Inhibition of atypical protein kinase Cι induces apoptosis through autophagic degradation of ß-catenin in esophageal cancer cells.

Atypical protein kinase Cι (PKCι) has been identified as an oncoprotein in esophageal squamous cell carcinomas. However, the mechanisms underlying the role of PKCι in this disease remain unclear. In the present work, we found that inhibition of PKCι expression by RNAi induced apoptosis via the down-regulation of ß-catenin in esophageal cancer cells. Furthermore, we found that PKCι regulated ß-catenin in an autophagy dependent way. Since down-regulation of ß-catenin induced by knockdown of PKCι could be rescued by autophagy inhibition; knockdown of PKCι activated autophagy and promoted the recruitment of ß-catenin into autophagosome. These results suggested that PKCι positively regulated ß-catenin through negatively regulated autophagy and depletion of PKCι promoted apoptosis via autophagic degradation of ß-catenin in esophageal cancer cells. These data provide new insights into PKCι signaling in human cancer.

Autophagy negatively regulates cancer cell proliferation via selectively targeting VPRBP.

There have been multiple lines of evidence suggesting that autophagy selectively targets signalling proteins and regulates cancer cell signalling in addition to bulk clearance of long-lived proteins and organelles. Protein degradation through autophagy requires receptor protein LC3B to sequester the substrates into the autophagosome. In the present study, we screened LC3B (light-chain 3B)-binding partners and identified autophagic substrates in cancer cells. With lung cancer NCI-H1975 and oesophageal cancer KYSE30 cell lines as models, we found that VPRBP (viral protein R-binding protein) was a novel LC3B-binding protein through GST (glutathione transferase)-LC3B pull-down combined with LC-MS/MS (liquid chromatography-tandem MS) methods. Co-immunoprecipitation assay showed that VPRBP-LC3/p62 were in the same protein complex as the two cell lines. Induction of autophagy led to a down-regulation of VPRPB, which could be rescued by the inhibition of autophagy degradation by BFA1 (bafilomycin A1) and by the disruption of autophagy through ATG5-knockdown. We also found that induction of autophagy promotes VPRBP-LC3/p62 interaction. Immunohistochemical examination of human NSCLC (non-small cell lung cancer) tissues showed that VPRBP was positively correlated with p62 and negatively correlated with LC3B. Moreover, p62 and VPRBP were associated with poor prognosis in lung ADC (adenocarcinoma) (p62, P=0.019; VPRBP, P=0.005). Patients with low expression of both p62 and VPRBP showed the best prognosis.

Genomic alterations with impact on survival in esophageal squamous cell carcinoma identified by array comparative genomic hybridization.

Risk assessment of esophageal squamous cell carcinoma (ESCC) is currently based on clinicopathological parameters. To identify genomic markers that can predict overall survival in ESCC, we performed array comparative genomic hybridization (array CGH) on a screening set of 35 tumor samples from ESCC patients. Prognosis association of the genes selected on the basis of the array CGH results was further validated by real-time PCR in two independent sample sets (n = 151 and 84). Genomic analysis revealed seven high-level amplifications and two homozygous deletions. Gain of 11q13.2 and loss of 7q34 and 18q21.1-q23 were associated with poor outcome. Gain of 11q13.2 was an independent prognostic factor and was selected for further validation. In both validation sets of samples, copy number increase of CPT1A in 11q13.2 was correlated with short overall survival (P = 0.015, n = 151 and P = 0.044, n = 84). Multivariate analysis confirmed that CPT1A gain provided prognostic information in ESCC (HR, 1.643; 95% CI: 1.076-2.509; P = 0.022; HR, 2.488; 95% CI: 1.235-5.013; P = 0.011). Immunohistochemistry showed significant correlation between strong expression of CPT1A protein and poor outcome of ESCC patients (P = 0.018, n = 73). Our data suggest that gain of CPT1A may be a candidate prognostic factor.

[Screening and identification of anoikis-resistant gene UBCH7 in esophageal cancer cells].

