91ºÚÁÏÍø

Authors

Dakota D. D. Okwuone¹, Deri Morgan², Hannah M. Smith², Grace Millington², Kiersten L. Berggren³, Christopher E. Lominska², Sufi M. Thomas^1,4, Gregory N. Gan^1,2

¹Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS

²Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS

³The University of New Mexico Comprehensive Cancer Center, Albuquerque, NM

4Department of Otolaryngology, University of Kansas Medical Center, Kansas City, KS

Introduction

Poor 5-year overall survival in head and neck cancer (HNC) can be attributed to high rates of locoregional and distant metastasis. We have previously demonstrated that high phosphorylation/activation of MAPK-activated protein kinase 2 (MK2), a stress-activated kinase directly downstream of p38 MAPK, in head and neck squamous cell carcinoma (HNSCC) is associated with worse overall survival and inhibition of the MK2 pathway enhances in vivo HNSCC radiosensitivity (1). Our prior work also demonstrates that radiotherapy-induced epithelial-to-mesenchymal transition (EMT) gene expression can be suppressed in cells when MK2 is inhibited. Several studies have shown that EMT plays a prominent role in HNSCC tumor invasion, treatment resistance, and locoregional metastases to lymph nodes (2-4). In this study, we investigate the impact of MK2 activity on HNSCC tumor progression by examining its contribution to cancer cell morphology.

Methods

We used lentiviral-shRNA and the CRISPR-Cas9 system to knock down/out the MK2 gene in multiple human and murine HNSCC cell lines. We measured tumor cell migration and invasion in both classic 2D in vitro culture as well as with 3D spheroids. We performed unbiased analysis via RNAseq in human HNSCC cell lines comparing MK2 shRNA vs scramble cell lines. Substantial gene and pathway alterations were examined using a combination of immunoblot and RT-qPCR.

Results

Our data shows knocking down/out MK2 in multiple HNSCC cell lines significantly diminishes cell migration and invasion in 2D and 3D in vitro culture assays. RNAseq analysis demonstrated loss of MK2 leads to a significant increase in many cell adhesion molecules, including E-cadherin, and a decrease in N-cadherin, suggesting a shift from mesenchymal to a more epithelial phenotype. Furthermore, we note the expression of multiple matrix metalloproteinases and EMT markers (i.e., Snail) are substantially reduced in MK2 shRNA and knockout cell lines.

Conclusion

These results indicate that MK2 pathway activity promotes HNSCC motility and invasion. We also observe the loss of MK2 contributes to a shift in the epithelial:mesenchymal ratio potentially by suppressing cancer EMT plasticity. All in all, this study provides evidence suggesting tumor MK2 activation mediates HNC progression.

References

1. Berggren KL, Restrepo Cruz S, Hixon MD, et al. MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma. Oncogene. 2019;38:7329-7341. [PMID: 31417185] doi:10.1038/s41388-019-0945-9
2. Jimenez L, Jayakar SK, Ow TJ, et al. Mechanisms of invasion in head and neck cancer. Arch Pathol Lab Med. 2015;139:1334-1348. [PMID: 26046491] doi:10.5858/arpa.2014-0498-RA
3. Dudas J, Ladanyi A, Ingruber J, et al. Epithelial to mesenchymal transition: a mechanism that fuels cancer radio/chemoresistance. Cells. 2020;9. [PMID: 32059478] doi:10.3390/cells9020428
4. Puram SV, Tirosh I, Parikh AS, et al. Single-cell transcriptomic analysis of primary and metastatic tumor ecosystems in head and neck cancer. Cell. 2017;171:1611-1624.e24. [PMID: 29198524] doi:10.1016/j.cell.2017.10.044

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