Downregulated Chibby in laryngeal squamous cell carcinoma with increased expression in laryngeal carcinoma Hep-2 cells

Xu,Jue; Ren,Gang; Zhao,De-An; Li,Bo-An; Cai,Cheng-Fu; Zhou,Yi; Luo,Xian-Yang

doi:10.3892/or.2014.3451

Downregulated Chibby in laryngeal squamous cell carcinoma with increased expression in laryngeal carcinoma Hep-2 cells

Authors:
- Jue Xu
- Gang Ren
- De-An Zhao
- Bo-An Li
- Cheng-Fu Cai
- Yi Zhou
- Xian-Yang Luo
View Affiliations

Affiliations: Department of Otolaryngology, The First Hospital Affiliated to Huzhou University Medical College, Huzhou, Zhejiang 313000, P.R. China, Department of Otolaryngology, The First Hospital Affiliated to Huzhou University Medical College, Huzhou, Zhejiang 313000, P.R. China, Department of Otolaryngology, the 174th Hospital of PLA, Xiamen, Fujian 361003, P.R. China, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, P.R. China, Department of Otolaryngology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, P.R. China
Published online on: August 29, 2014 https://doi.org/10.3892/or.2014.3451
Pages: 1947-1956

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Chibby (Cby) inhibits Wnt/β-catenin-mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of β-catenin) to bind to β-catenin. This suggests that Cby could be a tumor suppressor protein. In the present study, we examined Cby expression in laryngeal squamous cell carcinoma (LSCC) and its function and mechanism in laryngeal carcinoma cell lines. Cby expression levels were investigated by immunohistochemistry in a panel of 36 LSCC patient cases. The expression of β-catenin, c-myc and cyclin D1 in Hep-2 were determined through RT-PCR and western blot analysis. Activity of Wnt/β-catenin signaling pathway after overexpression of Cby was measured by TCF/LEF luciferase reporter gene assay. Proliferation, clone forming ability, cell cycle distribution and cell apoptosis of Hep-2 cells were detected by MTT assay, plate colony forming assay, flow cytometry and TUNEL assay, respectively. This study showed that expression of Cby protein was strongly downregulated in LSCC tumor tissues in comparison to normal laryngeal mucosa samples. No significant correlation was found between the expression of Cby in tumor tissue and gender, age, clinical stage and tumor differentiation of laryngeal cancer patients. When Cby was overexpressed in Hep-2 cells, the expression of cyclin D1 was reduced and β-catenin activity was inhibited. Proliferation and plate colony forming assays revealed a significant inhibitory effect of Cby on growth and colony formation ability of Hep-2 cells after Cby overexpression in comparison to control and mock-infected cells. In addition, we also found that upregulated expression of Cby resulted in accumulation of numbers of cells in G0/G1 phase with concomitant decrease in S phase by cell cycle assay. TUNEL staining demonstrated that, compared with the control group, the rate of apoptosis in the plv-cs2.0-Cby group was significantly increased. Taken together, downregulation of Cby was observed in LSCC, but with no significant correlation to the clinicopathological features of LSCC patients. Overexpression of Cby effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. A better understanding of the mechanisms of Cby gene activation in LSCC could provide potential novel therapeutic targets for human laryngeal carcinoma.

