Jinghua Tsai Chang

Requirement of Aryl Hydrocarbon Receptor Overexpression for CYP1B1 Up-Regulation and Cell Growth in Human Lung Adenocarcinomas

Purpose: CYP1B1 and CYP1A1 expression is up-regulated by activation of the aryl hydrocarbon receptor (AhR) through binding of ligands such as cigarette smoke components. We examined the association between AhR, CYP1B1, and CYP1A1 expression in noninvasive bronchioloalveolar carcinomas (BAC) and lung adenocarcinomas and investigated the effects of AhR overexpression on cell physiology. Experimental Design: AhR, CYP1B1, and CYP1A1 expression was examined in 107 lung adenocarcinomas and 57 BAC by immunohistochemistry. AhR expression in lung adenocarcinoma H1355 cells was stably reduced by RNA interference (RNAi). AhR, CYP1B1, and CYP1A1 expression was examined using real-time reverse transcription-PCR. Cell physiology was evaluated by measuring anchorage-independent growth and intracellular reactive oxygen species. Results: Expression of AhR and CYP1A1 was associated in smoking adenocarcinoma patients, whereas expression of AhR and CYP1B1 was associated regardless of smoking status. The level of CYP1B1, but not CYP1A1, was positively associated with AhR overexpression in BAC. 2,3,7,8-Tetrachlorobenzo-p-dioxin–induced CYP1A1/1B1 expression was reduced in AhR RNAi clones. In the absence of 2,3,7,8-tetrachlorobenzo-p-dioxin, CYP1B1 mRNA levels were reduced in AhR RNAi clones, whereas CYP1A1 mRNA levels were barely detectable. Furthermore, anchorage-independent growth and intracellular oxidative stress were significantly reduced in AhR RNAi cells. Conclusions: In the absence of exogenous AhR ligands (such as cigarette smoke components), AhR overexpression up-regulated the expression of CYP1B1 in the early stage of lung adenocarcinoma. Elevated AhR expression in lung adenocarcinoma cells could increase intracellular oxidative stress and promote cell growth, implying that disrupting AhR expression might prevent the early development of lung adenocarcinomas.

Genetic Determinants of Pemetrexed Responsiveness and Nonresponsiveness in Non-small Cell Lung Cancer Cells

Background: Pemetrexed disodium (Alimta®), LY231514, is an antifolate that is able to simultaneously inhibit the synthesis of purines and pyrimidines. Pemetrexed has been approved for first- and second-line treatment in patients with non-small cell lung cancer (NSCLC). However, there is still a lack of clinical biomarkers for predicting the therapeutic response to pemetrexed. The aim of this study is to establish new biomarkers for pemetrexed treatment in NSCLC. Methods: Human NSCLC cell lines were exposed to pemetrexed. The antitumor effect was measured by growth inhibition with MTT assay and expression of cell cycle mediators with immunoblots. Using the Superarray cancer pathway gene array, 482 genes were screened for differential expression in A549 cells that were untreated or treated with pemetrexed. Results: A549 cells exhibited sensitivity but H1355 cells showed resistance to pemetrexed. To investigate the mechanisms of responsiveness and nonresponsiveness to pemetrexed in these cell lines, we measured the expression levels of thymidylate synthase (TS), dihydrofolate reductase (DHFR), reduced folate carrier, and folylpoly-gamma-glutamate synthetase genes. TS, DHFR, and reduced folate carrier gene expressions were significantly reduced in A549 and H1355 cells. Pemetrexed caused cell cycle arrest in the G1 phase and S phase in H1355 and A549 cells, respectively. Significantly higher expressions of many genes, especially lipocalin-2 (Lcn-2) and nm23-H1 proteins, were noted in A549 cells treated with pemetrexed in comparison with untreated cells. Furthermore, reverse transcriptase polymerase chain reaction and Western blot showed that Lcn-2 and nm23-H1 expressions increase in response to pemetrexed treatment in a dose-responsive manner in pemetrexed-sensitive A549 cells but not in resistant H1355 cells. Conclusions: Our results indicated that downregulation of TS and DHFR genes and upregulation of p21, p27, Lcn-2, and nm23-H1 genes may serve as new biomarkers for predicting responsiveness to pemetrexed.

Aryl hydrocarbon receptor activation and overexpression upregulated fibroblast growth factor-9 in human lung adenocarcinomas.

