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. 2022 Nov 1;30(6):593-602.
doi: 10.4062/biomolther.2022.108.

HPV-18 E7 Interacts with Elk-1 Leading to Elevation of the Transcriptional Activity of Elk-1 in Cervical Cancer

Sung-Ho Go  1 , Seung Bae Rho  2 , Dong-Wha Yang  1 , Boh-Ram Kim  3 , Chang Hoon Lee  3 , Seung-Hoon Lee  1
Affiliations

HPV-18 E7 Interacts with Elk-1 Leading to Elevation of the Transcriptional Activity of Elk-1 in Cervical Cancer

Sung-Ho Go et al. Biomol Ther (Seoul). .

Abstract

The human papillomavirus (HPV)-18 E7 (E7) oncoprotein is a major transforming protein that is thought to be involved in the development of cervical cancer. It is well-known that E7 stimulates tumour development by inactivating pRb. However, this alone cannot explain the various characteristics acquired by HPV infection. Therefore, we examined other molecules that could help explain the acquired cancer properties during E7-induced cancer development. Using the yeast two-hybrid (Y2H) method, we found that the Elk-1 factor, which is crucial for cell proliferation, invasion, cell survival, anti-apoptotic activity, and cancer development, binds to the E7. By determining which part of E7 binds to which domain of Elk-1 using the Y2H method, it was found that CR2 and CR3 of the E7 and parts 1-206, including the ETS-DNA domain of Elk-1, interact with each other. As a result of their interaction, the transcriptional activity of Elk-1 was increased, thereby increasing the expression of target genes EGR-1, c-fos, and E2F. Additionally, the colony forming assay revealed that overexpression of Elk-1 and E7 promotes C33A cell proliferation. We expect that the discovery of a novel E7 function as an Elk-1 activator could help explain whether the E7 has novel oncogenic activities in addition to p53 inactivation. We also expect that it will offer new methods for developing improved strategies for cervical cancer treatment.

Keywords: Cervical cancer; Elk-1; HPV-18 E7; Protein-protein interaction; Transcriptional activity; Yeast two-hybrid.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors have declared that no competing interest exists.

