XiaoJuan Sun

Nicotine Stimulates PPARβ/δ Expression in Human Lung Carcinoma Cells through Activation of PI3K/mTOR and Suppression of AP-2α

We previously showed that nicotine stimulates non-small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) has also been shown to induce NSCLC cell growth. Here, we explore the potential link between nicotine and PPARbeta/delta and report that nicotine increases the expression of PPARbeta/delta protein; this effect was blocked by an alpha7 nAChR antagonist (alpha-bungarotoxin), by alpha7 nAChR short interfering RNA, and by inhibitors of phosphatidylinositol 3-kinase (PI3K; wortmannin and LY294002) and mammalian target of rapamycin (mTOR; rapamycin). In contrast, this effect was enhanced by PUN282987, an alpha7 nAChR agonist. Silencing of PPARbeta/delta attenuated the stimulatory effect of nicotine on cell growth, which was overcome by transfection of an exogenous PPARbeta/delta expression vector. Of note, nicotine induced complex formation between alpha7 nAChR and PPARbeta/delta protein and increased PPARbeta/delta gene promoter activity through inhibition of AP-2alpha as shown by reduced AP-2alpha binding using electrophoretic gel mobility shift and chromatin immunoprecipitation assays. In addition, silencing of Sp1 attenuated the effect of nicotine on PPARbeta/delta. Collectively, our results show that nicotine increases PPARbeta/delta gene expression through alpha7 nAChR-mediated activation of PI3K/mTOR signals that inhibit AP-2alpha protein expression and DNA binding activity to the PPARbeta/delta gene promoter. Sp1 seems to modulate this process. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth.

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.

Activation of Peroxisome Proliferator–Activated Receptor β/δ Induces Lung Cancer Growth via Peroxisome Proliferator–Activated Receptor Coactivator γ-1α

We previously demonstrated that a selective agonist of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), GW501516, stimulated human non-small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase alpha (AMPKalpha), a major regulator of energy metabolism. This was mediated through specific activation of PPARbeta/delta, as a PPARbeta/delta small interfering RNA inhibited the effect. However, AMPKalpha did not mediate the growth-promoting effects of GW501516, as silencing of AMPKalpha did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator-activated receptor coactivator gamma (PGC)-1alpha, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1alpha, consistent with PGC-1alpha being upstream of PI3-K/Akt. Of note, an activator of AMPKalpha, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPKalpha is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARbeta/delta stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPKalpha may oppose this effect.

Fish oil inhibits human lung carcinoma cell growth by suppressing integrin-linked kinase.

We previously showed that synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligands inhibit non–small cell lung carcinoma (NSCLC) cell growth through multiple signaling pathways. Here, we show that dietary compounds, such as fish oil (which contains certain kinds of fatty acids like ω3 and ω6 polyunsaturated fatty acids), also inhibit NSCLC cell growth by affecting PPARγ and by inhibiting the expression of integrin-linked kinase (ILK). Exogenous expression of ILK overcame, whereas silencing ILK enhanced the inhibitory effect of fish oil on cell growth. The inhibitor of p38 mitogen-activated protein kinase, SB239023, abrogated the inhibitory effect of fish oil on ILK expression, whereas the inhibitor of extracellular signal-regulated kinase, PD98059, had no effect. Transient transfection experiments showed that fish oil reduced ILK promoter activity, and this effect was abolished by AP-2α small interfering RNA and SB239023 and by deletion of a specific portion of the ILK gene promoter. Western blot analysis and gel mobility shift assay showed that fish oil significantly induced AP-2α protein expression and AP-2 DNA-binding activity in the ILK gene promoter and that this was dependent on PPARγ activation. Blockade of AP-2α abrogated the effect of fish oil on ILK expression and on cell growth, whereas exogenous expression of AP-2α enhanced cell growth in the setting of fish oil exposure. Taken together, these findings show that fish oil inhibits ILK expression through activation of PPARγ-mediated and p38 mitogen-activated protein kinase–mediated induction of AP-2α. In turn, this leads to inhibition of NSCLC cell proliferation. This study unveils a novel mechanism by which fish oil inhibits human lung cancer cell growth. (Mol Cancer Res 2009;7(1):108–17)

Rosiglitazone inhibits α4 nicotinic acetylcholine receptor expression in human lung carcinoma cells through peroxisome proliferator-activated receptor γ-independent signals

