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Activation of Wnt-β-catenin pathway in basal–parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma

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Abstract

In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.

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Acknowledgements

The authors thank the Director of Chittaranjan National Cancer Institute, Kolkata, India. We are grateful to Professor (Dr.) H. zur Hausen and Professor (Dr. Mrs.) E.M. de Villiers for their generous gift of HPV-16/18 plasmids. This work was supported by CSIR (Council of Scientific and Industrial Research, Government of India)-JRF/NET grant [File No.09/030(0059)/2010-EMR-I] to Mr C.Chakraborty, grant [Sr. No. 2121130723] from UGC (University Grants Commission, Government of India) to Mr. Sudip Samadder, grant [SR/SO/HS-116/2007] from the Department of Science and Technology (DST), Government of India and grant [No. 60(0111)/14/EMR-II of dt.03/11/2014] from CSIR (Council of Scientific and Industrial Research, Government of India) to Dr. C. K. Panda.

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Correspondence to Chinmay Kumar Panda.

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Fig. S1 Schematic Distribution of the samples used for the study. Supplementary material 1 (TIFF 2030 kb)

Fig. S2 Laser capture microdissection of normal cervical epithelium.Supplementary material 2 (TIFF 1077 kb)

11010_2017_3216_MOESM3_ESM.tif

Fig. S3 Kaplan–Meier 5-year survival probability curves with cumulative survival of CACX patients by Wnt3a protein expression. Supplementary material 3 (TIFF 1396 kb)

Table S1 Primers for promoter methylation analysis APC. Supplementary material 4 (DOCX 11 kb)

Table S2 Primers for mRNA expression analysis of APC. Supplementary material 5 (DOCX 11 kb)

Table S3 Primers for deletion analysis of APC. Supplementary material 6 (DOCX 11 kb)

11010_2017_3216_MOESM7_ESM.docx

Table S4 Expression/methylation status of APC, β-catenin, p-β-catenin (Y654) and Wnt3a in different normal cervical epithelium. Supplementary material 7 (DOCX 33 kb)

11010_2017_3216_MOESM8_ESM.docx

Table S5 Relation between deletion (%) and methylation (%) of APC in CIN and CACX. Supplementary material 8 (DOCX 12 kb)

Table S6 Allelic status of APC in CIN and CACX. Supplementary material 9 (DOCX 74 kb)

11010_2017_3216_MOESM10_ESM.docx

Table S7 Correlation with expression/methylation/deletion status of APC, β-catenin, p-β-catenin and Wnt3a in CIN and CACX. Supplementary material 10 (DOCX 28 kb)

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Chakraborty, C., Samadder, S., Roychowdhury, A. et al. Activation of Wnt-β-catenin pathway in basal–parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma. Mol Cell Biochem 443, 121–130 (2018). https://doi.org/10.1007/s11010-017-3216-5

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