ShouWei Han

Rosiglitazone suppresses human lung carcinoma cell growth through PPARγ-dependent and PPARγ-independent signal pathways

Peroxisome proliferator-activated receptors γ (PPARγ) exert diverse effects on cancer cells. Recent studies showed that rosiglitazone, a synthetic ligand for PPARγ, inhibits cell growth. However, the exact mechanisms underlying this effect are still being explored, and the relevance of these findings to lung cancer remains unclear. Here, we report that rosiglitazone reduced the phosphorylation of Akt and increased phosphatase and tensin homologue (PTEN) protein expression in non–small cell lung carcinoma (NSCLC) cells (H1792 and H1838), and this was associated with inhibition of NSCLC cell proliferation. These effects were blocked or diminished by GW9662, a specific PPARγ antagonist. However, transfection with a CMX-PPARγ2 overexpression vector restored the effects of rosiglitazone on Akt, PTEN, and cell growth in the presence of GW9662. In addition, rosiglitazone increased the phosphorylation of AMP-activated protein kinase α (AMPKα), a downstream kinase target for LKB1, whereas it decreased phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream target of mammalian target of rapamycin (mTOR). Of note, GW9662 did not affect the phosphorylation of AMPKα and p70S6K protein. The inhibitory effect of rosiglitazone on NSCLC cell growth was enhanced by the mTOR inhibitor rapamycin; however, it was blocked, in part, by the AMPKα small interfering RNA. Taken together, these findings show that rosiglitazone, via up-regulation of the PTEN/AMPK and down-regulation of the Akt/mTOR/p70S6K signal cascades, inhibits NSCLC cell proliferation through PPARγ-dependent and PPARγ-independent signals. [Mol Cancer Ther 2006;5(2):430–7]

Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways.

The Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, differentiation, and survival. However, the role of the Akt/mTOR/p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC) cell growth and survival. Herein, we explore the role of the Akt/mTOR/p70S6K pathway in fibronectin-induced NSCLC cell growth. We found that fibronectin stimulated the phosphorylation of Akt, an upstream inducer of mTOR, and induced the phosphorylation of p70S6K1 and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of mTOR in NSCLC cells (H1792 and H1838), whereas it inhibited the phosphatase and tensin homologue deleted on chromosome 10, a tumor suppressor protein that antagonizes the phosphatidylinositol 3-kinase/Akt signal. In addition, treatment with fibronectin inhibited the mRNA and protein expression of LKB1 as well as the phosphorylation of AMP-activated protein kinase (AMPKalpha), both known to down-regulate mTOR. Rapamycin, an inhibitor of mTOR, blocked the fibronectin-induced phosphorylation of p70S6K and 4E-BP1. Akt small interfering RNA (siRNA) and an antibody against the fibronectin-binding integrin alpha5beta1 also blocked the p70S6K phosphorylation in response to fibronectin. In contrast, an inhibitor of extracellular signal-regulated kinase 1/2 (PD98095) had no effect on fibronectin-induced phosphorylation of p70S6K. Moreover, the combination of rapamycin and siRNA for Akt blocked fibronectin-induced cell proliferation. Taken together, these observations suggest that fibronectin-induced stimulation of NSCLC cell proliferation requires activation of the Akt/mTOR/p70S6K pathway and is associated with inhibition of LKB1/AMPK signaling.

Up-regulation of p21 Gene Expression by Peroxisome Proliferator-Activated Receptor γ in Human Lung Carcinoma Cells

