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Ferroptosis is a new form of programmed cell death with characteristic accumulation of reactive oxygen species (ROS) resulting from iron accumulation and lipid peroxidation. Ferroptosis is involved in many diseases, including cancer, and induction of ferroptosis has shown attractive antitumour activities. In this review, we summarize recent findings on the regulatory mechanisms of key regulators of ferroptosis, including the catalytic subunit solute carrier family 7 member 11 (SLC7A11), the glutathione peroxidase 4 (GPX4), p53 and non-coding RNAs, the correlations between ferroptosis and iron homeostasis or autophagy, ferroptosis-inducing agents and nanomaterials and the diagnostic and prognostic value of ferroptosis-associated genes in TCGA data.

Bing Yuan,1,* Feng Liao,1,* Zhi-Zhou Shi,2 Yuan Ren,1 Xiao-Li Deng,1 Ting-Ting Yang,1 Deng-Yuan Li,1 Ru-Fang Li,1 Dan-Dan Pu,1 Yu-Jue Wang,1 Yan Tan,1 Zhen Yang,1 Yun-Hui Zhang1 1Department of Pulmonary and Critical Care Medicine, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, People’s Republic of China; 2Medical School, Kunming University of Science and Technology, Kunming, Yunnan, People’s Republic of China*These authors contributed equally to this work.Correspondence: Zhen YangDepartment of Pulmonary and Critical Care Medicine, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, People’s Republic of ChinaEmail 49387127@qq.comYun-Hui ZhangDepartment of Pulmonary and Critical Care Medicine, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, People’s Republic of ChinaEmail yunhuizhang3188@126.comObjective: Lung cancer is the first leading cause of cancer-related deaths both worldwide and in China and threatens human health and quality of life. New drugs and therapeutic methods are urgently needed. Our study evaluated the roles of dihydroartemisinin (DHA) in lung cancer and further explored its underlying mechanisms.Methods: CCK-8, colony formation and trypan blue exclusion assays were used to detect the cell viability, colony formation ability and cell death. qRT-PCR and Western blotting assays were applied to analyze the expressions of key molecules.Results: DHA inhibited the proliferation and colony formation abilities and enhanced the cell death and induced ferroptosis of lung NCI-H23 and XWLC-05 cancer cells. DHA reduced PRIM2 expression and silencing PRIM2 mimicked the inhibitory roles on proliferation and colony formation and promotive roles on cell death and ferroptosis of DHA in lung NCI-H23 and XWLC-05 cancer cells. We further found that DHA treatment and loss of PRIM2 reduced the GSH level and increased the cellular lipid ROS and mitochondrial MDA levels, and further downregulated the expressions of SLC7A11 and β-catenin in lung cancer cells, respectively. Exogenetic overexpression of PRIM2 recovered the inhibitory effects of DHA on proliferation and colony formation in lung NCI-H23 cancer cells, meanwhile loss of PRIM2 sensitizes NCI-H23 cells to DHA therapy. In vivo experiment further showed that DHA treatment significantly suppressed the tumor growth and downregulated PRIM2 and SLC7A11.Conclusion: Our study suggested that DHA inhibited the proliferation, colony formation and enhanced cell death and induced ferroptosis of lung cancer cells by inactivating PRIM2/SLC7A11 axis. Loss of PRIM2 induced ferroptosis might developed to be a novel therapeutic method in lung cancer therapy.Keywords: dihydroartemisinin, lung cancer, proliferation, ferroptosis, PRIM2, SLC7A11

miR-99a is down-regulated in esophageal squamous cell carcinoma (ESCC), however the role and underlying mechanism are still unknown. We aim to explore the role and mechanism of miR-99a down-regulation in ESCC. The expression of miR-99a in ESCC tissues and cell lines was detected by Human miRNA Microarrays and Real-time PCR. The effects of miR-99a on cell proliferation, migration and invasion were determined by Cell Counting Kit-8 (CCK-8) assay, transwell migration and invasion assay. Target gene of miR-99a were analyzed by target prediction software and validated by Real-time PCR and Western blotting assay. Our microarray results and four Gene Expression Omnibus (GEO) datasets showed lower expression level of miR-99a in ESCC tissues. Overexpression of miR-99a using mimics significantly suppressed cell proliferation, and decreased expressions of CCND1, CCNA2 and CCNE1. We also found that enhanced miR-99a significantly inhibited migration, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells, and down-regulated EMT associated transcription factor Slug, and MMPs including MMP2, MMP7 and MMP13. TargetScan predicted insulin-like growth factor 1 receptor (IGF1R) as the cadidate target gene of miR-99a, and western blotting confirmed the negative correlation between miR-99a and IGF1R. Importantly, we further found that knockdown of IGF1R also significantly inhibited the proliferation, migration, invasion and slug-induced EMT of ESCC cells, and reduced the cell cycle regulatory proteins and MMPs. In conclusion, our findings suggested that loss of miR-99a in ESCC promoted the tumor cell proliferation, migration, invasion and slug-induced EMT through activating IGF1R signaling pathway.

