MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Impact of G2 checkpoint defect on centromeric instability.

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G(2) checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G(2) to M phase transition, impaired G(2) checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G(2) checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G(2) checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G(2) checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G(2) checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G(2) checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells.

Id-1 induces cell invasiveness in immortalized epithelial cells by regulating cadherin switching and Rho GTPases.

Epithelial-mesenchymal transition (EMT), characterized by cadherin switching, contributes to cancer metastasis. Our recent study showed that Id-1 (inhibitor of differentiation-1) promotes metastasis in esophageal cancer cells, but whether the invasive and metastatic dynamics can be induced early in the carcinogenesis process is still unclear. Immortalization is regarded as the initial stage in the malignant transformation of normal cells. In this study, we investigated the role and mechanisms of Id-1 in inducing EMT and cell invasiveness in immortalized esophageal epithelial cells. We found that immortalized epithelial cells expressed higher endogenous levels of Id-1 compared with normal cells. Ectopic Id-1 expression inhibited the differentiation of immortalized esophageal epithelial cells and promoted cadherin switching, which was accompanied by increased adhesiveness to extracellular matrix, cell motility, migratory potential and matrix metalloproteinase-dependent invasiveness. GTPase activity assays showed that over-expression or short-hairpin RNA knockdown of Id-1 led to corresponding changes in Rac1 activity, whereas RhoA activity was significantly decreased with Id-1 depletion. Inhibitors targeting Rac1, RhoA, and Rho kinase suppressed the invasiveness of Id-1-expressing NE2-hTERT cells. Knockdown of N-cadherin in Id-1-over-expressing cells inhibited cell invasiveness and down-regulated RhoA activity. These data suggest that the Id-1-induced invasive potential may be regulated through the N-cadherin-RhoA axis and Rac1 activation.

Microtubule breakage is not a major mechanism for resolving end-to-end chromosome fusions generated by telomere dysfunction during the early process of immortalization.

Telomeres, the terminal chromosomal structure crucial for maintaining genomic integrity, shorten with deoxyribonucleic acid replications in most human somatic cells. Chromosomes carrying critically short telomeres tend to form end-to-end fusions, which are subject to breakage during cell division. However, it remains obscure how such telomere-mediated fusions are resolved during the process of immortalization, which is an early and indispensable step toward cancer. It has been hypothesized that the breakage could occur at either the microtubule or chromatid, causing numerical or structural chromosome instability, respectively. In this paper, we show that although the distributions of chromosomal segment losses or gains involved in structural aberrations were significantly correlated with the profiles of critically short telomeres in human epithelial cells undergoing immortalization, no such association was detected for whole-chromosome losses or gains in either metaphase or interphase cells. By distinguishing between homologues, we further showed that the specific homologues with critically short telomeres and frequent end-to-end fusions were not preferentially involved in respective whole-chromosome losses or gains. Our data therefore demonstrate that microtubule breakage is not a major mechanism for resolving chromosomal end-to-end fusions in human cells undergoing immortalization. An important implication of this finding is that microtubule-kinetochore attachment is stronger than the chromosome structure.

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells.

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

Role of MEK/ERK pathway in the MAD2-mediated cisplatin sensitivity in testicular germ cell tumour cells.

Testicular germ cell tumour (TGCT) is the most common malignancy in young males. Although most TGCTs are sensitive to cisplatin-based chemotherapy, significant numbers of TGCT patients still relapse and die each year because of the development of resistance to cisplatin. Previously, we first reported that a key regulator of the mitotic checkpoint, mitotic arrest deficient-2 (MAD2), was a mediator of cisplatin sensitivity in human cancer cells. In this study, we investigated whether MAD2 played a role in cellular sensitivity to cisplatin in TGCT cells and the underlying molecular mechanisms responsible. Using 10 TGCT cell lines, we found that increased MAD2 expression was correlated with cellular sensitivity to cisplatin, which was associated with activation of the MEK pathway. Treatment of cells expressing high levels of MAD2 with an MEK inhibitor, U0126, led to cellular protection against cisplatin-induced apoptosis. Inactivation of MAD2 by transfecting a dominant-negative construct in TGCT cells with high levels of MAD2 resulted in the suppression of MEK pathway and resistance to cisplatin-induced cell death. These results support previous suggestion on the involvement of mitotic checkpoint in DNA damage response in human cancer cells and demonstrate a possible molecular mechanism responsible for the MAD2-mediated sensitivity to cisplatin in TGCT cells. Our results also suggest that downregulation of MAD2 may be an indicator for identification of TGCT cancer cells that are potentially resistant to cisplatin-based therapy.

Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells.

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis.

The association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cells.

