Abstract
Human papillomavirus (HPV) is the key epidemiologic factor of cervical cancer, but additional cofactors are mandatory. Estrogen has been considered as one of those. Here, the aim was to study the effects of steroid hormones on HPV16 E6–E7, estradiol receptors ERα and ERβ, and progesterone receptor (PR) in HPV16-positive cervical carcinoma cell lines SiHa and CaSki grown as epithelial and fibroblast spheroid co-cultures. The spheroid co-cultures were exposured to 17β-estradiol or progesterone from day 7 onwards. mRNA levels of HPV16 E6–E7, ERα, ERβ and PR normalized against GAPDH were analyzed with quantitative reverse transcription–qPCR (RT-qPCR). 17β-estradiol and progesterone decreased HPV16 E6–E7 mRNA expression in CaSki and increased in SiHA co-cultures. In CaSki co-cultures, ERβ expression was blocked after 17β-estradiol exposure while in SiHa cells it slightly increased ERβ expression. PR expression was seen only in CaSki spheroids and it vanished after exposure to steroid hormones. Fibroblasts expressed all three hormone receptors as monolayers but ERβ expression decreased and ERα and PR vanished after co-culturing. Cell culturing platform changes both oncogene and hormone receptor expression in HPV16 positive cervical cancer cell lines. This needs to be considered when in vitro results are extrapolated to in vivo situations.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10616-017-0137-9/MediaObjects/10616_2017_137_Fig1_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10616-017-0137-9/MediaObjects/10616_2017_137_Fig2_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10616-017-0137-9/MediaObjects/10616_2017_137_Fig3_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10616-017-0137-9/MediaObjects/10616_2017_137_Fig4_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10616-017-0137-9/MediaObjects/10616_2017_137_Fig5_HTML.jpg)
Similar content being viewed by others
References
Atula S, Grenman R, Syrjänen S (1997) Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro. Exp Cell Res 235:180–187
Berger C, Qian Y, Chen X (2013) The p53-estrogen receptor loop in cancer. Curr Mol Med 13:1229–1240
Björnström L, Sjöberg M (2004) Estrogen receptor-dependent activation of AP-1 via non-genomic signalling. Nucl Recept 2:3
Chandrasekaran S, Giang UB, Kind MR, DeLouise LA (2011) Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane. Biomaterials 32:7159–7168
Chow LT (2015) Model systems to study the life cycle of human papillomaviruses and HPV-associated cancers. Virol Sinica 30:92–100
Chow LT, Broker TR (1997) In vitro experimental systems for HPV: epithelial raft cultures for investigations of viral reproduction and pathogenesis and for genetic analyses of viral proteins and regulatory sequences. Clin Dermatol 15:217–227
Chuang CK, Liao SK (2000) Human bladder carcinoma cells with an unusual pattern of in vitro growth: transition from nonproliferative spheroids to active monolayer growth upon interaction with tumor-derived fibroblasts. Anticancer Res 20:749–760
Chung SH, Lambert PF (2009) Prevention and treatment of cervical cancer in mice using estrogen receptor antagonists. Proc Natl Acad Sci USA 106:19467–19472
Chung SH, Wiedmeyer K, Shai A, Korach KS, Lambert PF (2008) Requirement for estrogen receptor alpha in a mouse model for human papillomavirus-associated cervical cancer. Cancer Res 68:9928–9934
Chung SH, Franceschi S, Lambert PF (2010) Estrogen and ERalpha: culprits in cervical cancer? Trends Endocrinol Metab 21:504–511
Chung SH, Shin MK, Ks Korach, Lambert PF (2013) Requirement for stromal estrogen receptor alpha in cervical neoplasia. Horm Cancer 4:50–59
Doorbar J (2016) Model systems of human papillomavirus-associated disease. J Pathol 238:166–179
Elson DA, Riley RR, Lacey G, Thordarson G, Talamantes FJ, Arbeit JM (2000) Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice. Cancer Res 60:1267–1275
Gascard P, Tlsty TD (2016) Carcinoma-associated fibroblasts: orchestrating the composition of malignancy. Genes Dev 30:1002–1019
Giersich JM, Coeytaux RR, Urrutia RP, Havrilesky LJ, Moorman PG, Lowery WJ, Dinan M, McBroom AJ, Hasselblad V, Sanders GD, Myers ER (2013) Oral contraceptive use and risk of breast, cervical, colorectal, and endometrial cancers: a systematic review. Cancer Epidemiol Biomark Prev 22:1931–1943
Hemmings C (2013) Is carcinoma a mesenchymal disease? The role of the stromal microenvironment in carcinogenesis. Pathology 45:371–381
Hietanen S, Syrjänen K, Syrjänen S (1998) Characterization of keratin and cell cycle protein expression in cell lines from squamous intraepithelial lesions progressing towards a malignant phenotype. Br J Cancer 77:766–775
Hwang JY, Lin BY, Tang FM, Yu WC (1992) Tamoxifen stimulates human papillomavirus type 16 gene expression and cell proliferation in a cervical cancer cell line. Cancer Res 52:6848–6852
