LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells.

The epithelial-to-mesenchymal transition (EMT) is a critical process by which cancer cells acquire malignant features. However, the molecular mechanism and functional implications of EMT and the mesenchymal-to-epithelial transition (MET) in tumor progression remain elusive. In this study, we established two aggressive cancer cell lines from the human oral cancer cell line SAS, mesenchymal-like SAS-m4 and epithelial-like SAS-δ. SAS-δ is a revertant cell obtained by inducing MET in SAS-m4. SAS-δ, but not SAS-m4, exhibited abnormal cell growth, including piled-up overgrowth and invasive tumor formation in the tongues of nude mice, suggesting that SAS-δ represented more advanced cancer cells than the parental SAS cells. EMT-related transcriptional factor SLUG is phosphorylated at T208 and partly stabilized by the Hippo pathway kinases, LATS1 and LATS2. Depletion of SLUG promoted the invasive activity of SAS-δ by increasing the protein levels of LATS1/2 and the proportion of the phosphorylated form among total SLUG protein. Our results suggest that the LATS1/2-SLUG axis regulates the transition of SAS cells to the advanced stage via repeated switching between EMT and MET. Therefore, an anti-SLUG-pT208 antibody would be valuable not alone as a malignant tumor marker antibody but also as a prognostic tool for patients with malignant disease.

Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation.

Activation of the epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of cancer and is associated with a poor prognosis in patients. The monoclonal antibody cetuximab, which displays EGFR extracellular domain-specific binding, has proven effective in the treatment of locally advanced disease and relapsed/metastatic disease. However, the effects of cetuximab are weaker than those of EGFR tyrosine kinase inhibitors (TKIs). This study investigates differences in the effects on cell growth of cetuximab and EGFR TKI AG1478 at the molecular level using oral squamous cell carcinoma (OSCC) cell lines. First, we found that there were EGFR-inhibitor-sensitive (EIS) and EGFR-inhibitor-resistant cell lines. The EIS cell lines expressed not only EGFR but also ErbB3, and both were clearly phosphorylated. The levels of phosphorylated ErbB3 were unaffected by cetuximab but were reduced by AG1478. EGFR ligand treatment increased the levels of phosphorylated EGFR but not phosphorylated ErbB3. Moreover, when EIS cell lines that were only capable of anchorage-dependent growth were grown in suspension, cell growth was suppressed and the levels of phosphorylated focal adhesion kinase (FAK), Src, and ErbB3 were significantly reduced. The levels of phosphorylated ErbB3 were unaffected by the FAK inhibitor PF573228, but were reduced by Src inhibition. Finally, combining cetuximab and a Src inhibitor produced an additive effect on the inhibition of EIS cell line growth.

LATS1/2 kinases trigger self-renewal of cancer stem cells in aggressive oral cancer.

Cancer stem cells (CSCs), which play important roles in tumor initiation and progression, are resistant to many types of therapies. However, the regulatory mechanisms underlying CSC-specific properties, including self-renewal, are poorly understood. Here, we found that LATS1/2, the core Hippo pathway-kinases, were highly expressed in the oral squamous cell carcinoma line SAS, which exhibits high capacity of CSCs, and that depletion of these kinases prevented SAS cells from forming spheres under serum-free conditions. Detailed examination of the expression and activation of LATS kinases and related proteins over a time course of sphere formation revealed that LATS1/2 were more highly expressed and markedly activated before initiation of self-renewal. Moreover, TAZ, SNAIL, CHK1/2, and Aurora-A were expressed in hierarchical, oscillating patterns during sphere formation, suggesting that the process consists of four sequential steps. Our results indicate that LATS1/2 trigger self-renewal of CSCs by regulating the Hippo pathway, the EMT, and cell division.

Molecularly-targeted therapy for the oral cancer stem cells.

Human cancer tissues are heterogeneous in nature and become differentiated during expansion of cancer stem cells (CSCs). CSCs initiate tumorigenesis, and are involved in tumor recurrence and metastasis. Furthermore, data show that CSCs are highly resistant to anticancer drugs. Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In this review, cetuximab and lapatinib-resistant oral squamous cell carcinoma (OSCC) cells proliferation and migration signal transduction passway is discussed by introducing our research.

