Hsei-Wei Wang

Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition

The epithelial–mesenchymal transition (EMT), one of the main mechanisms underlying development of cancer metastasis, induces stem-like properties in epithelial cells. Bmi1 is a polycomb-group protein that maintains self-renewal, and is frequently overexpressed in human cancers. Here, we show the direct regulation of BMI1 by the EMT regulator, Twist1. Furthermore, Twist1 and Bmi1 were mutually essential to promote EMT and tumour-initiating capability. Twist1 and Bmi1 act cooperatively to repress expression of both E-cadherin and p16INK4a. In patients with head and neck cancers, increased levels of both Twist1 and Bmi1 correlated with downregulation of E-cadherin and p16INK4a, and was associated with the worst prognosis. These results suggest that Twist1-induced EMT and tumour-initiating capability in cancer cells occurs through chromatin remodelling, which leads to unfavourable clinical outcomes.

Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma

The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.

Rapid generation of mature hepatocyte‐like cells from human induced pluripotent stem cells by an efficient three‐step protocol

Liver transplantation is the only definitive treatment for end‐stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient‐specific disease modeling. Here, we describe an efficient and rapid three‐step protocol that is able to rapidly generate hepatocyte‐like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC‐derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte‐like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low‐density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte‐like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte‐like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases. (Hepatology 2012)

K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

ABSTRACT The Kaposi’s sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, ∼50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman’s disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.

SNAIL Regulates Interleukin-8 Expression, Stem Cell–Like Activity, and Tumorigenicity of Human Colorectal Carcinoma Cells

BACKGROUND & AIMS Some cancer cells have activities that are similar to those of stem cells from normal tissues, and cell dedifferentiation correlates with poor prognosis. Little is known about the mechanisms that regulate the stem cell-like features of cancer cells; we investigated genes associated with stem cell-like features of colorectal cancer (CRC) cells. METHODS We isolated colonospheres from primary CRC tissues and cell lines and characterized their gene expression patterns by microarray analysis. We also investigated the biological features of the colonosphere cells. RESULTS Expanded CRC colonospheres contained cells that expressed high levels of CD44 and CD166, which are markers of colon cancer stem cells, and had many features of cancer stem cells, including chemoresistance and radioresistance, the ability to initiate tumor formation, and activation of epithelial-mesenchymal transition (EMT). SNAIL, an activator of EMT, was expressed at high levels by CRC colonospheres. Overexpression of Snail in CRC cells induced most properties of colonospheres, including cell dedifferentiation. Two hundred twenty-seven SNAIL-activated genes were up-regulated in colonospheres; gene regulatory networks centered around interleukin (IL)-8 and JUN. Blocking IL-8 expression or activity disrupted SNAIL-induced stem cell-like features of colonospheres. We observed that SNAIL activated the expression of IL8 by direct binding to its E3/E4 E-boxes. In CRC tissues, SNAIL and IL-8 were coexpressed with the stem cell marker CD44 but not with CD133 or CD24. CONCLUSIONS In human CRC tissues, SNAIL regulates expression of IL-8 and other genes to induce cancer stem cell activities. Strategies that disrupt this pathway might be developed to block tumor formation by cancer stem cells.

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposis sarcoma‐associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin‐ΔEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin‐ΔEx3 contain a mitochondrial‐targeting sequence, an N‐terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl‐2 homology)‐like domain. These suggested that K7 is a new viral anti‐apoptotic protein and survivin‐ΔEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl‐2 resides. K7 does not associate with Bax, but does bind to Bcl‐2 via its putative BH2 domain. In addition, K7 binds to active caspase‐3 via its BIR domain and thus inhibits the activity of caspase‐3. The BH2 domain of K7 is crucial for the inhibition of caspase‐3 activity and is therefore essential for its anti‐apoptotic function. Furthermore, K7 bridges Bcl‐2 and activated caspase‐3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti‐apoptotic complex that presents effector caspases to Bcl‐2, enabling Bcl‐2 to inhibit caspase activity. These data also suggest that survivin‐ΔEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor‐of‐apoptosis protein).

Mesenchymal Stem Cells from Human Umbilical Cord Express Preferentially Secreted Factors Related to Neuroprotection, Neurogenesis, and Angiogenesis

Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases such as infarcted myocardia and strokes because of their ability to promote endogenous angiogenesis and neurogenesis via a variety of secreted factors. MSCs found in the Wharton’s jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. We isolated MSCs from Wharton’s jelly and bone marrow (WJ-MSCs and BM-MSCs, respectively) and compared their secretomes. It was found that WJ-MSCs expressed more genes, especially secreted factors, involved in angiogenesis and neurogenesis. Functional validation showed that WJ-MSCs induced better neural differentiation and neural cell migration via a paracrine mechanism. Moreover, WJ-MSCs afforded better neuroprotection efficacy because they preferentially enhanced neuronal growth and reduced cell apoptotic death of primary cortical cells in an oxygen-glucose deprivation (OGD) culture model that mimics the acute ischemic stroke situation in humans. In terms of angiogenesis, WJ-MSCs induced better microvasculature formation and cell migration on co-cultured endothelial cells. Our results suggest that WJ-MSC, because of a unique secretome, is a better MSC source to promote in vivo neurorestoration and endothelium repair. This study provides a basis for the development of cell-based therapy and carrying out of follow-up mechanistic studies related to MSC biology.

MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells

Asymmetrical cell division (ACD) maintains the proper number of stem cells to ensure self-renewal. In cancer cells, the deregulation of ACD disrupts the homeostasis of the stem cell pool and promotes tumour growth. However, this mechanism is unclear. Here, we show a reduction of ACD in spheroid-derived colorectal cancer stem cells (CRCSCs) compared with differentiated cancer cells. The epithelial–mesenchymal transition (EMT) inducer Snail is responsible for the ACD-to-symmetrical cell division (SCD) switch in CRCSCs. Mechanistically, Snail induces the expression of microRNA-146a (miR-146a) through the β-catenin–TCF4 complex. miR-146a targets Numb to stabilize β-catenin, which forms a feedback circuit to maintain Wnt activity and directs SCD. Interference with the Snail–miR-146a–β-catenin loop by inhibiting the MEK or Wnt activity reduces the symmetrical division of CRCSCs and attenuates tumorigenicity. In colorectal cancer patients, the SnailHighNumbLow profile is correlated with cetuximab resistance and a poorer prognosis. This study elucidates a unique mechanism of EMT-induced CRCSC expansion.

Functional Module Analysis Reveals Differential Osteogenic and Stemness Potentials in Human Mesenchymal Stem Cells from Bone Marrow and Wharton's Jelly of Umbilical Cord

Mesenchymal stem cells (MSCs) found in bone marrow (BM)-MSCs are an attractive source for the regeneration of damaged tissues. Alternative postnatal, perinatal, and fetal sources of MSCs are also under intensive investigation. MSCs from the Whartons jelly matrix of umbilical cord (WJ)-MSCs have higher pancreatic and endothelial differentiation potentials than BM-MSCs, but the underlying mechanisms are poorly understood. We compared the gene expression profiles, enriched canonical pathways, and genetic networks of BM-MSCs and WJ-MSCs. WJ-MSCs express more angiogenesis- and growth-related genes including epidermal growth factor and FLT1, whereas BM-MSCs express more osteogenic genes such as RUNX2, DLX5, and NPR3. The gene expression pattern of BM-MSCs is more similar to osteoblasts than WJ-MSCs, suggesting a better osteogenic potential. In contrast, WJ-MSCs are more primitive because they share more common genes with embryonic stem cells. BM-MSCs are more sensitive to environmental stimulations because their molecular signatures altered more significantly in different culture conditions. WJ-MSCs express genes enriched in vascular endothelial growth factor and PI3K-NFκB canonical pathways, whereas BM-MSCs express genes involved in antigen presentation and chemokine/cytokine pathways. Drylab results could be verified by wetlab experiments, in which BM-MSCs were more efficient in osteogenic and adipogenic differentiation, whereas WJ-MSCs proliferated better. WJ-MSCs thus constitute a promising option for angiogenesis, whereas BM-MSCs in bone remodeling. Our results reveal systematically the underlying genes and regulatory networks of 2 MSCs from unique ontological and anatomical origins, as well as the resulted phenotypes, thereby providing a better basis for cell-based therapy and the following mechanistic studies on MSC biology.

Mesenchymal Stem Cells Promote Formation of Colorectal Tumors in Mice

BACKGROUND & AIMS Tumor-initiating cells are a subset of tumor cells with the ability to form new tumors; however, they account for less than 0.001% of the cells in colorectal or other types of tumors. Mesenchymal stem cells (MSCs) integrate into the colorectal tumor stroma; we investigated their involvement in tumor initiation. METHODS Human colorectal cancer cells, MSCs, and a mixture of both cell types were injected subcutaneously into immunodeficient mice. We compared the ability of each injection to form tumors and investigated the signaling pathway involved in tumor initiation. RESULTS A small number (≤ 10) of unsorted, CD133⁻, CD166⁻, epithelial cell adhesion molecule⁻(EpCAM⁻), or CD133⁻/CD166⁻/EpCAM⁻ colorectal cancer cells, when mixed with otherwise nontumorigenic MSCs, formed tumors in mice. Secretion of interleukin (IL)-6 by MSCs increased the expression of CD133 and activation of Janus kinase 2-signal transducer and activator of transcription 3 (STAT3) in the cancer cells, and promoted sphere and tumor formation. An antibody against IL-6 or lentiviral-mediated transduction of an interfering RNA against IL-6 in MSCs or STAT3 in cancer cells prevented the ability of MSCs to promote sphere formation and tumor initiation. CONCLUSIONS IL-6, secreted by MSCs, signals through STAT3 to increase the numbers of colorectal tumor-initiating cells and promote tumor formation. Reagents developed to disrupt this process might be developed to treat patients with colorectal cancer.