Catalytic-independent Roles And Mechanisms Of AKR1C1 In Promoting Lung Cancer

IntroductionLung cancer patients diagnosed with distant metastasis and drug resistance will both succumb to a decreased survival rate.Therefore,continued research into metastasis and resistance is required to improve outcomes in lung cancer.Increasing evidence shows existence of transcriptional reprogramming in lung cancer metastasis.STAT3?Signal transducer and activator of transcription 3?,activated in more than 50%lung cancer tissues,could promote metastasis via targeting genes.However,underlying mechanisms are still not clear for STAT3 activation which inversely correlates with lung cancer survival.More recently,scientists focus on autophagy in tumor drugs resistance.SQSTM1?Sequestosome 1?,as the first autophagy receptor discovered,is elevated in more than 31%lung cancer tissues.It is also reported SQSTM1 participates in different tumor drugs resistance.But it is still unknown for modulation of SQSTM1 in lung cancer drugs resistance.Collectively,functional research for STAT3 and SQSTM1 will be intriguing avenues of investigation during lung cancer development.AKR1C1?Aldo-keto reductase 1,family member,C1?overexpression is observed in lung cancer and predicates the overall survival of lung cancer patients.It's documented that AKR1C1 causes resistance to cisplatin and doxorubicin in lung cancer cells.And in our previous data,AKR1C1 drives lung cancer cells metastasis.Thus,these will benefit lung cancer patients to identify novel biological roles of AKR1C1 and uncover the underlying mechanisms in lung cancer progression.Section 1AKR1C1 catalytic-independently promotes the metastasis of lung cancer.ObjectiveAberrant activation of STAT3 exists in most lung cancer.Nuclear accumulation of phosphorylated STAT3 is critical for its activity to transcribe pro-metastasis genes.The aim of this section is to explore catalytic-independent roles of AKR1C1 in lung cancer metastasis.MethodsImmunohistochemistry and H&E?Hematoxylin and eosin staining?were used to assess proteins expression in tissues.Metastatic foci in the livers of nude mice were detected by PET-CT.Transwell assay and scratching assay were to evaluate invasion and motility of lung cancer cells.Changes in NADPH?Reduced form of nicotinamide adenine dinucleotide phosphate?absorbance?at 340 nm?reflect enzymes activity of AKR1Cs.Protein levels in cells were determined by western blot.Microarray analysis,qRT-PCR?Real-Time Quantitative Reverse Transcription PCR?and luciferase assay were carried out to explore transcription activity of STAT3.Nuclear fractionations seperation and immunofluorescence were implemented to observe STAT3 translocation.Kinase function of AKR1Cs was explored by kinase assay in vitro.Immunoprecipitation was used to determine protein-protein interactions.Chromatin immunoprecipitation was to evaluate the association of STAT3 at the promoter regions of its target genes.Results1.Lung cancer cells are conferred metastatic potential by AKR1C1 both in vitro and in vivo.?1?AKR1C1 expression in lymph node metastatic tumors was significantly elevated compared to the corresponding primary tumors.?2?AKR1C1 knockdown hindered invasion of NCI-H460 and A549.?3?Images from PET-CT showed increased metastatic foci for NCI-H1299-AKR1C1 cells and decreased metastatic foci for NCI-H460-shAKR1C1 cells.2.AKR1C1 promotes lung cancer cells metastasis in a canonical reductase activity-independent manner.?1?AKR1C1 stood out among the three members of the AKR1C family for its ability to promote the invasion of NCI-H1299 cells.?2?5BPSA?3-bromo-5-phenylsalicylic acid?imposed little impact on the pro-metastasis ability of AKR1C1 in NCI-H1299.3.AKR1C1 augments STAT3 transcription via modulating JAK2/STAT3?Janus kinase2/Signal transducer and activator of transcription 3?interaction.?1?A microarray analysis indicated a shift in the transcriptional profiles including STAT3 signaling pathway.?2?qRT-PCR analyses were further utilized to confirm the reduction of the mRNA levels of a variety of STAT3 target genes.?3?siAKR1C1 decreased phospharylation and translocation of