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Objective: ATP-binding cassette, subfamily G, member 2 (ABCG2) is a new member of the superfamily of ATP-binding cassette transporter proteins and known to be not only a member of the membrane transporters implicated in multidrug resistance, but also a molecular determinant of the side population phenotype, characteristics of which are reminiscent of stem cells. The aim of the current study was to clarify the significance of ABCG2 expression in esophageal squamous cell carcinoma (ESCC). Methods: We immunohistochemically investigated paraffin sections of 100 ESCC tumors and assessed the expression level of ABCG2 mRNA in 33 specimens by quantitative RT-PCR. Results: In the immunohistochemical study, ABCG2 expression was detectable in 61% of patients and the proportion of ABCG2-positive cells was variable (0–100%). Interestingly, the presence of ABCG2-positive cells in the tumor, regardless of their amount, was associated with poorer survival (p = 0.0088). Moreover, it was revealed to be an independent prognostic factor along with the extent of the primary tumor and positive lymph node metastasis in multivariate analysis using Cox’s regression model. In the quantitative RT-PCR study, higher tumor ABCG2 mRNA expression was associated with poorer survival (p = 0.017). Conclusions: The absolute presence of ABCG2-positive cells in the tumor is a single independent prognostic factor, suggesting the underlying roles in malignant characteristics of ESCC other than drug resistance.

To elucidate the possible involvement of gankyrin in ESCC progression and the effect of its down-regulation in ESCC, we investigated the expression of gankyrin in ESCC tissues comparing it with the corresponding normal esophageal epithelia and tested a short-hairpin RNA (shRNA) expression vector for gankyrin in ESCC cell lines. Gankyrin protein expression in 11 ESCC cell lines (KYSE series) was examined by RT-PCR and western blot. The expression of gankyrin mRNA in 30 ESCC tissues was compared with the corresponding normal epithelia by Real-time PCR. Expression of gankyrin protein was immunohistochemically analyzed in the ESCC of 103 patients. A gankyrin-shRNA vector was stably transfected into KYSE 170 cells to assess the role of gankyrin in cell motility, invasion and proliferation in vitro and tumor formation in vivo. Gankyrin expression increased in all 11 ESCC cell lines. Real-time PCR revealed that gankyrin expression was higher in the cancerous tissue for all 30 patients. In immunohistochemistry, gankyrin overexpression was correlated with lower survival rate (p 5 0.0001), extent of the primary tumor, lymph node metastasis, distant lymph node metastasis and stage (p 5 0.0072, p 5 0.0004, p 5 0.0172 and p 5 0.0002, respectively). A shRNA vector against gankyrin repressed growth, cell motility, invasiveness in vitro and tumor formation in vivo. Gankyrin overexpression is associated with poor prognosis. It may play an important role in ESCC tumor progression and could be a potentially important therapeutic gene target in ESCC. ' 2007 Wiley-Liss, Inc.Key words: gankyrin; oncogene; esophageal cancer; clinical significance; novel; cell cycle Despite considerable advances in surgical techniques, perioperative care and neoadjuvant chemoradiotherapy, esophageal squamous cell carcinoma (ESCC) still remains one of the most lethal cancers and the seventh leading cause of cancer death worldwide. 1,2 Gene study is widely performed in ESCC 3-5 and new oncogenes associated with the progression of ESCC have been identified and targeted to improve the survival of patients with this type of refractory cancer.Gankyrin is a novel oncoprotein with 7 ankyrin repeats. 6-8 Overexpression of gankyrin increases the phosphorylation and degradation of retinoblastoma (RB1) by its RB1-binding motif, suggesting that it promotes tumorigenicity and cancer cell proliferation. 9 Furthermore, binding to CDK4 and counteracting the inhibitory function of the INK4 proteins, including tumor suppressors p16 INK4A and p18 INK4B , gankyrin acts as an accelerator for cell cycle progression. 9-11 Gankyrin also binds to MDM2, facilitating MDM2-p53 interaction and increasing its polyubiquitilation and degradation. 12 These findings suggest the importance of gankyrin in the cell cycle control and tumorigenesis. However, the physiological and physiopathological role of gankyrin in the cell cycle progression of normal cells remains unknown.Overexpression of gankyrin has been found ubiquitously in hepatocellular carcinoma tissues, liver regeneration and f...

Purpose: We previously reported that the expression of Aurora-A was frequently up-regulated in human esophageal squamous cell carcinoma (ESCC) tissues as well as cell lines and the upregulation contributed to a poor prognosis. In this study, we assessed the possibility of Aurora-A suppression as a therapeutic target for ESCC using ESCC cell lines. Experimental Design: We established subclones using vector-based short hairpin RNA (shRNA). Then, we investigated the effect of Aurora-A suppression on proliferation and cell cycle changes in vitro. Next, chemosensitivity against docetaxel was investigated by tetrazolium saltb ased proliferation assay (WSTassay) and cell number determinations, and furthermore, the type of cell death induced by docetaxel was analyzed by flow cytometry. Finally, to examine the effect of Aurora-A shRNA on proliferation and chemosensitivity against docetaxel in vivo, a s.c. tumor formation assay in nude mice was done. Results: We established two genetically different stable cell lines (510 A and 1440 A) in which levels of Aurora-A were reduced. Cell growth was inhibited by 38.7% in 510 A and by 24.3% in 1440 A in vitro compared with empty vector^transfected controls (510 m and 1440 m), and this growth inhibition was mediated through G 2 -M arrest as confirmed by flow cytometry. Next, in a WSTassay, the IC 50 for Aurora-A shRNA-transfected cells was lower than that of empty vectort ransfected cells (510 A, 2.7 Â 10 À7 mol/L; 510 m, 4.8 Â 10 À7 mol/L; 1440 A, 2.6 Â 10 À7 mol/L; 1440 m, 4.9 Â 10 À7 mol/L). In addition, 0.3 nmol/L docetaxel induced a notable level of apoptosis in Aurora-A shRNA-transfected cells compared with empty vector^transfected cells. In the assay of s.c. tumors in nude mice, tumor growth in 510 A was inhibited by 36.1% compared with that in 510 m, and in tumors treated with docetaxel, the suppression of Aurora-A resulted in 44.0% tumor growth suppression in vivo. Conclusions: These results indicated that Aurora-A might play an important role in chemosensitivity to docetaxel, and the suppression of its expression might be a potential therapeutic target for ESCC.Several proteins strictly regulate the process of cellular division.

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