The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

口腔鳞状细胞癌（OSCC）的有效临床治疗仍然是一个挑战，需要开发有效的新药。苯乙双胍已被证明对不同肿瘤产生比二甲双胍更有效的抗肿瘤活性，然而，关于苯乙双胍对 OSCC 细胞的影响知之甚少。我们发现苯乙双胍抑制 OSCC 细胞增殖，促进 OSCC 细胞自噬和凋亡，在体内和体外均显着抑制 OSCC 细胞生长。 RNA-seq分析显示自噬途径是苯乙双胍的主要靶标，并确定了两个新靶标DDIT4（DNA损伤诱导转录物4）和NIBAN1（niban凋亡调节因子1）。我们发现苯乙双胍显着诱导 DDIT4 和 NIBAN1 的表达，从而促进 OSCC 自噬。此外，苯乙双胍引起的 DDIT4 和 NIBAN1 表达增强不会被 AMPK 的敲低所阻断，而是会被转录因子 ATF4（激活转录因子 4）的敲低所抑制，而转录因子 ATF4（激活转录因子 4）是由苯乙双胍处理 OSCC 细胞而诱导的。从机制上讲，这些结果表明，苯乙双胍触发内质网（ER）应激以激活PERK（蛋白激酶R样ER激酶），从而磷酸化过渡起始因子eIF2，而eIF2磷酸化的增加导致ATF4翻译的增加。总之，我们发现苯乙双胍诱导其新靶标 DDIT4，尤其是 NIBAN1 促进自噬和凋亡细胞死亡，从而抑制 OSCC 细胞生长。我们的研究支持苯乙双胍未来用于 OSCC 治疗的潜在临床用途。