Phenformin Promotes Oral Squamous Cell Carcinoma Cell Autophagy Through Induction Of ATF4 To Enhance DDIT4 And NIBAN1 Expression

Research BackgroundOral squamous cell carcinoma(OSCC),the sixth most common epithelial malignancy in the world and one of the most common oral malignancies in China,has a high incidence,low survival rate,and is highly susceptible to local recurrence and distant metastasis.The current treatment for OSCC is mainly surgery,supplemented with radiotherapy,chemotherapy and herbal medicine.Despite the rapid improvements in treatment over the past decades,the high mortality rate has still made it a global public health problem.More and more evidences suggest autophagy plays an irreplaceable role in the development of OSCC.Autophagy,an evolutionarily ancient and highly conserved catabolic process,plays an important role in maintaining the homeostasis of the body’s internal environment by inducing type Ⅱ cell death and removing unwanted,redundant or cancerous cells.However,after the cells have become cancerous,autophagy levels and functions tend to change,and this change is not exactly the same among different tumors.It has been found that autophagy level in OSCC was significantly lower than those in paracancerous tissues,and tumor growth was significantly inhibited after increasing autophagy levels,suggesting that the malignant progression of OSCC is closely related to changes in autophagic activity,and autophagy may be a potential new target for treatment of OSCC.Phenformin,a derivative of metformin,has been found to have a wide range of antitumor effects,which are more pronounced than that of metformin.Metformin has been shown to cause massive death of preneoplastic cells after induction of carcinogenesis,which was found to be more closely related to induction of autophagy.This suggests that the anticancer effects of phenformin may not be limited to inhibition of proliferation and promotion of apoptosis,but also induction of autophagy.Our previous studies already showed phenformin could inhibit OSCC cell growth,but the underlying molecular mechanism has not been clarified yet.Therefore,the present study will focus on whether phenformin can exert its anti-oral squamous cell carcinoma effect through the regulation of autophagic formation and explore its potential molecular mechanisms.Aims(1)To clarify whether phenformin inhibits the growth of OSCC cells in vitro and in vivo.(2)To investigate whether phenformin suppresses OSCC cell growth mainly through promotion of autophagy.(3)To explore the potential molecular mechanism by which phenformin promotes autophagy in OSCC cells.Methods(1)CCK-8 assay was performed for analysis of the OSCC cell growth.(2)The proliferation,apoptosis and autophagy of OSCC cells in vitro or in vivo were evaluated by EdU cell proliferation kit,LC3 single-label fluorescence tracking system,qRT-PCR and Western blot techniques,respectively.(3)Subcutaneous tumorigenesis assay in nude mice was used to investigate the inhibitory effect of phenformin on the growth of OSCC,and immunofluorescence staining and immunohistochemical staining were used for analysis of apoptosis and autophagy in OSCC cells in vivo.(4)RNA sequencing was performed to analyze the differentially expressed genes in phenformin-treated group versus the control group and to identify the key genes induced by phenformin treatment,and the expression of identified gene induced by phenformin was validated by qRT-PCR,Western blot,immunofluorescence staining and immunohistochemical staining from in vivo experiment.(5)The transfection of specific siRNAs was used to suppress the expression level of the identified genes in OSCC cells,to study whether the low expression of the key genes inhibit the role of phenformin in regulating cellular autophagy which was examined by qRT-PCR and Western blot analysis.(6)The potential upstream and downstream pathways of key genes were explored by reviewing the literature and RNA-seq results,and were confirmed by qRT-PCR and Western blot analysis.Results(1)CCK-8 results showed that phenformin significantly inhibited the growth of OSCC cells,and the inhibitory effect became more significant with increasing concentration or treatment time;secondly,the IC50 of phenformin on OSCC cells was significantly lower than that of metformin,which indicated that the inhibitory effect of phenformin on the growth of OSCC cells was better than that of metformin.(2)In vitro experiments showed that the number of proliferating OSCC cells was significantly reduced and apoptosis rate,the expression of apoptosis index and autophagy indexes were significantly increased after phenformin treatment,which indicated that phenformin significantly inhibited the proliferation of OSCC cells and promoted their apoptosis and autophagy.(3)The results of in vivo experiments showed that the tumor weight in the Phen group was significantly lower than that in the Con group,and the expression of proliferation indexes in tumor tissues was significantly lower,and the expression of apoptosis and autophagy indexes was significantly higher,which indicated that phenformin inhibited the growth of OSCC,and also had the effect of inhibiting the proliferation of OSCC cells and promoting apoptosis and autophagy in vivo.(4)RNA sequencing analysis revealed that the function of differentially expressed genes between phenformin treated and the control group mainly associated with autophagy and mitochondrial autophagy,and identified top two key genes by hot map analysis:DDIT4 and NIBAN1,which expression induced by phenformin treatment were validated by qRT-PCR,Western blot and immunofluorescence staining and immunohistochemical staining in both in vitro and in vivo.(5)Low expression of DDIT4 or NIBAN1 suppressed induction of autophagy by phenformin in OSCC cells.(6)Western blot showed that phenformin inhibited the phosphorylation of mTOR pathway and suppressed the autophagy-promoting effect of phenformin after the application of mTOR pathway activator;secondly,knockdown of AMPKa using siRNA did not affect the expression of DDIT4 and NIBAN1,while knockdown of ATF4 expression significantly inhibited the expression of DDIT4 and NIBAN1 and the autophagy-promoting effect of phenformin,which indicated that phenformin promotes OSCC cells autophagy by regulating ATF4 to control DDIT4 and NIBAN1 expression to inhibit mTOR signaling pathways.ConclusionThe present study found that compared to metformin,phenformin has a stronger inhibitory effect on the growth of OSCC cells.Phenformin can inhibit OSCC cell growth through suppression of proliferation,promotion of apoptosis and autophagy.RNA sequencing results showed that phenformin-induced differential genes were mainly involved in regulating cellular autophagy pathway and screened two key genes of autophagy pathway:DDIT4 and NIBAN1,indicating that phenformin mainly acts through inducing autophagy in OSCC cells.The molecular mechanism studies demonstrated that phenformin promotes OSCC cells autophagy by regulating ATF4 to control DDIT4 and NIBAN1 expression to inhibit mTOR signaling pathways.This study provides a theoretical basis for the research and development of inducing OSCC cell autophagy to treat OSCC in clinics.