Yeu Su

Overexpression of the thymosin β-4 gene is associated with increased invasion of SW480 colon carcinoma cells and the distant metastasis of human colorectal carcinoma

Cell–matrix and cell–cell adhesive interactions play important roles in the normal organization and stabilization of the cell layer in epithelial tissue. Alterations in the expression and function of these adhesion systems that cause a switch to a migratory phenotype in tumor invasion and metastasis are critical for the malignant conversion of epithelial cells. Thymosin β-4 (Tβ-4) is the major actin-sequestering protein that has been shown to be upregulated in a wide variety of human carcinomas and has been implicated to be involved in altering the motility of certain tumors. We have recently demonstrated that the growth rate, colony formation in soft agar, and motility, all good indicators for malignant progression, of SW480 colon carcinoma cells are dramatically increased by enforced Tβ-4 expression. To test the hypothesis that overexpression of this G-actin sequestering peptide also promotes tumor invasion, we examined not only the invasion capability of Tβ-4-overexpressing SW480 cells, but also the expression levels of Tβ-4 as well as several proteins that participate in different stages of tumor progression in matched samples of human primary colorectal adenocarcinoma and liver metastases from several patients. A marked increase on the invasiveness in Tβ-4-overexpressing SW480 cells with increased levels and activity of matrix metalloproteinase-7 (MMP-7) was observed. Furthermore, the levels of Fas as well as the susceptibility to Fas ligand-mediated apoptosis in Tβ-4-overexpressing cells were significantly decreased. Interestingly, the levels of Tβ-4 mRNA, β-catenin, c-Myc, and MMP-7 in metastatic liver lesions were relatively higher, whereas the levels of E-cadherin and Fas were significantly lower than those in the matched primary colorectal tumors. These results suggest that upregulation of Tβ-4, by promoting the disruption of cell–cell adhesion and a consequential activation of the β-catenin signaling, could be a key event in the acquisition of growth advantages as well as invasive phenotypes in human colorectal carcinomas.

Overexpression of the thymosin β -4 gene is associated with malignant progression of SW480 colon cancer cells

Thymosin β-4 (Tβ-4), a small peptide originally isolated from calf thymus, modulates the formation of F-actin microfilaments by sequestering the monomeric G-actin. Recent studies have shown that overexpression of the Tβ-4 gene occurs not only in many human carcinomas but also in the highly metastatic melanomas and fibrosarcomas. However, little is known about the specific growth advantages acquired by different tumors from this genetic abnormality. To address the above questions, Tβ-4-overexpressing human colon carcinoma (SW480) cells were established by stable transfection and their phenotypic changes were monitored. We found that both the morphology and the cortical actin cytoskeleton of SW480 cells were altered by Tβ-4 overexpression. Moreover, both cellular level and that distributed over the intercellular junctions of the E-cadherin were decreased in the Tβ-4 overexpressers, which were accompanied by a twofold increase in their saturation densities. Meanwhile, these cells also exhibited an increased ability to form colonies in soft agar. Interestingly, a dramatic increase of growth rate was detected in the Tβ-4 overexpressers, which might be attributed to an accelerated proliferation induced by c-Myc that was activated by nuclear β-catenin. Finally, a motility increase of these cells was demonstrated by two independent migration assays, which was accompanied by an enhanced focal contact. Taken together, our data suggest that the drastic growth property and motility changes of the SW480 cells overexpressing Tβ-4 gene are due mainly to a deregulated cell–cell adhesion arisen from the downregulation of E-cadherin, plus uncontrolled cell proliferation owing to the upregulation of β-catenin, both resulted from a breakdown of actin microfilaments caused by the overexpression of this G-actin sequestering peptide.

Restoration of Bone Mass and Strength in Glucocorticoid-Treated Mice by Systemic Transplantation of CXCR4 and Cbfa-1 Co-Expressing Mesenchymal Stem Cells