Figures

Fig. 1
Fig. 1
Interaction of Elk-1 with HPV-18 E7 (E7) in vivo and in vitro. (A) Interactions between Elk-1 and E7 are represented by the relative activity of β-galactosidase expression. (B) The growth of transformants was measured by their ability to grow on Leu medium and by β-galactosidase expression. (C) GST fusion proteins from induced lysates of Escherichia coli expressing recombinant pGEX-4T-1 vectors encoding E7 were incubated with the in vitro-translated Elk-1 protein. The bound Elk-1 protein was detected using autoradiography. Input indicates one-fifth of the labeled Elk-1 used in the assay. (D) The amount of GSTand GST-E7 proteins. To show that the amounts of GST fusion proteins used in this assay were similar, proteins were separated on 12% SDS-PAGE gels and stained in Coomassie Blue solution. *p<0.05 versus control.
Fig. 2
Fig. 2
Interaction and co-localization of E7 and Elk-1. (A) HEK293a cells were transiently transfected with 5 μg of pCDNA3.1/Elk-1 (lanes 2-4) and 5 μg of pCDNA4-His/E7 (lanes 1, 3 and 4) in the absence or presence of RasV12 (lanes 4). Cells were harvested from 48 h post-transfection and whole-cell lysates were immunoprecipitated with an anti-His antibody (Santa Cruz Biotechnology). Immunoprecipitations were separated on 10% SDS-PAGE gels and blotted with an anti-Elk-1 antibody (Santa Cruz Biotechnology). (B) HeLa cells were transiently transfected with 10 μg of pCDNA3.1/Elk-1. Cells were harvested 48 h post-transfection and endogenous E7 was co-immunoprecipitated with the anti-Elk-1 antibody (Santa Cruz Biotechnology) from HeLa cells. Immunoprecipitations were separated on 10% SDS-PAGE gels and blotted with an anti-Elk-1 antibody(Santa Cruz Biotechnology). (C) HEK293a cells were transiently transfected with pCDNA3.1/Elk-1 and pEGFP-C1/E7. Cells were fixed and subjected to indirect immunofluorescence using an anti-Elk-1 antibody (a, Santa Cruz Biotechnology) followed by DAPI (d) and GFP antibody (b) counterstaining. A merged image of Elk-1 and GFP-E7 immunostaining is shown in (c). Images were representative of three independent experiments.
Fig. 3
Fig. 3
Mapping of the Elk-1-binding domain on E7. (A) Schematic representation of cDNA constructs indicating the full-length GST-E7 and each deletion construct. (B) β-galactosidase assays were performed in the presence of X-gal to determine the binding activity of the constructs. Positive interactions were revealed based on cell growth on Leu-depleted medium (upper panel), as well as the formation of blue colonies on medium containing X-gal (lower panel). (C) Results of protein interactions using a GST pull-down assay (each protein was detected by autoradiography). Coomassie Blue-stained SDS-PAGE gel showing the in vitro interactions between GST–E7 and Elk-1 proteins. Each protein was separated on a 12% SDS-PAGE gel.
Fig. 4
Fig. 4
Mapping of the interaction region of E7 on Elk-1. (A) Schematic representation of cDNA constructs indicating the full-length Elk-1 and deletion constructs. (B) Binding of E7 to the Elk-1 (FL) protein. Pull-down analysis of in vitro-translated E7 with full-length Elk-1 immobilized onto Ni-NTA agarose beads (lane 3). The beads on their own were used as a control (lane 2). Input indicates one-tenth of the labeled E7 used in the assay (lane 1). (C) Binding of E7 to Elk-1 derivative proteins. GST fusion proteins from induced lysates of E. coli expressing recombinant pGEX-4T-1 vectors encoding five types of Elk-1 deletion fragments were incubated with in vitro-translated E7 protein (lanes 3-7). The bound E7 protein was detected using autoradiography. Input indicates one-tenth of the labeled E7 used in the assay (lane 1). To show that the amount of GST and GST-Elk-1 derivative proteins used in this assay were similar, six proteins were separated on a 10% SDS-PAGE gel and stained in Coomassie Blue solution (lower panel). (D) β-galactosidase assays were performed in the presence of X-gal to determine the binding activity of the constructs. Positive interactions were revealed based on cell growth on Leu-depleted medium (upper panel), as well as the formation of blue colonies on medium containing X-gal (lower panel).
Fig. 5
Fig. 5
E7 enhances the transcriptional activity of Elk-1.Luciferase reporter assays for the activity ofegr-1 (A), c-fos (B), and the E2F (C) promoter were performed with the expression vectors encoding Elk-1, Elk-En, and E7, as indicated. At 48 h post-transfection, the cells were harvested. Luciferase activity was normalized with respect to the Renilla luciferase activity. Values are expressed as the means ± SD from three experiments. (D, E) Effects of E7 on the expression levels of egr-1 and c-fos in HeLa and C33A cell lines. HeLa (D) and C33A (E) cells were transiently transfected with pCDNA3.1/Elk-1 and pCDNA4-His/E7. Whole-cell extracts were collected at 48 h after transfection. Egr-1 andc-fos were detected by Westernhybridizationanalysis.
Fig. 6
Fig. 6
Effects of E7, Elk-1, and Elk-En on cell proliferation. (A) Colony formation in C33A cells treated with Elk-1, E7, and Elk-En in modified Eagle’s medium (MEM) containing 10% fetal bovine serum (FBS). Cell numbers were determined after incubation for 10 days. (B) Values are expressed as the means ± SD from two experiments.
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References

    1. Aarthy M., Kumar D., Giri R., Singh S. K. E7 oncoprotein of human papillomavirus: structural dynamics and inhibitor screening study. Gene. 2018;658:159–177. doi: 10.1016/j.gene.2018.03.026. - DOI - PubMed
    1. Ala M. Target c-Myc to treat pancreatic cancer. Cancer Biol. Ther. 2022;23:34–50. doi: 10.1080/15384047.2021.2017223. - DOI - PMC - PubMed
    1. Arbyn M., Simon M., Peeters E., Xu L., Meijer C. J., Berkhof J., Cuschieri K., Bonde J., Vanlencak A. O., Zhao F.-H., Rezhake R., Gultekin M., Dillner J., de Sanjosé S., Canfell K., Hillemanns P., Almonte M., Wentzensen N., Poljak M. 2020 list of human papillomavirus assays suitable for primary cervical cancer screening. Clin. Microbiol. Infect. 2021;27:1083–1095. doi: 10.1016/j.cmi.2021.04.031. - DOI - PubMed
    1. Azam H., Pierro L., Reina M., Gallagher W. M., Prencipe M. Emerging role for the Serum Response Factor (SRF) as a potential therapeutic target in cancer. Expert Opin. Ther. Targets. 2022;26:155–169. doi: 10.1080/14728222.2022.2032652. - DOI - PubMed
    1. Balsitis S., Dick F., Lee D., Farrell L., Hyde R. K., Griep A. E., Dyson N., Lambert P. F. Examination of the pRb-dependent and pRb-independent functions of E7 in vivo. J. Virol. 2005;79:11392–11402. doi: 10.1128/JVI.79.17.11392-11402.2005. - DOI - PMC - PubMed
-