Purpose: The peroxisome proliferator-activated receptor γ (PPARγ), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the regulation of cell growth and differentiation although the exact mechanism(s) of this activity has not been elucidated. In this study, we explored the role of PPARγ signaling on the control of gene expression of the cycle-dependent kinase inhibitor p21 in human lung carcinoma cells. Experimental Design: Using several human lung carcinoma cell lines (small and non-small carcinoma cells), we assayed for cell growth inhibition and apoptosis induction. We also assayed for p21 mRNA and protein expression by reverse transcription-PCR, real-time reverse transcription-PCR, and Western blot analysis. Nuclear protein binding activities to three response elements located in the p21 promoter [nuclear factor (NF)-κB, Sp1, and NF-interleukin 6 (IL6) CAAT/enhancer binding protein (C/EBP)] were measured by gel mobility shift assays. We used transient transfection assays with p21 promoter reporter gene constructs to determine the transcriptional regulation by PPARγ ligands. Finally, by using p21 antisense oligonucleotides, we tested the link between PPARγ activation and p21 signaling in cell growth inhibition assays and by Western blot analysis. Results: We showed that the PPARγ ligands PGJ2 and ciglitazone inhibit the growth and induce the apoptosis of several human lung carcinoma cell lines, whereas the PPARα agonist WY14643 has little effect. Treatment of lung carcinoma cells with the PPARγ ligands PGJ2, ciglitazone, troglizaone, and GW1929 elevated p21 mRNA and protein levels and reduced cyclin D1 mRNA levels. These results were supported by transient transfection assays, which indicated that PPARγ ligands increased p21 gene promoter activity in human lung carcinoma cells. In addition, p21 antisense oligonucleotides inhibited PPARγ ligand-induced p21 protein expression and significantly blocked lung carcinoma cell growth inhibition induced by PPARγ ligands. Finally, electrophoresis mobility shift experiments demonstrated that PPARγ ligands increased the nuclear binding activities of Sp1 and NF-IL6 (C/EBP), two transcription factors with regulatory elements in the promoter region of the p21 gene. Conclusion: PPARγ ligands inhibit human lung carcinoma cell growth and induce apoptosis by stimulating the cyclin-dependent kinase inhibitor p21 and by reducing cyclin D1 gene expression. The induction of p21 gene expression by PPARγ ligands may be mediated through increased Sp1- and NF-IL6 (C/EBP)-dependent transcriptional activation. These observations unveil a mechanism for p21 gene regulation in lung carcinoma that represents a potential target for therapy.

Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase-2 (COX-2) expression.

Tobacco use is the most important risk factor for the development of lung carcinoma. One characteristic shared by tobacco‐related lung diseases is altered lung connective tissue content and composition. In particular, tobacco results in increased expression of fibronectin (FN), a matrix glycoprotein implicated in lung development, injury and repair and in tumor cell invasion. We hypothesized that excessive deposition of FN in lung might promote lung carcinoma cell proliferation. Consistent with this hypothesis, we found that FN stimulated human lung carcinoma cell proliferation and diminished apoptosis in vitro, and that this effect was mediated through the integrin α5β1 and associated with upregulation of cyclooxygenase‐2 (COX‐2) mRNA and protein expression, and increased prostaglandin E2 (PGE2) biosynthesis. The stimulatory effect of FN on COX‐2 was blocked by the specific COX‐2 inhibitor NS‐398 and by inhibitors of protein kinase C (PKC), Calphostin C, and extracellular signal‐regulated kinases (Erks), PD98095. Electrophoretic mobility shift assays revealed that FN increased the nuclear binding activity of cyclic AMP response element binding protein (CREB) and CCAAT/enhancer‐binding protein (C/EBP), 2 proteins known to play important roles in the regulation of COX‐2 promoter activity. Transient transfection assays with wild‐type and mutated constructs of the human COX‐2 gene promoter revealed that the stimulatory effect of FN was prevented when either the CRE or the NF‐IL6 (C/EBP) sites were mutated. Taken together, the results indicate that FN stimulates human lung carcinoma cell proliferation and diminishes apoptosis by inducing COX‐2 gene expression and PGE2 biosynthesis. Activation of PKC and Erk and DNA‐protein interactions at CRE and NF‐IL6 (C/EBP) sites in the COX‐2 gene promoter appear to play key roles in this process. This work demonstrates that signaling through specific matrix‐binding β1 integrins (i.e., α5β1) resulting from exaggerated deposition in lung of the matrix glycoprotein fibronectin might promote lung carcinoma cell growth.

Peroxisome-proliferator-activated-receptor gamma (PPARγ) independent induction of CD36 in THP-1 monocytes by retinoic acid

Retinoic acid (RA) has been shown to regulate cellular growth and differentiation of a variety of cell types, including cells of the myelomonocytic lineage. We used the monocytic leukaemia cell line THP‐1, which differentiates to macrophages in response to phorbol 12‐myristate 13‐acetate (PMA), to investigate the regulation by RA of genes in the scavenger receptor type B family (CD36) in human monocyte/macrophages. Reverse transcription–polymerase chain reaction and flow cytometry demonstrated that, like PMA and the natural peroxisome‐proliferator‐activated receptor‐γ (PPARγ) ligand 15d‐PGJ2, RA induced CD36 gene expression in these cells. Moreover, RA plus 15d‐PGJ2 further enhanced CD36 protein and mRNA levels over that seen with the RA or PPARγ compounds alone. The PPARγ antagonist GW9662 was shown to block completely PPARγ‐ligand induction of CD36 gene expression, but had little effect on the action of RA. Our data indicated that RXR‐ and RAR‐specific ligands (LG153 and TTNPB, respectively) were each alone able to increase CD36 mRNA and surface protein levels. By using calphostin C, a specific protein kinase C (PKC) inhibitor, we demonstrated that induction of CD36 by PMA, as well as by PPARγ and RXR ligands were dependent upon PKC activation. In contrast, activation of CD36 through the RAR pathway was not affected by inhibition of PKC activity. Taken together, these data demonstrate that RA can up‐regulate CD36 expression in human monocytes/macrophages. This regulation appears to be predominantly mediated through the RAR/RXR pathway of action and, unlike previously described methods of CD36 modulation, is independent of PPARγ and PKC signalling. This study suggests a possible role for RA in physiological processes involving the scavenger receptor function in cells of the monocyte/macrophage lineage.

