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PDF file - 1061K, S Figure 1. Down-regulation of CRT expression reduces PTP1B mRNA and protein expression in KYSE510 cells. S2. PTP1B promotes ESCC cell invasion and migration in KYSE510 cells. KYSE510 cells were transfected with PTP1B-specific siRNA (siRNA PTP1B) or non-targeting siRNA control (siRNA cont). S3. CRT regulates cell motility through PTP1B in ESCC cells. (A-C) 1x105 KYSE150 cells were seeded on 24-well plates with three replicates. S4. Cancer-inflammatory response was minimal in mouse model. S5. CRT regulates PTP1B transcription through Stat5a. S6. The expression of CRT and PTP1B in 176 ESCC tissues was evaluated by immunohistochemistry. S7. Correlation of PTP1B expression with overall survival in patients with CRT high-expressing tumors. S8. Model of CRT-dependent signaling pathway in ESCC cell metastasis. Supplementary Table 1. The candidate genes altered in CRT-siRNA KYSE150 cells as compared with non-targeting siRNA cells detected by cDNA array

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.

Supplementary Figures S1-S5.

Calreticulin (CRT) is a Ca2+-binding chaperone protein that alters cellular Ca2+-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. Mol Cancer Res; 11(9); 986–94. ©2013 AACR.

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins including p27kip1, p21cip1, p57kip2, p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway. (Mol Cancer Res 2009;7(1):12–22)

Supplementary Data from Suppression of Anoikis by SKP2 Amplification and Overexpression Promotes Metastasis of Esophageal Squamous Cell Carcinoma

Purpose: We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1.Experimental Design: Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC).Results: Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10−5). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025).Conclusions: Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC. Clin Cancer Res; 20(23); 6153–62. ©2014 AACR.

Effect of ANO1 on cell proliferation. Knock-down the expression of ANO1 inhibited the cell proliferation of KYSE30 (A) and KYSE510 cells (B). The absorbance at 450 nm was measured.

Supplementary Table S2 Metabolic gene with CNV larger than 20%.

Table S1-Summary of HCC clinical cohorts and their omics datasets. Table S2-Primers and oligoes used in this study

Supplementary Table 2. Relationship between NRP1/CRT co-expression and lymph node metastasis in 216 ESCC patients

Supplementary Figure 2. Quantitative real-time PCR analysis of STAT5A and STAT5B expression in ESCC cells transiently transfected with non-silencing siRNA, STAT5A siRNA or STAT5B siRNA. Columns, mean; Bars, SD (n = 3); **, P < 0.01.

Amplification and overexpression of MCM7 in ESCC. A. Amplification of MCM7 in array CGH study. B. Validation of MCM7 amplification by FISH. Left: two MCM7 signals in peripheral blood cells; Middle: two MCM7 signals in ESCC cells; Right: more than four MCM7 signals in ESCC cells. C. MCM7 overexpressed in ESCC by semi-quantitative RT-PCR. D. MCM7 overexpressed in ESCC by Western blotting. E. MCM7 overexpressed in ESCC by IHC.

The lower level of miR-192-5p was associated with poor prognosis of HCC patients.

Metabolic activities are often accompanied by cell-cycle progression, yet known connections between these two processes remain limited. Here, we identified the isocitrate dehydrogenase 3β (IDH3β) as a novel substrate of anaphase-promoting complex/cyclosome (APC/C)-CDH1 and an important regulator of the cell cycle. In esophageal squamous cell carcinoma (ESCC), IDH3β was posttranslationally upregulated in late G1 phase, and overexpression of IDH3β accelerated G1–S transition, contributing to the promotion of cell proliferation in vitro and in vivo. α-Ketoglutarate (α-KG), a crucial metabolite in tricarboxylic acid (TCA) cycle, was dependent on IDH3β level and partially accounted for IDH3β-mediated cell growth. IDH3β expression increased PFKFB3 protein levels and enhanced glucose uptake, and high expression of IDH3β correlated with poor survival in patients with ESCC, suggesting a potential application of IDH3β in prognosis. Overall, our results highlight a new molecular connection between cell-cycle regulation and the TCA cycle in ESCC.Significance:These findings show that IDH3β is an APC/C-CDH1 substrate and is expressed in a cell-cycle–dependent manner, highlighting novel molecular cross-talk between the TCA cycle and cell cycle in cancer cells.

Supplementary Data. Supplementary Materials and Methods, Supplementary Figure Legends

Frequency plot comparison analysis. A. Frequency plot comparison of ESCC with or without lymph node metastasis. B. Frequency plot comparison of ESCC at different stages.