Anoikis is a kind of programmed cell death induced by loss of extracellular matrix (ECM) adhesion, which is one of key factors for homestasis. Resistance to anoikis is required for tumor cell metastasis. We have previously shown several anoikis-resistance genes in esophageal squamous cell carcinoma (ESCC). In order to find novel anoikis-resistant genes in ESCC, we constructed retroviral cDNA library using total RNA from ESCC cell lines. NIH 3T3 cells, which are sensitive to anoikis, were infected with the library constructed. The cells were cultured in soft agar, and the clones which can survive in detached states were selected. The cDNAs inserted into the anoikis-resistant NIH3T3 clones were amplified using retroviral specific primers. Sequencing analysis showed that a cDNA fragment inserted into the anoikis-resistant clone contains full coding sequence (ORF) of human UBCH7/UBE2L3 gene. By infection with retrovirus encoding UBCH7 ORF (pMSCV-UBCH7), forced expression of UBCH7 increased the anoikis-resistance of NIH3T3 cells. More importantly, knockdown of UBCH7 expression by siRNA transfection reduced the anoikis-resistant ability of esophageal cancer MLuC1 cells. The data suggest that UBCH7/UBE2L3 gene would be involved in anoikis-resistance in ESCC.

NRS2002 assesses nutritional status of leukemia patients undergoing hematopoietic stem cell transplantation.

OBJECTIVE: To discuss whether nutritional risk screening 2002 (NRS2002) is appropriate for nutritional risk screening for leukemia patients before and after hematopoietic stem cell transplantation (HSCT), and whether there are risk differences in other conditions, such as age, gender and matching degree; to find the methods and indicators of nutritional risk screening for these patients before and after HSCT, in order to give timely intervention to guarantee the successful completion of the entire transplantation process. METHODS: Nutritional risk of 99 leukemia patients was screened with NRS2002 before and after HSCT. The (χ) (2) test was applied to compare the risk differences between groups such as age, gender and matching degree, while the differences of other enumeration data, such as recent (1-3 months) weight loss, reduced food intake within one week and BMI, were compared by continuity correction. RESULTS: Of the 99 leukemia patients, 22 cases (22.2%) had nutritional risk before HSCT, while all patients had nutritional risk after HSCT; there is no significant difference in nutritional risk between male and female, and patients of less than 30 years old, not-full matched, recent (1-3 months) weight loss, reduced food intake within a week or BMI <18.5 were more likely to have nutritional risk; and 77 cases (77.8%) had weight loss, among which 49 patients (63.6%) had more than 5% weight loss within one month. CONCLUSIONS: This study showed that leukemia patients should receive the nutritional risk screening conventionally before and after HSCT, and NRS2002 was only appropriate for nutritional risk screening before HSCT. More attention should be paid to the patients less than 30 years old or not-full matched. Weight change was one of the important nutritional indicators for patients after HSCT.

The size and depth of lesions measured by endoscopic ultrasonography are novel prognostic factors of primary gastric diffuse large B-cell lymphoma.

Diffuse large B cell lymphoma is one of the predominant histological subtypes of primary gastric lymphomas. Factors that contribute to precise stratification and guide the treatment of this disease are still not well understood. We analyzed 73 primary gastric diffuse large B cell lymphoma patients retrospectively, and found that patients characterized by late stage, multiple localization, B symptoms, lower serum albumin level and elevated LDH level had a shorter overall survival through Univariate Cox regression analysis. Multivariate Cox regression analysis demonstrated that ALB ≤ 35g/L, staging ≥ IIE and multiple sites localization were independent adverse prognostic factors. Significantly, in 35 patients who received endoscopy at diagnosis, Kaplan-Meier analyses indicated that patients with large (≥3 cm) and deep lesions (≥11 mm) had an inferior OS (p = .01 and .039). These findings implicated that tumor size and depth are two indicators of prognosis under ultrasonography. Further randomized studies with large number of cases are needed.

[Role of inducible costimulatory molecule-mediated Th17 cell polarization in renal fibrosis in spontaneously hypertensive rats].

