Min-Liang Kuo

Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal–Epithelial Transition in Head and Neck Cancer Cells

The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC.

Pretreatment Epidermal Growth Factor Receptor (EGFR) T790M Mutation Predicts Shorter EGFR Tyrosine Kinase Inhibitor Response Duration in Patients With Non–Small-Cell Lung Cancer

PURPOSE Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations have excellent response to EGFR tyrosine kinase inhibitors (TKIs), but T790M mutation accounts for most TKI drug resistance. This study used highly sensitive methods to detect T790M before and after TKI therapy and investigated the association of T790M and its mutation frequencies with clinical outcome. PATIENTS AND METHODS Direct sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and next-generation sequencing (NGS) were used to assess T790M in the following two cohorts of patients with NSCLC: TKI-naive patients (n = 107) and TKI-treated patients (n = 85). Results were correlated with TKI treatment response and survival. RESULTS MALDI-TOF MS was highly sensitive in detecting and quantifying the frequency of EGFR-activating mutations and T790M (detection limits, 0.4% to 2.2%). MALDI-TOF MS identified more T790M than direct sequencing in TKI-naive patients with NSCLC (27 of 107 patients, 25.2% v three of 107 patients, 2.8%, respectively; P < .001) and in TKI-treated patients (before TKI: 23 of 73 patients, 31.5% v two of 73 patients, 2.7%, respectively; P < .001; and after TKI: 10 of 12 patients, 83.3% v four of 12 patients, 33.3%, respectively; P = .0143). The EGFR mutations and their frequencies were confirmed by NGS. T790M was an independent predictor of decreased progression-free survival (PFS) in patients with NSCLC who received TKI treatment (P < .05, multivariate Cox regression). CONCLUSION T790M may not be a rare event before or after TKI therapy in patients with NSCLC with EGFR-activating mutations. The pretreatment T790M mutation was associated with shorter PFS with EGFR TKI therapy in patients with NSCLC.

Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway.

Interleukin-6 (IL-6) has received particular attention in the pathogenesis of cervical cancer, although the underlying mechanism remains elusive. This study revealed that IL-6 promotes in vivo tumor growth of human cervical cancer C33A cells, but does not substantially alter their in vitro growth kinetics. The in vivo angiogenic assays showed that IL-6 increases angiogenic activity in human cervical cancer cells, an effect that is specifically associated with upregulation of vascular endothelial growth factor (VEGF). Also, using anti-VEGF antibody to block VEGF function significantly inhibited IL-6-mediated angiogenesis and tumor growth in nude mice, strongly supporting the critical role of VEGF in the IL-6-mediated cervical tumorigenesis. Accordingly, the signaling pathway downstream of IL-6/IL-6R responsible for the regulation of VEGF was investigated. Notably, pharmacological inhibition of PI3-K or MAPK failed to inhibit IL-6-mediated transcriptional upregulation of VEGF. Meanwhile, blocking STAT3 pathway with dominant-negative mutant STAT3D effectively abolished IL-6-induced VEGF mRNA. In transient transfections, a luciferase reporter construct containing the full-length 1.5-kb VEGF promoter or a 1.2-kb fragment lacking the known hypoxic-response element also exhibited the same degree of response to IL-6. Additionally, transient transfection of STAT3D downregulated the 1.2-kb VEGF promoter luciferase reporter stimulated by IL-6. Based on the above phenomenon combined with the concomitant increased tumor expression of IL-6 and VEGF in cervical cancer tissues, we conclude that IL-6 may promote cervical tumorigenesis by activating VEGF-mediated angiogenesis via a STAT3 pathway.

Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells

Curcumin, widely used as a spice and coloring agent in food, possesses potent antioxidant, anti-inflammatory and anti-tumor promoting activities. In the present study, curcumin was found to induce apoptotic cell death in promyelocytic leukemia HL-60 cells at concentrations as low as 3.5 micrograms/ml. The apoptosis-inducing activity of curcumin appeared in a dose- and time-dependent manner. Flow cytometric analysis showed that the hypodiploid DNA peak of propidium iodide-stained nuclei appeared at 4 h after 7 micrograms/ml curcumin treatment. The apoptosis-inducing activity of curcumin was not affected by cycloheximide, actinomycin D, EGTA, W7 (calmodulin inhibitor), sodium orthovanadate, or genistein. By contrast, an endonuclease inhibitor ZnSO4 and proteinase inhibitor N-tosyl-L-lysine chloro-methyl ketone (TLCK) could markedly abrogate apoptosis induced by curcumin, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) had a partial effect. The antioxidants, N-acetyl-L-cysteine (NAC), L-ascorbic acid, alpha-tocopherol, catalase and superoxide dismutase, all effectively prevented curcumin-induced apoptosis. This result suggested that curcumin-induced cell death was mediated by reactive oxygen species. Immunoblot analysis showed that the level of the antiapoptotic protein Bcl-2 was decreased to 30% after 6 h treatment with curcumin, and was subsequently reduced to 20% by a further 6 h treatment. Furthermore, overexpression of bcl-2 in HL-60 cells resulted in a delay of curcumin-treated cells entering into apoptosis, suggesting that bcl-2 plays a crucial role in the early stage of curcumin-triggered apoptotic cell death.

