M6PR- and EphB4-Rich Exosomes Secreted by Serglycin-Overexpressing Esophageal Cancer Cells Promote Cancer Progression.

Accumulating evidence shows that exosomes participate in cancer progression. However, the functions of cancer cell exosome-transmitted proteins are rarely studied. Previously, we reported that serglycin (SRGN) overexpression promotes invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the paracrine effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cell invasion and tumor angiogenesis, and used mass spectrometry to identify exosomal proteins involved. Cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo, while augmented exosomal EphB4 mediated the pro-invasive effect of SRGN Exo on ESCC cells in vitro. In addition, in vitro studies showed that manipulation of M6PR expression affected the viability and migration of ESCC cells. Both M6PR and EphB4 expression levels were positively correlated with that of SRGN in the serum of patients with ESCC. High level of serum M6PR was associated with poor overall survival rates. Taken together, this study presents the first proof that exosomal M6PR and EphB4 play essential roles in tumor angiogenesis and malignancy, and that serum M6PR is a novel prognostic marker for ESCC patients.

Proteoglycans and their functions in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant disease that has a poor prognosis. Its high lethality is mainly due to the lack of symptoms at early stages, which culminates in diagnosis at a late stage when the tumor has already metastasized. Unfortunately, the common cancer biomarkers have low sensitivity and specificity in esophageal cancer. Therefore, a better understanding of the molecular mechanisms underlying ESCC progression is needed to identify novel diagnostic markers and therapeutic targets for intervention. The invasion of cancer cells into the surrounding tissue is a crucial step for metastasis. During metastasis, tumor cells can interact with extracellular components and secrete proteolytic enzymes to remodel the surrounding tumor microenvironment. Proteoglycans are one of the major components of extracellular matrix. They are involved in multiple processes of cancer cell invasion and metastasis by interacting with soluble bioactive molecules, surrounding matrix, cell surface receptors, and enzymes. Apart from having diverse functions in tumor cells and their surrounding microenvironment, proteoglycans also have diagnostic and prognostic significance in cancer patients. However, the functional significance and underlying mechanisms of proteoglycans in ESCC are not well understood. This review summarizes the proteoglycans that have been studied in ESCC in order to provide a comprehensive view of the role of proteoglycans in the progression of this cancer type. A long term goal would be to exploit these molecules to provide new strategies for therapeutic intervention.

mTORC2-mediated PDHE1α nuclear translocation links EBV-LMP1 reprogrammed glucose metabolism to cancer metastasis in nasopharyngeal carcinoma.

EBV infection of preinvasive nasopharyngeal epithelium is believed to be an initiation step during pathogenesis of nasopharyngeal carcinoma (NPC), but the mechanisms remain poorly understood. Here we report a novel mechanism driving NPC metastasis through the EBV-encoded LMP1-mediated metabolic reprogramming, via activation of IGF1-mTORC2 signaling and nuclear acetylation of the Snail promoter by the PDHE1α, an enzyme involved in glucose metabolism. Mechanistically, EBV-LMP1 increases the cellular secretion of IGF1 which promotes phosphorylation of IGF1R to activate mTORC2/AKT signaling linking glucose metabolism to cell motility. LMP1 expression facilitates translocation of mitochondrial PDHE1α into the nucleus in a phosphorylation-dependent manner at Ser293 residue. Functionally, nuclear PDHE1α promotes H3K9 acetylation on the Snail promoter to enhance cell motility, thereby driving cancer metastasis. Importantly, the IGF1/mTORC2/PDHE1α/Snail axis correlates significantly with disease progression and poor prognosis in NPC patients. This study highlights the functional importance of IGF1-mTORC2-PDHE1α signaling mediated by EBV-LMP1 in NPC pathogenesis.

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

Significance of NF-κB activation in immortalization of nasopharyngeal epithelial cells.

NF-κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF-κB is common in NPC and may contribute to NPC development. The role of NF-κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF-κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF-κB signaling is a common phenotype in telomerase-immortalized NPE cell lines. Our results suggest that NF-κB activation promotes the growth of telomerase-immortalized NPE cells, and suppression of NF-κB activity inhibits their proliferation. Furthermore, we observed upregulation of c-Myc, IL-6 and Bmi-1 in our immortalized NPE cells. Inhibition of NF-κB downregulated expression of c-Myc, IL-6 and Bmi-1, suggesting that they are downstream events of NF-κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF-κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF-κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.

Water extract from Pleurotus pulmonarius with antioxidant activity exerts in vivo chemoprophylaxis and chemosensitization for liver cancer.

