MK2 promotes p16 negative head and neck cancer migration, invasion, and metastasis

Abstract

For patients with locally advanced, p16-negative head and neck squamous cell carcinoma (HNSCC), overall survival remains poor due to primary locoregional failure and distant metastasis following curative therapy. We aimed to understand how MAPKAPK2 (MK2) regulates HNSCC tumor cell migration and invasion, important first steps in cancer metastases. The TCGA database and HNSCC tissue microarrays were used to show that MK2 expression was associated with more advanced cancers and faster cancer recurrence rates. We observed that silencing of tumor MK2 in human cell lines (shRNA) caused a significant reduction in tumor cell migration-invasion in a complex HNSCC microphysiologic system used to recapitulate the tumor microenvironment. Murine cells (Ly2) with MK2 silenced (CRISPR-Cas9) also demonstrated reduced migration and invasion using 2D and 3D monoculture cell migration-invasions assays. Ly2 cells are orthotopic p16-negative murine metastatic cells that spontaneously metastasize, and we observed that MK2 inhibition via genetic (Cas9/CRISPR) or pharmacologic (PF-3644022) methods led to a significant reduction in the number of circulating tumor cells, fewer lymph node and lung metastases, and MK2 inhibited mice showed improved overall survival. Our findings suggest that HNSCC MK2 regulates tumor cell migration-invasion and may be a promising therapeutic target to reduce metastases.

Fig. 1.. Loss of tumor MK2 leads to reduced tumor cell migration and invasion.

A, B. We performed a TCGA analysis of 520 head and neck cancer specimens with 44 normal tissue control specimens and evaluated MK2 transcript expression between normal and tumor specimens. MK2 transcript levels were stratified based on normal tissue and increasing tumor grade. C. Immunohistochemistry (IHC) of phospho-MK2 (p-MK2) was performed on a head and neck cancer tissue microarray (TMA) derived from HPV-negative, p16-negative oropharyngeal squamous cell carcinoma patients undergoing primary surgery. Kaplan-Meier recurrence-free survival analysis performed on the median MK2 phosphorylation. Slides were digitally acquired using Aperio platform and objective quantification of IHC staining performed using HALO. D. SDS-PAGE was performed on RIPA-lysed cell extracts from scramble and shRNA cell lines followed by immunoblot. E. MK2 scramble and shRNA HNSCC cell line spheroids (Tu167, Cal27) were grown in an MPS (LumeNEXT) populated by human cancer-associated fibroblasts and lymphatic endothelial vasculature. Cells were pre-stained with Vybrant DiD and migration was evaluated on day 1 (d1) and d4. The extent of tumor cells migration (% cellular area change) was compared between days. F Close up view of Tu167 scramble and shRNA at d1 and d4 demonstrating extent of tumor migration-spread in the MPS. G. Quantification of tumor cell migration. A minimum of 3 independent experiments were performed with multiple biologic replicates as shown for Tu167 and Cal27. Statistical analysis performed. P-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 2.. Loss of tumor specific MK2 in murine Ly2 tumor cell migration and invasion.

A. Representative figure demonstrating natural HNSCC progression from primary tumor to lymph node and lung following orthotopic tumor implantation into the floor of mouth. Image developed using BioRender. The murine Ly2 tumor cell line was orthotopic implanted into the floor of mouth (FoM) of Balb/c mice. White arrows denote primary HNSCC tumor and red arrows highlight peri-tumoral lymph nodes. Representative H/E and anti-p40 stained primary, lymph node and lung metastatic tumors. B. Ly2 parental wild type (WT) and Cas9/CRISPR MK2 knockout (KO) cell lines were immunoblotted for p38-MK2-HSP27 pathway proteins and mesenchymal-epithelial markers. C. Scratch migration and invasion assays were performed to evaluate tumor cell migration and invasion using a 2D method. Spheroid dissemination and spheroid invasion assays were performed to evaluate tumor cell migration and invasion using a 3D method.

Fig. 3.. Loss of tumor MK2 reduces tumorigenesis as well as impairs distant lymph node and lung metastases, in vivo.

A MK2 parental and KO Ly2 cells were orthotopically implanted into Balb/c mice using 50,000 cell transoral injection into the FoM. B Orthotopic tumor engraftment into immunocompetent Balb/c mice and tumor size was directly compared between WT and MK2 KO tumors. Tumor volumetric measurements were performed three times per week. C. A Kaplan Meier survival study was performed comparing orthotopically implant MK2 WT vs KO tumors. Survival was evaluated in all implanted animals. D. Total number of metastatic lymph nodes per animal were enumerated. WT, n = 9; KO, n = 14. E. Similarly, all 5 lung lobes were uniformly evaluated for number of metastases per mouse. F. Representative images from MK2 WT tumors and MK2 KO tumors were obtained at 20X magnification for lymph node and lung. G. MK2 WT and KO tumor growth in NSG mice evaluated by tumor volume measured by calipers. G. Total Lung sections were performed like E and total number of lung metastases were compared between MK2 WT and KO tumors. Statistical analysis performed. P-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 4.. MK2 inhibition leads to reduced circulating tumor cells in orthotopically implanted Ly2 tumors.

A. WT parental Ly2 tumors were orthotopically implanted into the FoM of Balb/c mice. When tumors were ~100 mm³, animals were treated with excipient control (placebo) or 50 μM PF-3644022 via IP injection. Drug was delivered every other day 3x/week for 2 weeks. Tumor volume was measured three times per week. Image created with BioRender.com. B. On the day of euthanasia, tumors size was measured one final time. Treatment with MK2 inhibitor led to a significant reduction in tumor volume. Animals were euthanized by CO2 asphyxiation followed by terminal exsanguination via cardiac puncture and primary tumors and lungs were harvested for evaluation. C. Body weight was measured with each treatment. D. Tumor and surrounding lymph nodes removed were evaluated for extent of metastatic disease. E. Blood was collected in EDTA tubes and stored on ice. Samples were passed through anti-FAPα coated and anti-EpCAM coated microfluidics devices in the KUMC BME Core Facility, washed in isotonic solution, and then FAPα and EpCAM expressing CTCs were eluted and immunophenotyped. Total number of EpCAM and FAPα expressing CTCs were enumerated between treatment arms. F. Lung metastases were evaluated from H/E-stained sections derived FFPE lung specimens from each treatment group. G. Representative images from a placebo drug treated and PF-3644022 treated mice. H/E stains performed to assess lung metastases. Student t-test performed. P value * <0.05.