Xiao-Chun Xu

B-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients. Expression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells. BCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins. The current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer.

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.

Background: The murine model of 4-nitroquinoline 1-oxide (4-NQO)-induced oral and esophageal cancer is frequently used to assess the effects of different cancer prevention! therapy agents in vivo, but the molecular mechanisms in those 4-NQO-induced carcinogenesis are unknown. This study investigated aberrant expression of cell growth-critical genes in 4-NQO-induced oral and esophageal cancer tissues in mice compared to those present in the human disease for association with survival of patients. Materials and Methods: C57LB6/129Sv mice were given 4-NQO in their drinking water to induce oral and esophageal cancer. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry were used to detect gene expression in the cancer tissues from mice and in 4-NQO-treated human esophageal cancer cell lines and esophageal cancer tissues. Methylation-specific PCR and DNA sequencing were performed to assess methylation of the Rarb2 promoter in murine tissues. Kaplan-Meier analysis was performed to associate gene expression in esophageal cancer tissues with survival data for patients with esophageal cancer. Results: 4-NQO dose-dependently induced pre-malignant and malignant lesions in the oral cavity and esophagus in mice that pathologically and morphologically mimicked human oral and esophageal cancer. Molecularly, 4-NQO inhibited Rar beta(2) but induced expression of phosphorylated extracellular-signal-regulated kinase-1 and -2 (p-ERK1/2) and Cox2 proteins and Rar beta(2) gene promoter methylation in murine tumors. In vitro treatment with 4-NQO altered expression of RAR beta(2), p-ERK1/2, and COX2 in human esophageal cancer cells. In tissues from 90 patients with esophageal cancer, expression of p-ERK1/2 and COX2 was up-regulated, and p-ERK1/2 expression was associated with advanced clinical tumor stage and consumption of hot beverages, while COX2 expression was associated with tumor de-differentiation in esophageal cancer. Furthermore, expression of p-ERK1/2 was associated with a worse overall survival rate of patients (p=0.014), whereas the association of COX2 expression with worse overall survival rate did not reach statistical significance (p=0.19). Knockdown of COX2 expression using transient transfection of a COX2 antisense expression vector inhibited Ki67 expression, an indicator of cell proliferation, in human esophageal cancer cells. Conclusion: 4-NQO-induced cancer in oral cavity and esophagus of mice not only pathologically and morphologically mimicked human oral and esophageal cancer, but also shared some molecular alterations (e.g. aberrant expression of Rarb2, p-ERK1/2, and Cox2). This study further demonstrated that targeting of the altered RAR beta(2)-led gene pathway could effectively suppress the development of this deadly type of cancer.

BACKGROUND: 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.METHODS: FXR expression in esophageal adenocarcinoma tissues was assessed by immunohistochemistry. Knockdown of FXR expression in esophageal cancer cells in vitro and in nude mice xenografts was suppressed by FXR small hairpin RNA (shRNA) and guggulsterone (a natural FXR inhibitor). Esophageal cancer cells were treated with bile acids to demonstrate their effects on growth‐promoting genes.RESULTS: FXR was expressed in 48 of 59 esophageal adenocarcinoma tissues (81.3%), and this overexpression was associated with higher tumor grade, larger tumor size, and lymph node metastasis; however, was inversely associated with retinoic acid receptor‐β₂ (RAR‐β₂) expression. Knockdown of FXR expression suppressed tumor cell growth in vitro and in nude mouse xenografts. Guggulsterone reduced the viability of esophageal cancer cells in a time‐dependent and dose‐dependent manner, whereas this effect was diminished after knockdown of FXR expression. Guggulsterone induced apoptosis through activation of caspase‐8, caspase‐9, and caspase‐3 in tumor cells. FXR mediated bile acid‐induced alterations of gene expression, eg, RAR‐β₂ and cyclooxygenase‐2 (COX‐2).CONCLUSIONS: Inhibition of FXR by FXR shRNA or guggulsterone suppressed tumor cell viability and induced apoptosis in vitro, and it reduced tumor formation and growth in nude mouse xenografts. FXR also mediated bile acid‐induced alterations of cell growth‐related genes in esophageal cancer cells. Cancer 2013. © 2012 American Cancer Society.

MiRNAs, endogenous oligonucleotide RNAs, play an important role in mammary gland carcinogenesis and tumor progression. Detection of their expression and investigation of their functions could lead to discovery of novel biomarkers for breast cancer.

To determine the effects of curcumin, (-)-epigallocatechin-3-gallate (EGCG), lovastatin, and their combinations on inhibition of esophageal cancer. Esophageal cancer TE-8 and SKGT-4 cell lines were subjected to cell viability methyl thiazolyl tetrazolium and tumor cell invasion assays in vitro and tumor formation and growth in nude mouse xenografts with or without curcumin, EGCG and lovastatin treatment. Gene expression was detected using immunohistochemistry and Western blotting in tumor cell lines, tumor xenografts and human esophageal cancer tissues, respectively. These drugs individually or in combinations significantly reduced the viability and invasion capacity of esophageal cancer cells in vitro. Molecularly, these three agents reduced the expression of phosphorylated extracellular-signal-regulated kinases (Erk1/2), c-Jun and cyclooxygenase-2 (COX-2), but activated caspase 3 in esophageal cancer cells. The nude mouse xenograft assay showed that EGCG and the combinations of curcumin, EGCG and lovastatin suppressed esophageal cancer cell growth and reduced the expression of Ki67, phosphorylated Erk1/2 and COX-2. The expression of phosphorylated Erk1/2 and COX-2 in esophageal cancer tissue specimens was also analyzed using immunohistochemistry. The data demonstrated that 77 of 156 (49.4%) tumors expressed phosphorylated Erk1/2 and that 121 of 156 (77.6%) esophageal cancers expressed COX-2 protein. In particular, phosphorylated Erk1/2 was expressed in 23 of 50 (46%) cases of esophageal squamous cell carcinoma (SCC) and in 54 of 106 (50.9%) cases of adenocarcinoma, while COX-2 was expressed in 39 of 50 (78%) esophageal SCC and in 82 of 106 (77.4%) esophageal adenocarcinoma. The combinations of curcumin, EGCG and lovastatin were able to suppress esophageal cancer cell growth in vitro and in nude mouse xenografts, these drugs also inhibited phosphorylated Erk1/2, c-Jun and COX-2 expression.

Background: Retinoid 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. Methods: Immunohistochemistry 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. Results: The 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. Conclusion: The 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.

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.

MicroRNAs (miRNAs) play an important role in the regulation of cell growth, differentiation, apoptosis, and carcinogenesis. Detection of their expression may lead to identifying novel markers for breast cancer.