Metformin, an anti-diabetic drug, is known to inhibit leukemia cell growth mainly through activation of AMP-activated protein kinase (AMPK), resulting in inhibition of mTORC1 signaling. Meanwhile, TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in leukemia cell growth have been reported by us and others, the roles of TYRO3 have not been well elucidated.
We examined the involvement of TYRO3 in anti-leukemia effects of metformin.
Four myeloid leukemia cell lines (OCI-AML2, OCI-AML3, AA, and K562) expressing TYRO3 protein were used. AA is a human cell line derived from pure erythroid leukemia. All cell lines were cultured with increasing concentrations of metformin. To knockdown TYRO3 expression, small interfering RNA (siRNA) against TYRO3 or control siRNA was transfected using an electroporation system. Cell growth was assessed with a colorimetric assay after culturing for three days. Expression levels and the activation of various signaling proteins in lysates from cultured cells were examined using immunoblotting. Expression levels of mRNA were examined by quantitative RT-PCR.
Treatment with metformin suppressed the growth of these four cell lines in a dose-dependent manner. Immunoblotting revealed that metformin increased phosphorylated AMPK while decreased phosphorylation of mTOR and the downstream proteins such as 4E-BP1 and P70S6K without affecting expression levels of those proteins in general. Moreover, metformin treatment decreased expression levels of TYRO3 and c-MYC and phosphorylation of TYRO3, STAT3, and ERK1/2. In K562 cells treated with metformin, decrease in phosphorylated TYRO3 was more evident than decrease in TYRO3 expression levels. In OCI-AML2, OCI-AML3, and K562 cells, which express AXL, metformin treatment also decreased expression levels and phosphorylation of AXL. Quantitative RT-PCR analysis showed that expression of TYRO3 and MYC mRNA decreased by metformin treatment. Next, siRNA experiments showed that TYRO3 knockdown suppressed the cell growth to approximately 50% of the control. It also suppressed phosphorylation of STAT3 and ERK1/2, which are downstream molecules of TYRO3 signaling, while it did not obviously suppress the phosphorylation of mTOR.
We revealed that metformin treatment suppressed TYRO3 expression in myeloid leukemia cells for the first time. We also found that TYRO3 is involved in leukemia cell growth because TYRO3 knockdown suppressed the growth. From these findings, we showed that growth inhibition by metformin might be partly due to its suppression of TYRO3. In leukemia cells expressing AXL, suppression of AXL expression might also be involved in growth inhibition by metformin. It is widely speculated that growth inhibition by metformin is mainly due to inhibition of mTORC1 signaling through AMPK activation and some AMPK-independent mechanisms. We showed here that suppression of TYRO3 and/or AXL by metformin might also be involved in growth inhibition, although the individual effects of AMPK-mTORC1 and TYRO3/AXL pathways are not clearly distinguished because mTOR is one of the downstream molecules of TYRO3/AXL pathway. In clinical settings in the future, metformin treatment might be effective for patients with leukemia cells expressing TYRO3 and/or AXL proteins as well as mTOR. Detection of expression and activation of these proteins can be done to select the appropriate patients for administration of metformin.