Esophageal cancer is a common cause of cancer-related deaths worldwide. Squamous cell carcinoma (SCC) is the major histological type of esophageal cancer in developing countries including China, and the prognosis is very poor. Many microRNAs are involved in several important biological and pathologic processes, and promote tumorigenesis. To better understand the prognostic and therapeutic roles of microRNAs in ESCC, we reviewed the diagnosis and prognosis associated oncogenic microRNAs (e.g. miR-21 and miR-17-92 cluster) and tumor suppressor microRNAs (e.g. miR-375, miR-133a and miR-133b), and diagnosis and prognosis associated oncogenic target genes (e.g. PDCD4 and CCND1) and tumor suppressor target genes (e.g. EZH2 and PDK1). We also summarized the prognostic microRNA and target gene pairs (e.g. miR-296 and CCND1, miR214 and EZH2). Taken together, our review highlights the opportunities and challenges for microRNAs in the molecular diagnosis and target therapy of ESCC.

The roles and mechanisms of mini-chromosome maintenance complex component 7 (MCM7) amplification and overexpression in esophageal carcinogenesis were investigated. By analyzing the TCGA datasets, we found that MCM7 was amplified in approximately 12% of esophageal squamous cell carcinomas (ESCCs), and in more than 4% of head and neck squamous cell carcinomas and stomach carcinomas. Overexpression of MCM7 was further verified in three independent GEO datasets of esophageal cancer. Knockdown of MCM7 using two siRNAs significantly inhibited cell proliferation, colony formation and migration of KYSE510 and EC9706 cells in vitro. Noteworthy, we further found that silencing of MCM7 suppressed the phosphorylation of AKT1 and mTOR both in KYSE510 and EC9706 cells, and reduced the cell cycle regulatory proteins cyclin D1, cyclin E2 and CDK2. Taken together, our findings suggested that MCM7 promoted tumor cell proliferation, colony formation and migration of ESCC cells via activating AKT1/mTOR signaling pathway.

Lung cancer is the most common cause of cancer-associated mortality globally. Long non-coding RNAs (lncRNAs) are transcripts with a length of >200 nucleotides, which are not translated into proteins. Growing evidence has indicated that certain lncRNAs are associated with various biological processes in cancer. However, the functions of KCNK15 and WISP2 antisense RNA 1 (KCNK15-AS1) in lung cancer carcinogenesis and progression have remained elusive. The present study indicated that KCNK15-AS1 was overexpressed in lung adenocarcinoma tissues compared with paracancerous normal tissues, and the high expression of KCNK15-AS1 was significantly associated with poor prognosis compared with the patients with low expression (P

The aim of the present study was to identify the candidate target genes of genomic aberrations in esophageal squamous cell carcinoma (ESCC). Array comparative genomic hybridization (CGH) and quantitative polymerase chain reaction were applied to analyze the copy number changes and expression level of candidate genes, respectively. Integrative analysis revealed that homozygous deletions of cyclin-dependent kinase inhibitor (CDKN) 2A and CDKN2B and gains of fascin actin-bundling protein 1 (FSCN1) and homer scaffolding protein 3 (HOMER3) occurred frequently in ESCC. The results demonstrated that the homozygous deletion of CDKN2A or CDKN2B was significantly associated with lymph node metastasis. Notably, the expression of CDKN2A and CDKN2B was lower in dysplasia than in normal esophageal epithelium. We also observed that the copy number increase of FSCN1 was significantly associated with pT, pN and pStage, and that the gain of HOMER3 was significantly linked with pN and pStage. We further revealed that FSCN1 and HOMER3 were overexpressed in ESCC, and that their overexpression was correlated with copy number increase. In conclusion, CDKN2A, CDKN2B, FSCN1 and HOMER3 are candidate cancer-associated genes and may play a tumorigenic role in ESCC.

