The current study shows a novel selective antitumor activity of PRMT5 inhibitor AMI-1 additive to cisplatin in lung adenocarcinoma cells. Micromolar concentration of AMI-1 mediates anti-survival effect through G1arrest and reduction of histone-4 dimethylation. Interestingly, AMI-1 is cyto-protective on normal epithelial cells.
Epigenetic targeting drugs can potentiate the effect of conventional chemotherapeutic agents in solid tumors 19. Overexpression of PRMT5 has been observed in solid and hematological malignancies, e.g. Mantle Cell Lymphoma (MCL) which depends on PRMT5 activity for their survival 20. PRMT5 mediates methylation of several proteins including E2F-1. Among the E2F family of transcription factors, E2F-1 has been implicated in opposing fates of cell cycle progression and apoptosis, depending on the cellular context 21. Dysregulation of E2F activity occurs in many tumors as a result of inactivation of the retinoblastoma tumor suppressor pRb 22−24. Arginine methylation of E2F-1 by PRMT5 regulates its biochemical and functional properties, including growth control. PRMT5 depletion increases E2F-1 protein expression and is associated with increased apoptosis 25. In line with this study, we showed that inhibition of PRMT5 activity in A549 cells by treatment with 10 µM AMI-1 reduced cell viability, induced growth arrest and apoptosis
Methylation of histone arginine residues by PRMT5 is a well-known mechanism of transcriptional repression. Among the type II PRMT family members, PRMT5 is the sole member capable of targeting histones 25 and is more commonly associated with transcriptional repression 25−26. PRMT5 preferentially methylates the N-terminal tails of histones with primary methylation sites being H4 arginine 3 and H3 arginine 25. We demonstrated that combined AMI-1 and cisplatin administration resulted in marked methylation reduction of histone-4, with no effect on β-catenin. In our study, β-catenin was tested as a potential target of PRMT5. In a previous study on hepatocellular cancer, there was a significant downregulation of β-catenin upon PRMT5 silencing 27.
AMI-1 is one of the first reported small molecule inhibitor of PRMT activity. The IC50 for PRMT1 enzyme inhibition is 8.81 µM 17. It specifically inhibits PRMT-mediated epigenetic modification of arginine methylation without affecting lysine methylation of cellular proteins in vitro 20. AMI-1 was first discovered for its ability to inhibit type I PRMT activity, however, later studies also indicated its ability to inhibit type II PRMT5 activity 17. Most studies that exploited the anti-tumor potential of AMI-1 by targeting PRMT5 used a high dose of AMI-1 (in the mM range) 27−29. The highlight of our study is that we demonstrated a potential anti-tumor activity of AMI-1 on A549 cells at a concentration as low as 10 µM. Furthermore, the dose of AMI-1 used in our study was found to be safe to normal bronchial epithelial cell line. These factors prompted us to focus our attention more on AMI-1. Other inhibitors of PRMT5 have also been identified. YQ36286 was investigated as a first-in-class oral inhibitor of PRMT5 selectivity acting against several histone methyltransferases. The IC50 of its enzymatic activity was achieved at low nanomolar concentrations. Selective small molecules PRMT5 inhibitors were also explored recently for anti-lung cancer activity 30.
PRMT5 is highly co-expressed with a number of functionally related genes including PRMT1; which mediates arginine mono- and asymmetric dimethylation of many proteins. It is the main enzyme that mediates methylation of histone H4; a specific tag for epigenetic transcriptional activation 31. Cyclin-dependent kinase 4 (CDK4) is also co-expressed with PRMT5. CDK4 phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition 32.
The success of cisplatin therapy in clinical practice is limited by the occurrence of innate and acquired drug resistance. The mechanisms underlying cisplatin resistance are multiple. Pre-, post-target, on- and off-target types of resistance have been identified; indicating the lack of a single mechanism-based strategy that may overcome cisplatin resistance 33. Various proteins, genes or pathways were found to be involved in resistance against cisplatin 34−35.
c-FLIP (CFLAR) is a regulator of the extrinsic apoptotic pathway that decreases caspase 8 activation. Non-small cell lung cancer (NSCLC) is characterized by over expression of FLIP and high cytoplasmic expression is further indicative of poor prognosis 36. Clinically, c-FLIP has been proposed as a prognostic marker in NSCLC 36 and stage II and III colorectal cancer 37. Chemotherapeutic agents are known to down-regulate the gene and protein expression of CFLAR and silencing it facilitates chemotherapeutic-induced apoptosis. Suppression of c-FLIP expression was also found to render resistant human bladder cancer cells more sensitive to cisplatin treatment 38. Recent studies have demonstrated PRMT1/5 to fine tune the degradation of anti-apoptotic protein CFLARL in human lung cancer cells 39. In this study, PRMT5 protected NSCLC cells from caspase activation and apoptosis induced by anti-cancer drugs and knocking down its expression led to CFLAR downregulation and activating chemotherapeutic-induced apoptosis. These studies suggest a plausible explanation for the observed additive effect of PRMT5 inhibitor, AMI-1, on cisplatin-induced cellular apoptosis and cell cycle progression.
In the current study, PRMT5 inhibitor AMI-1 showed two important properties that make it a potential therapeutic adjuvant to cisplatin to combat chemotherapeutic resistance, namely; increasing G1 cell arrest and induction of apoptosis. Also, bioinformatic pathway analyses of differentially expressed genes in PRMT5 silenced A549 cells were in line with our in vitro experiments, revealing increase in apoptosis and decrease in DNA damage, cell cycle progression, and Retinoblastoma activity pathways. A recent study suggested that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G1 cell cycle arrest and apoptosis 40. Thus, AMI-1 has a high potential to overcome cisplatin resistance. Altogether, our results showed a potential antineoplastic activity of PRMT5 inhibitor in lung cancer cells in combination with cisplatin. Further investigations are needed to explore the affected cellular pathways and to identify target genes that underwent epigenetic modification in response to this combination treatment.