Migration of oral squamous cell carcinoma cells are induced by HGF/c-Met signalling via lamellipodia and filopodia formation.

The activation of receptor tyrosine kinases (RTKs) results in cellular effects including cell proliferation, survival, migration and invasion; RTKs also play an important role in tumourigenesis. It has been reported that EGFR signalling controls the migration of oral squamous cell carcinoma (OSCC) SAS and HSC3 cells but not of HSC4 cells, although the proliferation of HSC4 cells is regulated by EGF/EGFR. In the present study, we investigated the roles of EGFR and the c-Met signalling pathway in cell migration via filopodia and lamellipodia formation, which may be prerequisites for migration. To explore the role of c-Met in cell migration, we inhibited c-Met RTK activity using the c-Met inhibitor SU11274 and activated c-Met using hepatocyte growth factor (HGF) in three OSCC cell lines HSC4, SAS and Ca9-22 and investigated migration potency using a wound healing assay. We showed that inhibition of c-Met significantly suppressed, and activation of c-Met significantly promoted, the migration of OSCC cells. Additionally, the migration of SAS and Ca9-22 cells was inhibited by the EGFR inhibitors AG1478 and cetuximab and promoted by EGF treatment. Moreover, migration potency was correlated with lamellipodia formation. Furthermore, western blot analyses demonstrated that SU11274 decreased and HGF increased lamellipodin protein levels as well as phosphorylated c-Met levels. Collectively, we demonstrated that c-Met signalling induced lamellipodia formation by upregulating lamellipodin, thereby promoting the migration of OSCC cells.

Regulation of cell migration via the EGFR signaling pathway in oral squamous cell carcinoma cells.

Cell migration potency is essential in cancer metastasis and is often regulated by extracellular stimuli. Oral squamous cell carcinoma cell lines include those that are sensitive, as well as resistant, to the effects of the epidermal growth factor receptor (EGFR) inhibitor cetuximab on cell migration. In the present study, the molecular differences in the EGFR response to cell migration between the SAS cetuximab-sensitive and HSC4 cetuximab-resistant cell lines was examined. Treatment with the EGFR inhibitors AG1478 and cetuximab reduced the migration potency of SAS cells, but not HSC4 cells. The migration of the two cell lines was inhibited under serum-free culture conditions, and the addition of EGF to the serum-free medium promoted the migration of SAS cells, but not HSC4 cells. In addition, SAS cell migration was reduced by the mitogen-activated protein kinase kinase and protein kinase B (Akt) inhibitors PD98059 and MK2206, whereas HSC4 cell migration was only inhibited by MK2206. EGF induced an increase in extracellular signal-regulated kinase phosphorylation levels in HSC4 cells, and stimulated Akt phosphorylation levels in SAS cells. Furthermore, the staining of actin filaments with phalloidin was significantly increased by the inhibition of EGFR in SAS cells, but was not observed as altered in HSC4 cells. Conversely, the addition of EGF to the culture medium decreased the accumulation of actin filaments in SAS cells. The results suggest that the EGF-EGFR signaling pathway has an important role in SAS cell migration via the modulation of actin dynamics, and that HSC4 cell migration is regulated by a serum component other than EGFR.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage­dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Interaction between basigin and monocarboxylate transporter 2 in the mouse testes and spermatozoa.

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 (MCT1), the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa.

Cetuximab-resistant oral squamous cell carcinoma cells become sensitive in anchorage-independent culture conditions through the activation of the EGFR/AKT pathway.

