Fibroblast Activation Protein Protects Bortezomib Induced Apoptosis In Multiple Myeloma Cells Through β-Catenin Signaling Pathway
Authors: Zi Fuming , He Jingsong , He Donghua , Li Yi , Yang Li , Wang Lijuan , Yang Yang , Zhao Yi , Wu Wenjun , Huang He , Yi Qing , Cai Zhen 
Published: October 2019

Abstract

Background
Multiple myeloma(MM) is a malignant plasma cells proliferative disease which is characterized by increased blood calcium level, renal insufficiency, anemia, and bone lesions(CRAB). The intricate cross-talk with the bone marrow microenvironment plays an important role in facilitating the growth and survival of myeloma cells. Fibroblast activation protein(FAP) is a vital transmembrane protein expressed in 90% epithelial tumor stroma, which is reported to be involved in mediating drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of tumor cells. The present study is aimed to investigate the roles of FAP may play in regulation of apoptosis in MM cells induced by bortezomib and the potential signaling pathway that FAP may be participated in.

Methods
Bone marrow mesenchymal stem cells(hBMMSCs) from MM patients and normal donors and the cell line of hBMMSCs were analyzed for expression of the FAP protein by semiquantitative real time-polymerase chain(qRT-PCR), flow cytometry(FCM) and immunofluorescence(IF). Tumor cell-conditioned medium (TCCM) from supernatant of MM cell lines were added to hBMMSCs to observe the effect of TCCM for the expression of FAP. We further studied the function and mechanism of FAP in bortezomib induced apoptosis of myeloma cells by silencing FAP with small interfering RNA(siRNA). Apoptotic cells of MM cells were detected by APC-CD138/annexin V-FITC using flow cytometry analysis. Western blotting was used to elucidate the signaling pathway that FAP may be involved in mediating apoptosis of MM cells induced by bortezomib.

Results
There was no significant difference in the expression of FAP in hBMMSCs isolated from MM patients and normal donors(p>0.05) as determined by qRT-PCR, FCM and immunofluorescence. hBMMSCs stimulated by TCCM for 7 days displayed an elevated expression of FAP as detected by qRT-PCR(p<0.05). In the presence of 30nM bortezomib, MM cell lines RPMI8226 or CAG cells co-cultured with hBMMSCs in which FAP was knockdown or not by siRNA for 48h demonstrated that FAP is capable of protecting RPMI8226 and CAG cells induced by bortezomib from apoptosis as determined by FCM(NC siRNA vs FAP siRNA, p<0.05). Further study showed that the activity of β-catenin was significantly elevated in RPMI8226 cells after co-cultured with hBMMSC in the presence of bortezomib. Knockdown FAP can reduce the expression of β-catenin and its downstream target proteins, such as c-myc, survivin, cyclin D1 in RPMI8226 cells detected by western blot.

Conclusions
Taken together, our data indicated that the expression level of FAP was no difference between the hBMMSCs isolated from MM patients and normal donors. The expression of FAP can be increased by TCCM stimulation. Further study demonstrated that FAP can protect MM cells from apoptosis induced by bortizomib, which is likely through β-catenin signaling pathway.

Disclosures:
No relevant conflicts of interest to declare.