Inhibition of Cellular MEK/ERK Signaling Suppresses Murine Papillomavirus Type 1 Replicative Activities and Promotes Tumor Regression

Background:

Persistent infections by high-risk human papillomavirus (hrHPV) pose a major healthcare problem as they trigger epithelial neoplasms that may become malignant¹. Although prophylactic vaccines can protect individuals against future HPV infections, they cannot clear pre-existing infections². Improved knowledge of HPV infection, transmission and pathogenesis is needed to design effective antiviral strategies that can curb ongoing HPV infections and their related diseases. However, HPV cannot infect non-human species and many animal papillomavirus models fail to fully recapitulate HPV pathogenesis and tumorigenesis³.

The recent discovery of a murine papillomavirus (MmuPV1) provided a major advancement to the study of HPV-induced pathogenesis and carcinogenesis. MmuPV1 has a similar genome organization to many hrHPVs and can induce tumors in mice³. In this preprint, the authors used a preclinical MmuPV1 infection model to test the efficacy of an antiviral therapy, specifically a MEK inhibitor, to induce papillomavirus-induced tumor growth. They also reported that a MEK inhibitor may be a potent therapy to suppress MmuPV1 genome amplification and viral oncogene transcription. The findings from this preprint are congruent to a previous publication by the same authors where they demonstrated MEK signaling as a key driver of HPV oncogene transcription in cell lines and three-dimensional organotypic cultures⁴.

Key findings:

1) MEK inhibitors suppressed tumor growth in a MmuPV1-infected preclinical model

The authors used a MmuPV1 infection mouse model to induce MmuPV1-driven papilloma (tumor) formation. After 6 weeks of papilloma growth, the mice were treated with vehicle or MEK inhibitor (cobimetinib or trametinib). MEK inhibitors prevented papilloma growth otherwise seen in the vehicle control. The extent of papillary projections and epidermis thickness were much lower in mice treated with MEK inhibitors compared to the vehicle control, suggesting reduced epidermis outgrowth. Consistent with these phenotypes, MEK inhibitor-treated papillomas showed reduced proliferative markers compared to the vehicle control. Overall, these findings indicate that MEK signaling drives papilloma growth and maintenance in a MmuPV1-infected mouse model

2) MEK inhibitor-induced papilloma growth inhibition is not dependent on T cells or neutrophils

Previous data suggest that neutrophils, CD4+ and CD8+ T cells may suppress papillomavirus-driven tumor growth^5-7. Although the mice used in this preprint have an intact immune system, they lack CD4+ and CD8+ T cells. This prompted the authors to investigate if MEK inhibitors contribute to MmuPV1-driven papilloma regression by promoting neutrophil infiltration. Neutrophils in the proximity of the tumors were stained using myeloperoxidase as a marker. However, the authors observed no significant differences in neutrophil infiltration between MEK inhibitor-treated and control mice. Therefore, MEK signaling promotes papilloma growth in a mechanism independent of T cell or neutrophil infiltration.

3) MEK inhibitors reduced MmuPV1 viral genome replication, gene transcription and transmission in vivo

Using in vitro cell lines, organotypic tissue models and patients’ biopsies, the authors previously demonstrated that MEK/ERK signaling drives transcription of HPV E6 and E7 genes⁴. This prompted them to investigate if MEK inhibitors reduce MmuPV1-induced papilloma growth by inhibiting viral replication and transcription. In situ hybridization of various probes against MmuPV1 suggested reduced viral genomic DNA and E6/E7 transcripts in MEK inhibitor-treated tumors compared to vehicle control.  The authors also observed that vehicle-treated mice developed secondary facial lesions, which was not the case for MEK inhibitor-treated mice. Therefore, they hypothesized that the MEK inhibitor reduces MmuPV1 transmission due to suppressed viral replication and lower viral early gene transcription. To test this hypothesis, they infected keratinocytes with MmuPV1 virions in vitro and treated the cells with increasing concentrations of MEK inhibitor. Indeed, inhibition of MEK signaling reduced the expression of MmuPV1 E1^E4 mRNAs, a gene that is expressed early in the viral life cycle. Therefore, MEK signaling reduces secondary MmuPV1-induced tumor formation by reducing virion transmission from primary tumors and inhibiting transcription of early viral genes in secondary lesions.