// Li Shang 1, * , Jia-Jie Hao 1, * , Xue-Ke Zhao 3, * , Jian-Zhong He 4, * , Zhi-Zhou Shi 1 , Hui-Juan Liu 5 , Li-Fei Wu 6 , Yan-Yi Jiang 1 , Feng Shi 1 , Hai Yang 1 , Yu Zhang 1 , Yi-Zhen Liu 1 , Tong-Tong Zhang 1 , Xin Xu 1 , Yan Cai 1 , Xue-Mei Jia 5 , Min Li 6 , Qi-Min Zhan 1 , En-Min Li 4 , Li-Dong Wang 3 , Wen-Qiang Wei 2 , Ming-Rong Wang 1 1 State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China 2 Department of Cancer Epidemiology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China 3 Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450000, China 4 Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China 5 Department of Histology and Embryology, Anhui Medical University, Hefei 230032, China 6 Department of Gastroenterology, Anqing City Hospital, Affiliated Anqing Hospital of Anhui Medical University, Anqing 246003, China * These authors have contributed equally to this work Correspondence to: Ming-Rong Wang, e-mail: wangmr2015@126.com Wen-Qiang Wei, e-mail: weiwq@cicams.ac.cn Li-Dong Wang, e-mail: ldwang2007@126.com En-Min Li, e-mail: nmli@stu.edu.cn Keywords: ANO1, esophageal squamous cell carcinoma, precancerous lesions, biomarker, prognosis Received: September 17, 2015 Accepted: March 01, 2016 Published: March 21, 2016 ABSTRACT Objectives: Anoctamin 1 (ANO1) has been found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous study. Herein we showed the clinical relevance of ANO1 alterations with ESCC and esophageal precancerous lesion progression. Results: ANO1 was detected in 38.1% (109/286) and 25.4% (77/303) of tumors in the two cohorts, but in none of morphologically normal operative margin tissues. ANO1 expression was significantly associated with a shorter overall survival (OS), especially in patients with moderately differentiated and stage IIA tumors. In 499 iodine-unstained biopsies from the endoscopic screening cohort in 2005-2007, all the 72 pathologically normal epithelial mucosa presented negative immunostaining, whereas ANO1 expression was observed in 3/11 tumors and 5/231 intraepithelial lesions. 7/8 ANO1-positive cases had developed unfavorable outcomes revealed by endoscopic follow-up in 2012. Analysis of another independent cohort of 148 intraepithelial lesions further confirmed the correlation between ANO1 expression and progression of precancerous lesions. 3/4 intraepithelial lesions with ANO1 expression had developed ESCC within 4-9 years after the initial endoscopic examination. Methods: Immunohistochemistry (IHC) was performed to examine ANO1 expression in surgical ESCC specimens and two independent cohorts of esophageal biopsies from endoscopic screening in high-incidence area of ESCC in northern China. Association between ANO1 expression, clinico-pathologic parameters, and the impact on overall survival was analyzed. Conclusions: Positive ANO1 is a promising biomarker to predict the unfavorable outcome for ESCC patients. More importantly, it can predict disease progression of precancerous lesions.

Background At present no objective prognostic biomarkers have been established in esophageal squamous cell carcinoma (ESCC). Objective To identify the genomic biomarkers associated with clinicopathological factors and prognosis of ESCCs. Methods Real-time PCR was used to analyze the copy number change and mRNA expression of genes. The survival curves were plotted according to Kaplan-Meier method and checked by log-rank test. Results We revealed the copy number increase of CACNA1C (12p13.33) and MRPL21 (11q13.2) as well as decrease of VIPR2 (7q36.3) and MAP3K7 (6q15) in ESCC by Real-time PCR, and also found that MRPL21 was significantly overexpressed and VIPR2 was underexpressed in ESCC. Gain of CACNA1C was significantly associated with differentiation (P = 0.043), and loss of VIPR2 was significantly linked with good prognosis (P = 0.016). Most importantly, we revealed that loss of MAP3K7 was significantly correlated with good prognosis in ESCC patients (n = 159, P = 0.004), especially in Grade II and pN0 patients (n = 76, P = 0.005 and n = 74, P = 0.024). Multivariate analysis confirmed that MAP3K7 loss provided prognostic information in ESCC (HR, 0.454; 95% CI: 0.208-0.995; P = 0.048). Conclusions Loss of MAP3K7 may be a candidate prognostic factor in ESCC.

