Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis. In this study, we have analyzed the level of miR-10b and cell motility and invasiveness in several human esophageal squamous cell carcinoma cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility. Additionally, we identified KLF4, a known tumor suppressor gene that has been reported to suppress esophageal cancer cell migration and invasion, as a direct target of miR-10b. MicroRNAs, a class of small noncoding RNAs, have been identified as a new kind of gene expression regulators through targeting mRNAs for translational repression or cleavage (1-3). Lately, emerging evidence suggests important roles for miRNAs 2 in apoptosis (4), hematopoietic development (5), cell proliferation (6), skin morphogenesis (7), and neural development (8). Deregulation of miRNAs has also been reported in a variety of tumors, including breast cancer, leukemia, lung cancer, and colon cancer (9 -11), which indicated a significant correlation between miRNAs and human malignancy. miRNA expression profiling in esophageal cancers revealed a distinct miRNA signature (12, 13), and some miRNAs showed correlation with several clinicopathologic parameters (13). Furthermore, miR-21 is reported to regulate the proliferation and invasion in ESCC (14), and the miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression (15), providing evidence of a causal role for miRNAs in esophageal cancer development.Recent studies show that miRNAs may act as activators or inhibitors of tumor metastasis by acting on multiple signaling pathways involved in metastasis (16 -18). Ma et al. (16) found that miR-10b initiates invasion and metastasis in breast cancer. miR10b, induced by the prometastatic transcription factor TWIST1, proceeds to inhibit translation of mRNA of HOXD10, a transcription factor already known for its roles in cell motility (19), resulting in increased expression of a pro-metastatic gene, RHOC. This study has provided the first evidence for a role of miRNA in tumor metastasis. Subsequently several additional miRNAs have been reported to act on various steps of metastasis (17, 18).Krüppel-like factor 4 (KLF4), a zinc finger protein of the Krüp-pel-like factor family, plays a role in cell cycle regulation, differentiation, and rises in response to DNA damage, serum starvation, and contact inhibition (20, 21). In line with these studies, the loss of KLF4 expression has been reported in several human tumors, including colorectal, stomach, esophageal, and bladder cancers (22-25), which indicates its tumor suppressor role. However, KLF4 also exhibits oncogenic properties. Overexpression of KLF4 could b...

MicroRNAs have key roles in tumor metastasis. Here, we describe the regulation and function of miR-34a and miR-34c (miR-34a/c) in breast cancer metastasis. Expression analysis verified that miR-34a/c expression is significantly decreased in metastatic breast cancer cells and human primary breast tumors with lymph node metastases. Overexpression of miR-34a/c could inhibit breast cancer cell migration and invasion in vitro and distal pulmonary metastasis in vivo. Further studies revealed that Fos-related antigen 1 (Fra-1 or Fosl1) is a downstream target of miR-34a/c as miR-34a/c bound directly to the 3'untranslated region of Fra-1, subsequently reducing both the mRNA and protein levels of Fra-1. Silencing of Fra-1 recapitulated the effects of miR-34a/c overexpression, whereas enforced expression of Fra-1 reverses the suppressive effects of miR-34a/c. Moreover, significant downregulation of miR-34a in metastatic breast cancer tissues was found to be inversely correlated with Fra-1 expression. Our results demonstrate that miR-34a/c functions as a metastasis suppressor to regulate breast cancer migration and invasion through targeting Fra-1 oncogene and suggest a therapeutic application of miR-34 in breast cancer.

The tumor suppressor P53 pathway plays a crucial role in preventing carcinogenesis and genetic variations of this pathway may be associated with cancer susceptibility. We tested this hypothesis by examining the contribution of functional polymorphisms in P53 and MDM2 to risk of esophageal squamous cell carcinoma (ESCC). DNA from 758 ESCC patients and 1,420 controls were genotyped for P53 codon 72Arg>Pro and MDM2 309T>G polymorphisms. Odds ratios (OR) and 95% confidence intervals (CI) of ESCC were estimated by logistic regression. We observed an increased risk of ESCC associated with the P53 Pro/Pro (OR, 1.83; 95% CI, 1.43-2.35; P < 0.001) or MDM2 GG (OR, 1.49; 95% CI, 1.16-1.91; P = 0.002) genotype, compared with the P53 Arg/Arg or MDM2 TT genotype, respectively. Interaction between these P53 and MDM2 polymorphisms increased risk of ESCC in a multiplicative manner, with the OR being 3.10 (95% CI, 2.07-4.69) for subjects carrying both P53 Pro/Pro and MDM2 GG genotypes. Significant interactions were observed between these polymorphisms and smoking, with risk being the highest (OR, 5.29; 95% CI, 2.91-9.61) in smokers having both P53 Pro/ Pro and MDM2 GG genotypes. The MDM2 GG genotype was also associated with risk of developing poorly differentiated and advanced ESCC compared with the GT or TT genotype (OR for high-grade and stages III-IV versus low-grade and stages I-II = 1.60; 95% CI, 1.00-2.64; P = 0.049). The P53 and MDM2 polymorphisms may be genetic determinants for the development of ESCC. (Cancer Res 2005; 65(20): 9582-7)

