Cuiqi Zhou

Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo

IntroductionBreast cancer is the leading cause of cancer death in women worldwide. Elevated expression of c-Myc is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against c-Myc in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumor effects elicited by a decrease in the protein level of c-Myc by RNAi and its possible mechanism of effects in MCF-7 cells.MethodA plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc to reduce its expression in MCF-7 cells. Western blot analysis was used to measure the protein level of c-Myc. We assessed the effects of c-Myc silencing on tumor growth by a growth curve, by soft agar assay and by nude mice experiments in vivo. Standard fluorescence-activated cell sorter analysis and TdT-mediated dUTP nick end labelling assay were used to determine apoptosis of the cells.ResultsOur data showed that plasmids expressing siRNA against c-myc markedly and durably reduced its expression in MCF-7 cells by up to 80%, decreased the growth rate of MCF-7 cells, inhibited colony formation in soft agar and significantly reduced tumor growth in nude mice. We also found that depletion of c-Myc in this manner promoted apoptosis of MCF-7 cells upon serum withdrawal.Conclusionc-Myc has a pivotal function in the development of breast cancer. Our data show that decreasing the c-Myc protein level in MCF-7 cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, and imply the therapeutic potential of RNAi on the treatment of breast cancer by targeting overexpression oncogenes such as c-myc, and c-myc might be a potential therapeutic target for human breast cancer.

β-Catenin/TCF pathway upregulates STAT3 expression in human esophageal squamous cell carcinoma

Precise roles of beta-catenin/TCF pathway involved in esophageal tumorigenesis remain elusive. Here we found STAT3 overexpression in esophageal cancer cells and tissues, and its overexpression in esophageal squamous cell carcinoma (ESCC) tissues correlated with beta-catenin cytoplasmic/nuclear accumulation. A functional TCF binding element was detected in STAT3 promoter which specifically bound to TCF4. Transfected beta-catenin induced STAT3 transcriptional activity dose-dependently, and also enhanced STAT3 mRNA and protein levels. These inductions were specifically abolished by dominant-negative TCF4. These results suggest that STAT3 is a target of beta-catenin/TCF pathway and might participate in esophageal tumorigenesis.

Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by β‐catenin /TCF pathway in human esophageal squamous cell carcinoma

Overexpression of human pituitary tumor transforming gene (PTTG) is wildly detected in many tumors, including esophageal cancer. Besides overexpression of PTTG in esophageal squamous cell carcinoma (ESCC) tissues and cells, we detected accumulation of cytoplasmic β‐catenin in ESCC. In our study, a putative TCF4‐binding element (TBE) was identified in PTTG promoter region. The activity of PTTG promoter containing the TBE was activated by S37Aβ‐catenin and inhibited by dominant‐negative TCF. Furthermore, the activation by S37Aβ‐catenin was mostly abrogated among PTTG promoter region without the TBE or with a mutant one. By using biotin‐streptavidin pull‐down assay, we also found that the TBE among PTTG promoter bound to TCF‐4 protein. Moreover, levels of PTTG mRNA and protein were increased by S37Aβ‐catenin. Finally, it is noticeable that we detected a correlation between β‐catenin localization and PTTG expression in 69 primary ESCC (p<0.01). In brief, our study shows that overexpression of PTTG in ESCC is likely due to the activation of β‐catenin/WNT signaling. As a target gene of β‐catenin/TCF pathway, PTTG may play an important role in tumorigenesis of human ESCC.

Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating β -catenin/TCF pathway

Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of β-catenin and promoted the transcriptional activity of β-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or ΔN TCF4, respectively. Also, we found that EB1 affected the interaction between β-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of β-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the β-catenin/TCF pathway.

PTTG Overexpression Promotes Lymph Node Metastasis in Human Esophageal Squamous Cell Carcinoma

Human pituitary tumor transforming gene (PTTG) overexpression correlates with metastasis in multiple tumors, and yet its molecular mechanisms of action remain elusive. We detected PTTG overexpression in 66% (111 of 169) of primary esophageal squamous cell carcinoma (ESCC) tumor tissues by in situ hybridization. PTTG overexpression correlated with lymph node metastasis (P < 0.05). Ectopic PTTG overexpression in a representative ESCC cell line, EC9706, increased in vitro cell migration and invasion and promoted in vivo lymph node metastasis. Suppressing PTTG expression by siRNA decreased cell motility in both PTTG-HA/EC9706 and KYSE150 cells. By using mass spectrometric analysis, we identified that PTTG up-regulated S100A4 and galectin-1 secretion and down-regulated tissue inhibitor of metalloproteinase-2 secretion to the culture media. PTTG induced S100A4 and galectin-1 mRNA and protein expression as assessed by Western blot and reverse transcription-PCR. Attenuating galectin-1 expression by siRNA constrained PTTG-HA/EC9706 cell motility (P < 0.05). PTTG activated E-box transcription and induced c-Myc protein expression in EC9706 cells, which in turn may act on an E-box motif within the galectin-1 promoter. Chromatin immunoprecipitation assays further confirmed specific c-Myc binding to galectin-1 promoter. PTTG-induced galectin-1 transactivation and expression were mediated by c-Myc, and both inductions were suppressed by c-Myc RNAi cotranfection. These findings elucidate the molecular mechanisms of PTTG overexpression in promoting tumor metastasis, whereby up-regulated PTTG modulates expression and secretion of metastasis-related factors to facilitate cell motility.

