that PTPN6 is located in a chromosomal region that is frequently damaged in childhood leukemia. Oka et al (10) observed the loss of mRNA and protein expression of PTPN6 in natural killer T-cell lymphomas and 95% of several other types of malignant lymphomas, while only 60% of less malignant forms were negative. The loss of expression of PTPN6 is likely associated with malignant transformation and increased invasiveness. In subsequent studies, promoter hypermethylation of PTPN6 was identified in several hematological malignancies (11,12) and in solid tumors, including nasopharyngeal carcinoma (13) and breast ductal carcinoma (14). The PTPN6 gene has two promoter regions that are 7 kb apart, and has 3 different transcripts. The longer transcript, driven by the P1 promoter, is expressed primarily in non-hematopoietic cells, whereas the shorter transcript, driven by the P2 promoter (P2), is only expressed in cells of the hematopoietic lineage (15). PTPN6 expression driven by P1 in non-hematopoietic cells is low compared with the expression regulated by P2 in hematopoietic cells (16). It has been suggested that downregulation of PTPN6 is primarily due to DNA hypermethylation of CpG islands in the PTPN6 P2 (10,11). However, the mechanism and methylation status of PTPN6 in ESCC have not yet been fully elucidated. The aims of the present study were to investigate the expression of PTPN6 in ESCC tissues and esophageal cancer cell lines, elucidate the role of CpG hypermethylation in the inactivation of PTPN6, and improve the understanding of the functional and prognostic significance of PTPN6 in ESCC tumorigenesis and progression.
Materials and methods
Cell culture and treatment
A total of 5 human esophageal cancer cell lines (Eca109, Kyse150, Kyse170, Yes-2 and TE1) and a human normal esophageal epithelial cell (HEEpiC) line were purchased from American Type Culture Collection (Manassas, VA, USA). All the cell lines were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 5% CO2, and were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis for mycoplasma contamination. All the cell lines were seeded prior to drug treatment. Cells (1.5×105/ml) were treated with 5 µM DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for the first 48 h and, subsequently, the medium containing 5-Aza-dC was changed every 24 h. Control cells were cultured in RPMI-1640 medium with no drug treatment.
Patients and specimens
A total of 71 primary ESCC samples and corresponding adjacent normal tissues were collected by surgical resection between January 2008 and January 2011 at the Department of Thoracic Surgery of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University, and conformed to all relevant ethical regulations for human research subjects in accordance with Declaration of Helsinki. All the participants signed a written informed consent form. The patients comprised 51 males and 20 females, with a median age of 62 years (range, 39–78 years) (Table I). Freshly removed ESCC and paired adjacent non-cancerous esophageal tissues were divided into two groups, one of which was fixed in formalin at room temperature and embedded in paraffin, and the other was frozen and stored at −80°C for DNA and RNA isolation. Clinical data and clinicopathological characteristics were collected from medical records. The subjects were interviewed for information on demographic and exogenous risk factors, including smoking, alcohol consumption and family history.
RT-qPCR analysis
Total RNA was extracted from cell lines and frozen tumor tissues using TRIzol® reagent (Thermo Fisher Scientific, Inc.). The RT-for-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to synthesize single-stranded cDNA according to the protocol of the manufacturer. The mRNA expression levels were quantified using primers, cDNA template and Power SYBR-Green PCR Master Mix (Promega Corporation, Madison, WI, USA), according to the protocol of Power SYBR-Green PCR Master Mix The primers used for PTPN6 are listed in Table II. The PCR cycle conditions were: 94°C for 30 sec, followed by 40 cycles of 94°C for 10 sec, 60°C for 30 sec and 72°C for 1 min. The data were analyzed by the 2−ΔΔCq method (17) and the human GAPDH gene was used as an endogenous control.
