Clinical and prognostic significance of expression of phosphoglycerate mutase family member 5 and Parkin in advanced colorectal cancer

BACKGROUND Drugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer(CRC)cells.Phosphoglycerate mutase family member 5(PGAM5)activates serine/threonine PTEN-induced putative kinase 1/Parkin pathway-mediated mitophagy.However,there are few studies on the clinical and prognostic significance of expression of PGAM5 protein and mitophagy-related protein Parkin in patients.AIM To assess the clinical significance of PGAM5 and Parkin proteins,as biomarkers for diagnosis and prognosis of CRC,by studying their expression in advanced CRC tissues and their association with clinicopathological parameters.METHODS The expression of PGAM5 and Parkin in CRC tissues from 100 patients was determined by immunohistochemistry.Each case was evaluated by using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-4).The final staining score was calculated as the intensity score multiplied by the proportion score.Specimens were categorized as either high or low expression according to the Youden index,and the association between the expression of PGAM5 or Parkin and clinicopathological factors was ascertained.Additionally,we employed western blot to measure PGAM5 and Parkin protein expression in six matched pairs of CRC and adjacent non-tumor tissues.RESULTS Immunohistochemical and western blot findings showed that both PGAM5 and Parkin protein expression in tumor tissues was significantly higher than that in the adjacent tissues:PGAM5 and Parkin were mainly expressed in the cytoplasm of colonic epithelial cells.PGAM5 and Parkin protein levels were significantly positively correlated in advanced CRC tissues.Moreover,reduced Parkin protein expression was an independent prognostic factor for overall survival and progression-free survival in CRC patients as evinced by multivariate analysis.CONCLUSION The expression of PGAM5 protein and mitophagy-related protein Parkin has diagnostic significance for CRC and may become new biomarkers.Parkin may be a potential marker for the survival of CRC patients.

Phosphoglycerate mutase 1 prostate cancer cell growth, knockdown inhibits migration, and invasion

Phosphoglycerate mutase 1 （PGAM1） is upregulated in many cancer types and involved in cell proliferation, migration, invasion, and apoptosis. However, the relationship between PGAM 1 and prostate cancer is poorly understood. The present study investigated the changes in PGAM1 expression in prostate cancer tissues compared with normal prostate tissues and examined the cellular function of PGAM1 and its relationship with clinicopathological variables. Immunohistochemistry and Western blotting revealed that PGAM 1 expression was upregulated in prostate cancer tissues and cell lines. PGAM 1 expression was associated with Gleason score （P = 0.01） and T-stage （P = 0.009）. Knockdown of PGAM 1 by siRNA in PC-3 and 22Rv I prostate cancer cell lines inhibited cell proliferation, migration, and invasion and enhanced cancer cell apoptosis. In a nude mouse xenograft model, PGAM1 knockdown markedly suppressed tumor growth. Deletion of PGAM1 resulted in decreased expression of Bcl-2, enhanced expression of Bax, caspases-3 and inhibition of MMP-2 and MMP-9 expression. Our results indicate that PGAM1 may play an important role in prostate cancer progression and aggressiveness, and that it might be a valuable marker of poor prognosis and a potential therapeutic target for prostate cancer.

PGK1-coupled HSP90 stabilizes GSK3βexpression to regulate the stemness of breast cancer stem cells

Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.

Identification of a novel PHGDH covalent inhibitor by chemical proteomics and phenotypic profiling

The first rate-limiting enzyme of the serine synthesis pathway(SSP), phosphoglycerate dehydrogenase(PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A(WA), a natural small molecule,as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue(Cys295)of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain(SBD)of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species(ROS) were elevated, leading to the inhibition of tumor proliferation.This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH.

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1βand IL-6

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATPcompetitive inhibitor of PGK1 with an affinity of Kd= 99.08 nmol/L. DC-PGKI stabilizes PGK1in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition,DC-PGKI unveils that PGK1 regulates production of IL-1β and IL-6 in LPS-stimulated macrophages.Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2(nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1β and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium(DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.

Differential gene expression of phosphoglyceric kinase (PGK) and hypoxic adaptation in chicken

Four single-nucleotide polymorphisms (SNP) of the Phosphoglyceric Kinase (PGK) gene were discov- ered based on comparison of the sequences from an altiplano chicken breed (Tibetan chicken) and two lowland breeds (White Leghorn and Shouguang chicken). Gel-shift results indicate that one of these SNPs, an A→G mutation at position 59 in exon10, is able to bind hypoxia-induced factor-l (HIF-1), functioning as a hypoxia response element (HRE). The mutant gene results in M→T mutation at position 379 amino acid. The combined activity of this HRE and HIF-1 could increase correspondingly under a hypoxic stimulus. Hypoxia leads to increased death rates of chicken embryos; while the M→T mutation described herein is prevalent in healthy embryos grown under hypoxic conditions, thus it may repre- sent an adaptation to hypoxia. Fluorescence quantitative reverse transcription PCR results revealed that HIF-1 upregulates the transcript level of the glycolytic enzyme PGK in the brain and skeletal mus- cle of animals subjected to hypoxia. Thus, a large amount of ATP is produced by increased glycolysis, allowing the organism to meet energy metabolism demands. As such, we believe this SNP to be an adaptation to the external anoxic environment.