Cheng-Wen Wu

Coexpression of Oct4 and Nanog Enhances Malignancy in Lung Adenocarcinoma by Inducing Cancer Stem Cell–Like Properties and Epithelial–Mesenchymal Transdifferentiation

Epithelial-mesenchymal transition (EMT), a critical process of cancer invasion and metastasis, is associated with stemness property of cancer cells. Though Oct4 and Nanog are homebox transcription factors essential to the self-renewal of stem cells and are expressed in several cancers, the role of Oct4/Nanog signaling in tumorigenesis is still elusive. Here microarray and quantitative real-time PCR analysis showed a parallel, elevated expression of Oct4 and Nanog in lung adenocarcinoma (LAC). Ectopic expressions of Oct4 and Nanog in LACs increased the percentage of CD133-expressing subpopulation and sphere formation, enhanced drug resistance, and promoted EMT. Ectopic expressions of Oct4 and Nanog activated Slug and enhanced the tumor-initiating capability of LAC. Furthermore, double knockdown of Oct4 and Nanog suppressed the expression of Slug, reversed the EMT process, blocked the tumorigenic and metastatic ability, and greatly improved the mean survival time of transplanted immunocompromised mice. The immunohistochemical analysis demonstrated that expressions of Oct4, Nanog, and Slug were present in high-grade LAC, and triple positivity of Oct4/Nanog/Slug indicated a worse prognostic value of LAC patients. Our results support the notion that the Oct4/Nanog signaling controls epithelial-mesenchymal transdifferentiation, regulates tumor-initiating ability, and promotes metastasis of LAC.

EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic oncomiR and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)-dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer.

Antitumor and antimetastatic activity of IL-23.

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 ± 333 mm3, then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8+ T cells, but not CD4+ T cells or NK cells, were crucial for the antitumor activity of IL-23.

Targeting cancer stem cells: emerging role of Nanog transcription factor.

The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the therapeutic efficacy of targeting Nanog as a cancer treatment method, current animal experiments using siNanog or shNanog have shown the promising therapeutic potential of Nanog targeting in several types of cancer.

DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression

Epithelial cell adhesion molecule (Ep-CAM) is believed to have a critical role in carcinogenesis and cell proliferation. However, the association of Ep-CAM with cancer invasion and progression is less clear. We found that Ep-CAM was highly expressed on low-invasive cells compared with highly invasive cells. Forced expression of Ep-CAM decreased cancer invasiveness, and silencing Ep-CAM expression elevated cancer invasiveness. Ep-CAM expression was associated with promoter methylation. Treatment with a demethylating agent, and/or the histone deacetylase inhibitor reactivated Ep-CAM expression in Ep-CAM-negative cells and inhibited cancer invasiveness. Using a promoter–reporter construct, we demonstrated methylation of the promoter fragment drive Ep-CAM-silenced transcription. Additionally, silenced Ep-CAM gene in cancer cells was enriched for hypermethylated histone 3 lysine 9. When unmethylated and active, this promoter was associated with acetylated histone 3 lysine 9. Furthermore, we observed an increased association of Ep-CAM promoter with repression components as tumor invasiveness increased. In cancer tissues, Ep-CAM expression significantly correlated with tumor progression and associated with promoter methylation. Our data support the idea that modulation of Ep-CAM plays a pivotal role in tumor invasion and progression. Moreover, aberrant DNA methylation of Ep-CAM is implicated in enhancing invasive/metastatic proclivity of tumors.

Human kallikrein 8 protease confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness

The human kallikrein 8 (KLK8) gene, a member of the human tissue kallikrein gene family, encodes a serine protease. The KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. We found that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppresses their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA reduced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and staining of cells for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with the detection of circulating tumor cells by quantitative PCR for the human Alu sequence showed that KLK8 suppresses tumor growth and invasion in vivo. Finally, studies of clinical specimens from patients with non-small cell lung cancer showed that the time to postoperative recurrence was longer for early-stage patients (stages I and II) with high KLK8 expression (mean, 49.9 months) than for patients with low KLK8 expression (mean, 22.9 months). Collectively, these findings show that KLK8 expression confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness.

CD13 (aminopeptidase N) can associate with tumor-associated antigen L6 and enhance the motility of human lung cancer cells.

Cancer metastasis is a multiple‐step process that involves the regulated interaction of diverse cellular proteins. We recently reported that the expression of tumor‐associated antigen L6 (TAL6) promoted the invasiveness of lung cancer cells and was inversely correlated with disease‐free survival of squamous lung carcinoma patients. We now report that CD13 (aminopeptidase N) can associate with TAL6 and can enhance cancer cell migration. CD13 was shown by coimmunoprecipitation to associate in vitro with TAL6 on several cancer cell lines and to associate in vivo by antibody‐mediated copatching immunofluorescence. CD13 was selectively expressed on highly invasive CL1‐5 lung cancer cells as compared to poorly invasive CL1‐0 lung cancer cells. The role of CD13 aminopeptidase activity in regulating cell motility was investigated with chemical inhibitors, specific antibodies and a catalytically inactive CD13 protein. Inhibition of CD13 aminopeptidase activity by nontoxic concentrations of leuhistin modestly decreased the migration of CL1‐5 cells. In contrast, binding of CD13 by specific antibodies significantly reduced both the migration and the invasion of CL1‐5 cells. Poorly invasive CL1‐0 cells that stably expressed CD13 displayed significantly (p ≤ 0.0005) enhanced cell migration (300% of control). Expression of an enzymatically inactive CD13 mutant on CL1‐0 cells also significantly (p ≤ 0.0005) enhanced cell migration (200% of control). Our results show that TAL6 and CD13 can form a complex on lung cancer cells, that these molecules can modulate cell migration and invasion and that the influence of CD13 on cell motility did not strictly depend on its aminopeptidase activity.