Western blot analysis of PTPN6 protein expression in ESCC cell lines
Total protein from cultured cell lines was extracted using radioimmunoprecipitation assay reagent supplemented with protease inhibitors (Thermo Fisher Scientific, Inc.). The protein was quantified using BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China). Protein samples (20 µg/lane) were prepared for western blot analysis with 15% SDS-PAGE gels and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). To detect the expression of PTPN6, the membranes were incubated at 4°C overnight with the specific primary antibodies (1:1,000; rabbit anti-human monoclonal antibody; cat. no. ab32559; Abcam, Cambridge, UK). Subsequently, the secondary antibody (goat anti-rabbit IgG-HRP; 1:2,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) incubated with the membrane at room temperature for 1 h. To ensure equal loading in all the lanes, anti-β actin (1:1,000; cat. no. ab119716; Abcam) was used as the control. Consequently, the protein bands were analyzed by the Image Lab software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Immunohistochemical staining for the PTPN6 protein in ESCC tissues
PTPN6 protein expression was determined by immunostaining using the streptavidin-peroxidase method in tumor samples and corresponding adjacent normal sections. Specimens were embedded in paraffin and cut into 4-µm sections. Then descending alcohol series were used to deparaffinization and rehydration were used by descending alcohol series. Antigen retrieval was performed in a pressure cooker at 100°C for 5 min in Tris-EDTA buffer (pH 9.0). Rabbit anti-human monoclonal antibody for PTPN6 (1:100 dilution; cat. no., ab32559; Abcam) was applied to investigate the protein expression of PTPN6 at 4°C overnight. Following an overnight incubation, specimens were subjected to the streptavidin-peroxidase (SP) method using a standard SP kit (cat. no. PV-9001; OriGene Technologies, Inc., Beijing, China) according to the manufacturer's protocol. PBS (pH 9.0) was used as negative control of the primary antibody. The slides were examined using a light microscope (Olympus BX41; Olympus Corporation, Tokyo, Japan; magnification, ×200 and ×400) and scored by experienced pathologists in a double-blinded manner.
DNA extraction and sodium bisulfite treatment
Genomic DNA was isolated from esophageal cancer cell lines, frozen ESCC tumor samples and corresponding normal tissues using a DNA extraction kit (Shanghai Generay Biotech Co. Ltd., Shanghai, China). To assess the DNA methylation patterns, DNA was bisulfite-modified using an Epitect Fast Bisulfite Conversion kit (Qiagen GmbH, Hilden, Germany), which converts unmethylated cytosine residues to thymine, whereas methylated cytosine residues remain unaffected.
Methylated CpG site distribution via bisulfite genomic sequencing (BGS) assay
To analyze the DNA methylation pattern of the PTPN6 P2, a BGS assay was used to detect the methylated CpG site distribution in the esophageal cancer cell lines. Subsequently, the online MethPrimer program was used to detect the distribution of CpG islands (URL: http://www.urogene.org/methprimer/). A pair of primers (from-167 to-326 bp) was designed by Sangon Biotech Co., Ltd. (Shanghai, China) to recognize sodium bisulfite-converted genomic DNA. The primer sequence for BGS: Sense 5′-AGGGTTGTGGTGAGAAATTAATTAG-3′, and antisense 5′-TTACACACTCCAAACCCAAATAATAC-3′. The PCR products were purified using the QIAEXII Gel Extraction kit (Qiagen GmbH) and cloned into pGEM-T vectors (Promega Corporation). Up to 10 clones for each specimen were analyzed by bisulfite sequencing.
Methylation analysis of PTPN6 via bisulfite conversion-specific and methylation-specific polymerase chain reaction (BS-MSP) assay
The PTPN6 P2 was analyzed by the BS-MSP method as described above using bisulfite-treated genomic DNA. According to the distribution of the primary methylated CpG sites by the BGS assay, the MSP primers were designed by Sangon Biotech Co., Ltd., and the reaction conditions were summarized in Table II. According to the manufacturer's recommendations, genomic DNA methylated in vitro by CpG methyltransferase (Sss I) (New England BioLabs, Inc., Ipswich, MA, USA) and water blanks were applied as positive and negative controls, respectively. The BS-MSP products were analyzed on 2% agarose gel with ethidium bromide staining. All reactions were performed in duplicate for each of the samples.
