Li Wha Wu

A gp130-Src-YAP module links inflammation to epithelial regeneration

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

Interleukin-17 receptor A signaling in transformed enterocytes promotes early colorectal tumorigenesis

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

Enhanced expression of vascular endothelial growth factor‐A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis

Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre‐S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre‐S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up‐regulated by pre‐S mutants was identified using a growth factor array in HuH‐7 cells. Immunohistochemistry, reverse‐transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH‐7 cells and liver tissues. We demonstrate that vascular endothelial growth factor‐A (VEGF‐A) was up‐regulated by pre‐S mutants in HuH‐7 cells and further confirmed in GGHs by immunostaining. The VEGF‐A up‐regulation by pre‐S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre‐S mutants‐expressed HuH‐7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF‐A neutralization. Consistent with this notion, enhanced expression of VEGF‐A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV‐related nontumorous livers. Conclusion: The enhanced expression of VEGF‐A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection. (HEPATOLOGY 2009;49:1962–1971.)

A Novel Sialyltransferase Inhibitor Suppresses FAK/Paxillin Signaling and Cancer Angiogenesis and Metastasis Pathways

Increased sialyltransferase (ST) activity promotes cancer cell metastasis, and overexpression of cell surface sialic acid correlates with poor prognosis in cancer patients. To seek therapies targeting metastasis for cancer treatment, we developed a novel ST inhibitor, Lith-O-Asp, and investigated its antimetastatic and antiangiogenic effects and mechanisms. We found that cells treated with Lith-O-Asp showed a reduction of activity on various ST enzymes by in vitro and cell-based activity analyses. Lith-O-Asp inhibited migration and invasion abilities in various cancer cell lines and showed inhibitory effect on the angiogenic activity of human umbilical vein endothelial cells. Indeed, Lith-O-Asp treatment consequently delayed cancer cell metastasis in experimental and spontaneous metastasis assays in animal models. Importantly, Lith-O-Asp decreased the sialic acid modification of integrin-β1 and inhibited the expression of phospho-FAK, phospho-paxillin, and the matrix metalloprotease (MMP) 2 and MMP9. Lith-O-Asp attenuated the Rho GTPase activity leading to actin dynamic impairment. In addition, 2DE-MS/MS and immunoblotting analyses showed that Lith-O-Asp altered the protein expression level and phosphorylation status of various proteins involved in crucial metastasis and angiogenesis pathways such as vimentin and ribonuclease/angiogenin inhibitor RNH1. Furthermore, Lith-O-Asp treatment significantly inhibited the invasive ability exerted by ectopic overexpression of various ST enzymes catalyzing α-2,6- or α-2,3-sialylation. Our results provide compelling evidence that the potential pan-ST inhibitor, Lith-O-Asp, suppressed cancer cell metastasis likely by inhibiting FAK/paxillin signaling and expressing antiangiogenesis factors. Lith-O-Asp is worthy for further testing as a novel antimetastasis drug for cancer treatment.

Antiangiogenic activity of Tripterygium wilfordii and its terpenoids.

ETHNOPHARMACOLOGICAL RELEVANCE Tripterygium wilfordii Hook. f. (Celastraceae) has been traditionally used as folk medicine for centuries in China for the treatment of immune-inflammatory diseases. AIM OF THE STUDY This study aimed to assess the antiangiogenic activities which support the therapeutic use of Tripterygium wilfordii and its terpenoids for angiogenesis disease such as cancer. MATERIALS AND METHODS The ethanol extract of Tripterygium wilfordii and subsequent fractions were evaluated on an in vivo antiangiogenic zebrafish embryo model. RESULTS Three antiangiogenic terpenoids were isolated by bioassay-guided purification, namely, celastrol (4), cangoronine (5) and triptolide (7). Among them, triptolide manifested the most potent antiangiogenic activity against vessel formation by nearly 50% at 1.2 microM. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt)2 and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. CONCLUSIONS The discovery of an alternative pathway further confirms the value of ethnopharmacological investigations into traditional botanicals for leads for potential drug development.

Distinct regulation of genes by bFGF and VEGF-A in endothelial cells

A finely tuned balance of angiogenic inhibitors and inducers controls the activity of angiogenesis characterized by proliferation, migration and differentiation of endothelial cells. Among many angiogenic factors, basic fibroblast growth factor (bFGF) was first identified to be angiogenic whereas vascular endothelial growth factor A (VEGF-A) is an endothelial cell specific mitogen. In addition to being a specific mitogen, VEGF-A is also known as a vascular permeability factor. The majority of growth factors transduce their mitogenic signals from cell surface to nucleus where gene expression occurs. Whether these ligands utilize a distinct or a common molecular pathway to exert their biological effects on human endothelial cells remains elusive. We thus studied the expression profile of 884 human genes under the influence of either bFGF or VEGF-A alone in the context of human endothelial cells. A total of ninety-four genes were differentially regulated by more than two folds. The expression patterns of 32 genes are similar between the treatment of either factor alone whereas those of the remaining 62 genes are only regulated by one but not the other factor. Their function in the control of angiogenesis will be discussed and apoptotic signaling in the regulation of angiogenesis is also implicated.

ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction

The success of using glycolytic inhibitors for cancer treatment depends on studying the individual role of frequently deregulated glycolytic genes in cancer. This report aims to study the prognostic implication, and determine the cellular role and action mechanism of glycolytic ENO1 overexpression in head and neck cancer. The relationship of ENO1 mRNA expression in 44-pair clinical specimens with patient clinicopathologic characteristics was analysed by semi-quantitative RT-PCR, Kaplan-Meier survival curve and Cox model analyses. Following ectopic ENO1 expression or knockdown, we studied the proliferative, migratory, invasive, colony-forming and tumourigenic abilities of ENO1-genetically altered cells. DNA microarray analysis was used to identify downstream targets responsible for the ENO1 action in the cells. The expression of ENO1 mRNA was increased in 68% of tumour (T) specimens when compared to their normal (N) counterparts, and positively associated with clinical progression (p<0.05). High ENO1 expression (T/N2) was frequently observed in the patients with large primary tumours, late clinical stages or advanced neck metastasis. Moreover, high ENO1 patients had significantly poorer clinical outcomes than low expressers (T/N<2). Ectopic ENO1 expression stimulated cell transformation, invasion and tongue tumour formation. ENO1 knockdown abrogated the stimulation. Suppression of ENO1-induced proinflammatory CCL20 chemokine expression significantly attenuated its stimulatory effects on cell transformation and invasion. A concordant expression of ENO1 and CCL20 was validated both in ENO1-expressing cells and in clinical specimens. Together, we demonstrate a prognostic role of ENO1 overexpression in head and neck cancer and ENO1-mediated promotion of cell transformation and invasion partly via induced CCL20 expression.

Triptolide functions as a potent angiogenesis inhibitor.

Triptolide is a key anti‐inflammatory compound of the Chinese herbal medicine Tripterygium wilfordii Hook. f. (Celastraceae). It also possesses potent antitumor activity. In this study, we show that triptolide is an angiogenesis inhibitor based on various angiogenesis assays. The IC50 in in vitro assays was 45 nM, which was much lower than the plasma concentrations of triptolide in the rat or human administered with T. wilfordii extracts for treating inflammation. When dosed in vivo, triptolide potently inhibited angiogenesis at 100 nM in Matrigel plug assay. Triptolide at 0.75 mg/kg/day significantly blocked tumor angiogenesis and tumor progression in murine tumorigenesis assay. The underlying mechanism of triptolide correlated with downregulation of proangiogenic Tie2 and VEGFR‐2 expression in human umbilical vein endothelial cell by semiquantitative RT‐PCR and western blot analysis. Although Tie2 inhibition appeared to be a later event as compared with VEGFR‐2, Tie2 overexpression significantly attenuated the inhibitory effect of triptolide on endothelial proliferation and network formation. By contrast, Tie2 knockdown mimicked the inhibitory effect of triptolide on endothelial network formation. Our findings suggest that antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor‐mediated signaling pathways, and triptolide can be a promising antiangiogenic agent.

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein.

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.

Ubiquitin-conjugating enzyme Ubc13 controls breast cancer metastasis through a TAK1-p38 MAP kinase cascade.

Significance We demonstrate that ubiquitin-conjugating enzyme Ubc13, whose expression is elevated in primary and metastatic breast cancer (BCa), promotes metastatic spread of BCa cells by controlling their lung-colonizing ability while having little effect on primary tumor growth. Mechanistically, Ubc13 is required for TGFβ-induced non-SMAD signaling via TAK1 and p38, a pathway that is first activated in the primary tumor. An Ubc13- and p38-dependent metastatic gene signature was identified, explaining how p38 may control metastasis and providing a measure for monitoring the effectiveness of pharmacologic p38 inhibition, which inhibits the growth of established metastatic lesions. We suggest that p38 inhibition should be considered as a potential treatment for metastatic BCa. Metastatic spread is the leading cause of cancer mortality. Breast cancer (BCa) metastatic recurrence can happen years after removal of the primary tumor. Here we show that Ubc13, an E2 enzyme that catalyzes K63-linked protein polyubiquitination, is largely dispensable for primary mammary tumor growth but is required for metastatic spread and lung colonization by BCa cells. Loss of Ubc13 inhibited BCa growth and survival only at metastatic sites. Ubc13 was dispensable for transforming growth factor β (TGFβ)-induced SMAD activation but was required for activation of non-SMAD signaling via TGFβ-activating kinase 1 (TAK1) and p38, whose activity controls expression of numerous metastasis promoting genes. p38 activation restored metastatic activity to Ubc13-deficient cells, and its pharmacological inhibition attenuated BCa metastasis in mice, suggesting it is a therapeutic option for metastatic BCa.