We and others have shown previously that nicotine, a major component of tobacco, stimulates non-small cell lung carcinoma (NSCLC) proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor γ (PPARγ) has been shown to inhibit NSCLC cell growth, but the exact mechanisms responsible for this effect remain incompletely defined. Herein, we show that nicotine induces NSCLC cell proliferation in part through α4 nAChR, prompting us to explore the effects of rosiglitazone, a synthetic PPARγ ligand, on the expression of this receptor. Rosiglitazone inhibited the expression of α4 nAChR, but this effect was through a PPARγ-independent pathway, because GW9662, an antagonist of PPARγ, and the transfection of cells with PPARγ small interfering RNA failed to abolish the response. The inhibitory effect of rosiglitazone on α4 nAChR expression was accompanied by phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 and down-regulation of Akt phosphorylation. These signals mediated the inhibitory effects of rosiglitazone on α4 nAChR expression because chemical inhibitors prevented the effect. Rosiglitazone was also found to stimulate p53, a tumor suppressor known to mediate some of the effects of nicotine. Interestingly, p53 up-regulation was needed for rosiglitazone-induced inhibition of α4 nAChR. Thus, rosiglitazone inhibits α4 nAChR expression in NSCLC cells through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which triggers induction of p53. Finally, like others, we found that nicotine stimulated the expression of α4 nAChR. This process was also inhibited by rosiglitazone through similar pathways. [Mol Cancer Ther 2009;8(1):110–8]

Abstract 1230: Nicotine induces expression of integrin-linked kinase in human lung carcinoma cells through A7 nAChR-mediated inhibition of AP-2A

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We previously demonstrated that nicotine stimulates non small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated induction of fibronectin expression. However, the mechanisms by which this agent affects human lung cancer occurrence and progression remain incompletely elucidated. Here, we show that nicotine increases the expression of integrin-linked kinase (ILK), a unique intracellular adaptor and kinase that has been implicated in the regulation of cancer cell growth/survival. This effect was blocked by the α7 nAChR antagonist, α-bungarotoxin, and by α7 nAChR siRNA. Silencing of ILK blocked the stimulatory effect of nicotine on phosphorylation of the PI3-K downstream target Akt and on cell growth. Furthermore, nicotine increased ILK gene promoter activity through inhibition of transcription factor AP-2α protein expression. Silencing of AP-2α enhanced, while exogenous expression of AP-2α attenuated, the stimulatory effect of nicotine on ILK expression. These findings appear to be relevant in vivo since we found that nicotine treatment increased tumor growth in animal models. Collectively, our results demonstrate that nicotine increases ILK gene expression through α7 nAChR-mediated inhibition of AP-2α followed by stimulation of PI3-K/Akt signaling. In turn, this leads to increased NSCLC cell growth. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth/survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1230.

Abstract 1223: Fibronectin induced lung cancer cell proliferation is dependent on induction of ILK through integrin B1-mediated activation of p38 MAPK and PI3-K

Fibronectin is a multi-functional matrix glycoprotein protein that has been linked to tumor progression. We previously demonstrated that cellular fibronectin (cFN) increased non-small cell lung carcinoma (NSCLC) cell growth through signals mediated by the integrin α5β1. Here, we explore the role of integrin-linked kinase (ILK), an integrin β1 subunit cytoplasmic domain interactor in cFN induced cell growth. We found that cFN increases ILK expression and kinase activity, promotes protein-protein interactions between ILK and integrin β1. Silencing of ILK attenuated the effect of cFN on cell proliferation. Knockdown of the integrin β1 subunit, but not the integrin α5, blocked the effect of cFN on ILK expression. In addition, the inhibitor of p38MAPK, SB239063, and the inhibitor of PI3-K, wortmannin, abrogated the effect of cFN on ILK expression, whereas the inhibitor of ERK, PD98059, had no effect. Taken together, our results demonstrate that fibronectin induces the expression of ILK through integrin β1-mediated signals that include p38 MAPK and PI3-K. This in turn, promotes lung carcinoma cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1223.

The fibronectin EDA splicing variant induces epithelial-mesenchymal transition in lung cancer cells through integrin α9β1-mediated activation of PI3-K and Erk

Cellular fibronectin (cFN) is one of the main components of tissue extracellular matrices (ECM), and is involved in multiple physiologic and pathologic processes such as embryogenesis, wound healin...