PPARβ/δ agonist stimulates human lung carcinoma cell growth through inhibition of PTEN expression: the involvement of PI3K and NF-κB signals

Recent studies suggest that activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) promotes cancer cell survival. We previously demonstrated that a selective PPARbeta/delta agonist, GW501516, stimulated human non-small cell lung carcinoma (NSCLC) cell growth. Here, we explore the mechanisms responsible for this effect. We show that GW501516 decreased phosphate and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor known to decrease cell growth and induce apoptosis. Activation of PPARbeta/delta and phosphatidylinositol 3-kinase (PI3K)/Akt signaling was associated with inhibition of PTEN. GW501516 increased NF-kappaB DNA binding activity and p65 protein expression through activation of PPARbeta/delta and PI3K/Akt signals and enhanced the physical interactions between PPARbeta/delta and p65 protein. Conversely, inhibition of PI3K and silencing of p65 by small RNA interference (siRNA) blocked the effect of GW501516 on PTEN expression and on NSCLC cell proliferation. GW501516 also inhibited IKBalpha protein expression. Silencing of IKBalpha enhanced the effect of GW501516 on PTEN protein expression and on cell proliferation. It also augmented the GW501516-induced complex formation of PPARbeta/delta and p65 proteins. Overexpression of PTEN suppressed NSCLC cell growth and eliminated the effect of GW501516 on phosphorylation of Akt. Together, our observations suggest that GW501516 induces the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PPARbeta/delta, which stimulates PI3K/Akt and NF-kappaB signaling. Overexpression of PTEN overcomes this effect and unveils PPARbeta/delta and PTEN as potential therapeutic targets in NSCLC.

Prostaglandin E2 Stimulates Human Lung Carcinoma Cell Growth through Induction of Integrin-Linked Kinase: The Involvement of EP4 and Sp1

Cyclooxygenase-2-derived prostaglandin E(2) (PGE(2)) stimulates tumor cell growth and progression. However, the mechanisms by which PGE(2) increases tumor growth remain incompletely understood. In studies performed in non-small cell lung carcinoma (NSCLC) cells, we found that PGE(2) stimulates the expression of integrin-linked kinase (ILK). ILK small interfering RNA (siRNA) inhibited the mitogenic effects of PGE(2). In view of its perceived importance, we turned our attention to the mechanisms involved in PGE(2)-induced ILK expression and found that this effect was blocked by an antagonist of the PGE(2) receptor subtype EP4 and by EP4 siRNA. Furthermore, we showed that PGE(2) induction of ILK was associated with phosphorylation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt, which were abrogated by ILK siRNA. Transient transfection, gel mobility shift assays, and chromatin immunoprecipitation experiments showed that PGE(2) induced ILK promoter activity and increased Sp1, although it had no effect on nuclear factor-kappaB and AP-2 DNA-binding activity. Blockade of Sp1 abrogated the effect of PGE(2) on expression of ILK and promoter activity and on cell growth. In summary, our observations show that PGE(2) increases NSCLC cell growth through increased ILK expression, which is dependent on EP4 signaling and on induction of Sp1 protein and Sp1 DNA-binding activity in the ILK promoter. These studies suggest a novel molecular mechanism by which PGE(2) stimulates NSCLC cell growth and unveils a new molecular target for the development of therapies against NSCLC.

