Using proteomic approach to identify tumor-associated proteins as biomarkers in human esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. The lower survival rate of ESCC is attributed to late diagnosis and poor therapeutic efficacy; therefore, the identification of tumor-associated proteins as biomarkers for early diagnosis, and the discovery of novel targets for therapeutic intervention, seems very important for increasing the survival rate of ESCC. To identify tumor-associated proteins as biomarkers in ESCC, we have analyzed ESCC tissues and adjacent normal tissues by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The results showed that a total of 104 protein spots with different expression levels were found on 2DE, and 47 proteins were eventually identified by MALDI-TOF MS. Among these identified proteins, 33 proteins including keratin 17 (KRT17), biliverdin reductase B (BLVRB), proteasome activator subunit 1 (PSME1), manganese superoxide dismutase (MnSOD), high-mobility group box-1(HMGB1), heat shock protein 70 (HSP70), peroxiredoxin (PRDX1), keratin 13 (KRT13), and so on were overexpressed, and 14 proteins including cystatin B (CSTB), tropomyosin 2 (TPM2), annexin 1 (ANX1), transgelin (TAGLN), keratin 19 (KRT19), stratifin (SFN), and so on were down-expressed in ESCC. Biological functions of these proteins are associated with cell proliferation, cell motility, protein folding, oxidative stress, and signal transduction. In the subsequent study using immunoassay on ESCC serum samples and tissue-array slides, two representative proteins, HSP70 and HMGB1, were selected as examples for the purpose of validation. The results showed that both HSP70 and HMGB1 can induce autoantibody response in ESCC sera and have higher expression in ESCC tissues. Especially, the frequency of antibodies to HSP70 in ESCC sera was significantly higher than that in normal human sera. The preliminary results suggest that some of these identified proteins might contribute to esophageal cell differentiation and carcinogenesis, certain proteins could be used as tumor-associated antigen (TAA) biomarkers in cancer diagnosis, and further studies on these identified proteins should provide more evidence of how these proteins are involved in carcinogenesis of ESCC.     

RAD54 forms DNA repair foci in response to DNA damage in living plant cells

Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double‐strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA‐TELANGIECTASIA MUTATED–SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co‐localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.     

Inhibiting UHRF1 expression enhances radiosensitivity in human esophageal squamous cell carcinoma

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is a novel nuclear protein which is overexpressed in various cancers but not yet examined in esophageal squamous cell carcinoma (ESCC). The correlation between UHRF1 and the radioresistance in ESCC is still unclear. In the present study, the expression of UHRF1 was examined by immunohistochemistry in specimens of ESCC patients treated with radiotherapy. The results showed that UHRF1 was significantly overexpressed in ESCC specimens. Overexpression of UHRF1 correlated significantly with advanced T-stage, positive lymph node metastasis and poor differentiation. In addition, UHRF1 was associated with radiotherapy response, in which overexpression of UHRF1 was observed more frequently in the radioresistant group than in the effective group. At the molecular level, inhibition of UHRF1 by lentivirus-mediated shRNA targeting UHRF1 increased the radiosensitivity and apoptosis, while decreased radiation-induced G2/M phase arrest in TE-1 cells. Moreover, inhibition of UHRF1 resulted in higher residual γH2AX expression after irradiation, but not initial γH2AX. Further study showed that inhibition of UHRF1 down-regulated the endogenous expressions of DNA repair protein Ku70 and Ku80 in TE-1 cells, and significantly inhibited the increase of these proteins after irradiation. Above all, our data suggested that UHRF1 might play an important role in radioresistance of ESCC, and inhibition of UHRF1 can increase the radiosensitivity of TE-1 cells by altering cell cycle progression, enhancing apoptosis, and decreasing DNA damage repair capacity.     

L1CAM regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1

Glioblastomas (GBMs) are highly lethal brain tumours with current therapies limited to palliation due to therapeutic resistance. We previously demonstrated that GBM stem cells (GSCs) display a preferential activation of DNA damage checkpoint and are relatively resistant to radiation. However, the molecular mechanisms underlying the preferential checkpoint response in GSCs remain undefined. Here, we show that L1CAM (CD171) regulates DNA damage checkpoint responses and radiosensitivity of GSCs through nuclear translocation of L1CAM intracellular domain (L1-ICD). Targeting L1CAM by RNA interference attenuated DNA damage checkpoint activation and repair, and sensitized GSCs to radiation. L1CAM regulates expression of NBS1, a critical component of the MRE11-RAD50-NBS1 (MRN) complex that activates ataxia telangiectasia mutated (ATM) kinase and early checkpoint response. Ectopic expression of NBS1 in GSCs rescued the decreased checkpoint activation and radioresistance caused by L1CAM knockdown, demonstrating that L1CAM signals through NBS1 to regulate DNA damage checkpoint responses. Mechanistically, nuclear translocation of L1-ICD mediates NBS1 upregulation via c-Myc. These data demonstrate that L1CAM augments DNA damage checkpoint activation and radioresistance of GSCs through L1-ICD-mediated NBS1 upregulation and the enhanced MRN-ATM-Chk2 signalling.     