View Figures

View References

1	Mojica-Manosa P, Reidy J, Wilson K and Douglas W: Larynx squamous cell carcinoma: concepts and future directions. Surg Oncol Clin N Am. 13:99–112. 2004. View Article : Google Scholar : PubMed/NCBI
2	Almadori G, Bussu F, Cadoni G, Galli J, Paludetti G and Maurizi M: Molecular markers in laryngeal squamous cell carcinoma: towards an integrated clinicobiological approach. Eur J Cancer. 41:683–693. 2005. View Article : Google Scholar : PubMed/NCBI
3	Klaus A and Birchmeier W: Wnt signalling and its impact on development and cancer. Nat Rev Cancer. 8:387–398. 2008. View Article : Google Scholar : PubMed/NCBI
4	Clevers H: Wnt/β-catenin signaling in development and disease. Cell. 127:469–480. 2006.
5	Brown AM: Wnt signaling in breast cancer: have we come full circle? Breast Cancer Res. 3:351–355. 2001. View Article : Google Scholar : PubMed/NCBI
6	Lustig B and Behrens J: The Wnt signaling pathway and its role in tumor development. J Cancer Res Clin Oncol. 129:199–221. 2003.PubMed/NCBI
7	Polakis P: Wnt signaling and cancer. Genes Dev. 14:1837–1851. 2000.
8	Taipale J and Beachy PA: The Hedgehog and Wnt signalling pathways in cancer. Nature. 411:349–354. 2001. View Article : Google Scholar : PubMed/NCBI
9	Logan CY and Nusse R: The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 20:781–810. 2004. View Article : Google Scholar : PubMed/NCBI
10	Kimelman D and Xu W: Beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene. 25:7482–7491. 2006. View Article : Google Scholar : PubMed/NCBI
11	Tetsu O and McCormick F: b-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 398:422–426. 1999. View Article : Google Scholar : PubMed/NCBI
12	Shtutman M, Zhurinsky J, Simcha I, et al: The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway. Proc Natl Acad Sci USA. 96:5522–5527. 1999.
13	He TC, Sparks AB, Rago C, et al: Identification of c-MYC as a target of the APC pathway. Science. 281:1509–1512. 1998. View Article : Google Scholar : PubMed/NCBI
14	Pietruszewska W, Kobos J and Gryczyński M: Expression of beta-catenin protein in laryngeal squamous-cell carcinoma. Otolaryngol Pol. 58:949–956. 2004.(In Polish).
15	Si WF, Sun W, Liu H, Liu J, Sun Y and Chen Z: Expression and clinical significance of E-cadherin and beta-catenin proteins in human laryngeal cancer. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 22:459–461. 2008.(In Chinese).
16	Goulioumis AK, Varakis J, Goumas P and Papadaki H: Differential β-catenin expression between glottic and supraglottic laryngeal carcinoma. Eur Arch Otorhinolaryngol. 267:1573–1578. 2010.
17	Sanz Ortega J, Valor C, Saez MC, et al: 3p21, 5q21, 9p21 and 17p13 allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation. Histol Histopathol. 18:1053–1057. 2003.
18	Pećina-Šlaus N, Kljaić M and Nikuševa-Martić T: Loss of heterozygosity of APC and CDH1 genes in laryngeal squamous cell carcinoma. Pathol Res Pract. 201:557–563. 2005.PubMed/NCBI
19	Takemaru K, Yamaguchi S, Lee YS, Zhang Y, Carthew RW and Moon RT: Chibby, a nuclear β-catenin-associated antagonist of the Wnt/Wingless pathway. Nature. 422:905–909. 2003.
20	Takemaru K, Fischer V and Li FQ: Fine-tuning of nuclear β-catenin by Chibby and 14-3-3. Cell Cycle. 8:210–213. 2009.
21	Li FQ, Mofunanya A, Harris K and Takemaru K: Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity. J Cell Biol. 181:1141–1154. 2008.PubMed/NCBI
22	Greaves S: Small changes in Wnt signalling. Nat Cell Biol. 5:3872003. View Article : Google Scholar : PubMed/NCBI
23	Karakoula K, Suarez-Merino B, Ward S, et al: Real-time quantitative PCR analysis of pediatric ependymomas identifies novel candidate genes including TPR at 1q25 and CHIBBY at 22q12-q13. Genes Chromosomes Cancer. 47:1005–1022. 2008. View Article : Google Scholar : PubMed/NCBI
24	Schuierer MM, Graf E, Takemaru K, Dietmaier W and Bosserhoff AK: Reduced expression of β-catenin inhibitor Chibby in colon carcinoma cell lines. World J Gastroenterol. 12:1529–1535. 2006.
25	Kafri T, van Praag H, Ouyang L, Gage FH and Verma IM: A packaging cell line for lentivirus vectors. J Virol. 73:576–584. 1999.PubMed/NCBI
26	Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265–275. 1951.PubMed/NCBI
27	Pinto D and Clevers H: Wnt control of stem cells and differentiation in the intestinal epithelium. Exp Cell Res. 306:357–363. 2005. View Article : Google Scholar : PubMed/NCBI
28	Li FQ, Mofunanya A, Fischer V, Hall J and Takemaru K: Nuclear-cytoplasmic shuttling of Chibby controls β-catenin signaling. Mol Biol Cell. 21:311–322. 2010.PubMed/NCBI
29	Jonas JC, Laybutt R, Steil GM, Trivedi N, Weir GC and Henquin JC: Potential role of the early response gene c-myc in beta-cell adaptation to changes in glucose concentration. Diabetes. 50:S1372001. View Article : Google Scholar : PubMed/NCBI
30	Fields WR, Desiderio JG, Putnam KP, Bombick DW and Doolittle DJ: Quantification of changes in c-myc mRNA levels in normal human bronchial epithelial (NHBE) and lung adenocarcinoma (A549) cells following chemical treatment. Toxicol Sci. 63:107–114. 2001. View Article : Google Scholar
31	Liu T, Peng H, Wu Z and Cui C: Expression and significance of signal transducer and activator of transcription 3 and c-myc in laryngeal squamous cell carcinoma. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 24:648–651. 2010.(In Chinese).
32	Krecicki T, Fraczek M, Jelen M, Zatonski T, Szkudlarek T and Dus D: Expression of c-myc oncoprotein in laryngeal squamous cell carcinoma. Acta Otolaryngol. 124:634–637. 2004. View Article : Google Scholar : PubMed/NCBI
33	Long X, Hu S, Cao P, Liu Z, Zhen H and Cui Y: The expression of oncogene c-myc and its role on human laryngeal cancer. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 23:1127–1129. 2009.(In Chinese).
34	Fernando R, Foster JS, Bible A, et al: Breast cancer cell proliferation is inhibited by BAD: regulation of cyclin D1. J Biol Chem. 282:28864–28873. 2007. View Article : Google Scholar : PubMed/NCBI
35	Fu ZJ, Ma ZY, Wang QR, et al: Overexpression of CyclinD1 and underexpression of p16 correlate with lymph node metastases in laryngeal squamous cell carcinoma in Chinese patients. Clin Exp Metastasis. 25:887–892. 2008. View Article : Google Scholar : PubMed/NCBI
36	Papadimitrakopoulou V, Izzo JG, Liu DD, et al: Cyclin D1 and cancer development in laryngeal premalignancy patients. Cancer Prev Res (Phila). 2:14–21. 2009. View Article : Google Scholar : PubMed/NCBI