Jing-He Li

Epidermal growth factor-like domain-containing protein 7 (EGFL7) enhances EGF receptor-AKT signaling, epithelial-mesenchymal transition, and metastasis of gastric cancer cells.

Epidermal growth factor-like domain-containing protein 7 (EGFL7) is upregulated in human epithelial tumors and so is a potential biomarker for malignancy. Indeed, previous studies have shown that high EGFL7 expression promotes infiltration and metastasis of gastric carcinoma. The epithelial–mesenchymal transition (EMT) initiates the metastatic cascade and endows cancer cells with invasive and migratory capacity; however, it is not known if EGFL7 promotes metastasis by triggering EMT. We found that EGFL7 was overexpressed in multiple human gastric cancer (GC) cell lines and that overexpression promoted cell invasion and migration as revealed by scratch wound and transwell migration assays. Conversely, shRNA-mediated EGFL7 knockdown reduced invasion and migration. Furthermore, EGFL7-overexpressing cells grew into larger tumors and were more likely to metastasize to the liver compared to underexpressing CG cells following subcutaneous injection in mice. EGFL7 overexpression protected GC cell lines against anoikis, providing a plausible mechanism for this enhanced metastatic capacity. In excised human gastric tumors, expression of EGFL7 was positively correlated with expression levels of the mesenchymal marker vimentin and the EMT-associated transcription repressor Snail, and negatively correlated with expression of the epithelial cell marker E-cadherin. In GC cell lines, EGFL7 knockdown reversed morphological signs of EMT and decreased both vimentin and Snail expression. In addition, EGFL7 overexpression promoted EGF receptor (EGFR) and protein kinase B (AKT) phospho-activation, effects markedly suppressed by the EGFR tyrosine kinase inhibitor AG1478. Moreover, AG1478 also reduced the elevated invasive and migratory capacity of GC cell lines overexpressing EGFL7. Collectively, these results strongly suggest that EGFL7 promotes metastasis by activating EMT through an EGFR−AKT−Snail signaling pathway. Disruption of EGFL7−EGFR−AKT−Snail signaling may a promising therapeutic strategy for gastric cancer.

Activation of Akt/mTOR Pathway Is Associated with Poor Prognosis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck region, which frequently occurs in Southeast Asia, especially in the south of China. It is known that the mammalian target of rapamycin (mTOR) pathway plays a central role in regulating cellular functions, including proliferation, growth, survival, mobility, and angiogenesis. Aberrant expression of the mTOR signaling pathway molecules has been found in many types of cancer. However, whether the alterations of p-Akt, p-p70S6K and p-4EBP1 protein expression are associated with clinicopathological features and prognostic implications in NPC have not been reported. The purposes of the present study are to investigate the association between the expression of p-Akt, p-p70S6K and p-4EBP1 proteins and clinicopathological features in NPC by immunohistochemistry. The results showed that the positive percentage of p-Akt, p-p70S6K and p-4EBP1 proteins expression in NPC (47.2%, 73.0% and 61.7%, respectively) was significantly higher than that in the non-cancerous nasopharyngeal control tissue (33.3%, 59.1% and 47.0%, respectively). There was a significantly higher positive expression of p-Akt in undifferentiated non-keratinizing nasopharyngeal carcinoma than that in differentiated non-keratinizing nasopharyngeal carcinoma (P = 0.014). Additionally, positive expression of p-p70S6K and p-4EBP1 proteins, and positive expression of either of p-Akt, p-p70S6K and p-4EBP1 were significantly correlated inversely with overall survival rates of NPC patients (P = 0.023, P = 0.033, P = 0.008, respectively). Spearman’s rank correlation test showed that expression of p-Akt in NPC was significantly associated with expression of p-p70S6K (r = 0.263, P<0.001) and p-4EBP1(r = 0.284, P<0.001). Also there was an obviously positive association between expression of p-p70S6K and p-4EBP1 proteins in NPC (r = 0.286, P<0.001). Multivariate Cox regression analysis further identified positive expression of p-4EBP1 and p-p70S6K proteins were the independent poor prognostic factors for NPC (P = 0.043, P = 0.027, respectively). Taken together, high expression of p-p70S6K and p-4EBP1 proteins may act as valuable independent biomarkers to predict a poor prognosis of NPC.

Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil.

Chemokine-mediated activation of G protein-coupled receptors CXCR1/2 promotes tumor growth, invasion, inflammation and metastasis. Repertaxin, a CXCR1/2 small-molecule inhibitor, has been shown to attenuate many of these tumor-associated processes. The present study aimed to investigate the effects of repertaxin alone and in combination with 5-fluorouracil (5-FU) on the malignant behavior of gastric cancer and the potential mechanisms. Gastric cancer MKN45 cells were treated in vitro with repertaxin and 5-FU, either alone or in combination. MTT and colony formation assay were performed to assess proliferation. Cell cycle progression and apoptosis was completed by flow cytometry. Migration and invasion were also assessed by Transwell and wound-healing assay. Western blot analysis and quantitative RT-PCR were performed to determine expression of signaling molecules. MKN45 cells were also grown as xenografts in nude mice. Mice were treated with repertaxin and 5-FU, and tumor volume and weight, angiogenesis, proliferation and apoptosis were monitored. Combination of repertaxin and 5-FU inhibited MKN45 cell proliferation and increased apoptosis better than either agent alone. Similarly, enhanced effect of the combination was also observed in migration and invasion assays. The improved effect of repertaxin and 5-FU was also observed in vivo, as xenograft models treated with both compounds exhibited significantly decreased tumor volume and increased apoptosis. In conclusion, repertaxin inhibited malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil. These data provide rationale that targeting CXCR1/2 with small molecule inhibitors may enhance chemotherapeutic efficacy for the treatment of gastric cancer.

Proteomics-based analysis of differentially expressed proteins in the CXCR1-knockdown gastric carcinoma MKN45 cell line and its parental cell

C-X-C chemokine receptor types 1 (CXCR1), a cell-surface G-protein-coupled receptor has been found to be associated with tumorigenesis, development, and progression of some tumors. Previously, we have found that CXCR1 overexpression is associated with late-stage gastric adenocarcinoma. We also have demonstrated that knockdown of CXCR1 could inhibit cell proliferation in vitro and in vivo. In this study, we compared the changes of protein expression profile between gastric carcinoma MKN45 cell line and CXCR1-knockdown MKN45 cell line by 2D electrophoresis. Among the 101 quantified proteins, 29 spots were significantly different, among which 13 were down-regulated and 16 were up-regulated after CXCR1 knockdown. These proteins were further identified by mass spectrometry analysis. Among them, several up-regulated proteins such as hCG2020155, Keratin8, heterogeneous nuclear ribonucleoprotein C (C1/C2), and several down-regulated proteins such as Sorcin, heat shock protein 27, serpin B6 isoform b, and heterogeneous nuclear ribonucleoprotein K were confirmed. These proteins are related to cell cycle, the transcription regulation, cell adherence, cellular metabolism, drug resistance, and so on. These results provide an additional support to the hypothesis that CXCR1 might play an important role in proliferation, invasion, metastasis, and prognosis, and drug resistance of gastric carcinoma.

Suppression Of ß-catenin Nuclear Translocation By CGP57380 Decelerates Poor Progression And Potentiates Radiation-Induced Apoptosis in Nasopharyngeal Carcinoma.

