Investigation of the effects of DUSP1 protein on breast cancer

The Mitogen-Activated Protein Kinase (MAPK) family is a protein kinase family that phosphorylate the serine and threonine amino acids of target proteins. These protein kinases can be classified as three basics subfamilies: ERK, JNK, p38. The MAPK family is involved in the regulation of many vital cellular functions such as cell growth, cell proliferation and apoptosis. Dual-Specificity Phosphase (DUSP) family is the protein phosphatase family that dephosphorylate MAPKs. These proteins are called double specificity phosphatases because of their ability of dephosphorylating both tyrosine and serine/Treonin residues of target proteins. Dual-Specificity Phosphase 1 (DUSP1) protein regulates MAPK activation. In this way, it is involved in many cellular functions such as inflammation, apoptosis, cell growth, cell proliferation. In our study, triple negative breast cancer cell line, MDA-MB-231 were used. With the CRISPR/CAS9 method, the expression of DUSP1 protein in these cells is reduced. We performed proliferation and motility assays in these cells and evaluated the changes in the expressions of MAPK proteins. Proliferation assays were performed with Incucyte Live-Cell Analysis system. The doubling time of the cells and the maximum number of cells were calculated over the obtained proliferation curves. Protein expression analysis were evaluated by Western Blot method. The cell motility was measured by 'Transwell Migration Assay' and wound healing assay. In proliferation assays, the reducing the expersion level of DUSP1 by using CRISPR/Cas9 methodology inhibited the proliferation of MDA-MB-231 cells. In addition, BCI, which is an inhibitor of DUSP1, has an antiproliferative effect on MDA-MB-231 cells. The antiproliferative effect of BCI is lower in the DUSP1-CRISPR group than Control CRISPR group. This is most probably related to decreased DUSP1 expression in DUSP1 CRISPR group. The decrease in DUSP1 expression caused an increased antiproliferative effect of sisplatin in MDA-MB-231cells. In the same concentration of cisplatin, doubling times of DUSP1-CRISPR group were more than the control group's. Similar to the above results, it has been seen that BCI increased the antiproliferative effect of cisplatin in DUSP1-CRISPR group more than the control group. Similarly, this result supported the explanation that the less effectiveness of BCI in DUSP1-CRISPR group was due to the reduction of the DUSP1protein. Migration capacities of the cells in DUSP1-CRISPR group were evaluated by wound healing assay and transwell migration assay. As the result of these experiments, the migration capacity of MDA-MB-231 cells in DUSP1-CRISPR group significantly declined compared to Control CRISPR group. This result shows that the decrease in DUSP1 protein level reduces the mobility and metastasis ability of MDA-MB-231 cells. In experiments with p38 and JNK inhibitors, the p38 inhibitor SB202190, increased proliferation in both groups at similar levels. When cells treated together with p38 inhibitor and cisplatin, DUSP1-CRISPR group has led to a greater increase in proliferation than control group. This situation was thought to be caused by increased p38 protein activation in DUSP1-CRISPR group. Additionally, it is thought that p38 and JNK proteins can take a part in developing cisplatin resistance. According to Western Blot results, ratio of phosphorylated/total proteins of p38 and JNK were increased in DUSP1-CRISPR group. This increase has not been seen in ERK protein. Considering the target selectivity of DUSP1 protein, this result is compatible with the literature. As a result, the decrease in DUSP1 protein expression mediates anticancer activity in MDA-MB231 cancer cells by reducing cell mobility, migration and proliferation. In addition, our results suggest that p38 activity takes important role in DUSP1-inhibition mediated anticancer effect and also in susceptibility to cisplatin treatment in MDA-MB-231 cells.