Cell transfection
To determine the overexpression of PTPN6, Eca109 and Yes-2 cells in the logarithmic growth phase were cultured in 6-well plates. When the density of Eca109 and Yes-2 cells reached to 80%, the cells were transfected with PTPN6 expression plasmid (pcDNA3.1-PTPN6) or the empty vector (pcDNA3.1-EV) (Sangon Biotech Co., Ltd.) as control at a final concentration of 2.5 µg/µl using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Following transfection, the cells were incubated in RPMI-1640 medium for 4–6 h, followed by replacement with RPMI-1640 supplemented with 10% FBS. After 24 h, the transfected cells were extracted for subsequent experimentation.
Cell proliferation
The cells (1.0×105) were seeded into 96-well plates for the cell proliferation assays. The proliferation of Eca109 and Yes-2 cells transfected with PTPN6 was determined by MTS assay. The absorbance was measured at a wavelength of 492 nm, followed by incubation for 4 h in a humidified incubator containing 5% CO2 at 37°C. The proliferation rates were determined at 0, 24, 48, 72 and 96 h after transfection. All the experiments were performed in triplicate.
Colony formation assay
For the colony formation assay, 2,500 cells were seeded in 6-well plates and incubated with RPMI-1640 medium containing 10% FBS for 1 week. Colonies (>50 cells) were fixed in methanol for 15 min (at room temperature) and dyed with 0.5% crystal violet solution for 20 min, and the colony number was counted under an inverted microscope (DWI40CCB; Leica, Wetzlar, Germany; magnification, ×100).
Wound healing assay
Cells (5.0×105) in the logarithmic growth phase were inoculated in 6-well plates. Following transfection for 24 h, scratch wounds were created using a 200 µl pipette tip. The detached cells were removed by washing with PBS 3 times. RPMI-1640 medium was then added to the plates and images were observed after culture for 0, 12 and 24 h. The inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×100) was applied to measure the relative migration distance.
Cell invasion assay
The invasion of PTPN6-transfected Eca109 and Yes-2 cells was measured in 24-well Transwell chambers (Corning Incorporated, Corning, NY, USA). The Transwell chambers were coated with 20 µl Matrigel at 4°C and incubated at 37°C for 4 h. After 24 h transfection, 5,000 cells/well were seeded in the upper chambers, and the lower chambers were filled with RPMI-1640 medium supplemented with 10% FBS. Following incubation at 37°C for 24 h, invading cells located in the lower chamber were fixed in 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet at room temperature for 30 min. The number of cells that had invaded through the membrane to the lower surface was observed in 5 microscopic fields per filter under the inverted microscope (Leica Microsystems GmbH; magnification, ×100). The experiments were performed in triplicate.
Statistical analysis
Statistical analysis was performed with SPSS 22.0 software package (IBM Corp., Armonk, NY, USA). The RT-qPCR results are presented as the mean ± standard deviation. Student's t-test was applied to compare the expression means between different continuous variables. Pearson's χ2 test was applied to assess the status of gene methylation. For prognostic analysis of PTPN6 protein expression and methylation, survival curves were constructed using the Kaplan-Meier method and the log-rank or the Breslow tests. One-way analysis of variance was adopted to measure the comparison of multiple groups (the function of PTPN6 in esophageal cancer cell lines), and within-group variations were performed by Student Newman-Keuls test. All statistical tests were two-sided and P<0.05 was considered to indicate a statistically significant difference.
Results
mRNA and protein expression of PTPN6 is decreased in esophageal cancer cell lines and ESCC tissues
The mRNA expression of PTPN6 was first detected in 5 esophageal cancer cell lines and normal human esophageal epithelial cells. As demonstrated in Fig. 1A, the mRNA expression of PTPN6 was markedly decreased in the ESCC TE1, Eca109, Kyse150, Kyse170 and Yes-2 cell lines compared with that in the normal esophageal epithelial HEEpiC cells. This result was additionally confirmed by the results of western blot analysis (Fig. 1B).
