Emerging Potential Roles of Lysyl-Oxidase-like 2 Stimulates Cancer Associated Fibroblasts Promoting Myelofibrosis of Myeloproliferative Neoplasms

Backgroud and objective: Myeloproliferative neoplasms (MPNs) are a group of clonal haematological disorders that characteristic with a multipotent haematopoietic stem cell transformation, often associating with the progression of myelofibrosis in the evolution course of disease. Progressive myelofibrosis finally turned out a high risk factor of transformation to leukemia and bone marrow failure. Cancer Associated Fibroblasts (CAFs) are recently thought to be a critical mediator in several hematological malignancies tumor microenvironment and associate with fibrosis. Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that promote the crosslinking of collagens or elastins in the extracellular matrix and mediate epithelial-mesenchymal transition (EMT). Here, we want to verify CAFs mediating myelofibrosis and explore the potential role of LOXL2 association with CAFs in simulated vivo microenvironment.

Patients and methods:For bone marrow specimens, normal samples (n=19) and patients with polycythemia vera (PV) (n=21), essential thrombocythemia (ET) (n=32), and primary myelofibrosis (PMF) (n=9) were contrasted. Markers of CAFs including α-smooth actin(α-SMA), fibroblast activation protein(FAP), transforming growth factor-β1(TGF-β1) and LOXL2 were detected by quantitative reverse transcription-PCR(RT-PCR). we also detected α-SMA, FAP, LOXL2 and reticulin protein by western blot and immunohistochemical staining. For cell lines, α-SMA and FAP were measured after cocultured mesenchymal stem cell(MSCs) with recombinant human lysyl oxidase homolog 2 Protein(rhLOXL2) in hypoxic niche for 24, 48, 72 and 96 hours, respectively.

Results: Markers of CAFs displayed a differential pattern of expression in MPNs especially in PMF(P<0.010) compared with normal samples(P=0.023) according to RT-PCR. Among these markers, the expression of α-SMA is the highest. For western blot and immunohistochemical staining, we discovered the level of α-SMA, FAP and LOXL2 expression was associated with the grading of reticulin fibrosis in bone marrows. we also founded that α-SMA and FAP were significantly expressed after cocultured MSCs with rhLOXL2 in hypoxia for 96 hours, but there were no significant increase in gene and protein level expression with the prolonged exposure time.

Conclusion: Over expression of CAFs played a critical role in promoting bone marrow fibrosis. LOXL2 may be a key factor in stimulating MSC to CAFs and further promoting disease progression, which may provide a new target spot for further excavation of the pathogenesis of MPNs and looking for more effective targeted therapies.