Mucin mRNA Expression in Lung Adenocarcinoma Cell Lines and Tissues

The expression pattern of mucin genes was studied in 7 lung adenocarcinoma cell lines (CL1, CL2, CL3, NCL2, PC9, PC13, PC14) and 12 lung adenocarcinoma tissues. CL1 and PC13 are poorly differentiated cell lines with low mucin glycoprotein production. The other 5 cell lines are well differentiated and produce a higher amount of mucins. Total RNA was extracted from these cell lines. Northern blot analysis was performed by hybridization with specific antisense oligonucleotide probes recognizing mucin-specific tandem repeats of 4 mucin genes (MUC1, MUC2, MUC3, MUC4). RT-PCR was carried out to amplify the 3 and 5 nonrepetitive coding regions of MUC1 and the 5 nonrepetitive coding region of MUC2. All these cell lines expressed MUC1, MUC2, and MUC4 mRNA but in variable mounts. The poorly differentiated cell lines (CL1 and PC13) had a relatively low level of expression of MUC1, MUC2, MUC3 and MUC4. RT-PCR, with primers amplifying the MUC1 nonrepetitive coding region 5 end, 293 bp, and the 3 end, 522 bp, as well as the MUC2 nonrepetitive 5 coding region, 308 bp, revealed the presence of MUC1 and MUC2 mRNA in all the cell lines. Sequence analysis of the PCR products were very homologous, similar to previously published MUC1 and MUC2 cDNA sequences. The expression pattern of mucin genes is consistent with that of mucin glycoproteins as studied using biochemical and immunological methods. Northern blotting and RT-PCR analysis in 12 lung adenocarcinoma tissues with various grades of differentiation (6 poorly differentiated adenocarcinomas and 6 moderately to well-differentiated adenocarcinomas) showed heterogeneous expression of the 4 mucin genes in tissues without clear correlation with the differentiation grade. Therefore the clinical implications of the differential expression of the mucin genes need further investigation.

Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner

BackgroundDespite of the fact that mammalian genomes are far more spacious than prokaryotic genomes, recent nucleotide sequencing data have revealed that many mammalian genes are arranged in a head-to-head orientation and separated by a small intergenic sequence. Extensive studies on some of these neighboring genes, in particular homologous gene pairs, have shown that these genes are often co-expressed in a symmetric manner and regulated by a shared promoter region. Here we report the identification of two non-homologous brain disease-related genes, with one coding for a serine protease inhibitor (SERPINI1) and the other for a programmed cell death-related gene (PDCD10), being tightly linked together by an asymmetric bidirectional promoter in an evolutionarily conserved fashion. This asymmetric bidirectional promoter, in cooperation with some cis-acting elements, is responsible for the co-regulation of the gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10.ResultsWhile SERPINI1 is predominantly expressed in normal brain and down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues but its gene transcription becomes aberrant in different types of cancers. By measuring the luciferase activity in various cell lysates, their 851-bp intergenic sequence was shown to be capable of driving the reporter gene expression in either direction. A 175-bp fragment from nt 1 to 175 in the vicinity of PDCD10 was further determined to function as a minimal bidirectional promoter. A critical regulatory fragment, from nt 176-473 outside the minimal promoter in the intergenic region, was identified to contain a strong repressive element for SERPINI1 and an enhancer for PDCD10. These cis-acting elements may exist to help coordinate the expression and regulation of the two flanking genes.ConclusionFor all non-homologous genes that have been described to be closely adjacent in the mammalian genomes, the intergenic region of the head-to-head PDCD10-SERPINI1 gene pair provides an interesting and informative example of a complex regulatory system that governs the expression of both genes not only through an asymmetric bidirectional promoter, but also through fine-tuned regulations with some cis-acting elements.

Suppression of the invasion and migration of cancer cells by SERPINB family genes and their derived peptides

Apart from SERPINB2 and SERPINB5, the roles of the remaining 13 members of the human SERPINB family in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of each SERPINB gene effectively suppressed the invasiveness and motility of malignant cancer cells. Among all of the genes, the SERPINB1, SERPINB5 and SERPINB7 genes were more potent, and the inhibitory effect was further enhanced by co-expression of any two of them. In addition, single treatment of the synthetic peptides corresponding to the P5-P5 sequences of the reactive center loop (RCL) of SERPINB1, SERPINB5 or SERPINB7 markedly suppressed the invasive and migratory properties of the cancer cells in a dose-dependent manner. More significantly, combination treatment of these peptides in cancer cells further improved the suppressive effect by 20-40%. Here, we determined the expression of all SERPINB family members in lung and breast cancer patients, and identified those members with potent inhibitory ability toward invasion and migration, and designed RCL-derived peptides to suppress the malignancy of cancer cells. Forced re-expression of these anti-invasive SERPINB genes or application of the SERPINB RCL-peptides may provide a reasonable strategy against lethal cancer metastasis.