New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration

Abstract

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy.

A Potential Role of RUNX2- RUNT Domain in Modulating the Expression of Genes Involved in Bone Metastases: An In Vitro Study with Melanoma Cells

“…RUNX2 involvement in regulating the epithelial-mesenchymal transition (EMT) process has been demonstrated in melanoma [14]. Recently, we demonstrated that the RUNX2 RUNT domain affects EMT by increasing the expression of N-cadherin and reducing the expression of E-cadherin [21]. In addition, we found that the RUNT domain also promotes EMT by upregulating the expression of vimentin [21].…”

“…We used A375 (American Type Culture Collection; ATCC: CTRL-1619TM) and MELHO (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen) human melanoma cells. The RUNT KO cells were obtained using CRISPR/Cas9 as we previously described [21]. Cell lines were cultured under 5% CO 2 and in RMPI (1640 (Roswell Park Memorial Institute) growth medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), supplemented with antibiotics (1% penicillin/streptomycin) and 1% glutamine.…”

“…Total RNA extraction and RT were performed as previously reported [21]. PCRs were performed in a total volume of 25 µl using 20 ng of cDNA for each sample.…”

“…The migration ability assay was conducted with an EVOS™ FL Auto Imaging System (Thermo Fisher Scientific) under time-lapse protocol for 48 h. Distances between the cell front and the bone slice or the signed blank space for every well were measured at the beginning and at the end of each experiment. Relative migration distances (RMDs) were calculated using the following formula: RMD = (t0-t1)/t0, where t0 is the distance between the cell front and the bone slice at time zero of the assay and t1 is the same distance at the end, as previously reported [21]. The RMD of WT cells was normalized to the RMD of RUNX2 KO cells to evaluate the role of RUNX2 in migration in the presence or absence of bone fragments.…”

“…In addition, RUNX2 promotes esophageal carcinoma by activating the AKT and ERK signaling pathways [20]. Recently, we demonstrated that the RUNT domain, namely the RUNX2 DNA binding domain, is involved in different pathways leading to melanoma transformation [21]. Considering that RUNX2 induces osteogenic genes expression through the RUNT DNA binding domain, we hypothesized that the RUNT domain might also be responsible for the bone tropism of cancer.…”