Tumor-Derived LOXL2 Is Highly Expressed in Myelofibrosis and Activating Mesenchymal Stem Cells Differentiate into Cancer Associated Fibroblasts In Vitro

Abstract

Background and objective: The disease progression of Myeloproliferative Neoplasms (MPN) is often accompanied by myelofibrosis, progressive myelofibrosis could be a high risk factor affecting the evolution of MPN into leukemia and bone marrow failure. Recently, Cancer associated fibroblasts (CAF) and lysyl-oxidase-like protein 2 (LOXL2) are found closely related to the process of myelofibrosis, but underlying molecular mechanism of how they mediate myelofibrosis is not yet fully understood.Mesenchymal Stem Cells (MSC) could differentiate into CAF in solid tumor. Here,we aim to investigate whether LOXL-2 could stimulate MSC into CAF and promote myelofibrosis in micro-environmental hypoxia.

Methods and Results: Using a-SMA and FAP as marker for CAF. The mRNA and protein expression level of a-SMA ,LOXL2 and FAP in MPN with myelofibrosis were significantly higher than normal subjects (P <0.05).We further analyzed the expression of a-SMA, FAP and LOXL2 in pathological specimens of bone marrow biopsy , Results suggested that a-SMA, FAP and LOXL2 were low expression or no expression in normal controls but displayed a moderate increase in in different types of MPNs particularly in PMF. Further, we checked the MSC in morphology and differentiation function from MPNs and normal controls. There was no significant difference in BMMSCs morphology and Immunophenotyping,but we found that the osteogenic differentiation of MPN derived BMMSCs were significantly higher than the control group. We cultured MSCs with rhLOXL 1% oxygen concentration. Results showed that with the culture time, a-SMA and FAP were progressively increased, especially in 96h, (n = 3, P <0.05), and the addition of BAPN(the inhibitor of loxl2) could significantly decrease the expression of a-SMA and FAP. On the basis of the above results, we also examined the expression of FAK and Src signaling pathway proteins between the three groups of BMMSCs cultured for 96h, The data showed that the expression of p-FAK and p-Src increased with the stimulation of rhLOXL2 at 1% oxygen concentration, and BAPN decreased the expression of protein while the expression of pathway protein was not significant under normal oxygen.

Conclusions: Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. BMMSCs from MPNs could obtain CAFs phenotype under MPNs bone marrow microenvironment and LOXL2 may be stimulating BMMSCs differentiate into CAFs by activated FAK/Src signaling pathway.