Tumor-suppressing effect of metformin through regulation of myeloid-derived suppressor cells and M2 macrophages in the tumor microenvironment of colorectal cancer

Abstract

Various types of cells are associated with progression and regression of tumor in tumor microenvironment. Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells, in contrast to role of M1 macrophages for the rejection of the tumor. It has been reported that metformin, which is one of the most common diabetes drugs, has anti-inflammatory and anti-tumor effects. However, it is not known how metformin affects on inflammatory cells of tumor microenvironment and its mechanism. Thus, I determined the effect of a metformin on M2 macrophage and MDSC using THP-1 cells and the mouse colon cancer model. In flow cytometry analysis using the THP-1 cell, metformin decreased the fractions of MDSCs expressing CD33 and arginase and M2 macrophages expressing CD206 and CD163. Meanwhile, metformin caused the activation of p-AMPK and the inhibition of p-S6 in the western blotting analysis. Moreover, the fraction of MDSCs and M2 macrophages was decreased by AICAR (AMPK activator) and increased by Compound C (AMPK inhibitor) treatment, and the inhibitory effect of metformin on MDSCs and M2 macrophages was reversed by the treatment of Compound C and mevalonate. In addition, the treatment of rapamycin or simvastatin also decreased the fractions of MDSCs and M2 macrophages in the culture of THP-1 cells, which was reversed by mevalonate. Furthermore, the induction of PGE2, a mediator for M2 differentiation by PMA and/or mevalonate was inhibited by metformin treatment. Metformin treatment induced the inhibition of protein prenylation in mevalonate pathway and thus reduced the proportion of MDSCs/M2 macrophages. In the mouse colon cancer model of ApcMin/+ mouse treated with DSS, metformin reduced the number and volume of colorectal tumors. In immunohistochemistry stains, MDSCs expressing CD33 and arginase and M2 macrophages expressing CD206 and CD163 were significantly reduced by metformin treatment in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK-activation and mTOR inhibition leading to inhibition of the mevalonate pathway, which includes HMG-CoA reductase and protein prenylation. 암이 진행됨에 따라 형성되는 미세 종양 환경에는 다양한 세포들이 존재하며 각자 고유의 역할에 기여하며 종양 환경을 구성한다. 그 중에, Myeloid-derived suppressor cells (MDSCs)와 M2 대식세포는 항암제에 면역 저항성을 증가시키고 종양의 형성에 기여하며, 그에 반해 M1 대식 세포는 종양 억제에 관여하고 있다. 당뇨병 치료제로 널리 알려진 metformin은 다양한 보고에서 면역 억제와 종양 억제 효과를 보이고 있다고 알려진다. 하지만 metformin의 면역 세포에 대한 효과와 그 기작에 대해서는 아직 잘 알려져 있지 않은 상태이다. 혈액암 환자로부터 유래한 면역 세포인 THP-1에 metformin을 처리하여 대식세포 조절여부를 대해 알아 보았으며, 농도에 따라 MDSC (CD33, arginase)와 M2 macrophage (CD206, CD163)를 감소시킴을 확인하였다. Western blot analysis을 통하여, metformin의 처리가 p-AMPK를 활성화시키고 m-TOR를 억제시킴을 확인하였다. MDSC와 M2는 AMPK 활성제에 의해 감소되며 억제제인 Compound C에 의해 증가한다는 것을 확인하였다. 대식 세포에 대한 metformin의 억제 효과는 Compound C 와 mevalonate pathway의 매개체인 mevalonate에 의해서 반전되었다. HMG-CoA reductase (HMGCR) 억제제 (simvastatin)와 mTOR 억제제 (rapamycin) 또한 이들 대식 세포를 감소시켰으며, 역시 mevalonate에 의하여 억제효과가 반전되었다. DSS를 처리한 APCmin 마우스 대장암 동물 모델을 이용하여, metformin이 종양의 크기 및 숫자와 MDSC/M2 대식세포를 의미있게 억제함을 확인 하였다. 또한, metformin과 rapamycin이 HMGCR의 mRNA level을 낮춤을 확인하였고, mevalonate pathway를 통한 protein farnesylation, geranyl-geranylasion 억제제를 처리 하였을 때, MDSC/M2 대식세포를 효과적으로 억제함을 확인하였다. 결론적으로, 이러한 metformin의 MDSC/M2 대식세포 억제 효과는 metformin이 AMPK 활성화를 통해 mTOR 활성을 억제시키고, 이를 통한 mevalonate pathway의 protein prenylation 억제와 PGE2 억제를 통해 조절됨을 확인하였다.