The mRNA expression of PTPN6 in ESCC tumor tissues was significantly decreased compared with that in the corresponding normal tissues (P<0.01; Fig. 1C). PTPN6 mRNA expression was associated with Tumor Node Metastasis stage (18), pathological differentiation and lymph node (LN) metastasis (Fig. 1D). Immunohistochemical staining was used to assess the protein expression of PTPN6 in tumor tissues and corresponding normal tissues. Protein expression of PTPN6 was detected primarily in the cytoplasm and the nucleus of tumor or normal cells (Fig. 1E). The protein expression of PTPN6 in tumor tissues (32.4%, 23/71) was markedly decreased compared with that in corresponding normal tissues (77.5%, 55/71; P<0.01; Table III). When stratified for clinicopathological characteristics, PTPN6 protein expression was identified to be significantly associated with tumor-node-metastasis (TNM) stage, pathological differentiation and LN metastasis (P<0.05; Table IV).
 | Table III.Protein expression and methylation status of PTPN6 in ESCC tumor tissues and corresponding normal tissues. |
| Table IV.Immunohistochemical staining characteristics and methylation status of PTPN6 in ESCC tissues. |
Downregulation of PTPN6 is associated with poor ESCC patient survival
The 5-year survival rate in the positive and negative PTPN6 expression ESCC groups was 47.8 and 20.8%, respectively (P<0.05; log-rank test). As presented in Fig. 1F, patients with ESCC negative for protein expression of PTPN6 exhibited poor survival.
Upregulation of PTPN6 by 5-Aza-dC treatment in esophageal cancer cell lines
As demonstrated in Fig. 2A, the online MethPrimer program was used to detect the distribution of CpG islands in the PTPN6 promoter region and genomic sequence. A total of 1 CpG island was identified to be located in the promoter region. The Eca109, Kyse170 and Yes-2 cell lines, which exhibited a relatively low PTPN6 expression, were subsequently treated with 5-Aza-dC. As indicated in Fig. 2B, the mRNA expression level of PTPN6 was markedly upregulated in these 3 esophageal cancer cell lines following treatment with 5-Aza-dC, suggesting that aberrant methylation may be one of the mechanisms leading to PTPN6 silencing in esophageal cancer cell lines.
Methylation analysis of PTPN6 in esophageal cancer cell lines and tumor tissues
The methylation status of the CpG sites in the promoter region of PTPN6 was first verified by BGS assay in esophageal cancer cell lines, and frequent hypermethylation of the CpG sites in the promoter region of PTPN6 was detected in Eca109, Kyse170 and Yes-2 cells (Fig. 2C). In particular, fully methylated PTPN6 in the Eca109 and Yes-2 cell lines was detected by the BS-MSP assay (Fig. 2D). Following treatment with 5-Aza-dC, the aberrant methylation status of the cells was reversed in the 3 cell lines. The frequency of PTPN6 methylation in ESCC tumor tissues (63.4%, 45/71) was significantly higher compared with that in corresponding normal tissues (16.9%, 12/71; P<0.05; Table III and Fig. 2D). When stratified for clinicopathological characteristics, the methylation frequency of PTPN6 was associated with TNM stage, pathological differentiation and LN metastasis (P<0.05). However, the methylation status of PTPN6 in ESCC tumor tissues was not associated with age or sex (P>0.05; Table IV).
Association between PTPN6 expression and methylation status
As demonstrated in Fig. 2E, the mRNA expression level of PTPN6 in ESCC tissues with PTPN6 methylation was significantly decreased compared with that in ESCC tissues with unmethylated PTPN6 (P<0.05). Similarly, the protein expression of PTPN6 in ESCC tissues with PTPN6 methylation was significantly decreased compared with that in ESCC tissues with unmethylated PTPN6 (P<0.05; Table V).
| Table V.Association between PTPN6 protein expression and methylation status in patients with esophageal squamous cell carcinoma. |
Promoter hypermethylation of PTPN6 is associated with poor ESCC patient survival
As demonstrated in Fig. 2F, PTPN6 methylation was identified to be negatively associated with ESCC patient survival. In patients with ESCC with hypermethylation of PTPN6, the 5-year survival rate was 17.8% compared with 50.0% in patients with ESCC with unmethylated PTPN6 (P<0.05; log-rank test).
