BACH1-induced ferroptosis drives lymphatic metastasis by repressing the biosynthesis of monounsaturated fatty acids.

Esophageal squamous cell carcinoma (ESCC) is one of the fatal malignancies worldwide. It has an increased propensity to metastasize via lymphogenous routes in an early stage. The prognosis of patients with lymph node metastases (LNM) is often worse than that of patients without metastases. Although several factors have been found to influence metastasis, the mechanisms of preference for specific metastatic routes remain poorly understood. Herein, we provide evidence that the intrinsic hypersensitivity of tumor cells to ferroptosis may proactively drive lymphatic metastasis. Serum autoantibodies associated with LNM of early ESCC were screened using a whole-proteome protein array containing 19 394 human recombinant proteins, and an anti-BACH1 autoantibody was first identified. Pan-cancer analysis of ferroptosis-related genes with preferential lymphatic metastasis and preferential hematogenous metastasis based on The Cancer Genome Atlas data was performed. Only BACH1 showed significant overexpression in tumors with preferential lymphatic metastasis, whereas it was downregulated in most tumors with preferential nonlymphatic metastasis. In addition, it was found that the serum levels of autoantibodies against BACH1 were elevated in early-stage patients with LNM. Interestingly, BACH1 overexpression and ferroptosis induction promoted LNM but inhibited hematogenous metastasis in mouse models. Transcriptomic and lipidomic analyses found that BACH1 repressed SCD1-mediated biosynthesis of monounsaturated fatty acids, especially oleic acid (OA). OA significantly attenuated the ferroptotic phenotypes and reversed the metastatic properties of BACH1-overexpressing cells. OA addition significantly rescued the ferroptotic phenotypes and reversed the metastatic properties of BACH1-overexpressing cells. Importantly, the concentration gradient of OA between primary lesions and the lymph resulted in the chemoattraction of tumor cells to promote invasion, thus facilitating lymphatic metastasis. BACH1-induced ferroptosis drives lymphatic metastasis via the BACH1-SCD1-OA axis. More importantly, this study confirms that ferroptosis is a double-edged sword in tumorigenesis and tumor progression. The clinical application of ferroptosis-associated agents requires a great caution.

Noninvasive urinary protein signatures associated with colorectal cancer diagnosis and metastasis.

Currently, imaging, fecal immunochemical tests (FITs) and serum carcinoembryonic antigen (CEA) tests are not adequate for the early detection and evaluation of metastasis and recurrence in colorectal cancer (CRC). To comprehensively identify and validate more accurate noninvasive biomarkers in urine, we implement a staged discovery-verification-validation pipeline in 657 urine and 993 tissue samples from healthy controls and CRC patients with a distinct metastatic risk. The generated diagnostic signature combined with the FIT test reveals a significantly increased sensitivity (+21.2% in the training set, +43.7% in the validation set) compared to FIT alone. Moreover, the generated metastatic signature for risk stratification correctly predicts over 50% of CEA-negative metastatic patients. The tissue validation shows that elevated urinary protein biomarkers reflect their alterations in tissue. Here, we show promising urinary protein signatures and provide potential interventional targets to reliably detect CRC, although further multi-center external validation is needed to generalize the findings.

Acetylation-induced PCK isoenzyme transition promotes metabolic adaption of liver cancer to systemic therapy.

Sorafenib and lenvatinib are approved first-line targeted therapies for advanced liver cancer, but most patients develop acquired resistance. Herein, we found that sorafenib induced extensive acetylation changes towards a more energetic metabolic phenotype. Metabolic adaptation was mediated via acetylation of the Lys-491 (K491) residue of phosphoenolpyruvate carboxykinase isoform 2 (PCK2) (PCK2-K491) and Lys-473 (K473) residue of PCK1 (PCK1-K473) by the lysine acetyltransferase 8 (KAT8), resulting in isoenzyme transition from cytoplasmic PCK1 to mitochondrial PCK2. KAT8-catalyzed PCK2 acetylation at K491 impeded lysosomal degradation to increase the level of PCK2 in resistant cells. PCK2 inhibition in sorafenib-resistant cells significantly reversed drug resistance in vitro and in vivo. High levels of PCK2 predicted a shorter progression-free survival time in patients who received sorafenib treatment. Therefore, acetylation-induced isoenzyme transition from PCK1 to PCK2 contributes to resistance to systemic therapeutic drugs in liver cancer. PCK2 may be an emerging target for delaying tumor recurrence.