OBJECTIVE: To explore the role of inducible costimulatory molecule (ICOS) signaling pathway-mediated Th17 cells polarization in renal damage in essential hypertension. METHODS: Four-week-old spontaneously hypertensive rats (SHR) were randomly divided into control (SHR-C) group and intervention (SHR-I) group and subjected to intraperitoneal injections of PBS and ICOS monoclonal antibody for 2 weeks, respectively. Blood pressure of the rats was monitored using noninvasive tail artery blood pressure measuring instrument. The percentage of Th17 cells in the splenocytes was analyzed using flow cytometry, and the expression levels of IL-17A mRNA in the rat's kidneys were detected using RT-PCR. The levels of IL-17A and TGF-ß1 in the plasma and kidneys were dynamically detected using ELISA and immunohistochemistry, respectively. Renal pathological changes in the rats were detected using Masson staining. RESULTS: At the age of 10 and 30 weeks, the rats in SHR-C group had a significantly higher blood pressure than those in SHR-I group (P<0.05 or 0.01). In rats in SHR-C group, Th17 cells percentage in the splenocytes and IL-17A mRNA level in the kidney was significantly higher than those in SHR-I group from the age of 6 weeks (P<0.05). The expressions of IL-17A and TGF-ß1 in the plasma and kidney were significantly higher in SHR-C group than that in SHR-I group at 6 weeks (P<0.05). Compared with those in SHR-C group, the rats in SHR-I group showed significant alleviation of renal fibrosis from the age of 30 weeks (P<0.05). CONCLUSION: The ICOS signaling pathway-mediated Th17 cells polarization plays an important role in renal fibrosis in hypertensive rats.

ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis.

BACKGROUND: ANXA2 (Annexin A2) is a pleiotropic calcium-dependent phospholipid binding protein that is abnormally expressed in various cancers. We previously found that ANXA2 is upregulated in esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the functional significance of ANXA2 dysregulation and underlying mechanism in ESCC. METHODS: Proliferation, migration, invasion and metastasis assay were performed to examine the functional roles of ANXA2 in ESCC cells in vitro and in vivo. Real-time RT-PCR, immunoblotting, ChIP, reporter assay, confocal-immunofluorescence staining, co-immunoprecipitation and ubiquitination assay were used to explore the molecular mechanism underlying the actions of deregulated ANXA2 in ESCC cells. RESULTS: Overexpression of ANXA2 promoted ESCC cells migration and invasion in vitro and metastasis in vivo through activation of the MYC-HIF1A-VEGF cascade. Notably, ANXA2 phosphorylation at Tyr23 by SRC led to its translocation into the nucleus and enhanced the metastatic potential of ESCC cells. Phosphorylated ANXA2 (Tyr23) interacted with MYC and inhibited ubiquitin-dependent proteasomal degradation of MYC protein. Accumulated MYC directly potentiated HIF1A transcription and then activated VEGF expression. Correlation between these molecules were also found in ESCC tissues. Moreover, dasatinib in combination with bevacizumab or ANXA2-siRNA produced potent inhibitory effects on the growth of ESCC xenograft tumors in vivo. CONCLUSIONS: This study provides evidence that highly expressed p-ANXA2 (Tyr23) contributes to ESCC progression by promoting migration, invasion and metastasis, and suggests that targeting the SRC-ANXA2-MYC-HIF1A-MYC axis may be an efficient strategy for ESCC treatment.

MCMs expression in lung cancer: implication of prognostic significance.

Minichromosome Maintenance (MCM) proteins play essential roles in various cancers. We previously reported that MCM7 could be a prognostic biomarker in non-small cell lung cancer (NSCLC). The purpose of current study is to explore roles of other MCM proteins in NSCLC and their correlation with clinico-pathologic parameters of NSCLC patients. We evaluated the expression of MCM2, MCM5 and MCM6 immunohistochemically in 571 primary NSCLC samples. High expression of MCM2, MCM5 and MCM6 was detected in 42.2%, 38.3% and 52.9% of tumor tissues, respectively. The expression of MCM2, MCM5 and MCM6 was significantly associated with gender (P = 0.00004, 0.00004, 0.008), tumor type (P < 0.00001, < 0.00001, 0.00001) and smoking history (P = 0.009, 0.00043, 0.002). MCM2 and MCM5 were detected more in central-type lung cancer (P< 0.006, 0.016). Higher labeling index (LI) of MCM2 was observed more frequently in aged patients (P = 0.023) and in those at later stage (P = 0.001). Higher MCM5 LIs was detected more in patients with distant metastasis (P = 0.008). Kaplan-Meier curves indicated that early-stage (stage I/II) patients with higher MCM2 LIs had a poorer OS compared to those with lower LIs (P = 0.021). And lung squamous cell carcinoma (SCC) patients presenting high MCM5 expression had shorter OS (P = 0.015). Multivariate Cox regression analysis showed that MCM5 was an independent prognostic indicator (P = 0.035, HR = 1.586, 95%CI: 1.032-2.437). We reported for the first time that higher MCM5 LIs could be an independent adverse prognostic biomarker for SCC patients.

