Pharmacological Inhibition Of Bmi1 By PTC-209 Impaired Tumor Growth In Head Neck Squamous Cell Carcinoma

Bmi1(B lymphoma Mo-MLV insertion region 1 homolog)is a core component of PRC1(the polycomb repressive complex 1)and contributes to diverse fundamental cellular processes including cell proliferation,apoptosis and senescence.Overexpression of Bmi1 was frequently observed in diverse human malignancies and associated with tumor initiation and propagation,stem cell maintenance and poor prognosis,and therefore represented a novel diagnostic marker and a therapeutic target with great potentials.We utilized the publicly available databases including GENT,cBioPortal,Oncomine and TCGA to interrogate the mutational landscape and expression pattern of Bmi1 in HNSCC.We employed the PTC-209,a novel chemical inhibitor of Bmi1,to reveal its therapeutic efficacy against HNSCC and the potential underlying mechanisms.ObjectiveIn the present study,we aimed to reveal the frequencies of Bmi1 genetic amplification,mutation and deletion in HNSCC samples through further bioinformatics analyses in database,and we sought to determine the therapeutic effect of PTC-209 in vitro and a HNSCC xenograft model.Methods1.The detailed original data of Bmi1 DNA mutation and mRNA expression in HNSCC were downloaded from online databases GENT,cBioPortal,Oncomine and TCGA which included results from both squamous cell carcinnoma and normal tissue.The correlations between Bmi1 expression with clinicopathological parameters were analyzed.2.The HNSCC cell lines Cal27 and FaDu were treated with PTC-209 and the expression change of Bmi1 was detected by western blot and real-time RT-PCR assays;The protein–protein interaction was determined by protein immunoprecipitation,CHX and ubiquitination assay;The changes of cell proliferation,cell cycle,apoptosis,invasion and migration were analyzed by MTT,Annexin V-PI double staining,Transwell and wound-healing assays following PTC-209 treatment;The cancer stem cell subpopulations in Cal27 and FaDu cells were further assayed by tumorsphere formation,colony-forming and flow cytometry assays.3.The HNSCC cells were subcutaneous transplanted in nude mice to observed the anticancer effects of PTC-209 in vivo;Routine H&E staining?IHC staining were used to survey the expression of Bmi1 and ki67 in samples derived from animals in each group;IF staining was used to reveal the number of CD44-positive cells.Results1.Through online bioinformatics data mining and interrogation,we found that the mRNA level of Bmi1 varied remarkably among diverse human cancers and its abun-dance was frequently higher in cancers than those normal counterparts including HNSCC.2.(1)Bmi1 protein levels were significantly decreased in Cal27 and FaDu cells treated with PTC-209 in both dose-and time-dependent manners presumably by post-transcriptional repression and ubiquitin-proteasomal degradation;(2)The ubiquitination of Bmi1 protein was markedly increased following PTC-209 exposure;(3)Bmi1 transcripts and its well-established downstream target p16 were significantly downregulated upon PTC-209 treatment;(4)PTC-209 impaired cell proliferation,and had synergic anti-cancer effects when combined with cisplatin and 5-FU;(5)Cell cycle analysis revealed G1 arrest upon PTC-209 treatment:the percentages of cells in G1stage was increased;(6)The proportions of cell undergoing apoptosis were significantly increased(Cal27 3.16%vs 7.81%;FaDu 2.84%vs 9.03%;P<0.01)after PTC-209 exposure;(7)PTC-209 inhibited cell migration and invasion,and invasion-associated EMT markers E-cadherin was upregulated while N-cadherin and Vimentin were concomitantly downregulated following PTC-209 addition;(8)Colony formation,sphere formation,and ALDH~+subpopulation were significantly reduced upon PTC-209 exposure.3.PTC-209 administration significantly reduced tumor growth in a HNSCC xenograft model probably by Bmi1 inhibition and impaired cell proliferation.ConclusionPharmacological depletion of Bmi1 in HNSCC by PTC-209 induces anti-neoplastic effects both in vitro and in vivo xenograft animal model.Disruption of Bmi1 by selective and potent chemical inhibitors might represent a novel therapeutic strategy against HNSCC,especially for patients with aberrant Bmi1 overexpression.