MicroRNA-1470 (miR-1470) is overexpressed in esophageal squamous cell carcinoma (ESCC); however, its role and underlying molecular mechanism remain unknown. The aim of the present study was to explore the tumorigenic role and mechanism of miR-1470 overexpression in ESCC. The expression of miR-1470 in ESCC tissues and cell lines was detected using human miRNA microarrays and the reverse transcription-quantitative polymerase chain reaction, respectively. The effects of miR-1470 on cell proliferation, migration and senescence were determined using a Cell Counting Kit-8 assay, Transwell migration assay and β-galactosidase staining kit. Western blotting was used to analyze the expression levels of genes in the apoptosis signaling pathway. An increased expression level of miR-1470 was observed in ESCC tissues compared with that in paracancerous tissues. Knockdown of miR-1470 significantly suppressed proliferation, and down-regulated the cell cycle regulatory gene cyclin E1. It was also revealed that knockdown of miR-1470 significantly inhibited migration, and decreased the expression levels of matrix metalloproteinase 2 (MMP2), MMP13 and MMP14. Western blotting analysis revealed that knockdown of miR-1470 induced apoptosis by increasing B-cell lymphoma 2 (Bcl-2) expression. The results of the present study suggest that overexpression of miR-1470 in ESCC promotes cancer cell proliferation by accelerating the cell cycle and inhibiting apoptosis, and also enhances cancer cell migration by upregulating MMPs.

Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes and candidate copy number driving genes in esophageal squamous cell carcinoma (ESCC). We used array comparative genomic hybridization to identify recurrent genomic alterations and screened the candidate targets of selected amplification regions by quantitative and semi-quantitative RT-PCR. Thirty-four gains and 16 losses occurred in more than 50 % of ESCCs. High-level amplifications at 7p11.2, 8p12, 8q24.21, 11q13.2-q13.3, 12p11.21, 12q12 and homozygous deletions at 2q22.1, 8p23.1-p21.2, 9p21.3 and 14q11.2 were also identified. 11q13.2 was a frequent amplification region, in which five genes including CHKA, GAL, KIAA1394, LRP5 and PTPRCAP were overexpressed in tumor tissues than paracancerous normal tissues. The expression of ALG3 at 3q27.1 was higher in ESCCs, especially in patients with lymph node metastasis. Target gene identification of amplifications or homozygous deletions will help to reveal the mechanism of tumor formation and explore new therapy method.

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 β-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 β-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/β-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 β-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/β-catenin in anoikis resistance of ESCC cells. Clin Cancer Res; 17(13); 4285–95. ©2011 AACR.

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.

MicroRNAs (miRNAs) play important roles in the progression of human cancer including esophageal squamous cell carcinoma (ESCC). Although previous reports showed that miR-125b-5p was down-regulated in ESCC, the roles and mechanisms of loss of function of miR-125b-5p in ESCC were still unknown. Using microRNA microarray and GEO datasets, we found and confirmed that miR-125b-5p was down-regulated in ESCC tissues. In-vitro assays showed that ectopic miR-125b-5p expression repressed cell proliferation, migration and invasion, and induced cell senescence. We also found that miR-125b-5p reduced the expressions of cell cycle regulatory genes including CCNA2, CCND1 and CCNE1, and regulated the markers of epithelial to mesenchymal transition (EMT) including E-cadherin, N-cadherin and EMT associated transcription factor Slug, and also decreased the MMPs including MMP2, MMP7 and MMP13. Furthermore, the candidate target gene HMGA2 was negatively regulated by miR-125b-5p both in mRNA and protein levels. Importantly, knockdown of HMGA2 partially phenocopied the effects of miR-125b-5p overexpression on cell cycle regulators and EMT markers. In conclusion, our results suggested that overexpression of miR-125b-5p inhibited cell proliferation, migration and invasion partially by down-regulating HMGA2 in ESCC.

BACKGROUND: Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes in pancreatic cancer. MATERIALS AND METHODS: We used array comparative genomic hybridization (array CGH) to identify recurrent genomic alterations and validated the protein expression of selected genes by immunohistochemistry. RESULTS: Sixteen gains and thirty-two losses occurred in more than 30% and 60% of the tumors, respectively. High-level amplifications at 7q21.3-q22.1 and 19q13.2 and homozygous deletions at 1p33-p32.3, 1p22.1, 1q22, 3q27.2, 6p22.3, 6p21.31, 12q13.2, 17p13.2, 17q21.31 and 22q13.1 were identified. Especially, amplification of AKT2 was detected in two carcinomas and homozygous deletion of CDKN2C in other two cases. In 15 independent validation samples, we found that AKT2 (19q13.2) and MCM7 (7q22.1) were amplified in 6 and 9 cases, and CAMTA2 (17p13.2) and PFN1 (17p13.2) were homozygously deleted in 3 and 1 cases. AKT2 and MCM7 were overexpressed, and CAMTA2 and PFN1 were underexpressed in pancreatic cancer tissues than in morphologically normal operative margin tissues. Both GISTIC and Genomic Workbench software identified 22q13.1 containing APOBEC3A and APOBEC3B as the only homozygous deletion region. And the expression levels of APOBEC3A and APOBEC3B were significantly lower in tumor tissues than in morphologically normal operative margin tissues. Further validation showed that overexpression of PSCA was significantly associated with lymph node metastasis, and overexpression of HMGA2 was significantly associated with invasive depth of pancreatic cancer. CONCLUSION: These recurrent genomic changes may be useful for revealing the mechanism of pancreatic carcinogenesis and providing candidate biomarkers.