Transplantation of gene‐modified mesenchymal stem cells (MSCs) in animals for bone regeneration therapy has been evaluated extensively in recent years. However, increased endosteal bone formation by intravenous injection of MSCs ectopically expressing a foreign gene has not yet been shown. Aside from the clearance by lung and other tissues, the surface compositions of MSCs may not favor their bone marrow (BM) migration and engraftment. To overcome these hurdles, a gene encoding the chemokine receptor largely responsible for stromal‐derived factor‐1 (SDF‐1)‐mediated BM homing and engraftment of hematopoietic stem cells (HSCs), CXCR4, was transduced into mouse C3H10T1/2 cells by adenovirus infection. A dose‐dependent increase of CXCR4 surface expression with a parallel enhanced chemotaxis toward SDF‐1 in these cells after virus infection was clearly observed. Higher BM retention and homing of CXCR4‐expressing MSCs were also found after they were transplanted by intramedullary and tail vein injections, respectively, into immunocompetent C3H/HeN mice. Interestingly, a full recovery of bone mass and a partial restoration of bone formation in glucocorticoid‐induced osteoporotic mice were observed 4 wk after a single intravenous infusion of one million CXCR4‐expressing C3H10T1/2 cells. In the meantime, complete recovery of bone stiffness and strength in these animals was consistently detected only after a systemic transplantation of CXCR4 and Cbfa‐1 co‐transduced MSCs. To our knowledge, this is the first report to show unequivocally the feasibility of ameliorating glucocorticoid‐induced osteoporosis by systemic transplantation of genetically manipulated MSCs.

Fluvastatin and lovastatin but not pravastatin induce neuroglial differentiation in human mesenchymal stem cells

Recent studies have shown that statins, the most potent inhibitors of 3‐hydroxy‐2‐methylglutaryl coenzyme A (HMG‐CoA) reductase, stimulate bone formation in vitro and in rodents by activating the expression of bone morphogenetic protein‐2 (BMP‐2), one of the most critical osteoblast differentiation‐inducing factors. However, the effect of statins on mesenchymal stem cells (MSCs) is yet to be reported. The purpose of this study is to investigate the influence of fluvastatin, lovastatin, and pravastatin, three commonly prescribed lipid‐lowering agents, on the proliferation and differentiation of human MSCs. To our surprise, even though fluvastatin and lovastatin effectively suppressed the growth of human MSCs, a neuroglia rather than osteoblast‐like morphology was observed after treatment. Interestingly, such morphological change was inhibited by the co‐addition of geranylgeranyl pyrophosphate (GGPP). Immunofluorescence staining with antibodies against neuron‐, astrocyte‐, as well as oligodendrocyte‐specific markers confirmed the neuroglial identity of the differentiated cells. However, BMP‐2 is unlikely to play a positive role in neuroglial differentiation of MSCs since its expression was down‐regulated in fluvastatin‐treated cells. Taken together, our results suggest that fluvastatin and lovastatin induce neuroglial differentiation of human MSCs and that these cholesterol‐lowering agents might be used in conjunction with MSC transplantation in the future for treating neurological disorders and injuries.

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic proteins 2 (BMP2). Mitogen‐activated protein kinase (MAPK) cascades, such as extracellular signal‐regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun‐N‐terminal kinase (JNK), the other kinase of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first showed that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3‐E1 cell lines. We then showed that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss‐of‐function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN‐JNK1). Consistently, BMP2‐induced osteoblastic differentiation is reduced by JNK1 gain‐of‐function, such as enforced expression of a constitutively active JNK1 (CA‐JNK1). Most importantly, we showed that Runx2 is required for JNK1‐mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3‐E1 cells to the extent similar to that achieved by overexpression of wild‐type (WT) Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2‐induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation.

Colorectal carcinoma: from tumorigenesis to treatment

Abstract.Colorectal carcinoma (CRC) is a complicated and often fatal genetic disease. Fortunately, owing to rapid expansion of knowledge and technology development in oncology, much progress has been made regarding the diagnosis, understanding of the molecular genetics and malignant progression, as well as the novel regimens of CRC. In this review, we summarize the staging system, the most critical genetic and epigenetic alterations, the pleiotropic effects of MMP-7, the controversial roles of Hedgehog signaling, the intriguing involvement of thymosin β-4, and the possible contribution of the putative colon (cancer) stem cells in CRC tumorigenesis. Current treatments as well as several potentially applicable therapeutic strategies for CRC are also discussed.

Cbfb enhances the osteogenic differentiation of both human and mouse mesenchymal stem cells induced by Cbfa-1 via reducing its ubiquitination-mediated degradation.

