Baoxiang Guan

Evidence that the death receptor DR4 is a DNA damage-inducible, p53-regulated gene

DR4 (TRAIL‐R1), a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that triggers the apoptotic machinery upon binding to its ligand tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Although three other TRAIL receptors DR5, DcR1, and DcR2 are induced by DNA damage and are regulated by the wild‐type p53 tumor suppressor, it was not known whether these factors also affect DR4 expression. In this study, we found that DR4 expression is also enhanced by DNA damage whether induced by ionizing radiation or by chemotherapeutic agents. The induction was observed predominantly in cells containing wild‐type p53 and was similar to the regulation patterns of DR5 and Fas, two other members of the family which are known to be regulated by p53. Transfection of HPV 16 E6 gene into cells with wild‐type p53, which decreased the level of p53 protein, resulted in suppression of DR4 induction by DNA‐damaging agents. Conversely, introduction of exogenous wild‐type p53 through adenovirus infection has led to upregulation of endogenous DR4 in cells with mutant p53. Moreover, the transcription inhibitor actinomycin D abolished DNA‐damaging agent‐induced DR4 expression. Thus, DR4 appears to be a DNA damage‐inducible, p53‐regulated gene.

Prognostic significance of differentially expressed miRNAs in esophageal cancer

Altered microRNA (miRNA) expression has been found to promote carcinogenesis, but little is known about the role of miRNAs in esophageal cancer. In this study, we selected 10 miRNAs and analyzed their expression in 10 esophageal cancer cell lines and 158 tissue specimens using Northern blotting and in situ hybridization, respectively. We found that Let‐7g, miR‐21 and miR‐195p were expressed in all 10 cell lines, miR‐9 and miR‐20a were not expressed in any of the cell lines, and miR‐16‐2, miR‐30e, miR‐34a, miR‐126 and miR‐200a were expressed in some of the cell lines but not others. In addition, transient transfection of miR‐34a inhibited c‐Met and cyclin D1 expression and esophageal cancer cell proliferation, whereas miR‐16‐2 suppressed RAR‐β2 expression and increased tumor cell proliferation. Furthermore, we found that miR‐126 expression was associated with tumor cell dedifferentiation and lymph node metastasis, miR‐16‐2 was associated with lymph node metastasis, and miR‐195p was associated with higher pathologic disease stages in patients with esophageal adenocarcinoma. Kaplan‐Meier analysis showed that miR‐16‐2 expression and miR‐30e expression were associated with shorter overall and disease‐free survival in all esophageal cancer patients. In addition, miR‐16‐2, miR‐30e and miR‐200a expression were associated with shorter overall and disease‐free survival in patients with esophageal adenocarcinoma; however, miR‐16‐2, miR‐30e and miR‐200a expression were not associated with overall or disease‐free survival in squamous cell carcinoma patients. Our data indicate that further evaluation of miR‐30e and miR‐16‐2 as prognostic biomarkers is warranted in patients with esophageal adenocarcinoma. In addition, the role of miR‐34a in esophageal cancer also warrants further study.

Evidence that the human death receptor 4 is regulated by activator protein 1.

Death receptor 4 (DR4; also called TRAIL-R1), a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that triggers the apoptotic machinery upon binding to its ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Although several chemotherapeutic agents were reported to induce DR4 expression, the mechanism of this effect remains largely unknown. To begin to understand its regulation, we cloned a 1.8 Kb 5′-flanking region of the human DR4 gene and identified several putative binding sites for transcription factors including activator protein 1 (AP-1). Among the three putative AP-1 binding sites, the site located at −350/−344 is functionally active as evidenced by a combination of electrophoretic mobility shift and luciferase reporter assays. The AP-1 activator phorbol 12–myristate 13-acetate (TPA) enhanced the binding of this DR4 AP-1 binding site to protein(s) in a nuclear extract from TPA-treated cells, increased luciferase activity of a reporter construct containing this site and induced DR4 expression at the transcription level. These results indicate that AP-1 regulates DR4 expression via the AP-1 binding site located at −350/−344. AP-1 has been implicated in many critical cellular processes including apoptosis, and is a major target of the c-Jun NH3-terminal kinase signaling pathway that is activated by many anticancer drugs. Therefore, our findings may increase the understanding of the mechanisms underlying AP-1-mediated apoptosis as well as drug-induced apoptosis.

Inhibition of farnesoid X receptor controls esophageal cancer cell growth in vitro and in nude mouse xenografts

Gastroesophageal reflux is a risk factor for esophageal adenocarcinoma, and bile acid and its farnesoid X receptor (FXR) have been implicated in esophageal tumorigenesis. The authors investigated the role of FXR expression and activity in esophageal cancer initiation and growth.

Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na+/H+ exchanger-1 (NHE-1) ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues (66 of 101 cases [65.3%], but not in normal esophageal squamous cell epithelium (1 of 26 cases [3.8%]). Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone (a natural bile acid receptor inhibitor) showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.

Antitumor Effect of Retinoic Acid Receptor-β2 Associated with Suppression of Cyclooxygenase-2

Retinoic acid receptor-β2 (RAR-β2) is a putative tumor suppressor gene in various cancers. To determine the underlying molecular mechanisms, we transfected RAR-β2 cDNA into esophageal cancer TE-1 and TE-8 cells and found that RAR-β2 suppressed tumor cell growth in vitro and tumor formation in nude mice in TE-8 cells, whereas the stable transfection of RAR-β2 did not restore retinoid sensitivity or inhibit tumor formation in nude mouse in TE-1 cells. Molecularly, we revealed that RAR-β2 antitumor activity was associated with expression and suppression of cyclooxygenase-2 (COX-2) in these tumor cell lines. Moreover, antisense RAR-β2 cDNA induced COX-2 expression in TE-3 cells. Furthermore, when COX-2 expression is first blocked by using antisense COX-2 expression vector, the effect of RAR-β2 is diminished in these tumor cells. In addition, we analyzed expression of RAR-β2 and COX-2 mRNA in tissue specimens and found that RAR-β2 expression is associated with low levels of COX-2 expression in esophageal cancer tissues. Induction of RAR-β2 expression in oral leukoplakia tissues after the patients treated with 13-cis RA correlated with a reduction in COX-2 expression and clinical response. Our findings indicate that some of RAR-β2 antitumor activities are mediated by suppression of COX-2 expression in some of these esophageal cancer cells. After correlating antitumor effect of RAR-β2 with COX-2 expression in the published studies, we also found the association. Thus, further studies will determine whether manipulation of COX-2 expression in different cancers can antagonize RAR-β2 activity.

Tumor-suppressor activity of RRIG1 in breast cancer

BackgroundRetinoid receptor-induced gene-1 (RRIG1) is a novel gene that has been lost in several types of human cancers. The aim of this study was to determine whether RRIG1 plays a role in breast cancer, such as in the suppression of breast cancer cell growth and invasion.MethodsImmunohistochemistry was used to detect RRIG1 expression in breast tissue specimens. Gene transfection was used to restore or knock down RRIG1 expression in breast cancer cell lines for analysis of cell viability, colony formation, and migration/invasion potential. Reverse-transcription polymerase chain reaction and western blot assays were used to detect the changes in gene expression. The RhoA activation assay was used to assess RRIG1-induced inhibition of RhoA activity.ResultsThe immunohistochemical data showed that RRIG1 expression was reduced in breast cancer tissues compared with normal and atypical hyperplastic breast tissues. RRIG1 expression was inversely correlated with lymph node metastasis of breast cancer but was not associated with the status of hormone receptors, such as estrogen receptor, progesterone receptor, or HER2. Furthermore, restoration of RRIG1 expression inhibited proliferation, colony formation, migration, and invasion of breast cancer cells. Expression of RRIG1 also reduced phosphorylated Erk1/2 and Akt levels; c-Jun, MMP9, and Akt expressions; and RhoA activity. In contrast, knockdown of RRIG1 expression promoted breast cancer cell proliferation, colony formation, migration, and invasion potential.ConclusionThe data from the current study indicated that RRIG1 expression was reduced or lost in breast cancer and that restoration of RRIG1 expression suppressed breast cancer cell growth and invasion capacity. Future studies will determine the underlying molecular mechanisms and define RRIG1 as a tumor-suppressor gene in breast cancer.

Abstract B70: Inhibition of nuclear bile acid receptor FXR as a target in prevention of esophageal adenocarcinoma