Complete mitochondrial genome of the capped langur (Trachypithecus pileatus).

The complete mitochondrial sequence of the capped langur (Trachypithecus pileatus) has been determined using long amplification polymerase chain reaction (LA-PCR). The total sequence length is 16,526 bp and includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 D-loop locus. The base composition of H-strand is 31.9% A, 29.1% T, 26.2% C and 12.8% G, with an AT content of 55.3%. The arrangement of genes in T. pileatus is identical to that of other primate species. All genes are encoded on the heavy strand with the exception of ND6 and eight tRNA genes. The mitochondrial genome of T. pileatus presented here will contribute to a better understanding of the species' population genetics, helping to protect its genetic diversity and resolve phylogenetic relationships within the family.

A panel of protein markers for the early detection of lung cancer with bronchial brushing specimens.

BACKGROUND: To date, no robust biomarkers have been available in clinical practice that can provide an early diagnostic evaluation of lung cancer. The objective of this study was to identify potential biomarkers for the early detection of lung cancer using bronchial brushing specimens. METHODS: Immunocytochemistry was used to investigate the expression of 35 proteins in 880 bronchial brushing specimens from both outpatients and inpatients who had either lung cancer or benign lung lesions. An optimal panel was identified that had high sensitivity and considerable specificity for detecting lung cancer. Associations between protein expression and clinicopathologic parameters were assessed. RESULTS: Tumor protein 53 (TP53), TP63, Ki67, epidermal growth factor receptor (EGFR), minichromosome maintenance complex component 6 (MCM6), MCM7, uncharacterized proteins KIAA1522 and KIAA0317, and ubiquitin-protein ligase UHR1 (ICBP90) frequently presented high expression in bronchial brushing specimens from patients who had lung cancer compared with patients who had benign lung lesions. A 6-protein panel consisting of TP53, Ki67, MCM6, MCM7, KIAA1522, and KIAA0317 was identified as the best combination, with sensitivity of 81.1% (309 of 381 specimens) for detecting nonsmall cell lung cancer (NSCLC) and 86.8% (145 of 167 specimens) for detecting small cell lung cancer (SCLC) (specificity, 83.3%; 65 of 78 specimens). The combination of cytology and the protein panel significantly improved the sensitivity of bronchial brushing examination for detecting lung cancer (P<.00001), which increased from 49.1% to 81% in early stage NSCLC (stage I and II). In combined analyses, the protein panel was positively associated with patient sex (P=.00033), tumor type (P<.00001), tumor location (P<.00001), and lymph node metastasis (P=.028). CONCLUSIONS: The 6-protein panel is a potential biomarker for the early detection of lung cancer in bronchial brushings.

PKCι counteracts oxidative stress by regulating Hsc70 in an esophageal cancer cell line.

Using a glutathione S-transferase pull-down liquid chromatography-coupled tandem mass spectrometry approach and immunoprecipitation/immunoblot analysis, we found that heat shock cognate protein 70 (Hsc70) was involved in the complex formed by atypical protein kinase Cι (PKCι) and LC3 in the esophageal cancer cell line KYSE30. Further study indicated that Hsc70 was targeted by autophagic degradation, and knockdown of PKCι down-regulated Hsc70 by promoting autophagy. PKCι knockdown sensitized cells to oxidative stress-induced apoptosis, whereas forced PKCι expression counteracted the oxidative stress-induced apoptosis via Hsc70.