Downregulation of Survivin by RNAi Inhibits the Growth of Esophageal Carcinoma Cells

Esophageal squamous cell carcinoma ranks among one of the most frequent cause of cancer death in the world. Understanding of the molecular mechanisms involved in the pathogenesis of esophageal cancer becomes critical to develop more effective treatments. Elevated expression of survivin in esophageal carcinoma has been reported before and suppression of survivin expression leads to many tumor cells growth inhibition. We hypothesized that down-regulation of survivin would inhibit the growth of human esophageal cancer cells. RNA interference directed against survivin was introduced into a human esophageal squamous cell carcinoma cell line KYSE510. Stable clones were selected and western blot analysis was performed to detect the protein level of survivin. Tumor cell growth in vitro and in vivo was assessed by trypan blue exclusion and nude mice experiments. Annexin ¢?/propidium iodide staining followed by flow cytometric analysis and TUNEL assay were used to detect apoptosis in cell culture and in nude mice. We found that RNA interference could efficiently and stably suppress survivin expression in KYSE510 cells. Down-regulation of survivin resulted in significantly inhibition of tumor growth in vitro and in vivo. The mechanism appears to be increased induction of apoptosis. Our results suggest a potential role for the targeting of survivin in the treatments of esophageal carcinoma.

Accumulation of cytoplasmic β-catenin correlates with reduced expression of E-cadherin, but not with phosphorylated Akt in esophageal squamous cell carcinoma: Immunohistochemical study

Accumulation of β‐catenin in cytoplasm occurs frequently during the pathogenesis of esophageal squamous cell carcinoma (ESCC). The mechanism leading to this alteration, however, is largely unknown. In the present study, immunohistochemistry was performed for β‐catenin, E‐cadherin and Ser473 phosphorylated Akt (P‐Akt) in 44 tissue samples of ESCC and corresponding normal esophageal epithelium. Exon 3 of the β‐catenin gene was analyzed by using single‐strand conformation polymorphism and direct sequencing. In addition to the reduced expression of E‐cadherin and membranous β‐catenin observed in 65.9% and 68% of ESCC tested, respectively, cytoplasmic accumulation of β‐catenin was also detected in 68% (30/44) cases. However, only two cases were found to have the same β‐catenin gene mutation. The data showed that cytoplasmic accumulation of β‐catenin was significantly associated  with  reduced  expression  of  E‐cadherin  (P < 0.05) and that of membranous β‐catenin (P < 0.05). Furthermore, cytoplasmic β‐catenin was correlated significantly with lymph node metastasis (P < 0.05). In contrast, although strong staining of P‐Akt occurred in 14 of 44 cases (32%), there was no significant correlation between the positive staining of P‐Akt and cytoplasmic β‐catenin. Taken together these results suggest that the lost membranous β‐catenin might translocate to cytoplasm depending on reduced expression of E‐cadherin, while Akt seems unlikely to play a role in this process.

FRAT1 overexpression leads to aberrant activation of β-catenin/TCF pathway in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with a poor prognosis. Although aberrant activation of β‐catenin/T‐cell factor (TCF) pathway has been observed in ESCC, mechanisms underlying this phenomenon remain unknown. Frequently rearranged in advanced T‐cell lymphomas‐1 (FRAT1), overexpressed in some ESCC lines, is a positive regulator of β‐catenin/TCF pathway. However, little is known about the molecular relationship between FRAT1 and β‐catenin/TCF in ESCC. In this study, we analyzed freshly resected ESCC specimens and demonstrated that FRAT1 was overexpressed in approximately 74% of tumor samples compared with matched normal tissue. Overexpression of FRAT1 significantly promoted esophageal cancer cells growth, whereas suppression of FRAT1 level by RNAi markedly inhibited their growth. In addition, FRAT1 overexpression induced the nuclear accumulation of β‐catenin and promoted the transcriptional activity of β‐catenin/TCF. These effects were reversed by coexpression of GSK 3β or ΔN TCF4. Furthermore, accumulation of β‐catenin was correlated with FRAT1 overexpression in ESCC and the basal layer of normal esophageal epithelium. Finally, continued expression of c‐Myc is necessary and sufficient for maintenance of the growth state in cells expressing FRAT1. Taken together, these results support the novel hypothesis that aberrant activation of β‐catenin/TCF pathway in esophageal cancer appears to be due to upstream events such as FRAT1 overexpression, and c‐Myc may be an important element in oncogenesis of human ESCC induced by FRAT1.