We have previously shown that growth of the oral squamous cell carcinoma cell line SAS, is resistant to cetuximab in monolayer culture conditions, even though epidermal growth factor receptor (EGFR) was phosphorylated, but the growth of SAS aggregates was sensitive to cetuximab. In the present study, we demonstrate differences in the EGFR signaling pathways utilized by SAS cells in monolayer and suspension cultures at the molecular level. Cetuximab treatment of SAS cells in monolayer cultures inhibits the phosphorylation of EGFR and ERK, and reduces the cell migratory potency, but not cell proliferation. AG1478 treatment reduces the phosphorylation of EGFR, ERK and AKT, and affects cell growth in monolayer cultures. The phosphorylation levels of EGFR and AKT are significantly higher in SAS cell aggregates compared to monolayer cultures. Treatment with cetuximab and AG1478 reduces the growth of SAS aggregates and eliminates the phosphorylation of EGFR and AKT. Furthermore, proliferation of SAS aggregates is also inhibited by LY294002 and MK2206, which are inhibitors of PI3K and AKT, respectively. In addition, treatment with the lipid raft disruptor filipin III reduced the phosphorylation levels of EGFR and Akt in SAS aggregates, but not in SAS monolayer culture. These results suggest the possibility that ligands in the serum stimulate the phosphorylation of EGFR localized in lipid rafts leading to PI3K-AKT activation, which results in the growth of SAS aggregates, therefore resulting in the sensitivity of SAS aggregates to cetuximab.

Isolation and propagation of neural crest stem cells from mouse embryonic stem cells via cranial neurospheres.

The developmental fate of the multipotent neural crest (NC) is determined along with the neural axis in which NC cells are generated. Only the cranial NC can differentiate into mesectodermal derivatives such as osteoblasts, chondrocytes, and adipocytes in vivo. Here, we attempted to selectively differentiate mouse embryonic stem (ES) cells into cranial NC stem cells and propagate them to explore their developmental potential to differentiate into mesectodermal derivatives. Using aggregation cultures in feeder- and serum-free neural induction medium (NIM) without serum replacement and l-glutamine, we obtained NIM neurospheres composed of neuroepithelium. The NIM neurospheres expressed the rostral markers Otx1 and Otx2, but not nonrostral markers Hoxb4, Hoxb9, Lbx1, and TH, which characterize cranial neurospheres. Subsequently, AP2α, Sox9, p75, Snail, Slug, and Twist-positive NC cells were differentiated in 4-day adhesion cultures of cranial neurospheres. In addition, sphere clusters in adhesion cultures were differentiated into osteoblasts, while migrating cells were not. By taking advantage of the sphere-formation capability, we isolated and propagated NC stem cells from the sphere clusters and confirmed their multipotency. NC stem cells expressed NC and stem cell markers, and they maintained differentiation potency in the NC derivatives. These results show that cranial NC stem cells were obtained reproducibly and efficiently without special inducing factors, gene transfection, or fluorescence-activated cell sorting selection.

The apoptotic initiator caspase-8: its functional ubiquity and genetic diversity during animal evolution.

The caspases, a family of cysteine proteases, play multiple roles in apoptosis, inflammation, and cellular differentiation. Caspase-8 (Casp8), which was first identified in humans, functions as an initiator caspase in the apoptotic signaling mediated by cell-surface death receptors. To understand the evolution of function in the Casp8 protein family, casp8 orthologs were identified from a comprehensive range of vertebrates and invertebrates, including sponges and cnidarians, and characterized at both the gene and protein levels. Some introns have been conserved from cnidarians to mammals, but both losses and gains have also occurred; a new intron arose during teleost evolution, whereas in the ascidian Ciona intestinalis, the casp8 gene is intronless and is organized in an operon with a neighboring gene. Casp8 activities are near ubiquitous throughout the animal kingdom. Exogenous expression of a representative range of nonmammalian Casp8 proteins in cultured mammalian cells induced cell death, implying that these proteins possess proapoptotic activity. The cnidarian Casp8 proteins differ considerably from their bilaterian counterparts in terms of amino acid residues in the catalytic pocket, but display the same substrate specificity as human CASP8, highlighting the complexity of spatial structural interactions involved in enzymatic activity. Finally, it was confirmed that the interaction with an adaptor molecule, Fas-associated death domain protein, is also evolutionarily ancient. Thus, despite structural diversity and cooption to a variety of new functions, the ancient origins and near ubiquitous distribution of this activity across the animal kingdom emphasize the importance and utility of Casp8 as a central component of the metazoan molecular toolkit.