PTP1B contributes to calreticulin-induced metastatic phenotypes in esophageal squamous cell carcinoma.

UNLABELLED: Calreticulin (CRT) is a Ca(2+)-binding chaperone protein that alters cellular Ca(2+)-homeostasis in the endoplasmic reticulum (ER). Previously it was shown that CRT was overexpressed in esophageal squamous cell carcinoma (ESCC), and elevated CRT expression promoted the migration and invasion of ESCC cells. In the present study, the mechanisms underlying the role of CRT in esophageal carcinoma progression were investigated. Critically, depletion of CRT or protein-tyrosine phosphatase 1B (PTP1B) reduced ESCC cell migration and metastasis to the lung, whereas restoration of PTP1B protein levels rescued cell migration in CRT-silenced cells. Knockdown of CRT decreased PTP1B protein expression by reducing phosphorylation at the Y694 site of STAT5A, whereas knockdown of PTP1B reduced ERK1/2 phosphorylation at T204. Immunohistochemical analysis of CRT and PTP1B expression in ESCC patient tissues was strongly correlated. Importantly, PTP1B expression was associated with poor survival in patients with CRT overexpression. Overall, these data indicate a novel signaling pathway connecting CRT, STAT5A, PTP1B, and ERK1/2 in the regulation of ESCC cell migration. IMPLICATIONS: These findings suggest that PTP1B is a downstream effector of CRT signaling, promotes tumor progression, and can potentially be used as a new drug target for ESCC.

Consistent and differential genetic aberrations between esophageal dysplasia and squamous cell carcinoma detected by array comparative genomic hybridization.

PURPOSE: Our aim was to identify frequent genomic aberrations in both esophageal squamous cell carcinoma (ESCC) and esophageal dysplasia and to discover important copy number-driving genes and microRNAs (miRNA) in ESCC. EXPERIMENTAL DESIGN: We conducted array-based comparative genomic hybridization (array CGH) on 59 ESCC resection samples and 16 dysplasia biopsy samples. Expression of genes at 11q13.3 was analyzed by real-time PCR (RT-PCR) and immunohistochemistry (IHC). Integrated analysis was conducted to identify genes or miRNAs with copy number-expression correlations. RESULTS: Array CGH identified 11 amplifications and eight homozygous deletions in ESCC. Integrated analysis of array CGH data with matched gene expression microarray data showed that 90 overexpressed genes and 24 underexpressed genes were consistent with DNA copy number changes, including 12 copy number-driving miRNAs. In esophageal dysplasia, six gains, four losses, 12 amplifications, and four homozygous deletions were detected. Amplifications of 7p11.2 and 11q13.2-11q13.3 (CCND1) and homozygous deletion at 9p21.3 (CDKN2A) were consistent genomic changes in both dysplasia and carcinoma. ANO1 at 11q13.3 was overexpressed at the mRNA and protein levels in tumors, and higher mRNA expression was correlated with the copy number increase. In particular, ANO1 expression was elevated in moderate dysplasia compared with normal esophageal epithelium. IHC revealed that ANO1 overexpression was positively correlated with lymph node metastasis and advanced clinical stage. Knockdown of ANO1 significantly inhibited the proliferation of KYSE30 and KYSE510 cells. CONCLUSION: Copy number aberrations in both esophageal dysplasia and ESCC may be useful as potential biomarkers for early detection. In addition, ANO1 may be a candidate target gene in esophageal tumorigenesis.

Three methods assess nutritional status of leukemia patients before hematopoietic stem cell transplantation.