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage

Inhibition of cell division is critical for viability under DNA‐damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS‐induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C‐terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild‐type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C‐terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.     

Sibling rivalry in checkpoint control of cell cycle and DNA damage response

Comment on: Pabla N, et al. Proc Natl Acad Sci USA 2012; 109:197-202.     

Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma

Apoptosis is a natural form of cell death involved in many physiological changes in the cell. Defects in the process of apoptosis can lead to serious diseases. During some apoptotic pathways, proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin degradation together with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interacting partners like flap endonuclease 1, DNase I, and exonuclease III were already suggested, but also other interacting partners were proposed. We conducted a living-cell confocal fluorescence microscopy followed by an image analysis of fluorescence resonance energy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins during apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin degradation. The possible existence of a degradation complex consisting of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investigated.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.     

The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest,DNA damage response and autophagy in cancer cells

Chemotherapy- or radiotherapy-induced DNA damage activates the Chk1-dependent DNA damage response (DDR) and cell cycle checkpoints to facilitate cell survival. Numerous attempts have been made to identify specific Chk1 inhibitors to enhance the efficiency of chemotherapy or radiotherapy. In this study, we investigated the molecular mechanisms underlying the antitumor activity of LY2603618, a potent and selective small molecule inhibitor of Chk1 protein kinase, in human lung cancer cells. Treatment of cancer cells with LY2603618 caused cell cycle arrest in the G2/M phase. A marked induction of DDR, including the phosphorylation of ATM, Chk2, p53 and histone H2AX, was observed after LY2603618 treatment. LY2603618 inhibited Chk1 autophosphorylation (S296 Chk1) and increased DNA damage-mediated Chk1 phosphorylation (S345 Chk1). In addition, LY2603618-treated lung cancer cells transitioned from LC3-I to LC3-II, a hallmark of autophagy. Blocking autophagy with chloroquine (CQ) further enhanced LY2603618′s inhibitory effect on cell viability/proliferation. LY2603618 also significantly increased p38 and c-Jun N-terminal kinase (JNK) phosphorylation. Pretreatment with the JNK inhibitor reduced cleavage of caspase-3 and PARP levels in LY2603618-treated cells. These results suggest the following: (i) the biological consequences of LY2603618 in lung cancer cells is associated with both inhibition of Chk1 phosphorylation on S296 and activation of the DNA damage response network; and (ii) the anticancer property of LY2603618 might be increased by inhibiting autophagy.     

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.     

Gli1 interacts with YAP1 to promote tumorigenesis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the predominant esophageal cancer type in China. The aberrant activation of glioma-associated oncogene homolog1 (Gli1), a key factor in Hedgehog (Hh) signaling pathway, has been found in esophageal carcinoma. Moreover, Yes-associated protein 1 (YAP1), the major mediator of Hippo signaling pathway, has been linked to esophageal carcinoma progression. However, the precise roles and the underlying mechanism of both Gli1 and YAP1 in ESCC are unclear. Here, we found that Gli1 and YAP1 are overexpressed in ESCC and are associated with poor prognosis. In addition, we confirmed that knockdown of Gli1 or YAP1 suppresses ESCC cell growth, migration, and invasion in ESCC TE1 and EC109 cells. Significantly, Gli1 interacts with YAP1 in ESCC cells. Both Gli1 and YAP1 proteins are closely correlated with each other in human ESCC samples. Mechanistically, Gli1 upregulates YAP1 in a LATS1-independent manner. Conversely, YAP1 induces Gli1 by regulating phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Most importantly, we demonstrated that the interaction between Gli1 and YAP1 promotes ESCC tumor growth in vitro and in vivo. Our findings established a novel signaling mechanism by which the interaction between Gli1 and YAP1 promotes ESCC cell growth. This signaling regulation of the tumorigenesis provides a new therapeutic strategy for highly lethal ESCC.