The Antiapoptotic Gene mcl-1 Is Up-Regulated by the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway through a Transcription Factor Complex Containing CREB

ABSTRACT mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position −87 and CRE-2 at −70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of themcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation ofmcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

Interleukin-6 Inhibits Transforming Growth Factor-β-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Akt and Signal Transducers and Activators of Transcription 3 Pathways

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-β (TGF-β), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-β-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15 INK4B genes, indicating that IL-6 interferes with only a subset of TGF-β activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-β-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-β, with the former likely playing a major role in this anti-apoptosis.

Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-κB signal transduction pathway

This study is to investigate the molecular mechanism of radiation-enhanced cell invasiveness of hepatocellular carcinoma (HCC) correlating with clinical patients undergoing radiotherapy and subsequently developing metastasis. Three HCC cell lines (HepG2, Hep3B and Huh7) and normal hepatocyte cell line (CL-48) were irradiated with different doses. The effect of radiation on cell invasiveness was determined using the Boyden chamber assay. Radiation-enhanced invasion capability was evident in HCC cells but not in normal hepatocytes. Invasion was observed in gelatin-coated but not fibronectin-coated or type I collagen-coated membranes. Radiation upregulated matrix metalloproteinase-9 (MMP-9) mRNA level, MMP-9 protein level and MMP-9 activity. MMP-9 antisense oligonucleotides inhibited radiation-induced MMP-9 expression and thereby significantly inhibited radiation-induced HCC invasion. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt chemical inhibitors LY294002 and wortmannin suppressed radiation-induced MMP-9 mRNA expression. Transient transfection with dominant-negative Akt plasmid also showed that the PI3K/Akt-signaling pathway was involved in this radiation-induced MMP-9 expression. Moreover, nuclear factor-κB (NF-κB) decoy oligodeoxynucleotide suppressed radiation enhanced MMP-9 promoter activity completely. PI3K/Akt chemical inhibitors inhibited radiation-induced NF-κB-driven luciferase promoter activity. Taken together, our results indicated that sublethal dose of radiation could enhance HCC cell invasiveness by MMP-9 expression through the PI3K/Akt/NF-κB signal transduction pathway.

Tumor-Associated Macrophage-Induced Invasion and Angiogenesis of Human Basal Cell Carcinoma Cells by Cyclooxygenase-2 Induction

Tumor-associated macrophages (TAMs) and cyclooxygenase-2 (COX-2) are associated with invasion, angiogenesis, and poor prognosis in many human cancers. However, the role of TAMs in human basal cell carcinoma (BCC) remains elusive. We found that the number of TAMs infiltrating the tumor is correlated with the depth of invasion, microvessel density, and COX-2 expression in human BCC cells. TAMs also aggregate near COX-2 expressing BCC tumor nests. We hypothesize that TAMs might activate COX-2 in BCC cells and subsequently increase their invasion and angiogenesis. TAMs are a kind of M2 macrophage derived from macrophages exposed to Th2 cytokines. M2-polarized macrophages derived from peripheral blood monocytes were cocultured with BCC cells without direct contact. Coculture with the M2 macrophages induced COX-2-dependent invasion and angiogenesis of BCC cells. Human THP-1 cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages with M2 functional profiles. Coculture with PMA-treated THP-1 macrophages induced COX-2-dependent release of matrix metalloproteinase-9 and subsequent increased invasion of BCC cells. Macrophages also induced COX-2-dependent secretion of basic fibroblast growth factor and vascular endothelial growth factor-A, and increased angiogenesis in BCC cells.

H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.

mcl-1 Is an Immediate-Early Gene Activated by the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Signaling Pathway and Is One Component of the GM-CSF Viability Response

ABSTRACT mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, themcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor β chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between −197 and −69 is responsible for cytokine activation of the mcl-1 gene. Overexpression ofmcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.