Background At present no objective parameters to identify the risk of liver metastasis after surgery have been established in rectal cancer. Objective To identify the chromosomal aberrations that are correlated with liver metastasis of rectal cancer. Methods Primary tumor tissues of rectal carcinoma were analyzed by array-based comparative genomic hybridization (array-CGH). Genomic aberrations were identified by Genomic Workbench and MD-SeeGH. Results The most frequent gains in rectal cancer were at 20q11.21-q13.33, 8q11.21-q24.3, 13q12.11-q14.2 and losses in 5q13.2, 8p23.3-p22, 17p13.3-p13.2 and 18q11.2-q23. Seven amplifications at 6p21.1, 8q24.21, 8q24.3, 13q13.2 and 20q13.2-q13.32 and nine homozygous deletions at 1q31.3, 4q12-q13.1, 4q32.3-q33, 5q13.2, 8p23.2, 8q11.23, 16p13.2, 19p13.11 and 19q13.41 were identified. Both frequency plot comparison and SAM (Significance analysis of microarray) methods indicated that losses at 1p35.3, 4p14, 14q23.1-q32.11 and 18p11.32-p11.21 were more frequent in patients without liver metastasis. Conclusions These liver metastasis associated genomic changes may be useful to reveal the mechanism of metastasis and identify candidate biomarkers.

The aim of the present study was to screen and identify the chromosomal aberrations that are correlated with clinicopathological characteristics of rectal cancer using array-based comparative genomic hybridization (array-CGH). Forty-eight fresh frozen tumor tissues of rectal carcinoma were analyzed by array-CGH. The results showed that most frequent gains included 8q24.3, 20q11.21-q13.32, 20q13.33 and losses in 8p23.3-p12, 17p13.1-p12 and 18q11.2-q23 were noted. Fourteen amplifications and seven homozygous deletions were identified in the rectal cancer samples. Losses of 4p16.1-p15.31, 8p21.1-p12 and gains of 7p12.3-p12.1 and 13q33.1-q34 were associated with positive lymph node status and advanced clinical stage (stages III and IV). The 17q24.2-25.3 gain was more frequent in patients with distant metastasis. Integrated analysis indicated that overexpression of PDP1, TRIB1, C13orf27, FOXA2, PMEPA1 and PHACTR3 was associated with gains, and underexpression of FHOD, SMAD4 and BCL2 was associated with losses. Pathway enrichment analysis showed that pathways of nitrogen metabolism, oxidative phosphorylation, cell cycle, maturity onset diabetes of young, cytokine-cytokine receptor interaction, MAPK signaling pathway and dentatorubropallidoluysian atrophy were influenced by copy number changes.

Background Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC). Methods Using multiplex-fluorescence in situ hybridization (M-FISH) and oligo array-based comparative hybridization (array-CGH), we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. Results M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134) of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM) (P = 0.004 and 0.022) and advanced stages (P = 0.004 and 0.039). Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026). Conclusions The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

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. Mol Cancer Res; 9(4); 390–402. ©2011 AACR.

PLK1 is essential for the maintenance of genomic stability during mitosis. In our study, we found that overexpression of PLK1 was an independent prognostic factor (RR=4.253, p=0.020) and significantly correlated with survivin, an antiapoptotic protein, in esophageal squamous cell carcinoma (ESCC). Reverse transcription-polymerase chain reaction and fluorescence in situ hybridization (FISH) revealed upregulation of PLK1 mRNA and amplification of PLK1 gene, respectively. Depletion of PLK1 activated the intrinsic apoptotic pathway, which was substantiated by loss of mitochondrial membrane potential, reduction of Mcl-1 and Bcl-2 as well as activation of caspase-9. Coimmunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin and PLK1 depletion led to downregulation of survivin. Cotransfection of survivin constructs could partially reverse PLK1-depletion-induced apoptosis. These data suggest that PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC. Survivin is probably involved in antiapoptotic function of PLK1.