Embryonic stem cell (ES cell) lines were first generated by culturing mouse inner cell mass (ICM) on feeder layers in 1981 [1]. However, in large domestic animals, attempts to establish ES cell lines from ICM of blastocysts or the later epiblast have not been successful. This has hindered the efficient production of genetically modified livestocks by using ES-based approaches. Recently, it was found that ectopic expression of various combinations of transcription factors is able to reprogram somatic cells to a pluripotent state [2][3][4][5]. These induced pluripotent stem (iPS) cells show similarities to embryo-derived ES cells and can be used to produce viable mice through tetraploid complementation [6,7]. So far, iPS cells of several mammalian species have been successfully generated [2,3,[8][9][10][11][12]. In this letter, we report the first establishment of bovine iPS cells using defined transcription factors and a modified culture medium.cDNAs coding for the bovine OCT4 (also named POU5F1), SOX2, KLF4, MYC, LIN28, and NANOG genes were cloned into pMXs retroviral vector. The pMXs plasmids containing human OCT4, SOX2, KLF4, and c-MYC genes were all purchased from Addgene. GP2-293 cells were used as the packaging cell line for retroviral production. Bovine fibroblasts used in this study were derived from an E55 Western Shandong Yellow cattle fetus. Three sets of factors, termed b4TF, b6TF, and h4TF, were used to transduce cells by overnight retroviral infection, respectively. Whereas the former two included only bovine factors (b4TF: bOCT4, bSOX2, bMYC, bKLF4; b6TF: bOCT4, bSOX2, bKLF4, bMYC, bLIN28, bNANOG), the latter employed only human factors (hOCT4, hSOX2, hc-MYC, hKLF4). On day 2, the infected cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeders at a density of 1 × 10 4 cells per 100-mm dish. The next day after being seeded onto feeders, growing cells were cultured in iPS media ( Figure 1A and Supplementary information, Table S1). Bovine iPS (hereinafter named biPS) cells with a mouse ES-like morphology were detected after ~3 Figure 1B). On day 21-35, colonies were isolated mechanically using a 200 µl pipette and transferred to feeder-coated tissue culture dishes. The biPS cells were split with trypsin at a ratio of 1:10 every 4-5 days afterwards (Supplementary information, Data S1). A total of 26 b6TF-derived colonies have been expanded into biPS cell lines. These lines maintained good ES-like morphology for more than 16 passages. However, none of the colonies generated by b4TF or h4TF could be passaged more than six times. Importantly, we showed that the combination of six transcription factors (b6TF set) significantly increased the efficiency of iPS cell generation (by three-fold) compared to the other two combinations ( Figure 1C).We tested eight different types of biPS culture media (Supplementary information, Table S1) by assessing the numbers of ES-like colonies obtained from b6TF-transduced BEFs on day 28. Three out of the eight media could efficiently generat...

Purpose: Stomatin-like protein 2 (SLP-2) is a novel and unusual stomatin homologue of unknown functions. It has been implicated in interaction with erythrocyte cytoskeleton and presumably other integral membrane proteins, but not directly with the membrane bilayer.We show here the involvement of SLP-2 inhuman esophageal squamous cell carcinoma (ESCC), lung cancer, laryngeal cancer, and endometrial adenocarcinoma and the effects of SLP-2 on ESCC cells. Experimental Design: Previous work of cDNA microarray in our laboratory revealed that SLP-2 was significantly up-regulated in ESCC. The expression of SLP-2 was further evaluated in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma by semiquantitative reverse transcription-PCR, Western blot, and immunohistochemistry. Mutation detection of SLP-2 exons was done by PCR and automated sequencing. Antisense SLP-2 eukaryotic expression plasmids were constructed and transfected into human ESCC cell line KYSE450. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, clonogenecity assay, flow cytometry assay, nude mice tumorigenetic assay, and cell attachment assay were done to investigate the roles of SLP-2 gene. Results: All tumor types we tested showed overexpression of SLP-2 compared with their normal counterparts (P V 0.05). Moreover, immunohistochemistry analysis of mild dysplasia, severe dysplasia, and ESCC showed that overexpression of SLP-2 occurred in premalignant lesions. Mutation analysis indicated that no mutation was found in SLP-2 exons. KYSE450 cells transfected with antisense SLP-2 showed decreased cell growth, proliferation, tumorigenecity, and cell adhesion. Conclusions: SLP-2 was first identified as a novel cancer-related gene overexpressed in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma. Decreased cell growth, cell adhesion, and tumorigenesis in the antisense transfectants revealed that SLP-2 may be important in tumorigenesis.