α5β1-Integrin Expression Is Essential for Tumor Progression in Experimental Lung Cancer

The matrix glycoprotein, fibronectin, stimulates the proliferation of non-small cell lung carcinoma in vitro through α5β1 integrin receptor-mediated signals. However, the true role of fibronectin and its receptor in lung carcinogenesis in vivo remains unclear. To test this, we generated mouse Lewis lung carcinoma cells stably transfected with short hairpin RNA shRNA targeting the α5 integrin subunit. These cells were characterized and tested in proliferation, cell adhesion, migration, and soft agar colony formation assays in vitro. In addition, their growth and metastatic potential was tested in vivo in a murine model of lung cancer. We found that transfected Lewis lung carcinoma cells showed decreased expression of the α5 gene, which was associated with decreased adhesion to fibronectin and reduced cell migration, proliferation, and colony formation when compared with control cells and cells stably transfected with α2 integrin subunit in vitro. C57BL/6 mice injected with α5-silenced cells showed lower burden of implanted tumors, and a dramatic decrease in lung metastases resulting in higher survival as compared with mice injected with wild-type or α2 integrin-silenced cells. These observations reveal that recognition of host- and/or tumor-derived fibronectin via α5β1 is important for tumor growth both in vitro and in vivo, and unveil α5β1 as a potential target for the development of anti-lung cancer therapies.

Cytokine regulation by peroxisome proliferator-activated receptor gamma in human endometrial cells.

OBJECTIVE To determine whether peroxisome proliferator-activated receptor (PPAR)-gamma ligands can affect the expression of interleukin-6 (IL-6) and cytokines related to the pathogenesis of endometriosis. DESIGN In vitro study to determine whether PPARs are expressed in human endometrial cells and determine the effects of various PPAR-gamma ligands on IL-6 and other cytokine expression in these cells. SETTING Academic medical center. PATIENT(S) Women presenting for infertility workup. INTERVENTION(S) Endometrial cell cultures were treated with PPAR-gamma ligands. MAIN OUTCOME MEASURE(S) Interleukin-6, IL-8, colony stimulating factor-1 (CSF-1) and macrophage chemotactic factor (MCP-1) protein secretion, messenger RNA expression of IL-6, PPAR-alpha, -beta, and -gamma. RESULT(S) Using a human endometrial cell line (EM42), as well as primary stromal and epithelial endometrial cells, we show the presence of PPAR-alpha, -beta, and -gamma by reverse transcription-polymerase chain reaction (RT-PCR) in these cells. PPAR-gamma ligands stimulated IL-6 secretion and induced enhancement of IL-6 mRNA levels. These ligands also stimulated the secretion of IL-8 and CSF-1. CONCLUSION(S) PPAR-gamma may play a role in the pathogenesis of endometriosis related to the production of IL-6 and some other cytokines.

COX‐2 inhibitors suppress integrin α5 expression in human lung carcinoma cells through activation of Erk: Involvement of Sp1 and AP‐1 sites

Tumor cell expression of COX‐2 has been implicated in the progression of murine and human lung cancer. Inhibition of COX‐2 by nonsteroidal antiinflammatory drugs reduces the risk of cancer development in humans and suppresses tumor growth in animal models. However, the underlying mechanisms for this beneficial effect are not fully understood. Here we explore the potential link between the anticancer effects of COX‐2 inhibitors and the expression of the integrin α5β1. Expression of this integrin in carcinoma cells is associated with invasiveness and malignant progression. This, together with our studies showing that fibronectin, the ligand of α5β1, stimulates the growth of human lung carcinoma cells, and that this effect is mediated through α5β1‐dependent signals, has prompted us to examine the effects of COX‐2 inhibitors on α5β1 expression in human non small cell lung carcinoma (NSCLC) cells. We found that the selective COX‐2 inhibitors NS398 and Nimesulide decreased mRNA expression and protein production of the integrin α5 subunit. This effect was associated with inhibition of NSCLC cell adhesion to fibronectin. The COX‐2 inhibitors triggered the phosphorylation of extracellular signal‐regulated kinase (Erk) in a time‐dependent manner, and the inhibitor of Mek‐1/Erk PD98095 prevented their inhibitory effects on integrin α5 expression. Transient transfection assays showed that the COX‐2 inhibitors affected integrin α5 gene transcription by acting between −92 to −41 bp of the human integrin α5 gene promoter. Gel mobility shift assays showed that the COX‐2 inhibitors increased Sp1 DNA binding, but decreased that of AP‐1. These effects were accompanied by an increase in Sp1 protein and a decrease in c‐Jun protein expression, as well as inhibition of SAPK/JNK phosphorylation. The Sp1 inhibitor, Mithramycin A, also blocked the inhibitory effect of the COX‐2 inhibitors on α5 expression and promoter activity. Overall, these findings suggest that COX‐2 inhibitors suppress α5β1 integrin expression in NSCLC through effects on integrin α5 gene transcription mediated by Erk activation, increased Sp1, decreased AP‐1 DNA binding and inactivation of SAPK/JNK signals. Our observations unveil a new mechanism of action against NSCLC for COX‐2 inhibitors that relates to regulation of integrin α5 gene expression and, consequently, recognition of extracellular matrices (i.e., fibronectin) by tumor cells.