Avaliação do papel da via FAK/SRC na proliferação e apoptose em linhagem celular JAK2V617F positiva

Abstract

Chronic Myeloproliferative Neoplasms (NMPC), which includes Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal disorders characterized by excess of proliferation and apoptosis resistance, leading to accumulation of myeloid cells. The identification of the acquired JAK2V617F mutation in patients with PV, ET and PMF allowed a better understanding about this disease pathogenesis, as the constitutive activation of JAK2 tyrosine kinase plays an important role in increasing cell proliferation and resistance to apoptosis. However, there is still no pharmacological treatment that leads all patients to molecular remission, justifying the study of new molecular targets. In turn, the Focal Adhesion Kinase (FAK) protein has an important influence on the processes of proliferation, migration and cell survival and, as it is overexpressed in several neoplasms, it becomes a promising target in the development of drugs cancer. The present study proposes to evaluate the role of FAK in the proliferation and apoptosis of SET-2 cells, a JAK2V617F positive cell line. With this purpose, the following were analyzed: (1) interaction between FAK and SRC by immunoprecipitation; (2) the effect of the FAK inhibitor (PF 562,271) on the viability and cell proliferation by MTT; (3) the effect of the FAK inhibitor on apoptosis induction by evaluating the expression of cleaved PARP and cleaved Caspase 3 by Western Blotting, (4) the effect of JAK inhibitor (ruxolitinib) on the expression of p-SRC and SRC. In addition, a RNAseq dataset from SET-2 cells treated with INC424 (ruxolitinib) for 4h and 48h (GSE 69827), available in "Gene Expression Omnibus" was analyzed. The differential gene expression and correlation of PTK2 gene and other 65 genes related to proliferation, survival, apoptosis and adhesion were performed using the software R studio. The results indicate that the inhibitor of FAK PF 562,271 significantly reduced in 71.5%, 85.5%, 83.7% and 86.3% the cell viability at 5uM, 10uM, 25uM and 50uM, respectively. The inhibitor also induced apoptosis, since there was an increase in the expression of cleaved PARP and cleaved Caspase 3 after treatment with the inhibitor. The analysis of the RNAseq database showed that the JAK inhibitor modulated the expression of PTK2 gene and other genes involved in proliferation, apoptosis and cell adhesion processes. Regarding the genes related to cell adhesion, the genes PTK2, SRC, PXN, TLN1, DCC, ITPKC and FLNA showed increased expression and CRK, PTRH2, VCL, CASS4, ACTB, ZYX, VASP, CTNNB1 and BCAR1 showed decreased expression. In conclusion, the results indicate that FAK inhibition sensibilizes SET-2 to apoptosis, reducing cell viability and JAK inhibition with INC424 seems to have influence in PTK2 gene expression and other genes related to focal adhesion pathway.