IARC Working Group on the Evaluation of Carcinoagenic Risks to Human (2012) Biological agents. Volume 100 B. A review of human carcinogens. IARC Monogr Eval Carcinog Risks Hum 100:1–441
Jacquin E, Baraquin A, Ramanah R, Carcopino X, Morel A, Valmary-Deqano S, Bravo IG, de Sanjose D, Mouqing C, Pretet JL (2013) Methylation of human papillomavirus type 16 CpG sites at E2-binding site 1 (E2BS1), E2BS2, and Sp1-binding site in cervical cancer samples as determined by high-resolution melting analysis-PCR. J Clin Microbiol 51:3207–3215
Jia M, Dahlman-Wright K, Gustafsson JA (2015) Estrogen receptor alpha and beta in health and disease. Best Pract Res Clin Endocrinol Metab 29:557–568
Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392–401
Kilani RT, Tamimi Y, Hanel EG, Wong KK, Karmali S, Lee PW, Moore RB (2003) Selective reovirus killing of bladder cancer in a co-culture spheroid model. Virus Res 93:1–12
Kim CJ, Um SJ, Kim TY, Kim EJ, Park TC, Kim SJ, Namkoong SE, Park JS (2000) Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells. Int J Gynecol Cancer 10:157–164
Kunz-Schughart LA, Heyder P, Schroeder J, Knuechel R (2001) A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation. Exp Cell Res 266:74–86
Kwasniewska A, Postawski K, Gozdzicka-Jozefiak A, Kwasniewski W, Grywalska E, Zdunek M, Korobowicz E (2011) Estrogen and progesteron receptor expression in HPV-positive and HPV-negative cervical carcinomas. Oncol Rep 26:153–160
López-Romero R, Garrido-Guerrero E, Rangel-Lopez A, Manuel-Apolinar L, Pina-Sanchez P, Lazos-Ochoa M, Mantilla-Morales A, Bandala C, Salcedo M (2013) The cervical malignant cells display regulation of ER-α but retain the ER-β expression. Int J Clin Exp Pathol 6:1594–1602
Merne M, Syrjänen S (2003) The mesenchymal substrate influences the epithelial phenotype in a three-dimensional cell culture. Arch Dermatol Res 295:190–198
Mitrani-Rosenbaum S, Tsvieli R, Tur-Kaspa R (1989) Oestrogen stimulates differential transcription of human papillomavirus type 16 in SiHa cervical carcinoma cells. J Gen Virol 70:2227–2232
Nath S, Devi GR (2016) Three-dimensional culture systems in cancer reserach: focus on tumor spheroid model. Pharm Ther 163:94–108
Newfield L, Bradlow HL, Sepkovic DW, Auborn K (1998) Estrogen metabolism and the malignant potiential of human papillomavirus immortalized keratinocytes. Proc Soc Exp Biol Med 217:322–326
Nin DS, Huang W, Ali M, Yew CW, Kutateladze TG, Deng LW (2015) O-GlcNacylation of MLL5β is essential for MLL5β-AP-1 transcription complex assembly at the HPV16/18-long control region. J Mol Cell Biol 7:180–183
Peitsaro P, Johansson B, Syrjänen S (2002) Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique. J Clin Microbiol 40:886–891
Ruutu M, Wahlroos N, Syrjänen K, Johansson B, Syrjänen S (2006) Effects of 17beta-estradiol and progesterone on transcription of human papillomavirus 16 E6/7 oncogenes in CaSki and SiHa cell lines. Int J Gynecol Cancer 16:1261–1268
Seidl P, Huettinger R, Knuechel R, Kunz-Schughart LA (2002) Three-dimensional fibroblast-tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro. Int J Cancer 102:129–136
Smith JS, Green J, Berrington de Gonzalez A, Appleby P, Peto J, Plummer M, Franceschi S, Beral V (2003) Cervical cancer and use of hormonal contraceptives: a systematic review. Lancet 361:1159–1167
Son J, Wook Park J, Lambert PF, Chung S-H (2014) Requirement of estrogen receptor alpha DNA-binding domain for HPV oncogene-induced cervical carcinogenesis in mice. Carcinogenesis 35:489–496
Weiswald L, Bellet D, Dangles-Marie V (2015) Spherical cancer models in tumor biology. Neoplasia 17:1–15
Willberg J (2007) The dento-epithelial junction—epithelial cell adhesion and production of extracellular matrix molecules. Ann Univ Turku Med Odontol Ser D 748:33–34
Yuan F, Chen DZ, Liu K, Sepkovic DW, Bradlow HL, Auborn K (1999) Anti-estrogenic activities of indole-3-carbinol in cervical cells: implication for prevention of cervical cancer. Anticancer Res 19:1673–1680
Acknowledgements
We thank Mariia Valkama from her skillful technical assistance in cell cultures. We thank the following research grants: Government Special Foundation for the Turku University Central Hospital, Turku, Finland, Turku; University Foundation, Turku, Finland; Cancer Foundations of Western Finland, Turku, Finland.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have conflict of interest.
Rights and permissions
About this article
Cite this article
Ruutu, M., Rautava, J., Turunen, A. et al. 17β-estradiol and progesterone effect on human papillomavirus 16 positive cells grown as spheroid co-cultures. Cytotechnology 70, 235–244 (2018). https://doi.org/10.1007/s10616-017-0137-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-017-0137-9