PURα mediates epithelial-mesenchymal transition to promote esophageal squamous cell carcinoma progression by regulating Snail2.

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal cancers in the world. Dysregulation of purine-rich element binding protein alpha (PURα), which contributes to the initiation of PURΑ syndrome, is reportedly involved in the progression of multiple cancers, but its function and underlying mechanisms in ESCC progression remain unclear. Here, we first demonstrated that PURα promoted cell growth, migration and invasion in ESCC both in vitro and in vivo. An immunohistochemistry assay was then performed on 225 ESCC tissues, showing that high PURα expression was positively associated with lymph node metastasis and the AJCC stage, and the ESCC patients with positive PURα expression had worse survival. In addition, RNA sequencing implied that PURα induced epithelial-mesenchymal transition (EMT) in ESCC, which was further confirmed by qPCR, Western blotting and immunofluorescence analyses. Mechanistically, PURα enhanced the transcription of Snail2 by binding to its promoter region. Knockdown of Snail2 reversed PURα-induced EMT and inhibited the migration and invasion of ESCC cells. In conclusion, this study indicated that PURα promotes Snail2 transcriptional activity to induce EMT during ESCC progression.

Mutant p53 increases exosome-mediated transfer of miR-21-3p and miR-769-3p to promote pulmonary metastasis.

OBJECTIVE: Tumor metastasis is a complex, multistep process that depends on tumor cells and their communication with the tumor microenvironment. A p53 gain-of-function mutant has been shown to enhance the tumorigenesis, invasion, and metastasis abilities of tumor cells. This study aimed to investigate the roles of p53 R273H mutation in the tumor microenvironment. METHODS: The in vitro and in vivo effects of the p53 R273H mutant on the invasion and metastasis of HCT116 cells were investigated. Exosomes from wild-type and HCT116-TP53(R273H) cells were cocultured with mouse embryonic fibroblasts (MEFs). The roles of differentially expressed exosomal microRNAs identified by microarray analysis were investigated. The functions of the p53 R273H mutant in tumor cells were also investigated via gene expression microarray and quantitative polymerase chain reaction (qPCR) analyses. RESULTS: Introducing p53 R273H mutant into HCT116 cells significantly potentiated pulmonary metastasis in vivo. In the presence of exosomes derived from HCT116-TP53(R273H) cells, the exosomes were taken up by MEFs and became activated. Microarray analysis showed that the p53 R273H mutation increased the exosomal levels of miR-21-3p and miR-769-3p. Intriguingly, in clinical samples, miR-21-3p and miR-769-3p levels were significantly higher in patients with a p53 mutation than in those without this mutation. Furthermore, both miR-21-3p and miR-769-3p activated fibroblasts and exerted a synergistic effect via their target genes on the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. The activated fibroblasts excreted cytokine TGF-ß and may have reciprocally induced cancer cells to undergo epithelial-mesenchymal transition (EMT). Indeed, HCT116-TP53(R273H) cells showed increased expression of ZEB1 and SNAI2 and decreased transcription of several cell adhesion molecules. CONCLUSIONS: The mutant p53-exosomal miR-21-3p/miR-769-3p-fibroblast-cytokine circuit appears to be responsible for communication between tumor and stromal cells, with exosomal miRNAs acting as a bridge. miR-21-3p and miR-769-3p are potential predictive markers of pulmonary metastasis and candidate targets for therapeutic interventions.

A novel rapid quantitative method reveals stathmin-1 as a promising marker for esophageal squamous cell carcinoma.