Loss of E-cadherin (E-cad) has been associated with progression and poor survival in nasopharyngeal carcinoma (NPC). In this study, we investigated the role of methylation on E-cad inactivation in NPC cell lines, as well as in NPC tissue samples. Using 6 NPC cell lines, we found that methylation of the E-cad 5' CpG island promoter region was correlated with the loss of both mRNA and E-cad protein expression in these cell lines. In addition, using 29 NPC and 10 non-malignant nasopharyngeal samples, we also observed 5' CpG methylation of the E-cad gene in 52% (15 out of 29) NPC samples, but in only 10% (1 out of 10) of the non-malignant nasopharyngeal tissues. Our findings indicate that 5' CpG island methylation of the E-cad gene may play an important part in the inactivation of E-cad in NPC. Our results also suggest that reducing the methylation of the E-cad gene may be a potential therapeutic strategy for NPC.

Inhibitor of apoptosis proteins and ovarian dysfunction in galactosemic rats.

Galactosemia is a genetic disease with deficiency of galactose-1-uridyltransferase, resulting in the accumulation of galactose or galactose-1-phosphate in the blood and tissues. Rats were fed with normal rat chow and with a high-galactose diet for 4 weeks to give control and galactosemic groups, and their ovarian function was studied. The two groups of rats were injected with pregnant mare's serum gonadotrophin (PMSG) and were killed at different time points after human chorionic gonadotrophin (hCG) injection. The number of oocytes ovulated in the controls was significantly higher than in the galactosemic group. Morphometric studies of the ovaries also showed a higher number of corpora lutea in the controls. Western blot analysis of granulosa cells showed that the overall expressions of Fas and FasL were lower in the control group and their expressions of inhibitor of apoptosis proteins (IAPs) were higher than in the galactosemic group, especially at 8 h post hCG injection. TDT-mediated dUTP-biotin nick end-labeling (TUNEL) and immunohistochemical staining of ovarian sections with Ki-67 and IAPs showed more apoptotic granulosa cells in the galactosemic group and the expressions of IAPs in granulosa cells also confirmed the result of the Western blot. These findings support our hypothesis that ovarian dysfunction in galactosemic rats is due to increased apoptosis in granulosa cells of maturing follicles.

Expression of e-cadherin and beta-catenin in trophoblastic tissue in normal and pathological pregnancies.

E-cadherin and beta-catenin are cell-cell adhesion molecules, which are thought to play an important role in trophoblastic differentiation and remodelling during gestation. Their expression may be altered in pathological conditions with trophoblastic invasion. In this study, we used immunohistochemical methods to study the pattern of expression of E-cadherin and beta-catenin in villous trophoblastic tissue in normal and pathological pregnancies. In villous trophoblastic tissue, E-cadherin had a membranous distribution, whereas beta-catenin had a mixed-membranous and granular cytoplasmic distribution. The levels of expression of E-cadherin and beta-catenin correlated with each other. From first to third trimesters, the expression of both E-cadherin and beta-catenin showed a decreasing trend. In preeclampsia, there was an up-regulation of E-cadherin and beta-catenin expression. In placenta accreta, the level of expression of both did not differ from that in normal third-trimester placenta. In gestational trophoblastic diseases, there was a general trend of down-regulation of both E-cadherin and beta-catenin. Altered expression of E-cadherin and beta-catenin may play a role in the development of normal and pathological placentas.

Protection of sperm DNA against oxidative stress in vivo by accessory sex gland secretions in male hamsters.

Reactive oxygen species scavengers present in male accessory sex gland secretions might afford antioxidant protection to sperm DNA. This study was conducted to determine whether accessory sex gland secretions protect the genome and function of spermatozoa against oxidative damage in the uterus. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands removed; (ii) ampullary glands removed; (iii) ventral prostate gland removed and (iv) sham-operated controls. Ejaculated spermatozoa recovered from uteri 15-30 min after mating with experimental males and caput and cauda epididymal spermatozoa obtained from intact males were incubated in 0-20 mmol NADPH l(-1) for 2 h. These spermatozoa and untreated uterine spermatozoa were processed for two types of comet assay (single cell gel electrophoresis): alkaline comet assay (pH > 13) which revealed single-strand DNA breakage and neutral comet assay (pH 9) which revealed double-strand DNA breakage. In comparison with the sham-operated controls, spermatozoa that had not been exposed to accessory sex gland secretions had a higher incidence and more extensive single-strand DNA damage with increasing concentrations of NADPH. Spermatozoa from hamsters without ampullary glands and from hamsters without the ventral prostate glands were similar to those of the control group. After incubation with NADPH, the capacity of spermatozoa from hamsters without accessory glands and from sham-operated controls to fuse with oocytes in vitro was reduced. However, only hamsters without accessory glands showed a negative correlation between single-strand DNA damage and sperm-oocyte fusion. Cauda epididymal spermatozoa were less susceptible to NADPH treatment compared with caput epididymal spermatozoa. The results of the present study showed that male accessory sex gland secretions can preserve the integrity of the sperm genome.