Fig S1. The changing trend of α-KG, fumarate and malate were significantly correlated with S phase.Fig S2. IDH3β accumulated in late G1 phase and CDC20 was not the regulator of IDH3β. Fig S3. The interaction of IDH3β and CDH1 was strong in G1 and weak in G2/M. Fig .S4 IDH3β regulated the G1/S transition and cell proliferation. Fig .S5 Xenograft tumor formation of stable IDH3β overexpressed KYSE410 cells and control cells.Fig .S6 IDH3α, IDH3β and IDH3γ contribute to oxidative metabolism in TCA cycle.Fig .S7 IDH3β-dependent α-KG generation accounts for improving cell proliferation. Fig .S8 IDH3β enhances cytoplasmic PFKFB3 level in IDH3β overexpressed cell.

Tumorigenicity assay of HuH7 cells with altered miR-192-5p and PABPC4.

Supplementary Figures S1-S8; Supplementary Tables S1-S4.

The Up-stream regulation of miR-192-5p expression.

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.

Quantitative real-time PCR analysis，Proliferation assay and colony formation assay，Cell cycle analysis，Metabolite measurement.

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort (n = 241) for five groups of CSC+ HCC tissues, i.e., EpCAM+, CD90+, CD133+, CD44+, and CD24+ HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC+ HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC+ HCC from two independent cohorts (n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC+ HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC+ HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis.Significance:miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis.

Expression analysis of mature and precursor of miR-192-5p in HCC patients with different levels of multiple stem cell-related biomarkers, and miRNA sequence alignment analysis.

Supplementary Figure 3. Western blot analysis in ESCC cells treated with or without 100 μmol/L KN-93 for 24 hours.

Venn diagrams comparing differences in expression of our gene/microRNA expression assay and other microarray experiments. A. Comparison of differentially expressed genes in different experiments. B. Comparison of differentially expressed microRNAs in different experiments. â†', overexpressed; â†", underexpressed.

Genomic aberrations in esophgeal dysplasia and ESCC. A. Validation of 11q13.3 amplification by FISH. Left: 11q13.3 amplification detected by array CGH. Right: FISH results. B. Comparison of the number of aberrations between dysplasia and ESCC.

The distribution of CSC hepatic markers and the expression of CSC-miRNAs in HCC cohorts.

Supplementary Figure 1. Quantitative real-time PCR analysis of CRT expression in ESCC cells transiently transfected with non-silencing siRNA or CRT siRNA. Columns, mean; Bars, SD (n = 3); **, P < 0.01.

mRNA expression of ANO1 in ESCC cell lines. X. Name of ESCC cell lines. Y. The relative mRNA expression of ANO1 compared with GAPDH.

MiR-192-5p suppressed cell migration and cell invasion in HCC cells.

Supplementary Table 1. NRP1 expression positively correlates with CRT in ESCC

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. Clin Cancer Res; 19(21); 5867–78. ©2013 AACR.

It includes additional materials and methods, i.e., Plasmids, siRNAs, and LY294002 treatment; assays to examine cell malignancy features; qPCR and Western blot.

Supplement file: Materials and Methods

Supplementary Figure 3 from Amplification and Overexpression of CTTN (EMS1) Contribute to the Metastasis of Esophageal Squamous Cell Carcinoma by Promoting Cell Migration and Anoikis Resistance

Supplementary Figure 2 from Amplification and Overexpression of CTTN (EMS1) Contribute to the Metastasis of Esophageal Squamous Cell Carcinoma by Promoting Cell Migration and Anoikis Resistance

Supplementary Figure 1 from Amplification and Overexpression of CTTN (EMS1) Contribute to the Metastasis of Esophageal Squamous Cell Carcinoma by Promoting Cell Migration and Anoikis Resistance

Supplementary Figure Legends 1-3 from Amplification and Overexpression of CTTN (EMS1) Contribute to the Metastasis of Esophageal Squamous Cell Carcinoma by Promoting Cell Migration and Anoikis Resistance

Gain of chromosome 11q13 is a common event in esophageal squamous cell carcinoma (ESCC). The cortactin gene (CTTN, also EMS1), located at 11q13, plays a pivotal role in coupling membrane dynamics to cortical actin assembly. This gene has been implicated in the motility of several types of cells. In the present study, we found that the amplification and overexpression of the CTTN gene was associated with lymph node metastasis in ESCC. Functional analysis by small interfering RNA–mediated silencing of CTTN revealed that in addition to the effect on cell migration, CTTN influenced cell invasiveness by anoikis resistance. In vivo assay showed that inhibition of CTTN expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, we showed for the first time that the protective role of CTTN in anoikis resistance was correlated with the activation of the phosphatidylinositol 3-kinase/Akt pathway. Overall, the data suggest that CTTN is an oncogene in the 11q13 amplicon and exerts functions on tumor metastasis in ESCC. (Cancer Res 2006; 66(24): 11690-9)