Stathmin-1 is a microtubule depolymerization protein that regulates cell division, growth, migration, and invasion. Overexpression of stathmin-1 has been observed to be associated with metastasis, poor prognosis, and chemoresistance in various human cancers. Our previous studies found that serum stathmin-1 was significantly elevated in patients with esophageal squamous cell carcinoma (ESCC) by ELISAs. Here, we constructed high-affinity monoclonal antibodies and then developed a competitive AlphaLISA for rapid, accurate quantitation of stathmin-1 in serum. Compared to ELISA, our homogeneous AlphaLISA showed better sensitivity and accuracy, a lower limit of detection, and a wider linear range. The measurements of nearly 1000 clinical samples showed that serum stathmin-1 level increased dramatically in patients with squamous cell carcinoma (SCC), especially in ESCC, with a sensitivity and a specificity of 81% and 94%, respectively. Even for early stage ESCC, stathmin-1 achieved an area under the receiver operating characteristic curve (AUC) of 0.88. Meanwhile, raised concentrations of stathmin-1 were associated with lymph node metastasis and advanced cancer stage. Notably, various types of SCC showed significantly higher AUCs in serum stathmin-1 detection compared to adenocarcinoma. Furthermore, we confirmed that stathmin-1 was enriched in the oncogenic exosomes, which can explain the reason why it enters into the blood to serve as a tumor surrogate. In conclusion, this large-scale and systematic study of serum stathmin-1 measured by our newly established AlphaLISA showed that stathmin-1 is a very promising diagnostic and predictive marker for SCC in the clinic, especially for ESCC.

Novel carbazole sulfonamide microtubule-destabilizing agents exert potent antitumor activity against esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide due to its chemoresistance and poor prognosis. Currently, there is a lack of effective small molecule drugs for the treatment of ESCC. Microtubules are an attractive target for cancer therapy since they play a central role in various fundamental cell functions. We investigated the anti-ESCC activity and mechanisms of the small molecule tubulin ligands, SL-3-19 and SL-1-73, which are two carbazole sulfonamide derivatives, in vitro and in vivo for the first time. These drugs were previously screened from a small molecule library with over 450 compounds and optimized for high aqueous solubility [1,2]. Here, we reveal the promising activities of these compounds against esophageal cancer. Mechanistically, both SL-3-19 and SL-1-73 inhibited ESCC cell growth by inducing cell apoptosis and arresting the cell cycle at G2/M phase in a dose-dependent manner. These drugs effectively inhibited microtubule assembly, greatly disrupted microtubule maturation by down-regulating acetylated α-tubulin, and significantly disrupted the vascular structure by obstructing the formation of capillary-like tubes in vitro. Consistent with their in vitro activities, SL-3-19 and SL-1-73 inhibited the growth of ESCC xenografts and inhibited the microvessel density in vivo. In summary, SL-3-19 and SL-1-73 are novel microtubule-destabilizing agents that have a potential antitumor effect on ESCC both in vitro and in vivo, and SL-3-19 had a higher activity than SL-1-73, with a low IC50 value and an observable antitumor activity in vivo. These results indicate that SL-3-19 may be a new therapeutic candidate for ESCC treatment.

Overexpression of stathmin plays a pivotal role in the metastasis of esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is a serious malignant tumor that affects human health. We analyzed the correlation between serum stathmin level and ESCC and elucidated the molecular mechanisms of stathmin's promotion of ESCC cell invasion and metastasis. METHODS: Stathmin level in ESCC and healthy control serum were detected by enzyme-linked immunosorbent assay (ELISA), and the clinical parameters were analyzed. We established ESCC cells with stathmin overexpression or knockdown and then evaluated the effects of stathmin on invasion and metastasis in ESCC. Differentially expressed genes were analyzed by Human Transcriptome Array and confirmed by RT-PCR. The expression levels of the integrin family, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) were detected by immunoblotting. RESULTS: Serum levels of stathmin were significantly higher in ESCC than in control serum and associated with lymph node metastasis, tumor stage and size. Furthermore, we found that stathmin promoted migration and invasion of ESCC cells in vitro and in vivo. In addition, we confirmed that the activation of the integrinα5ß1/FAK/ERK pathway is increased in stathmin-overexpression cells and accelerates cell motility by enhancing cell adhesion ability. CONCLUSION: Stathmin may predict a potential metastasis biomarker for ESCC.

Environmental factors, seven GWAS-identified susceptibility loci, and risk of gastric cancer and its precursors in a Chinese population.