We had previously reported that aryl hydrocarbon receptors (AhRs) are overexpressed in lung adenocarcinomas. Benzo[a]pyrene (BaP), an AhR agonist, increased FGF‐9 expression in human lung adenocarcinoma cells. Similarly, several AhR agonists increased FGF‐9 mRNA levels, and BaP‐induced FGF‐9 expression was prevented by cotreatment with AhR antagonist in human lung adenocarcinoma cells. Furthermore, AhR agonists increased transcriptional activity of FGF‐9 promoter. Modulation of AhR expression via RNA interference or transient overexpression respectively reduced or increased both constitutive and BaP‐induced FGF‐9 expression in human lung cells. These results suggested that AhR activation and overexpression increased FGF‐9 expression in lung cells. FGF‐9 increased growth of lung fibroblasts but not that of lung adenocarcinoma cells. However, conditioned media collected from FGF‐9‐treated fibroblasts increased cell growth of lung adenocarcinoma cells. Furthermore, lung adenocarcinoma cells expressed FGF receptor 2 and cotreatment with anti‐FGF receptor 2 prevented the interaction between fibroblasts and tumor cells. It is likely that FGF‐9‐stimulated fibroblasts secreted unknown factors, which activated FGF receptor 2 and subsequently promoted growth of lung adenocarcinoma cells. We further compared AhR and FGF‐9 expression in 146 non‐small cell lung cancer (NSCLC) cases by immunohistochemistry. FGF‐9 expression was more common in adenocarcinomas than in squamous cell carcinomas. Furthermore, FGF‐9 and AhR expression were well correlated in lung adenocarcinomas. These results suggest that AhR expression correlated positively with FGF‐9 expression in lung adenocarcinomas, which might promote tumor growth by modulating communication between tumor cells and fibroblasts. Preventing AhR overexpression or disturbing FGF‐9 function may reduce the development of lung adenocarcinomas.

Induction of tubulin by Docetaxel is associated with p53 status in human non small cell lung cancer cell lines

Docetaxel (DOC), a member of the taxane family of anticancer drugs, binds to tubulin and produces unnaturally stable microtubules that induce cell death. DOC is used clinically alone or in combination with other compounds to treat advanced stages of cancer. We have treated the human lung cancer cell lines A549 and H1299 and human cervical cancer HeLa cells with low concentrations of DOC to characterize the response of β‐tubulin isotypes and p53 genes. The relationship between p53 function and DOC, acting through a microtubule‐based mechanism, was examined. We found that after 18‐hr treatment with DOC, β‐tubulin gene transcription was enhanced in p53‐null H1299 cells but not in A549 cells. Also, p53 RNA was strongly induced in the A549 cells. In addition, β‐tubulin levels also increased in the H1299 cells after the DOC treatment. Further demonstrating an association of DOC treatment with p53 and β‐tubulin, inhibition of p53 expression by interference RNA in A549 cells showed increasing β‐tubulin gene expression with DOC treatment. We also selected a clone from the H1299 cells that stably expressed p53, examined the β‐tubulin expression after DOC treatment and found an inhibition of β‐tubulin induction in these p53‐expressing cells. Our data suggest that the initial response of cells to DOC treatment involves p53; alternatively, in the absence of p53, tubulins may be transactivated. Selection of the DOC‐resistant A549 cells showed β‐tubulin expression was increased, in contrast to the initial response to the DOC treatment. From the initial and selection responses of β‐tubulin in cancer cells, it appears that there is a p53‐associated β‐tubulin expression as a result of the DOC treatment.

Up-regulation of osteopontin expression by aryl hydrocarbon receptor via both ligand-dependent and ligand-independent pathways in lung cancer.

