Studies On The Phospholipase D1-mediated Cancer Cell Migration And The Mechanisms Of Biguanides Intervention

Phospholipase D(PLD)is a ubiquitously expressed enzyme in both prokaryotic and eukaryotic cells,which is responsible for the hydrolysis of membrane phospholipids such as hydrolysis of phosphatidylcholine into phosphatidic acid(PA)and choline.Recently,it has been found overexpressed and hyperactivated PLDs in many types of cancer tissues,especially PLD1 and PLD2.It has been found cancer patients with high expression of PLD1 have a poor prognosis and a reduced survival period,which suggests PLDs serve as a novel target of cancer therapy.Given that current activity inhibitors of PLDs have limited impacts on their expression,screening for inhibitors of PLDs expression might also be an alternative approach.Biguanides,especially metformin(Met),have become the drug of the first choice for Type 2 diabetes in clinics.Increasing studies have started to pay attention to the anticancer effects of biguanides over the past few years,although their anticancer mechanisms still remain unclear.To investigate whether PLDs engage in the anticancer mechanisms of biguanides and establish the connections among biguanides,PLD,and cancer,the study is carried out as follow:In this study,a PLD variant whose enzyme activity could be controlled by blue light was designed based on the LOV2 optogenetic element.PLD-derived PA was found essential for serum-induced m TORC1 activation by this PLD variant,which suggests the significance of PLD for cell growth.PLDs have been shown to engage in the regulation of cellular activity via the interaction of their protein scafolds(such as PH and PX domain)with other proteins,or their enzymatic activity for PA production.This PLD variant might be an approach for the investigations on the functional differences between PLDs and PA,and the spatiotemporal regulatory roles of them.It has been demonstrated that cancer cell migration was inhibited by silencing of PLD1 expression or inhibition of PLD1 activity.However,overexpression of PLD1 enhanced cancer cell proliferation and migration.Besides,Met has been shown to repress cell viability and migration in a dose-and time-dependent manner.To investigate whether PLDs are involved in the regulation of cell function for biguanides,the PLDs expression was detected in various cancer cell lines in response to Met treatment.The results showed that Met significantly reduced PLD1 expression in various cancer cell lines,while the PLD2 protein expression was intact in these tested cells after Met treatment.This action of biguanides was not AMPK-dependent.Moreover,PLD1 expression was inhibited in presence of the clinical dose of Met(40 μM)and Phenformin(Phe)(4 μM)for an elongated treatment.The above results demonstrated that PLD1 was a novel target and inhibition of PLD1 might be a novel anticancer mechanism.The further results showed that biguanides did not reduce PLD1 gene transcription and their co-treatment with protein degradation inhibitors would also decrease PLD1 protein expression which means protein degradation did not involve in the biguanide-induced PLD1 downregulation.However,similar to biguanides,the m TOR inhibitors also decreased the expressions of PLD1,asparagine synthetase(ASNS),and phosphoserine aminotransferase(PSAT1).The action of biguanides on PLD1 expression was partially rescued after the activation of the m TOR sigaling pathway.Further study revealed that the biguanide-induced PLD1 downregulation was negated in the 4E-BP1-knockdown or EIF4E-overexpressed cancer cells,which indicates the involvement of the m TOR signaling pathway and its downstream effectors.Subsequently,the RNA co-immunoprecipitation(RIP)technique was used to detect the PLD1 expression in precipitation component and cytoplasm for assessment of PLD1 m RNA translation.The results showed that the combination between EIF4 E and 4E-BP1 was increased and the PLD1 m RNA and ASNS m RNA(positive control)translation was inhibited after biguanide treatments.This regulation was further validated by the result that Phe treatment reduced the number of m RNA translational clusters with m RNA translation imaging technique.The RNA-seq technique was uesed to analyze the differential genes whose m RNA translation process were inhibited.The results showed the translational process of PLD1 and ASNS m RNA was inhibited,which were consistent with the results of q PCR.Moreover,the differential genes were closely related to cancer and cell metabolism according to KEGG pathway analysis.In summary,PLDs were engaged in cancer process via their enzymatic activities and protein scaffold and were considered as a novel anticancer target.This study revealed the regulatory mechanism of biguanides in PLD1 downregulation that biguanides reduced PLD1 protein biosynthesis via the inhibition of the m TOR signaling pathway and downstream translation process,which eventually reduced the PLD1 biosynthesis,and established the connection among biguanides,PLD1,and cancer.Biguanides-induced PLD1 downregulation might serve as a novel anticancer mechanism for cancer prevention and treatment.