Gene-environment interactions may increase gastric cancer (GC) risk. Seven susceptibility loci identified by genome-wide association studies (GWASs) suggest that genetic factors play a role in gastric carcinogenesis. Meanwhile, Helicobacter pylori (H. pylori) infection, smoking, and alcohol drinking are also important environmental factors for gastric cancer. However, studies to explore the role of gene-environment interactions in gastric carcinogenesis, and particularly the relationship between the seven susceptibility loci and their potential interactions with H. pylori infection, smoking, and alcohol drinking in risk of GC, and severe intestinal metaplasia (IM)/dysplasia, have been inconclusive. A total of 1273 subjects in a Chinese population were recruited, and genotyping was carried out using the competitive allele-specific PCR (KASP) method. Unconditional logistic regression was applied to model the associations between genetic polymorphisms and the disease risk. Effect modifications by H. pylori infection, smoking and alcohol drinking were evaluated. PSCA rs2294008/rs2976392 showed a significant, multiplicative interaction with H. pylori infection in risk of GC. Meanwhile, PRKAA1 rs13361707 had an additive interaction with H. pylori infection. SLC52A3 rs13042395 showed an interaction with alcohol drinking in risk of GC. Moreover, three SNPs, MUC1 rs4072037, ZBTB20 rs9841504 and PRKAA1 rs13361707, were associated with precancerous gastric lesions (severe IM/dysplasia). Our data suggest that genetic predisposition factors identified by GWAS may interact with environmental risk factors, Particularly for H. pylori infection and alcohol consumption, to increase the risk of GC.

NDRG1 overexpression promotes the progression of esophageal squamous cell carcinoma through modulating Wnt signaling pathway.

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however ß-catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and ß-catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and  ß-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

A novel TP53 pathway influences the HGS-mediated exosome formation in colorectal cancer.

Tumor-derived exosomes are important for cell-cell communication. However, the role of TP53 in the control of exosome production in colorectal cancer (CRC) is controversial and unclear. The features of exosomes secreted from HCT116 TP53-wild type (WT), TP53-knockout (KO) and constructed TP53 (R273H)-mutant (MT) cells were assessed. The exosomes from the MT and KO cells exhibited significantly reduced sizes compared with the WT cells. A comprehensive proteomic analysis of exosomal proteins was performed using the isobaric tag for relative and absolute quantitation (iTRAQ)-2D-LC-MS/MS strategy. A total of 3437 protein groups with ≥2 matched peptides were identified. Specifically, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) was consistently down-regulated in the exosomes from the MT and KO cells. Functional studies demonstrated that low HGS levels were responsible for the decreased exosome size. TP53 regulated HGS expression and thus HGS-dependent exosome formation. Furthermore, the HGS expression was gradually increased concomitant with CRC carcinogenesis and was an independent poor prognostic factor. In conclusion, a novel HGS-dependent TP53 mechanism in exosome formation was identified in CRC. HGS may serve as a novel prognostic biomarker and a candidate target for therapeutic interventions.

Novel circulating peptide biomarkers for esophageal squamous cell carcinoma revealed by a magnetic bead-based MALDI-TOFMS assay.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application.

Aberrant expression of peroxiredoxin 1 and its clinical implications in liver cancer.

AIM: To investigate the expression characteristics of peroxiredoxin 1 (PRDX1) mRNA and protein in liver cancer cell lines and tissues. METHODS: The RNA sequencing data from 374 patients with liver cancer were obtained from The Cancer Genome Atlas. The expression and clinical characteristics of PRDX1 mRNA were analyzed in this dataset. The Kaplan-Meier and Cox regression survival analysis was performed to determine the relationship between PRDX1 levels and patient survival. Subcellular fractionation and Western blotting were used to demonstrate the expression of PRDX1 protein in six liver cancer cell lines and 29 paired fresh tissue specimens. After bioinformatics prediction, a putative post-translational modification form of PRDX1 was observed using immunofluorescence under confocal microscopy and immunoprecipitation analysis in liver cancer cells. RESULTS: The mRNA of PRDX1 gene was upregulated about 1.3-fold in tumor tissue compared with the adjacent non-tumor control (P = 0.005). Its abundance was significantly higher in men than women (P < 0.001). High levels of PRDX1 mRNA were associated with a shorter overall survival time (P = 0.04) but not with recurrence-free survival. The Cox regression analysis demonstrated that patients with high PRDX1 mRNA showed about 1.9-fold increase of risk for death (P = 0.03). In liver cancer cells, PRDX1 protein was strongly expressed with multiple different bands. PRDX1 in the cytosol fraction existed near the theoretical molecular weight, whereas two higher molecular weight bands were present in the membrane/organelle and nuclear fractions. Importantly, the theoretical PRDX1 band was increased, whereas the high molecular weight form was decreased in tumor tissues. Subsequent experiments revealed that the high molecular weight bands of PRDX1 might result from the post-translational modification by small ubiquitin-like modifier-1 (SUMO1). CONCLUSION: PRDX1 was overexpressed in the tumor tissues of liver cancer and served as an independent poor prognostic factor for overall survival. PRDX1 can be modified by SUMO to play specific roles in hepatocarcinogenesis.