Chemoprophylaxis and chemosensitization are promising strategies to combat human cancers. Natural antioxidant agents show great promise in cancer therapy, and the use of edible mushrooms against cancer is receiving more interest globally. In this study, the radical scavenging activities including diphenyl-1-picrylhydrazyl, superoxide anion radical, hydroxyl radical, and hydrogen peroxide were compared among hot water extracts from 3 edible mushrooms, among which Pleurotus pulmonarius (Pp) possessed the highest antioxidant potential. Oral administration of Pp 2 wk in advance could markedly inhibit the incidence and size of tumor (Huh7 liver cancer cells) with an inhibition rate of 93.1% in nude mice. No obvious side effect was observed in the Pp-treated mice as indicated by their body weight and histological analysis of major organs. The cancer prevention by Pp treatment might be explained by the inhibition of cancer cell proliferation indicated by reduction of ki-67 staining and the inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling pathway in the Pp-treated mice. Furthermore, a significant synergistic effect was observed when the mice were treated with a combination of low dose of cisplatin and Pp. Taken together, these results suggest the potential application of Pp as an adjuvant in the chemotherapy of liver cancer.

Enhanced IL-6/IL-6R signaling promotes growth and malignant properties in EBV-infected premalignant and cancerous nasopharyngeal epithelial cells.

Nasopharyngeal carcinoma (NPC) is etiologically associated with Epstein-Barr virus (EBV) infection. However, the exact role of EBV in NPC pathogenesis remains elusive. Activation of signal transducer and activator of transcription 3 (STAT3) is common in human cancers including NPC and plays an important role in the pathogenesis and progression of human cancers. Interleukin-6 (IL-6), a major inflammatory cytokine, is a potent activator of STAT3. In this study, we report that EBV-infected immortalized nasopharyngeal epithelial (NPE) cells often acquire an enhanced response to IL-6-induced STAT3 activation to promote their growth and invasive properties. Interestingly, this enhanced IL-6/STAT3 response was mediated by overexpression of IL-6 receptor (IL-6R). Furthermore, IL-6R overexpression enhanced IL-6-induced STAT3 activation in uninfected immortalized NPE cells in vitro, and promoted growth and tumorigenicity of EBV-positive NPC cell line (C666-1) in vivo. Moreover, it is shown for the first time that IL-6R was overexpressed in clinical specimens of NPC. IL-6 expression could also be strongly detected in the stromal cells of NPC and a higher circulating level of IL-6 was found in the sera of advance-staged NPC patients compared to the control subjects. Therefore, IL-6R overexpression, coupled with enhanced IL-6/STAT3 signaling may facilitate the malignant transformation of EBV-infected premalignant NPE cells into cancer cells, and enhance malignant properties of NPC cells.

Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells.

Undifferentiated nasopharyngeal carcinomas (NPCs) are commonly present with latent EBV infection. However, events regulating EBV infection at early stages of the disease and the role of EBV in disease pathogenesis are largely undefined. Genetic alterations leading to activation of cyclin D1 signaling in premalignant nasopharyngeal epithelial (NPE) cells have been postulated to predispose cells to EBV infection. We previously reported that loss of p16, a negative regulator of cyclin D1 signaling, is a frequent feature of NPC tumors. Here, we report that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1. Furthermore, overexpression of cyclin D1 is closely associated with EBV infection. Therefore we investigated the potential role of cyclin D1 overexpression in dysplastic NPE cells in vitro. In human telomerase reverse transcriptase-immortalized NPE cells, overexpression of cyclin D1 or a p16-resistant form of CDK4 (CDK4(R24C)) suppressed differentiation. This suppression may have implications for the close association of EBV infection with undifferentiated NPC. In these in vitro models, we found that cellular growth arrest and senescence occurred in EBV-infected cell populations immediately after infection. Nevertheless, overexpression of cyclin D1 or a p16-resistant form of CDK4 or knockdown of p16 in the human telomerase reverse transcriptase-immortalized NPE cell lines could counteract the EBV-induced growth arrest and senescence. We conclude that dysregulated expression of cyclin D1 in NPE cells may contribute to NPC pathogenesis by enabling persistent infection of EBV.

Expression of Epstein-Barr virus-encoded LMP1 and hTERT extends the life span and immortalizes primary cultures of nasopharyngeal epithelial cells.

Cell immortalization is regarded as an early and pre-requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein-Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV-encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c-Myc, Bmi-1, and Id-1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT-immortalized nasopharyngeal epithelial cells. The LMP1/hTERT-immortalized NP446 cells were non-tumorigenic in immunosuppressed nude mice and retained anchorage-dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV-encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma.

Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization.

Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-beta effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-alpha. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.

Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells.

Berberine is an active ingredient extracted from Coptidis rhizoma which has been used for centuries as a traditional Chinese medicine for treatment of inflammatory diseases. Recent studies have indicated that berberine has anticancer properties. Berberine arrested cell growth and inhibited cell migration in various cancer cell lines. In this study, we examined the effects of berberine on HONE1 cells, which have been commonly used as a cell model for nasopharyngeal carcinoma. We observed the inhibitory effects of berberine on HONE1 cells at a high dosage (>150 microM). Berberine effectively induced the mitotic arrest of HONE1 cells at 300 microM which was associated with apoptosis. Berberine had differential intracellular localization at low and high doses. At a low dose (50 microM), berberine was localized in the mitochondria while at a high dose (300 microM), berberine was localized in the nucleus which may have induced mitotic arrest. Berberine effectively inhibited cell migration and invasion at low doses. Using a specific GST pull-down assay of activated Rho GTPases, we demonstrated that berberine suppressed the activation of Rho GTPases including RhoA, Cdc42 and Rac1. This indicates a novel function of berberine in the suppression of Rho GTPase signaling to mediate its inhibitory action on cell migration and motility. The potential of berberine to inhibit cancer metastasis in cancer warrants further investigation.

Id1 overexpression induces tetraploidization and multiple abnormal mitotic phenotypes by modulating aurora A.

The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.

Establishment and characterization of a human non-small cell lung cancer cell line.

Lung cancer is one of the most common causes of cancer death worldwide. Although many efforts have been made to explore the mechanisms involved in the development of lung cancer, the genetic events involved in the pathogenesis of lung cancer are still unclear. For a better mechanistic scope of study, a well-established cellular model is essential. We report the establishment of a squamous cell carcinoma (SCC) cell line of human lung, SCC-37. Chromosomal abnormalities and global genomic alterations of SCC-37 were studied by spectral karyotyping (SKY) and comparative genomic hybridization (CGH), respectively. Results showed that SCC-37 was a hypodiploid with complex chromosomal rearrangements. Some of the alterations, such as the gain of 1q25-qter in SCC-37, have been correlated to the tumor recurrence of non-small cell lung cancer (NSCLC). Other interesting findings include the amplification of 3q25-qter and 12q13, suggesting the existence of important oncogenes in the amplicons. This cell line may thus provide a useful cellular resource for studying the pathogenesis of SCC of the lung in the future.

Physical status of HPV-16 in esophageal squamous cell carcinoma.

BACKGROUND: Infection with high-risk human papillomavirus (HPV) has been implicated as one of the risk factors of esophageal squamous cell carcinoma (ESCC). Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of the HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. OBJECTIVES AND STUDY DESIGN: To investigate the viral integration status of HPV-16 infection in ESCC, 35 HPV-positive ESCC specimens collected from Chinese patients were subject to real-time quantitative PCR for determination of physical status of HPV-16 by analyzing the ratios of E2 to E6 genes. RESULTS: Our results showed that only 8.6% (3/35) of the HPV-16 positive specimens harbored exclusively the episomal form (i.e. E2/E6 ratio > or = 1), whereas the remaining 91.4% contained either only the integrated form (5.7%, with E2/E6 ratio = 0) or a mixture of episomal and integrated forms of viral molecules (85.7%, with E2/E6 ratios > 0 but < 1). Amongst the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/integrated E6 ratios of less than 1, indicating a predominance of integrated form of viral genes in these lesions. CONCLUSION: Our finding of frequent integration of viral DNA in the host genome suggests that integration HPV-16 is common in ESCC from Chinese patients and implies that HPV infection may play a role in the pathogenesis of ESCC.

Human papillomavirus infection and loss of heterozygosity in esophageal squamous cell carcinoma.

The incidence/mortality rates of esophageal squamous cell carcinoma (ESCC) vary widely in different parts of China. Human papillomavirus (HPV) infection is considered a possible risk factor. Loss of heterozygosity (LOH) analysis on 87 ESCC specimens collected from three different areas of China showed lower frequency of LOH at marker D3S1621 in Linxian, an area with exceptionally high incidence of ESCC but low HPV infection rate. HPV-positive ESCC from Hong Kong, but not Sichuan, had higher frequency of LOH at D5S82 (APC, MCC), D6S497 (p21/Waf-1, HLA) and D13S260 (BRCA2) than HPV-negative samples. Our results suggest that different genetic pathways of carcinogenesis may be associated with geographic differences in risk factors.