Autocrine and Paracrine Regulatory Mechanisms of Growth Arrest-Specific Gene 6 Contribute to Disease Progression of Multiple Myeloma
Authors: Furukawa Miki , Ohkawara Hiroshi , Ikeda Kazuhiko , Ito Emi , Imai Jun-ichi , Yanagisawa Yuka , Honma Reiko , Watanabe Shinya , Waguri Satoshi , Ogawa Kazuei , Takeishi Yasuchika 
Published: October 2019

Abstract
Multiple myeloma (MM) is a lethal hematologic neoplasm characterized by proliferation of malignant plasma cells in part due to abnormal surrounding microenvironment, but the mechanism of disease progression remains largely unknown in MM. Thus, we screened molecules differentially expressed in bone marrow (BM) cells of MM patients using microarray analysis, which revealed significant upregulation of Gas6 (growth arrest-specific gene 6), a vitamin K-dependent protein with a structural homology with protein S. Since upregulation of Gas6 and its tyrosine kinase receptors have been reported to associate with various malignancies, we hypothesized that Gas6 contributed to the pathogenesis of MM.
Gas6 was overexpressed in the BM of MM patients and in various MM cell lines. We found that the serum levels of Gas6 protein were markedly increased in 18 symptomatic MM patients compared to 16 healthy volunteers using human Gas6 ELISA. High levels of soluble Gas6 were detected in the supernatants of in vitro cultured MM cell lines RPMI-8226 and AMO-1 using Gas6 ELIZA. Analysis of apoptosis by flow-cytometry (FCM) with annexin V showed that exogenous Gas6 (100-200 ng/mL) inhibited the apoptosis of MM cells. MTT cell proliferation assay showed that exogenous Gas6 induced cell proliferation of MM cells with increased extracellular signal regulated kinase (ERK) phosphorylation. These results suggest that the presence of an autocrine loop of Gas6 and its receptor with ERK signaling enhanced the proliferative and anti-apoptotic effects on MM cells.
Bone marrow stromal cell (BMSC) line HS-5 was found to secrete high levels of soluble Gas6 into culture supernatants using Gas6 ELIZA. Reduction of MM cell apoptosis by HS-5 cell-conditioned medium (CM) was suppressed by Gas6 neutralizing antibody (20 microg/mL) using FCM with annexin V. HS-5 cell-CM induced a marked increase in cell proliferation of MM cells, which was completely inhibited by Gas6 neutralizing antibody (0.2-20 microg/mL) using MTT cell proliferation assay. Our results showed a critical role of Gas6 in the pathology of apoptosis inhibition and cell proliferation of MM cells through paracrine mechanisms.
Interleukin (IL)-6 is identified as a major growth and anti-apoptotic factor of MM cells through autocrine mechanisms. Conversely, BMSCs provide efficient support for MM cell survival by paracrine IL-6 stimulation. As determined by western blotting and real-time PCR, HS-5 cell-CM induced Gas6 upregulation, which was suppressed by IL-6 neutralizing antibody (1-10 microg/mL in a dose-dependent manner in MM cells. Meanwhile, HS-5 cell-CM induced the increased IL-6 expression, which was reversed by Gas6 neutralizing antibody in MM cells. Our findings indicate that Gas6 possessed both autocrine and paracrine functions similar to IL-6 between MM cells and BMSCs.
Intercellular cell adhesion molecule-1 (ICAM-1) enhanced adhesion of MM cells to BMSCs and subsequently resulted in MM disease progression. Our immnoblotts showed that HS-5 cell-CM induced ICAM-1upregulation, and the ICAM-1 upregulation was reversed by Gas6 neutralization antibody, indicating that Gas6 stimulation increased ICAM-1 synthesis and subsequently accelerated the proliferation of MM cells.
The present study offers new insights into autocrine and paracrine actions of Gas6 in concert with IL-6 between MM cells and BMSCs on apoptosis inhibition and cell proliferation of MM cells. We suggest that Gas6-related signaling pathways may be an attractive therapeutic target for the treatment of MM.

Disclosures
No relevant conflicts of interest to declare.