YM155, a survivin suppressant, triggers PARP-dependent cell death (parthanatos) and inhibits esophageal squamous-cell carcinoma xenografts in mice.

Here we demonstrated that sepantronium bromide (YM155), a survivin suppressant, inhibited esophageal squamous-cell carcinoma (ESCC) growth in mice bearing human ESCC xenografts without affecting body weight. In cell culture, YM155 decreased survivin levels and caused PARP-1 activation, poly-ADP polymer formation, and AIF translocation from the cytosol to the nucleus. Genetic knockdown of PARP-1 or AIF abrogated YM155-induced parthanatos cell death. Furthermore, FOS, JUN and c-MYC gene transcription, which is stimulated by activated PARP-1, was increased following YM155 treatment. Our data demonstrate that YM155 did not trigger apoptosis, but induced parthanatos, a cell death dependent on PARP-1 hyper-activation, and support clinical development of YM155 in ESCC.

SELEX aptamer used as a probe to detect circulating tumor cells in peripheral blood of pancreatic cancer patients.

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15+/CD45- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.

A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces cell autophagy and apoptosis in esophageal squamous cell carcinoma cells via up-regulation of BNIP3 and N-myc downstream-regulated gene-1.

The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate is an antioxidant found in the testa of peanut seeds. Previous studies have shown that ethyl-3,4-dihydroxybenzoate can effectively reduce breast cancer cell metastasis by inhibiting prolyl-hydroxylase. In this study, we investigated the cytotoxic effect of ethyl-3,4-dihydroxybenzoate on esophageal squamous cell carcinoma cells in vitro and identified key regulators of ethyl-3,4-dihydroxybenzoate-induced esophageal cancer cell death through transcription expression profiling. Using flow cytometry analysis, we found that ethyl-3,4-dihydroxybenzoate induced S phase accumulation, a loss in mitochondrial membrane permeabilization, and caspase-dependent apoptosis. Moreover, an expression profile analysis identified 46 up- and 9 down-regulated genes in esophageal cancer KYSE 170 cells treated with ethyl-3,4-dihydroxybenzoate. These differentially expressed genes are involved in several signaling pathways associated with cell cycle regulation and cellular metabolism. Consistent with the expression profile results, the transcriptional and protein expression levels of candidate genes NDRG1, BNIP3, AKR1C1, CCNG2 and VEGFA were found to be significantly increased in treated KYSE 170 cells by reverse-transcription PCR and western blot analysis. We also found that protein levels of hypoxia-inducible factor-1α, BNIP3, Beclin and NDRG1 were increased and that enriched expression of BNIP3 and Beclin caused autophagy mediated by microtubule-associated protein 1 light chain 3 in the treated cells. Autophagy and apoptosis were activated together in esophageal cancer cells after exposed to ethyl-3,4-dihydroxybenzoate. Furthermore, knock-down of NDRG1 expression by siRNA significantly attenuated apoptosis in the cancer cells, implying that NDRG1 may be required for ethyl-3,4-dihydroxybenzoate-induced apoptosis. Together, these results suggest that the cytotoxic effects of ethyl-3,4-dihydroxybenzoate were mediated by the up-regulation of NDRG1, BNIP3, Beclin and hypoxia-inducible factor-1α, initiating BNIP3 and Beclin mediated autophagy at an early stage and ultimately resulting in esophageal cancer cell apoptosis.

Receptor interactive protein kinase 3 promotes Cisplatin-triggered necrosis in apoptosis-resistant esophageal squamous cell carcinoma cells.