Core‐binding factors are a small family of heterodimeric transcription factors that play critical roles in development. Whereas Cbfa‐1, one of the three α subunits in the family, is essential for osteogenesis, Cbfb, the only β subunit, forms heterodimers with different Cbfas to increase their DNA binding affinity by inducing conformational changes. Although defective bone formation was found in both Cbfa‐1 and Cbfb knockout animals, the precise role of the latter in osteogenesis remains unclear. To dissect the contribution of Cbfb in osteogenic differentiation of mesenchymal stem cells (MSCs), recombinant adenoviruses carrying Cbfb (AdHACbfb) and Cbfa‐1 (AdCbfa‐1) were generated and used to infect both the mouse C3H10T1/2 cells and human bone marrow‐derived MSCs. Although Cbfb alone failed to trigger osteogenesis of MSCs, it markedly enhanced the gene expression and enzyme activity of alkaline phosphatase as well as osteocalcin activation in those cells overexpressing Cbfa‐1. Enhancement of the osteogenic differentiation‐inducing effect of Cbfa‐1 by Cbfb resulted from an increase in stability of the former due to the suppression of ubiquitination‐mediated proteasomal degradation by the latter. Taken together, in addition to defining the role of Cbfb in osteogenic differentiation of MSCs, our results also suggest that the Cbfa‐1 and Cbfb coexpressing MSCs might be an appropriate strategy for bone repairing and regeneration therapies.

Thymosin beta‐4 directs cell fate determination of human mesenchymal stem cells through biophysical effects

Change of actin filament organization at the early stage of cell differentiation directs cell fate commitment of mesenchymal stem cells (MSCs). Thymosin beta‐4 (Tβ4), a major G‐actin sequestering peptide, is known to regulate the cytoskeleton. The study investigated the ways in which Tβ4 regulates cell fate determination in MSCs upon differentiation induction. It was found that Tβ4 decreased F‐actin formation, reduced the F‐actin/G‐actin ratio, and inhibited osteogenic differentiation; such actin reorganization was not associated with the change of Runt‐related transcription factor 2 gene expression during early osteogenic induction. Besides, Tβ4 reciprocally facilitated adipogenic differentiation. Tβ4 treatment was found to up‐regulate gene as well as promote surface expression of adipocyte adhesion molecule during early adipogenic differentiation, which accompanied acceleration of adipocyte phenotypic maturation but was not associated with differential expression of peroxisome proliferator‐activated receptor gamma during the first week of adipogenic induction. In summary, Tβ4 initiated cell fate determination of MSCs through biophysical effects exerted by cytoskeleton reorganization and altered cell‐cell adhesion rather than direct regulation of lineage‐determining transcriptional factors. Such findings suggest that Tβ4, a ubiquitous peptide, may be involved in osteoporosis when its intracellular concentration is elevated. Further investigation of targeting Tβ4 for future osteoporosis treatment is warranted.

Imipramine blue halts head and neck cancer invasion through promoting F-box and leucine-rich repeat protein 14-mediated Twist1 degradation.

The unique characteristic of head and neck squamous cell carcinoma (HNSCC) is that local invasion rather than distant metastasis is the major route for dissemination. Therefore, targeting the locally invasive cancer cells is more important than preventing systemic metastasis in HNSCC and other invasive-predominant cancers. We previously demonstrate a specific mechanism for HNSCC local invasion: the epithelial–mesenchymal transition (EMT) regulator Twist1 represses microRNA let-7i expression, leading to the activation of the small GTPase Rac1 and engendering the mesenchymal-mode movement in three-dimensional (3D) culture. However, targeting the EMT regulator is relatively difficult because of its transcription factor nature and the strategy for confining HNSCC invasion to facilitate local treatment is limited. Imipramine blue (IB) is a newly identified anti-invasive compound that effectively inhibits glioma invasion. Here we demonstrate that in HNSCC cells, a noncytotoxic dose of IB represses mesenchymal-mode migration in two-and-a-half-dimensional/3D culture system. IB suppresses EMT and stemness of HNSCC cells through inhibition of Twist1-mediated let-7i downregulation and Rac1 activation and the EMT signalling. Mechanistically, IB inhibits reactive oxygen species-induced nuclear factor-κB pathway activation. Importantly, IB promotes degradation of the EMT inducer Twist1 by enhancing F-box and leucine-rich repeat protein 14 (FBXL14)-mediated polyubiquitination of Twist1. Together, this study demonstrates the potent anti-invasion and EMT-inhibition effect of IB, suggesting the potential of IB in treating local invasion-predominant cancers.

Construction of a cell-based high-flux assay for the rev protein of HIV-1

The Rev of HIV-1 is essential for the replication of the viruses and is therefore a very attractive target for the development of antiviral drugs. To establish a cell-based high-flux assay system for random screening of Rev inhibitors, cells carrying both the Rev-expressing gene and a Rev-inducible SeAP gene were generated by permanent transfection. SeAP produced by these cells was 5-10-fold higher than that synthesized by cells not carrying the Rev gene. Northern blot analysis demonstrated that the increase in SeAP was due mainly to an increase in SeAP transcripts, indicating the effect of Rev proteins synthesized by the transfected cells. The assay system reported in this study should be useful for screening novel Rev inhibitors.