Nuclear localization of β-catenin is essential for the progression of various human cancers via transcriptional upregulation of downstream genes. The MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis has been reported to activate Wnt/β-catenin signaling, and CGP57380, an inhibitor of MNK kinases, inhibits the proliferation of multiple cancers. In this study, we showed that β-catenin signaling (including β-catenin, cyclin D1, c-Myc, and MMP-7) and p-eIF4E expression were elevated in nasopharyngeal carcinoma (NPC) compared with non-cancerous nasopharyngeal epithelial tissues, and was associated with clinical characteristics of NPC patients. Lymph node metastasis, gender, aberrant β-catenin expression, and elevated levels of MMP-7 and cyclin D1 were independent prognostic factors. Significantly, expression of p-eIF4E was positively correlated with β-catenin, and targeting the MNK-eIF4E axis with CGP57380 downregulated β-catenin in the nucleus, which in turn decreased proliferation, cell cycle progression, migration, invasion, and metastasis of NPC in vitro and in vivo. CGP57380 also potentiated radiation-induced apoptosis in NPC. Moreover, CGP57380 upregulated β-catenin in the cytoplasm thus blocking epithelial-mesenchymal transition (EMT), a key mechanism in cancer cell invasiveness and metastasis. Mechanistically, inhibition of β-catenin nuclear translocation by CGP57380 was dependent on AKT activation. Notably, identification of the MNK/eIF4E/β-catenin axis might provide a potential target for overcoming the poor prognosis mediated by β-catenin in NPC.

CXCR1 promotes malignant behavior of gastric cancer cells in vitro and in vivo in AKT and ERK1/2 phosphorylation.

CXCR1 is a member of the chemokine receptor family, which was reported to play an important role in several cancers. The present study investigated the influence of CXCR1 stable knockdown or overexpression on the malignant behavior of gastric cancer cells in vitro and in vivo and the potential mechanisms. MKN45 and BGC823 cells were stably transfected with plasmid pYr-1.1-CXCR1-shRNA (knockdown) and pIRES2-ZsGreen1-CXCR1 (overexpression), respectively. Malignant behavior was evaluated in vitro for changes in proliferation by MTT and colony forming assays; cell cycle and apoptosis by flow cytometry; and migration and invasion using transwell and wound-healing assays. Proliferation, cell cycle, apoptosis, migration and invasion-related signaling molecule expression were measured by real-time RT-PCR and western blot analysis. CXCR1 knockdown and overexpressing xenografts were monitored for in vivo tumor growth. Stable knockdown of CXCR1 inhibited MKN45 cell proliferation, migration and invasion, but were reversed in BGC823 cells stably overexpressing CXCR1. In addition, MKN45 cells stably transfected with CXCR1 shRNA inhibited AKT and ERK1/2 phosphorylation, protein expression of cyclin D1, EGFR, VEGF, MMP-9, MMP-2 and Bcl-2, and increased protein expression of Bax and E-cadherin (all P<0.05). In vivo CXCR1-shRNA-MKN45 cells transplanted into nude mice formed smaller tumors than non-transfected or scrambled-shRNA cells (both P<0.05). In contrast BGC823 cells overexpressing CXCR1 formed larger tumors in mice than cells carrying an empty expression plasmid or non-transfected cells (both P<0.05). CXCR1 promoted gastric cancer cell proliferation, migration and invasion. The present study provides preclinical data to support CXCR1 as a novel therapeutic target for gastric cancer.

Fatty acid synthase–associated protein with death domain: a prognostic factor for survival in patients with nasopharyngeal carcinoma

Fatty acid synthase-associated protein with death domain (FADD) is a key adaptor protein that bridges a death receptor (eg, death receptor 5) to caspase 8 to form the death-inducing signaling complex during apoptosis. The expression and prognostic impact of FADD in nasopharyngeal carcinoma (NPC) have not been well studied. This study focused on detecting FADD expression and analyzing its prognostic impact on NPC. FADD expression was assessed on pretreatment tumor tissues of 248 cases of NPC patients and 76 cases of noncancerous nasopharyngeal control tissue. The results showed that the positive percentage of FADD expression in NPC (63.7%, 158/248) was significantly higher than that in the noncancerous nasopharyngeal control tissues (28.9%, 22/76) (P < .0001). The positive expression of FADD in the NPC with cervical lymph node metastasis was significantly higher than those without lymph node metastasis (P = .009). Furthermore, FADD expression was more pronouncedly increased in metastatic NPC than the matched primary NPC tissues (P = .017). Both univariate and multivariate survival analysis indicated that increased FADD expression was significantly correlated inversely with overall survival in NPC patients (P = .003 and P = .007, respectively). Taken together, high expression of FADD may be an independent biomarker for poor prognosis in NPC.