Why I like this preprint:

The oncogenic effects of HPV are well-known for decades, but it is still unclear what governs HPV genome amplification and viral gene transcription. I really appreciate the comprehensive approach that the authors used to validate their findings – ranging from an in vitro to in vivo study. It is really amazing to see such consistent results across various experimental models. I hope that HPV-related diseases can be combated in the near future!

Questions for the authors:

• (a) Your previous paper suggests that EGFR/MEK/ERK signaling activates AP1 transcription factor that promotes HPV E6 and E7 gene transcription. Have you tested the antiviral efficacy of EGFR and/or JNK inhibitors in the MmuPV1 infection model? (b) Although EGFR promotes HPV oncogene transcription, concurrent radiation and EGFR inhibition did not provide substantial curative benefit for HPV-related head and neck cancer in clinical trials⁸. Why do you think this is the case?
• The HPV genome is commonly integrated in host chromosomes of HPV-related cancers. How effective will MEK inhibitors be in treating these cancers, given that the HPV genome remains integrated in host cells despite suppression of early viral gene transcription?
• You have provided strong evidence that MEK/ERK signaling governs HPV oncogene transcription and viral genome amplification. What are your next steps in expanding this research?

References

1          Kombe Kombe, A. J. et al. Epidemiology and Burden of Human Papillomavirus and Related Diseases, Molecular Pathogenesis, and Vaccine Evaluation. Frontiers in Public Health 8, doi:10.3389/fpubh.2020.552028 (2021).

2          Joura, E. A. et al. A 9-Valent HPV Vaccine against Infection and Intraepithelial Neoplasia in Women. New England Journal of Medicine 372, 711-723, doi:10.1056/NEJMoa1405044 (2015).

3          Spurgeon Megan, E. & Lambert Paul, F. Mus musculus Papillomavirus 1: a New Frontier in Animal Models of Papillomavirus Pathogenesis. Journal of Virology 94, e00002-00020, doi:10.1128/JVI.00002-20 (2020).

4          Luna, A. J. et al. MEK/ERK signaling is a critical regulator of high-risk human papillomavirus oncogene expression revealing therapeutic targets for HPV-induced tumors. PLOS Pathogens 17, e1009216, doi:10.1371/journal.ppat.1009216 (2021).

5          Trimble, C. L. et al. Human Papillomavirus 16-Associated Cervical Intraepithelial Neoplasia in Humans Excludes CD8 T Cells from Dysplastic Epithelium. The Journal of Immunology 185, 7107-7114, doi:10.4049/jimmunol.1002756 (2010).

6          Handisurya, A. et al. Strain-Specific Properties and T Cells Regulate the Susceptibility to Papilloma Induction by Mus musculus Papillomavirus 1. PLOS Pathogens 10, e1004314, doi:10.1371/journal.ppat.1004314 (2014).

7          Cladel, N. M. et al. Mouse papillomavirus infection persists in mucosal tissues of an immunocompetent mouse strain and progresses to cancer. Scientific Reports 7, 16932, doi:10.1038/s41598-017-17089-4 (2017).

8          Mehanna, H. et al. Radiotherapy plus cisplatin or cetuximab in low-risk human papillomavirus-positive oropharyngeal cancer (De-ESCALaTE HPV): an open-label randomised controlled phase 3 trial. The Lancet 393, 51-60, doi:10.1016/S0140-6736(18)32752-1 (2019).

 

Tags: cancer, hpv, papillomavirus

doi: https://doi.org/10.1242/prelights.34671

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