Cisplatin-based chemotherapy is currently the standard treatment for locally advanced esophageal cancer. Cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, but the mechanism by which programmed necrosis is induced remains unknown. In this study, we provide evidence that cisplatin induces necrotic cell death in apoptosis-resistant esophageal cancer cells. This cell death is dependent on RIPK3 and on necrosome formation via autocrine production of TNFα. More importantly, we demonstrate that RIPK3 is necessary for cisplatin-induced killing of esophageal cancer cells because inhibition of RIPK1 activity by necrostatin or knockdown of RIPK3 significantly attenuates necrosis and leads to cisplatin resistance. Moreover, microarray analysis confirmed an anti-apoptotic molecular expression pattern in esophageal cancer cells in response to cisplatin. Taken together, our data indicate that RIPK3 and autocrine production of TNFα contribute to cisplatin sensitivity by initiating necrosis when the apoptotic pathway is suppressed or absent in esophageal cancer cells. These data provide new insight into the molecular mechanisms underlying cisplatin-induced necrosis and suggest that RIPK3 is a potential marker for predicting cisplatin sensitivity in apoptosis-resistant and advanced esophageal cancer.

Identification of tumor antigens that elicit a humoral immune response in the sera of Chinese esophageal squamous cell carcinoma patients by modified serological proteome analysis.

Our aim was to identify novel tumor-associated antigens from the esophageal squamous cell carcinoma (ESCC) cell line EC0156, and related autoantibodies in sera from patients with ESCC. We used modified serological proteome analysis, involving one- and two-dimensional electrophoresis, Western blot, and MALDI-TOF/TOF-MS to identify 6 ESCC-associated antigens. From these, 105 kDa heat shock protein (HSP105) and triosephosphate isomerase (TIM) were further evaluated and we determined they could induce autoantibody responses in ESCC sera and are highly expressed in ESCC tissues. Anti-HSP105 and anti-TIM autoantibodies were found in 39.1% (18/46) and 34.8% (16/46) of patients with ESCC, respectively, but only in two controls. A receiver operating characteristic curve constructed with HSP105 and TIM gave a sensitivity of 54.3% and 95% (38/40) specificity in discriminating ESCC from matched controls. Interestingly, we found that autoantibodies against TIM in ESCC serum mainly reacted with glycosylated but not deglycosylated TIM. The preliminary results suggest the potential utility of screening autoantibodies in sera for use as biomarkers for cancer diagnosis.

Proteome atlas of human chromosome 8 and its multiple 8p deficiencies in tumorigenesis of the stomach, colon, and liver.

Chromosome 8, a medium-length euchromatic unit in humans that has an extraordinarily high mutation rate, can be detected not only in evolution but also in multiple mutant diseases, such as tumorigenesis, and further invasion/metastasis. The Chromosome-Centric Human Proteome Project of China systematically profiles the proteomes of three digestive organs (i.e., stomach, colon, and liver) and their corresponding carcinoma tissues/cell lines according to a chromosome organizational roadmap. By rigorous standards, we have identified 271 (38.7%), 330 (47.1%), and 325 (46.4%) of 701 chromosome 8-coded proteins from stomach, colon, and liver samples, respectively, in Swiss-Prot and observed a total coverage rate of up to 58.9% by 413 identified proteins. Using large-scale label-free proteome quantitation, we also found some 8p deficiencies, such as the presence of 8p21-p23 in tumorigenesis of the above-described digestive organs, which is in good agreement with previous reports. To our best knowledge, this is the first study to have verified these 8p deficiencies at the proteome level, complementing genome and transcriptome data.

Oncogenic but non-essential role of N-myc downstream regulated gene 1 in the progression of esophageal squamous cell carcinoma.

N-myc downstream regulated gene 1 (NDRG1/Cap43/Drg-1) has previously been shown to be dysregulated in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the role of NDRG1 in the neoplastic progression of ESCC using ectopic gain-of-function and loss-of-function approaches. Stable transfectants of the KYSE30 ESCC cell line with altered NDRG1 levels were generated by lentiviral transduction. Although no measurable effects on in vitro cell proliferation were observed with altered NDRG1 expression, the ectopic overexpression of NDRG1 was positively linked to recognized markers of metastasis, angiogenesis and apoptotic evasion. Accordingly, in the nude mouse xenograft model system, NDRG1 overexpression promoted the in vivo growth of KYSE30 derived xenografts, which could be attributed to the reduced apoptotic and enhanced angiogenic activities associated with this gene. These processes were mediated in part by increased NFκB activity in NDRG1 overexpressing cells. Nevertheless, no significant phenotypic changes were observed in response to NDRG1 knock-down, suggesting that this gene might not be essential for the neoplastic progression of ESCC. Taken together, our results suggest that NDRG1 may play positive but dispensable roles in the progression of esophageal squamous cell carcinoma.