BACKGROUND: Some leukemia patients before hematopoietic stem cell transplantation (HSCT) have nutritional risk or undernutrition, which was one of the main reasons that caused series of complications during transplantation. The aim of this study was to find out some appropriate methods to learn about the nutritional status of leukemia patients before HSCT. METHODS: Nutritional status of patients with leukemia was assessed with three common methods of nutritional assessment (nutritional risk screening 2002 (NRS2002), mini nutritional assessment (MNA) and subjective global assessment (SGA)) before HSCT. The assessment results of NRS2002 and MNA were compared by paired χ(2) test. The consistency was analyzed by Kappa test. RESULTS: In this study, 13 of 50 patients (26.0%) with leukemia had nutritional risk before HSCT assessed by NRS2002, including 7 patients (14.0%) with undernutrition. Of 50 patients assessed by SGA, only 1 case (2.0%) was mild or moderate undernutrition, and the remaining 49 patients (98.0%) were well-nutrition. Assessed by MNA, 1 case (2.0%) was undernutrition, 11 cases (22.0%) were potential undernutrition, and 38 cases (76.0%) were well-nutrition. Paired χ(2) test results showed that the difference between NRS2002 and MNA was statistically significant (χ(2) = 13.64, P < 0.05); Kappa test results showed that they were consistent between NRS2002 and MNA (Kappa = 0.62, P < 0.05). CONCLUSIONS: It is important to know the nutritional status of patients with leukemia before HSCT, and NRS2002 should be the first choice of nutritional assessment for patients with leukemia. If NRS2002 and MNA used at the same time, the accuracy and comprehensiveness of the assessment can be improved.

Prognostic significance of MCM7 expression in the bronchial brushings of patients with non-small cell lung cancer (NSCLC).

PURPOSE: To identify potential biomarkers for the prognosis of non-small cell lung cancer (NSCLC) patients by using bronchial brushing specimens. METHODS: The expression of MCM7, Ki67 and EGFR was evaluated in 494 NSCLC tissues and 174 bronchial brushings using immunohistochemical and immunocytochemical techniques. Associations between protein expression and clinico-pathologic parameters were assessed, and the impact on overall survival (OS) was analyzed. RESULTS: High expression of MCM7, Ki67 and EGFR was detected in 33.3%, 23.5% and 12.7% of tissues and in 52.4%, 52.7% and 20.6% of bronchial brushings, respectively. Expression of MCM7 and Ki67 was associated with squamous cell carcinoma (SCC) in both tissues and bronchial brushings (MCM7: P = 0.0007, 0.00003; Ki67: P < 0.00001, 0.00001). Overexpression of MCM7 in tumor tissues was detected more frequently in poorly differentiated tumors (P = 0.0120) and non-bronchioloalveolar carcinomas (non-BACs) (P = 0.0238). EGFR overexpression was observed in tissues of larger tumors (P = 0.00004) and in bronchial brushings at later stage (P = 0.0262). Kaplan-Meier curves indicated that patients with overexpression of MCM7 or Ki67 had a poorer OS compared to those with low expression for all stages (P < 0.00001, 0.0233) and early-stages (P < 0.00001, 0.0032). In particular, the patients with MCM7 overexpression in bronchial brushings had a poorer prognosis (P = 0.0045). Multivariate Cox regression analysis showed that MCM7 was an independent prognostic indicator both in tissue samples and bronchial brushings. CONCLUSIONS: Our data suggest that MCM7 and Ki67 in tumor tissues may be potential markers of a poor prognosis for NSCLC patients. MCM7 in bronchial brushings also showed an independent prognostic value, which may be useful when biopsies are unavailable.

Genomic profiling of rectal adenoma and carcinoma by array-based comparative genomic hybridization.

BACKGROUND: Rectal cancer is one of the most common cancers in the world. Early detection and early therapy are important for the control of death caused by rectal cancer. The present study aims to investigate the genomic alterations in rectal adenoma and carcinoma. METHODS: We detected the genomic changes of 8 rectal adenomas and 8 carcinomas using array CGH. Then 14 genes were selected for analyzing the expression between rectal tumor and paracancerous normal tissues as well as from adenoma to carcinoma by real-time PCR. The expression of GPNMB and DIS3 were further investigated in rectal adenoma and carcinoma tissues by immunohistochemistry. RESULTS: We indentified ten gains and 22 losses in rectal adenoma, and found 25 gains and 14 losses in carcinoma. Gains of 7p21.3-p15.3, 7q22.3-q32.1, 13q13.1-q14.11, 13q21.1-q32.1, 13q32.2-q34, 20p11.21 and 20q11.23-q12 and losses of 17p13.1-p11.2, 18p11.32-p11.21 and 18q11.1-q11.2 were shared by both rectal adenoma and carcinoma. Gains of 1q, 6p21.33-p21.31 and losses of 10p14-p11.21, 14q12-q21.1, 14q22.1-q24.3, 14q31.3-q32.1, 14q32.2-q32.32, 15q15.1-q21.1, 15q22.31 and 15q25.1-q25.2 were only detected in carcinoma but not in adenoma. Copy number and mRNA expression of EFNA1 increased from rectal adenoma to carcinoma. C13orf27 and PMEPA1 with increased copy number in both adenoma and carcinoma were over expressed in rectal cancer tissues. Protein and mRNA expression of GPNMB was significantly higher in cancer tissues than rectal adenoma tissues. CONCLUSION: Our data may help to identify the driving genes involved in the adenoma-carcinoma progression.