Upregulation of PTPN6 inhibits esophageal cancer cell proliferation and invasion in vitro
The function of PTPN6 was then investigated in esophageal cancer cell lines. The construct containing PTPN6 transcripts (pcDNA3.1-PTPN6) was transfected into Eca109 and Yes-2 cells. As indicated in Fig. 3A and B, significant upregulation of PTPN6 was detected in pcDNA3.1-PTPN6-transfected Eca109 and Yes-2 cells. Transfection of PTPN6 led to a marked inhibition of Eca109 and Yes-2 cell proliferation, as detected by the MTS assay (Fig. 3C). The results were additionally verified with the colony formation assay (Fig. 3D). Furthermore, the wound healing assay was performed, starting 24 h after pcDNA3.1-PNPN6 transfection. Overexpression of PTPN6 effectively decreased the area of the scratch covered (Fig. 3E). Similarly, the Transwell assay confirmed a decrease in the migration ability of pcDNA3.1-PTPN6-transfected cells (Fig. 3F). These results indicate that PTPN6 inhibited the proliferation, migration and invasion of Eca109 and Yes-2 cells in vitro.
Discussion
The PTPN6 gene is located on human chromosome 12p13 and encodes a Mr 68,000 non-receptor type protein-tyrosine phosphatase. The PTPN6 gene has been considered as a candidate tumor suppressor in hematological and solid malignancies, and promoter methylation may be an important epigenetic mechanism involved in silencing its expression. However, the detailed roles of PTPN6 and its promoter methylation status in the pathogenesis of primary ESCC remain elusive. In the present study, significant downregulation of PTPN6 and frequent hypermethylation of the CpG sites within the P2 were detected in esophageal cancer cell lines and ESCC tissues. The mRNA expression level of PTPN6 was significantly upregulated in 5-Aza-dC-treated esophageal cancer cells. In addition, the methylation status and expression of PTPN6 were associated with TNM stage, pathological differentiation and LN metastasis in patients with ESCC. Additional study verified that aberrant hypermethylation of the P2 exhibited higher tumor specificity and was associated with the expression level of PTPN6. Survival analysis demonstrated that downregulation and hypermethylation of PTPN6 were associated with poor ESCC patient survival. Furthermore, upregulation of PTPN6 inhibited the proliferation and invasion of esophageal cancer cells in vitro.
Genomic DNA methylation is an important epigenetic event in humans, and the alterations of methylation patterns may serve important roles in tumorigenesis (19–21). As aberrant DNA methylation is one of the earliest molecular changes during the transformation process from normal to cancerous cells (22), detection of an aberrant DNA methylation pattern may have potential applications in the early detection of malignancies. Transcriptional silencing of PTPN6 due to promoter hypermethylation has been previously demonstrated in several hematopoietic cell lines, leukemia and lymphoma (10,23,24). A high frequency of promoter hypermethylation was also observed in endometrial carcinoma, and was identified to be associated with patient age and tumor differentiation. PTPN6 promoters were completely methylated in endometrial carcinoma cell lines, and this methylation status was reversed by 5-Aza-dC treatment (25). In the present study, downregulation of PTPN6 was detected in esophageal cancer cell lines and ESCC tissues, and P2 hypermethylation may be one of the important epigenetic mechanisms silencing this gene in ESCC. Furthermore, the expression and methylation status of PTPN6 were associated with TNM stage, pathological differentiation and LN metastasis in patients with ESCC, and were associated with patient survival, indicating that detection of the P2 methylation status may be a promising biomarker for predicting the prognosis of ESCC.
