The Antidiabetic Biguanide Metformin Induces Growth Arrest in Multiple Myeloma Cells in Vitro, overcoming the Effect of Stromal Cells
Authors: Ooi Melissa G. , McMillin Douglas W. , Negri Joseph M. , Delmore Jake , Munshi Nikhil C. , Chauhan Dharminder , Hideshima Teru , Richardson Paul G. , Mitsiades Constantine S. , Anderson Kenneth C. , Mitsiades Nicholas 
Published: April 2020

Abstract
Obesity and insulin resistance are associated with increased risk for several malignancies, including multiple myeloma (MM), possibly due to hyperinsulinemia and the resulting increased circulating free IGF-I levels that can have a growth-promoting and anti-apoptotic effect on MM cells. It has been proposed that improving insulin sensitivity, with a low-fat diet or with the use of the biguanide metformin, could suppress circulating insulin and free IGF-I levels and lower cancer risk. Metformin has been widely used as an antidiabetic agent since the 1950s and has an excellent safety profile. Interestingly, epidemiologic studies have suggested that patients with type 2 diabetes mellitus who are treated with metformin have lower cancer incidence and lower cancer-related mortality than diabetics who receive sulfonylurea monotherapy or insulin. Even more intriguing is the recent finding that metformin can have direct anticancer activity in vitro against prostate and breast carcinoma cell lines. In breast carcinoma cells, metformin inhibits mammalian target of rapamycin (mTOR) activity and suppresses translation initiation and global protein synthesis, via an AMP-activated protein kinase (AMPK)-dependent pathway. We investigated the direct effects of metformin in vitro in a panel of 17 MM cell lines and sublines using MTT, flow cytometry, annexin V-PI, as well as compartment-specific bioluminescence (CS-BLI) assays in the absence or presence of bone marrow stromal cells (BMSCs). We found that metformin suppressed the growth of all MM cell lines and sublines, including those that are resistant to conventional and several investigational anti-MM agents. The anti-MM activity of metformin was achieved with clinically relevant sub-mM or low mM levels, and was in the same order of magnitude or stronger than its activity reported in solid tumor cell line models. The anti-MM activity of metformin did not involve apoptotic/necrotic cell death, but inhibition of proliferation (G0/G1 arrest). Western blot analyses showed that metformin treatment of RPMI-8226/S MM cells led to increased phosphorylation of AMPK; suppression of p70S6K phosphorylation; and decreased cyclin D1 levels. These results are consistent with the known effect of metformin in triggering AMPK activity and thereby suppressing the function of its downstream targets mTOR and p70S6K. Co-culture with BMSCs did not abrogate the anti-MM effect of metformin, but further sensitized MM cell to this agent, at concentrations which did not affect BMSC viability. This sensitization was more prominent in MM cell lines that respond to BMSCs with increased proliferation/survival (e.g. MM-1S, MM-1R, KMS-11, L363) than in cell lines independent/unresponsive to stromal stimulation (e.g. OPM-2). To further probe the mechanistic basis for this stroma-induced sensitization to metformin, we studied the transcriptional signature of genes suppressed by mTOR inhibition, a downstream effect of metformin, in MM cells cultured alone vs. co-cultured with BMSCs. We observed that stroma triggers a significant increase in the average expression of mTOR-responsive genes in stroma responsive MM cell lines, but not in stroma unresponsive lines, in which this transcriptional signature is constitutively upregulated. This observation suggests that the increased metformin sensitivity of MM cells co-cultured with BMSCs may reflect the ability of metformin to counteract the upregulation of p70S6K/mTOR pathway activity triggered by stroma. Our studies demonstrate a direct, growth-suppressive effect of metformin in MM cells in vitro, which can be potentiated by interactions with the bone marrow microenvironment. Taken together with the beneficial effect of this antidiabetic agent on circulating insulin and free IGF-I levels and its favorable safety profile, these data support the clinical evaluation of metformin in patients with MM.