The secreted glycol-phosphoprotein OPN not only plays important roles in immune responses and tissue remodeling but is also intimately involved in tumorigenesis. It is up-regulated in various cancers and correlated with poor prognosis. It is evident by enhancing growth and migration of cancer cells. However, the mechanisms that participate in up-regulation of OPN in lung cancer are largely unknown. Up-regulation of aryl hydrocarbon receptor (AhR), a transcription factor activated by xenobiotics, has been observed in lung cancer as well as premalignant lesions. In this study we demonstrated that AhR positively regulates OPN expression in lung cancer. We observed positive correlation of OPN and AhR expression in lung cancer specimen. Knockdown or overexpression of AhR exhibited down- or up-regulation of OPN expression in lung cancer cells. We identified an OPN promoter region between positions -268 and +435 that was activated by both ligand-independent and ligand-activated AhR. However, this region does not contain AhR response element/dioxin response element (DRE/XRE). Further truncations and internal deletions of the promoter revealed that the ligand-independent and ligand-activated AhR function through different regions of OPN promoter. The region between -268 and -100 was required for ligand-independent AhR activity. This region contains several cis-elements including AP2, C/EBP, SP1 and AP1 sites. On the other hand, the ligand-activated AhR up-regulates OPN activity through two regions of OPN promoter; one contains NFκB site at +137 and the other is between positions -100 and +126. This study suggested that both overexpression of un-induced AhR (in cases of non-smokers with high level of AhR) and ligand-activated AhR (such as smokers) contribute to up-regulation of OPN that in turn leads to lung tumorigenesis.

Aryl Hydrocarbon Receptor is a Target of 17-allylamino-17-demethoxygeldanamycin and Enhances its Anticancer Activity in Lung Adenocarcinoma Cells

We have demonstrated that aryl hydrocarbon receptor (AhR) is overexpressed in lung adenocarcinoma (AD). AhR is usually associated with heat shock protein 90 (Hsp90) in the cytoplasm. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, is currently under evaluation for its anticancer activity in clinical trials. Here we investigated whether AhR plays a role in 17-AAG-mediated anticancer activity by functioning as a downstream target or by modulating its anticancer efficacy. AhR expression in lung AD cells was modulated by siRNA interference or overexpression. Tumor growth was determined with colony formation in vitro or in vivo. Anticancer activity of 17-AAG was determined by measuring cell viability, cell cycle distribution, and expression of cell cycle regulators. Proteins and mRNA levels were examined by immunoblotting and the real-time reverse transcription-polymerase chain reaction, respectively. In this study, AhR overexpression positively modulated growth of lung AD cells, at least partially, via RelA-dependent mechanisms. Although treatment with 17-AAG reduced AhR levels and AhR-regulated gene expression in lung AD cells, AhR expression increased anticancer activity of 17-AAG. In addition, 17-AAG treatment reduced cell viability, CDK2, CDK4, cyclin E, cyclin D1, and phosphorylated Rb levels in AhR-expressing lung AD cells. NAD(P)H:quinone oxidoreductase (NQO1), which is regulated by AhR, was shown to increase anticancer activity of 17-AAG in cells. Knockdown of NQO1 expression attenuated the reduction of cell cycle regulators by 17-AAG treatment in AhR overexpressed cells. We demonstrated that AhR protein not only functions as a downstream target of 17-AAG, but also enhances anticancer activity of 17-AAG in lung AD cells.

Integrin β3 and CD44 levels determine the effects of the OPN-a splicing variant on lung cancer cell growth

Osteopontin (OPN), a phosphorylated glycoprotein, is frequently overexpressed in cancer. Among the three OPN isoforms, OPN-a is the most highly expressed in lung cancer cell lines and lung tumors. Overexpression of OPN-a greatly reduced CL1-5 lung adenocarcinoma cell growth, but had no effect on growth in A549 lung adenocarcinoma cells. Examination of the expression of integrins and CD44, which are possible OPN-a receptors, revealed that differences in integrin β3 levels might explain this discrepancy between CL1-5 and A549 cells. When integrin β3 was ectopically expressed in A549 cells, OPN-a inhibited their growth, whereas OPN-a increased cell growth following integrin β3 knockdown in CL1-5 cells. This OPN-a-induced increase in growth appeared to result from activation of the CD44/NFκB pathway. Our results demonstrated that OPN-a inhibits growth of cells with high integrin β3 levels and increases growth via activation of the CD44/NFκB pathway in cells with low integrin β3 levels. Thus, OPN-a, integrin β3, and CD44 interact to affect lung cancer cell growth, and this study may aid in the development of cancer treatment strategies involving these molecules.

A novel exon 15‐deleted, splicing variant of Slit2 shows potential for growth inhibition in addition to invasion inhibition in lung cancer

The axon guidance cue molecule Slit2 has been shown to suppress cancer cell invasion. However, the role of Slit2 in growth inhibition is still controversial. The authors identified a novel exon 15 (AKEQYFIP)‐deleted slit2, located at the end of the second leucine‐rich repeat (LRR2). Because LRR2 interacts with Robo1 receptor to inhibit invasion, they hypothesized that exon 15 plays an important role in modulating Slit2 function.