It has been suggested that epigenetic silencing of PTPN6 in myeloproliferative neoplasms and K562 cells causes constitutive activation of JAK/STAT signaling (26). The reversal of PTPN6 expression by 5-Aza-dC treatment caused decreased expression levels of p-STAT3, p-JAK3 and JAK3, but not of the STAT3 protein (27). The JAK/STAT signaling pathway is one of the most important signaling cascades that regulate immune response, cell growth, differentiation and other cellular biological activities (28). The silence of PTPN6 may result in JAK or STAT activation in cancer cells (11,24). However, the role of PTPN6 in ESCC has not been fully elucidated. In the present study, it was confirmed that upregulation of PTPN6 inhibited the proliferation and invasion of esophageal cancer cells in vitro, indicating that PTPN6 may serve as a tumor suppressor gene by inhibiting the proliferation and invasion of cancer cells. However, the specific regulated pathway of PTPN6 in ESCC requires additional investigation.
In summary, PTPN6 may serve as tumor suppressor gene in ESCC and inhibit esophageal cancer cell proliferation and invasion. The P2 is frequently methylated in esophageal cancer cells and ESCC tissues, and this may be one of the epigenetic mechanisms implicated in PTPN6 silencing in ESCC. Furthermore, PTPN6 may serve as a potential prognostic marker for predicting survival in patients with ESCC.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
All data generated and analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
LL conducted the analyses, participated in the overall conceptualization of the study, wrote the final manuscript and performed the computational analyses. JL conceptualized and supervised the study. SZ and XL participated in the analysis of results. All authors have read and approved the final version of this manuscript.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of The Fourth Hospital of Hebei Medical University, and conformed to all relevant ethical regulations for human research subjects. All the participants signed a written informed consent form.
Patient consent for publication
All the participants signed a written informed consent form.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
P2 |
promoter 2 |
|
ESCC |
esophageal squamous cell carcinoma |
|
PTPs |
protein tyrosine phosphatases |
|
PTPN6 |
protein tyrosine phosphatase, non-receptor type 6 |
References
|
Lambert R and Hainaut P: The multidisciplinary management of gastrointestinal cancer. Epidemiology of oesophagogastric cancer. Best Pract Res Clin Gastroenterol. 21:921–945. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Enzinger PC and Mayer RJ: Esophageal cancer. N Engl J Med. 349:2241–2252. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Guohong Z, Min S, Duenmei W, Songnian H, Min L, Jinsong L, Hongbin L, Feng Z, Dongping T, Heling Y, et al: Genetic heterogeneity of oesophageal cancer in high-incidence areas of southern and northern China. PLoS One. 5:e96682010. View Article : Google Scholar : PubMed/NCBI | |
|
Pennathur A, Gibson MK, Jobe BA and Luketich JD: Oesophageal carcinoma. Lancet. 381:400–412. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Meeusen B and Janssens V: Tumor suppressive protein phosphatases in human cancer: Emerging targets for therapeutic intervention and tumor stratification. Int J Biochem Cell Biol. 96:98–134. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Elson A: Stepping out of the shadows: Oncogenic and tumor-promoting protein tyrosine phosphatases. Int J Biochem Cell Biol. 96:135–147. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Tonks NK: Protein tyrosine phosphatases-from housekeeping enzymes to master regulators of signal transduction. FEBS J. 280:346–378. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Kleppe M, Soulier J, Asnafi V, Mentens N, Hornakova T, Knoops L, Constantinescu S, Sigaux F, Meijerink JP, Vandenberghe P, et al: PTPN2 negatively regulates oncogenic JAK1 in T-cell acute lymphoblastic leukemia. Blood. 117:7090–7098. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Plutzky J, Neel BG, Rosenberg RD, Eddy RL, Byers MG, Jani-Sait S and Shows TB: Chromosomal localization of an SH2-containing tyrosine phosphatase (PTPN6). Genomics. 13:869–872. 1992. View Article : Google Scholar : PubMed/NCBI | |
|