Incidence of esophageal adenocarcinoma is increasing in the United States and other Western countries. Frequent gastroesophageal reflux or gastroesophageal reflux disease, resulting in Barrett esophagus, may be responsible for the increase. The main function of bile acid is to facilitate the formation of micelles for promotion of the processing and absorption of dietary fat. As surfactants or detergents, bile acids are potentially toxic to the cells, so their concentrations in the small intestine are tightly regulated. However, patients with frequent gastroesophageal reflux will have reflux damage caused by acid and bile acid-containing juice in the distal esophagus, with the result that normal squamous cells around the gastroesophageal junction will change to a new cell phenotype (incomplete intestinal metaplasia), because this type of cell is more resistant to acid and bile-caused injuries; therefore, Barrett esophagus is formed. This study aimed to determine the tumor-promoting effects of bile acid on esophageal cancer cells and the underlying molecular mechanisms. The data showed that different bile acids (i.e., chenodeoxycholic acid, deoxycholic acid, and lithocholic acid) induced COX-2 but inhibited RAR-β2 expression through farnesoid X receptor (FXR) expression in esophageal cancer cell lines. FXR is a nuclear receptor for bile acids functioning as a signaling molecule in the liver and the intestines and frequent gastroesophageal reflux induces FXR expression in esophageal cells. Indeed, the ex vivo data demonstrated that FXR was highly expressed in esophageal adenocarcinoma (81%) tissues and was associated with reduced RAR-β2 expression. Knockdown of FXR expression using FXR shRNA antagonized the effects of bile acid in gene expression and suppressed tumor cell viability in vitro and in nude mouse xenografts. Moreover, guggulsterone, a FXR inhibitor, was able to reduce the viability of esophageal cancer cells in time- and dose-dependent manner in vitro. Apoptosis induced by guggulsterone was through activation of caspase 8, 9, and 3. In conclusion, suppression of FXR expression using FXR shRNA or its inhibitor guggulsterone was able to suppress tumor cell growth in vitro and in vivo and induce apoptosis in vitro. This study demonstrated that inhibition of FXR could be further evaluated as a target in prevention of esophageal adenocarcinoma. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B70.

Abstract 5676: Farnesoid X receptor-mediated carcinogenic effects of bile acid in esophageal cancer: A potential chemoprevention target

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC OBJECTIVES: The incidence of esophageal adenocarcinoma is increasing in the United States. Frequent gastroesophageal reflux or gastroesophageal reflux disease, resulting in Barrett esophagus, may be responsible for the increase. In the present study, we determined the carcinogenic effects of bile acid in esophageal cancer and then explored the use of the farnesoid X receptor (FXR) inhibitor guggulsterone to control tumor cell viability and gene expression. METHODS: Cell culture, Western blot, RT-PCR, gene transfection, and immunohistochemistry assays were used. RESULTS: We found that bile acid inhibited RAR-β2 and RRIG1 but induced COX-2 expression in a dose- and time-dependent manner and that FXR, the bile acid receptor, was responsible. Furthermore, bile acid also induced FXR expression in esophageal cancer cell lines. Knockdown of FXR expression using FXR shRNA antagonized the effects of bile acid in gene expression. We then demonstrated that guggulsterone treatment was able to reduce the viability of esophageal cancer cells and induce RAR-β2 and RRIG1 expression but suppress COX-2 and MMP-9 expression. In addition, ex vivo data demonstrated that FXR was highly expressed in both esophageal squamous cell carcinoma (71%) and adenocarcinoma (81%) tissues and was associated with reduced RAR-β2 and RRIG1 expression. CONCLUSIONS: Suppression of FXR activity using FXR shRNA or its inhibitor guggulsterone was able to antagonize bile acid in regulating gene expression and cancer viability. 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 5676.

Abstract 3783: RRIG1 suppress growth and invasion of prostate cancer cell lines through inactivation of Src and RhoA

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In our previous studies, we have demonstrated that expression of retinoid receptor-induced gene 1 (RRIG1) was significantly reduced in different human cancers and that restoration of RRIG1 expression suppressed tumor cell growth in vitro and in vivo. In prostate cancer, we have showed that PC3 cells do not express RRIG1 but 22RV1 express high levels of RRIG1 mRNA. In this study, we first analyzed RRIG1 expression in prostate cancer tissue specimens and then transfected RRIG1 cDNA into PC3 cells and RRIG1 antisense cDNA into 22RV1 cells to restore or knockdown RRIG1 expression in these cells, respectively. We found that expression of RRIG1 protein was significantly downregulated in prostate cancer tissues compared to the normal tissues. Moreover, stably RRIG1-transfected prostate cancer cells grew much slower than that of the controls, whereas the antisense RRIG1-transfected 22RV1 cells grew faster than that of the controls. After that, we assessed the ability of migration and invasion of these stable cells using Boyden chamber assay and found that RRIG1 transfection reduced tumor cell migration and invasion in PC3 cells compared to the control transfections. In contrast, the antisense RRIG1 transfection promoted the tumor cell migration and invasion in 22RV1 cells. Molecularly, we found that RRIG1 was binding to Src protein and suppressed Src phosphorylation as well as RhoA activation. Consequently, RRIG1 reduced expression of phosphorylated Erk1/2 and AKT as well as expression of c-Jun, COX-2, and cyclinD1. The results from the current study demonstrated a tumor-suppressive effect of RRIG1 in prostate cancer, suggesting that RRIG1 gene may play a role in suppressing prostate tumorigenesis and progression. 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 3783.