Abstract A56: Metformin targets Rho GTPases to inhibit neuroblastoma cell growth; implications in the treatment of neuroblastoma
Authors: Kumar Ambrish , Al-Sammarraie Nadia , DiPette Donald J. , Singh Ugra S. 
Published: October 2015

Abstract
In recent years, oncologists are paying considerable attention to metformin (N', N'-dimethylbiguanide) in the treatment of various types of cancers, as some population-based studies have shown low cancer incidences and mortalities among diabetic-patients treated with metformin. In the present study, using neuroblastoma cells, we explored the anti-tumor activity and the underlying mechanisms of metformin. Neuroblastoma is a malignant cancer of the postganglionic sympathetic nervous system; it develops in the adrenal gland and metastasizes to liver, bone, bone marrow, lymph nodes, neck and chest. It is the most common cancer in infants younger than one and second most common tumors in children. In the United States of America, it accounts for 7% of all childhood cancers, and is responsible for 15% of all cancer deaths in children younger than 15 years. Various genetic and cytological alterations allow cells to develop drug resistance and help neuroblastoma tumors to escape most available therapies.
Using human neuroblastoma cells of different genotypes (SH-SY5Y, MYCN-nonamplified; and SK-N-BE(2), MYCN-amplified cells), we generated subcutaneous xenograft mice models, and metformin (50, 100 and 250 mg/kg body weight per mice) was given daily by oral gavage to tumor-bearing mice. Our in vivo results demonstrated that metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors in both mice models. Immunofluorescence and Western blot results indicated reduction in tumor growth was due to the increased DNA fragmentation and apoptotic cell death that occur via activation of caspase-3. Metformin also inhibited the initiation of spheroid formation in 3-D cultures (hanging drop assay). In xenograft tumors, metformin affected the phosphorylation of mitogen-activated protein kinases (MAPKs). Metformin induced the phosphorylation of JNK1/2 and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Since ERK1/2 is involved in cell proliferation and JNK1/2 promotes cell death, our observations clearly indicated that MAP kinases are involved in metformin-induced cell death.
Rho-GTPases (RhoA, Rac1 and Cdc42), a family of small G-proteins, act as molecular switches to regulate cellular functions- cell division, motility and cell survival. These small G-proteins exist either as a GTP-bound active form or an inactive GDP-bound form. The aberrant expression and/or activity of Rho-GTPases are associated with the progression of various cancers. Our glutathione-S-transferase (GST)-pull down assays showed that metformin significantly increased activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) while it decreased activation of RhoA (GTP-RhoA) in these tumors. Infection of neuroblastoma cells by adenoviruses expressing dominant negative Rac1, Cdc42 (Rac1-N17 and Cdc42-N17, respectively) and constitutively active RhoA (RhoA-V14), or incubation with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These gain-in and loss-off function studies suggested that metformin impairs Rho-GTPase signaling to initiate apoptosis via JNK pathway.
Together, our results reported the inhibitory effects of metformin against neuroblastoma, and demonstrated the role of Rho-GTPases in metformin-mediated apoptotic cell death. The cytotoxic effects of metformin against MYCN-nonamplified and MYCN-amplified multidrug resistant neuroblastoma cells in our studies further signifies that metformin can be a promising drug candidate for neuroblastoma therapy.
Citation Format: Ambrish Kumar, Nadia Al-Sammarraie, Donald J. DiPette, Ugra S. Singh. Metformin targets Rho GTPases to inhibit neuroblastoma cell growth; implications in the treatment of neuroblastoma. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A56.