Oka T, Yoshino T, Hayashi K, Ohara N, Nakanishi T, Yamaai Y, Hiraki A, Sogawa CA, Kondo E, Teramoto N, et al: Reduction of hematopoietic cell-specific tyrosine phosphatase SHP-1 gene expression in natural killer cell lymphoma and various types of lymphomas/leukemias: Combination analysis with cDNA expression array and tissue microarray. Am J Pathol. 159:1495–1505. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Chim CS, Fung TK, Cheung WC, Liang R and Kwong YL: SOCS1 and SHP1 hypermethylation in multiple myeloma: Implications for epigenetic activation of the Jak/STAT pathway. Blood. 103:4630–4635. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Amin HM, Hoshino K, Yang H, Lin Q, Lai R and Garcia-Manero G: Decreased expression level of SH2 domain-containing protein tyrosine phosphatase-1 (Shp1) is associated with progression of chronic myeloid leukaemia. J Pathol. 212:402–410. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Challouf S, Ziadi S, Zaghdoudi R, Ksiaa F, Ben Gacem R and Trimeche M: Patterns of aberrant DNA hypermethylation in nasopharyngeal carcinoma in Tunisian patients. Clin Chim Acta. 413:795–802. 2010. View Article : Google Scholar | |
|
Hachana M, Trimeche M, Ziadi S, Amara K and Korbi S: Evidence for a role of the Simian Virus 40 in human breast carcinomas. Breast Cancer Res Treat. 113:43–58. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Banville D, Stocco R and Shen SH: Human protein tyrosine phosphatase 1C (PTPN6) gene structure: Alternate promoter usage and exon skipping generate multiple transcripts. Genomics. 27:165–173. 1995. View Article : Google Scholar : PubMed/NCBI | |
|
Tsui FW, Martin A, Wang J and Tsui HW: Investigations into the regulation and function of the SH2 domain-containing protein-tyrosine phosphatase, SHP-1. Immunol Res. 35:127–136. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Yamasaki M, Miyata H, Miyazaki Y, Takahashi T, Kurokawa Y, Nakajima K, Takiguchi S, Mori M and Doki Y: Evaluation of the nodal status in the 7th edition of the UICC-TNM classification for esophageal squamous cell carcinoma: Proposed modifications for improved survival stratification: Impact of lymph node metastases on overall survival after esophagectomy. Ann Surg Oncol. 21:2850–2856. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Warnecke PM and Bestor TH: Cytosine methylation and human cancer. Curr Opin Oncol. 12:68–73. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Parris TZ, Kovács A, Hajizadeh S, Nemes S, Semaan M, Levin M, Karlsson P and Helou K: Frequent MYC coamplification and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplified breast carcinomas. Oncogenesis. 3:e952014. View Article : Google Scholar : PubMed/NCBI | |
|
Vasiljević N, Scibior-Bentkowska D, Brentnall AR, Cuzick J and Lorincz AT: Credentialing of DNA methylation assays for human genes as diagnostic biomarkers of cervical intraepithelial neoplasia in high-risk HPV positive women. Gynecol Oncol. 132:709–714. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
El-Osta A, Baker EK and Wolffe AP: Profiling methyl-CpG specific determinants on transcriptionally silent chromatin. Mol Biol Rep. 28:209–215. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Oka T, Ouchida M, Koyama M, Ogama Y, Takada S, Nakatani Y, Tanaka T, Yoshino T, Hayashi K, Ohara N, et al: Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas. Cancer Res. 62:6390–6394. 2002.PubMed/NCBI | |
|
Chim CS, Wong KY, Loong F and Srivastava G: SOCS1 and SHP1 hypermethylation in mantle cell lymphoma and follicular lymphoma: Implications for epigenetic activation of the Jak/STAT pathway. Leukemia. 18:356–358. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Sheng Y, Wang H, Liu D and Zhang C, Deng Y, Yang F, Zhang T and Zhang C: Methylation of tumor suppressor gene CDH13 and SHP1 promoters and their epigenetic regulation by the UHRF1/PRMT5 complex in endometrial carcinoma. Gynecol Oncol. 140:145–151. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang MY, Fung TK, Chen FY and Chim CS: Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm. J Cell Mol Med. 17:1282–1290. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Han Y, Amin HM, Frantz C, Franko B, Lee J, Lin Q and Lai R: Restoration of shp1 expression by 5-AZA-2′-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma. Leukemia. 20:1602–1609. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H: Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene. 24:6406–6417. 2005. View Article : Google Scholar : PubMed/NCBI |
