Abstract 3222: Metformin enhances cisplatin cytotoxicity of lung cancer through Stat3 pathway
Authors: Lin Chien-Chung , Yeh Hsuan-Heng , Su Wu-Chou 
Published: August 2012

The antitumorigenic effects of metformin were mostly interpreted as attributing to the activation of LKB1-AMPK pathway and the studies investigating the AMPK-independent mechanism of metformin remained limited. In lung cancer, cisplatin-based chemotherapy remained the first line regimen for advanced lung cancer patients especially those without EGFR mutation. Previous study demonstrated the linkage between IL6-Stat3 autocrine and chemoresistance. In this study, we first showed that lung cancer line S3C (PC14PE6/AS2 cells with expressing plasmids containing constitutively-active Stat3) was more resistant to cisplatin than PC14PE6/AS2 cell. Treating lung cancer cells with Stat3 inhibitor (JSI-124) or metformin combined with cisplatin, the proliferation of cancer cells were inhibited additionally. And metformin inhibited Stat3 phosphorylation in PC14PE6/AS2 and A549 cells. To investigate the upstream mechanism associated Stat3 phosphorylation, we examined the secretion of IL-6 which was inhibited by metformin. And VEGF secretion, representing Stat3 downstream pathway, was inhibited by metformin in vitro and in vivo. Conversely, the secretion of VEGF was not inhibited in S3C cells after metformin treatment. However, using another AMPK activator-AICAR, the enhancement of cisplatin cytotoxicity is not obvious. In contrast to metformin, AICAR induced AMPK phosphorylation but Stat3 phosphorylation was not inhibited. To verify the LKB-AMPK independent effect of metformin on Stat3 phosphorylation, PC14PE6/AS2 cells were transfected with siRNA targeting LKB1 or AMPK. We demonstrated the inhibition of Stat3 phosphorylation of metformin even under the knockdown of LKB1-AMPK pathway. We also found that MTOR downstream signal including P70S6K and 4EBP1 were inhibited by metformin. Though previous study demonstrated MTOR pathway modulated Stat3 pathway, we found P70S6K and 4EBP1 phosphorylation were inhibited after treatment with metformin at 8hr and Stat3 phosphorylation change was detected after 24hr. Using MTOR inhibitor (rapamycin), the Stat3 tyrosine phosphorylation was not inhibited. Finally, we used subcutaneous tumor xenografts to evaluate the effect of combination of metformin with cisplatin in vivo. Body weight, blood sugar, and tumor volume were recorded in four groups including control, metformin, cisplatin, and metformin combined with cisplatin. Body weights and blood sugar were not different between combination treatment group and control group. In combination group, tumor stopped growing at 18th day and the tumor sizes were obvious less than the other three groups. Immunohistochemical staining of ki67 verified the lower proliferation in combination group. Our study demonstrated that metformin, independent of LKB1-AMPK pathway, inhibits Stat3 phosphorylation which contributes the enhancement of cisplatin cytotoxicity in lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3222. doi:1538-7445.AM2012-3222