PLK1 Is transcriptionally activated by NF-κB during cell detachment and enhances anoikis resistance through inhibiting ß-catenin degradation in esophageal squamous cell carcinoma.

PURPOSE: To investigate the molecular mechanisms through which polo-like kinase-1 (PLK1) takes part in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells. EXPERIMENTAL DESIGN: The role of PLK1 in cell anoikis resistance was examined by ectopic gene expression and siRNA-mediated knockdown. Glutathione S-transferase pull-down and co-immunoprecipitation assays were utilized to investigate PLK1-interacting proteins. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter gene assays were carried out to identify the transcription factors responsible for PLK1 expression during anoikis resistance. RESULTS: We found that detachment of ESCC cells triggers the upregulation of PLK1. Elevated PLK1 expression contributes to protection against anoikis in cancer cells through the regulation of ß-catenin expression. Moreover, we showed that, through direct binding to the PLK1 promoter, the NF-κB subunit RelA transcriptionally activates PLK1, which inhibits the ubiquitination and degradation of ß-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/ß-catenin expression. In addition, RelA gene amplification and protein overexpression was significantly correlated with PLK1 expression in ESCC tissues. CONCLUSIONS: Our findings suggest that upregulation of PLK1 triggered by cell detachment is regulated by RelA at the transcriptional level. PLK1 protects esophageal carcinoma cells from anoikis through modulation of ß-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/ß-catenin in anoikis resistance of ESCC cells.

Atypical protein kinase Cι (PKCι) promotes metastasis of esophageal squamous cell carcinoma by enhancing resistance to Anoikis via PKCι-SKP2-AKT pathway.

Protein kinase Cι (PKCι) is an atypical PKC isoform and participates in multiple aspects of the transformed phenotype in human cancer cells. We previously reported that frequent amplification and overexpression of PKCι were correlated with lymph node metastasis in primary esophageal squamous cell carcinomas (ESCC). In the present study, short interfering RNA-mediated silencing of PKCι revealed that this enzyme was required for cell migration, invasion, and resistance to anoikis. In vivo experiments showed that PKCι suppression decreased tumor growth in esophageal cancer xenografts and lung metastases in nude mice. At the molecular level, knockdown of PKCι in suspended ESCC cells caused a decrease in S-phase kinase-associated protein 2 (SKP2) that had been reported to promote resistance to anoikis via the PI3K/AKT pathway. AKT phosphorylation was abolished after PKCι suppression, but AKT activation could be refreshed by PKCι upregulation, suggesting that PKCι enhanced cell resistance to anoikis via the PKCι-SKP2-PI3K/AKT pathway. Addition of the proteasome inhibitor MG132 prevented the decrease of SKP2 in PKCι silenced cells, and polyubiquitin-SKP2 was elevated after PKCι depletion, showing that PKCι might regulate the expression of SKP2 through the ubiquitin-proteasome pathway in suspended cells. Furthermore, overexpression of SKP2 in PKCι-downregulated cells restored cell resistance to anoikis. Most importantly, PKCι expression significantly correlated with SKP2 in 133 ESCC tissues (P = 0.031). Taken together, our data show that PKCι promotes tumorigenicity and metastasis of human esophageal cancer and that SKP2 is a candidate downstream effector of PKCι signaling in ESCC.