STAT3.?4?AKR1C1 overexpression also reinforced the nuclear pY705-STAT3 levels and enhanced the association of STAT3 at the promoter regions of its target genes upon IL-6 stimulation.?5?In 40 lymph node metastatic tumors,pY705-STAT3 positively correlated with AKR1C1 expression.?6?Further studies showed that siJAK2 impeded pY705-STAT3 level as well as invasion ability enhanced by AKR1C1.?7?In NCI-H460 cells,shAKR1C1 disrupted JAK2/STAT3 interaction.4.AKR1C1 catalytic-independently activates STAT3.?1?The other two family members,AKR1C2 and AKR1C3,share similar catalytic activities,but show minimal binding affinity with STAT3.?2?Enzymatic mutants of AKR1C1 could still be precipitated by STAT3 and showed a comparable ability activiting STAT3 target genes with that of wild-type AKR1C1.?3?5BPSA had no effect on STAT3 transcription.ConclusionsAKR1C1 interacts with STAT3 and facilitates its phosphorylation,thus reinforcing the binding of STAT3 to the promoter regions of target genes,which ultimately promotes tumor metastasis.Further studies showed that AKR1C1 might facilitate the interaction of STAT3 with its upstream kinase JAK2.Intriguingly,AKR1C1 exerts these pro-metastatic effects in a catalytic-independent manner.Section 2AKR1C1 catalytic-independently interacts with SQSTM1 and modulates its functions.ObjectiveIn response to oxidative stress,autophagy receptor SQSTM1 oligomerizes and delivers cargo into autolysosomes.LC-MS/MS analysis showed potential interaction of AKR1C1 and SQSTM1.This section will focus on AKR1C1/SQSTM1 interaction,uncovering catalytic-independent roles of AKR1C1 and providing functional insights into AKR1C1/SQSTM1 interaction.MethodsImmunoprecipitation and GST pulldown were used to explore AKR1C1/SQSTM1interaction.Different mutants were constructed by coloning.Expression levels of LC3B?Microtubule associated protein 1 light chain 3 beta?and other proteins were examined by western blot.Immunofluorescence was carried out to observe LC3B puncta and SQSTM1 puncta.Non-reduced SDS PAGE was establised to evaluate SQSTM1 oligomers.Results1.Identification of SQSTM1 as an AKR1C1-interacting partner.?1?Immunoprecipitation showed that precipitated SQSTM1 could interact with exogeneous AKR1C1 as well as endogeneous AKR1C1.?2?Purified GST-AKR1C1could interact with SQSTM1 from cell lysate.2.Enzymatic activity of AKR1C1 doesn't involve in binding with SQSTM1.?1?Inspite of similar catalytic activity,SQSTM1 interacted with AKR1C1 not AKR1C2/AKR1C3.?2?R47H&L54V and R170H&Q172L mutation for AKR1C1abolished interaction between AKR1C1 and SQSTM1.?3?Enzymatic mutants of AKR1C1 had comparable affinity for SQSTM1 as that of wild-type AKR1C1.?4?5BPSA didn't interfere interaction between GST-AKR1C1/SQSTM1 in GST pulldown assay.3.ROS?Reactive oxygen species?plays a vital role in AKR1C1 and SQSTM1interaction.?1?5BPSA could impeded the binding between AKR1C1 and SQSTM1 in293T cells.?2?H₂O₂?Hydrogen peroxide?could reinforce interaction between AKR1C1 and SQSTM1 which could be rescued by antioxidants.?3?AKR1C1/SQSTM1 complex accumulated gradually after autophagy induction by HBSS?Hank's balanced salt solution?treatment.4.AKR1C1 accelerates autophagy flux.?1?AKR1C1 overexpression led to LC3B-?accumulation in Hela and NCI-H1299 cells.?2?AKR1C1 overexpression markedly increased levels of LC3B-?,as well as LC3B-GFP puncta in comparison to those exposed to CQ?Chloroquine?alone.?3?AKR1C1 overexpression in NCI-H1299 cells also increased levels of LC3B-?,as well as LC3B-GFP puncta in comparison to those exposed to autophagy induction alone.5.AKR1C1 promotes SQSTM1 oligomerisation.?1?AKR1C1 enhanced the ubiquitination of SQSTM1 complex.?2?AKR1C1 augmented interaction of SQSTM1-Myc and SQSTM1-Flag.?3?AKR1C1 further increased SQSTM1 puncta stimulated by H₂O₂.?4?In lung cancer cells,shAKR1C1 impinged on SQSTM1puncta and oligomers in response to H₂O₂.ConclusionsIn a catalytic-independent manner,AKR1C1 directly interacts with SQSTM1 in response to ROS.Further studies showed that AKR1C1 may be an autopgagy inducer by mudulating oligomerization of SQSTM1 followed by increased affinity with autophagic substrates.