Cyclin D1 expression is correlated with cell differentiation and cell proliferation in oral squamous cell carcinomas.

The present study conducted an immunohistochemical investigation of cyclin D1 and Ki-67 expression in oral squamous cell carcinoma (SCC) to evaluate the correlations between cell differentiation, cell proliferation and metastasis, and the effect of anticancer drug medication and cyclin D1 expression. Cyclin D1 and Ki-67 were detected clearly in the nuclei of 35 SCC samples. No correlation between cyclin D1 protein expression and oral SCC differentiation was found. By contrast, the majority of metastatic foci (90%) exhibited strong cyclin D1 expression, whereas weak expression was observed in metastatic foci with pre-operative adjuvant therapy. Additionally, cyclin D1 and Ki-67 were expressed in basal to suprabasal cells of well-differentiated oral SCC, whereas cyclin D1-positive and Ki-67-negative cells were present in the highly-differentiated region, according to a double-immunostaining method. These results indicate that the expression of cyclin D1 protein plays a role in cell differentiation and cell proliferation in well-differentiated oral SCC.

Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency.

Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. In the present study, we characterized cetuximab-resistant oral squamous cell carcinoma (OSCC) cell lines. The human OSCC cell lines HSC3, HSC4 and SAS were used in the present study. Effects of inhibitors including cetuximab on growth in cells were assessed by MTT assays. Southern blotting and immunofluorescence analysis were performed to examine protein expression and localization. Sphere formation was used to characterize stem cell-like properties. Floating aggregation culture was used for anchorage-independent growth. Cetuximab inhibited proliferation of HSC3 and HSC4 cells, but not SAS cells. Proliferation of all three cell lines was inhibited by the EGFR/ErbB2/ErbB4 inhibitor II. The EGFR inhibitor AG1478 strongly inhibited HSC3 and HSC4 proliferation, but that of SAS cells only moderately. EGFR proteins were localized on cell surface and phosphorylated in all three cell lines. SAS cells could proliferate in serum-free monolayer culture and formed spheres from single cells in floating culture. HSC3 and HSC4 could not proliferate under serum-free culture conditions and could not form spheres. Growth of SAS spheres required serum, and was inhibited by both AG1478 and cetuximab. Thus, cetuximab-resistant SAS cells not only engaged in EGFR-independent growth but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent but is also affected by the growth microenvironment.

NANOG expression correlates with differentiation, metastasis and resistance to preoperative adjuvant therapy in oral squamous cell carcinoma.

NANOG protein, a transcription factor expressed in embryonic stem cells, is overexpressed in tumor development. Although studies investigating the function of NANOG in cancer have shown that it plays several roles, such as in cell proliferation, invasion and metastasis, the overall function of NANOG in cancer cells has remained elusive. In the present study, NANOG expression in oral squamous cell carcinoma (OSCC) was examined to determine its potential clinical significance. The expression of NANOG protein was assessed in 60 patients with OSCC by immunohistochemistry, and its correlation with clinicopathological factors and metastasis was evaluated. NANOG protein levels in human OSCC cell lines were determined by western blotting and immunofluorescence staining. NANOG protein expression was identified in 52 cases (86.7%) and expression levels were higher in primary foci of poorly differentiated OSCC than in those of well-differentiated OSCC, indicating that NANOG expression is associated with OSCC differentiation. Regardless of the differentiation levels of primary foci, NANOG expression levels in metastatic foci were extremely high. In addition, NANOG expression in metastatic foci was maintained at high levels following preoperative adjuvant therapy. Furthermore, NANOG protein was detected at an identical level in human OSCC cell lines. These data indicate that NANOG-expressing OSCC cells tend to metastasize and that metastatic tumors expressing NANOG may be resistant to preoperative adjuvant therapy, including chemoradiation. Thus, assessment of NANOG expression may assist the strategy for treatment of OSCC metastasis.

Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a.

Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Distinct DNA methylation dynamics of spermatogenic cell-specific intronless genes is associated with CpG content.

In mammals, DNA methylation is restricted to cytosines of CpG dinucleotides, which are frequently found in short genomic regions including gene promoters. Methylation within CpG-rich regions around promoters tends to repress gene expression; thus, the CpG islands of housekeeping genes are normally unmethylated. We previously described a testis-specific single-exon gene containing a CpG-rich sequence that is methylated and thus repressed in somatic cells, whereas its expression in spermatogenic cells requires that it be hypomethylated. However, the relationship among the specific expression of spermatogenic genes, their methylation dynamics, and their CpG frequencies are poorly understood. Here, we analyzed the methylation patterns of the sphort genomic region around the transcription start site in spermatogenic cell-specific single-exon genes of various CpG contents. By using UniGene and Ensembl database analyses of the mouse genome and reverse transcription-PCR, we identified 39 single-exon genes that are exclusively expressed in spermatogeniccells. Regardless of their specific expression characteristics, genes containing higher (7 to 14 CpGs in 200 bp; mean = 12) and lower (2 to 6 CpGs in 200 bp; mean = 3.1) number ofCpG were hypo- and hyper-methylated, respectively, in all cell types examined, including spermatogeniccells. We found that genes with intermediate number of CpG (2 to 11 CpGs in 200 bp; mean = 6.9) are methylated in somatic cells, but not in male germ cells. These results suggest that DNA methylation dynamics of spermatogenic cell-specific single-exon genes are associated with CpG content, and the methylation status are stably maintained throughout male germ cell development.

Heparin-binding epidermal growth factor-like growth factor is a potent regulator of invasion activity in oral squamous cell carcinoma.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) has been shown to stimulate the growth of various cell types in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared with normal tissue, its contribution to cancer progression remains obscure. The objective of this study was to explore the effects of HB-EGF on proliferation, invasion activity and MMP-9 levels of an oral squamous cell carcinoma cell line, HSC3, in vitro. MTT assays, Matrigel invasion assays and RT-PCR in combination with RNA interference (RNAi) were used in this study. An RNAi-mediated decrease in HB-EGF expression reduced invasion activity and MMP-9 mRNA levels, but not proliferation, in HSC3 cells. The addition of purified HB-EGF to cell culture medium upregulated MMP-9 mRNA levels in HSC3 cells. Furthermore, the TACE inhibitor TAPI-2 or EGFR inhibitor AG1478 decreased MMP-9 mRNA levels in HSC3 cells. These data indicate that HB-EGF released from HSC3 cells by TACE stimulates EGFR in an autocrine manner, which in turn activates invasion activity via MMP-9 upregulation.

Generation of iPS cells using a BacMam multigene expression system.

Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64-98%. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15-24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.

A novel function of EpCAM in oral squamous cell carcinoma cells under anchorage-independent conditions.

Epithelial cell adhesion molecule (EpCAM), involved in Ca2+-independent homotypic cell-cell adhesion in epithelial tissues, is overexpressed in several cancer types. Although studies investigating the function of EpCAM in cancers have shown that it plays a role in cell proliferation, invasion and metastasis, the overall function of EpCAM in cancer cells has remained elusive. Here, we report a novel function of EpCAM in multicellular aggregates (MCAs). EpCAM inhibition using RNA interference (RNAi) did not affect cell morphology, proliferation or expression of certain genes, including cyclin D1 in monolayer cultures of the human oral squamous cell carcinoma cell lines HSC-3 or HSC-4. However, in HSC-4 cells cultured as MCAs, suppression of EpCAM significantly reduced the expression levels of cyclin D1. Nuclear localization of the cyclin D1 protein was observed in MCAs of HSC-4 cells but not in MCAs of EpCAM knockdown HSC4 cells, suggesting that EpCAM regulates cyclin D1 expression and localization in HSC-4 cells under anchorage-independent conditions. We propose that targeting EpCAM might result in more efficient therapies under certain conditions of oral squamous cell carcinoma.