| BackgroundLung cancer is the leading cause of cancer related death in both man and women worldwide.In China,the incidence of lung cancer ranked No.1 in men and No.2 in women(second to breast cancer).There are two major pathological types of lung cancer:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC accounts for approximately 85%of all lung cancer cases,can be further categorized into three major types:lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large-cell carcinoma.Although remarkable progress has been made in early screening and individualized therapy in recent years,the prognosis for patients with lung cancer is still very poor,with a 5-year survival rate less than 20%.Gene mutations,cigarette smoking and environmental pollutants are the main causes of human lung cancer.Mutations in several cancer driver genes,including EGFR,KRAS and ALK,play an important role in lung cancer initiation and progression.Targeted therapies for EGFR and ALK gene mutations bring the significant improvement in the patient outcome.About 75%of male lung cancer deaths and 18%of female lung cancer deaths are caused by somking.In addition,environmental pollutants and hazards,such as hexavalent chromium[Cr(?)],are known to contribute to many adverse health effects including human cancer.In recent years,the epigenetic changes caused by Cr(?)have received extensive attention in the field of cancer research.Cr(?)compounds are extensively used in many industrial processes,including chrome plating,chrome pigment production,stainless steel manufacturing and leather tanning.Numerous epidemiological studies have reported that chromate workers chronically exposed to Cr(?)through inhalation displayed high incidences of lung cancer.In 1990,Cr(?)compounds were classified as class ? human carcinogens via inhalation by the International Agency for Research on Cancer.Multiple mechanisms have been proposed to be involved in Cr(?)-induced carcinogenesis,including DNA damage,genomic instability,oxidative stress,and epigenetic modulation.However,the molecules and pathways contributing to Cr(?)-induced lung carcinogenesis have not been fully investigated yet.Recently,the research team of Dr.Hong Sun at New York University School of Medicine established several Cr(?)-transformed human bronchial epithelial cell lines as an in vitro system to investigate the mechanism underlying Cr(?)-induced lung carcinogenesis.To identify potential genes and molecules contributed to Cr(?)-induced malignant cell transformation,gene expression profiling was performed using Affymetrix Microarray.409 differentially expressed genes(DEGs)were identified in Cr(?)-transformed cells compared to control cells.Hedgehog-interacting protein(HHIP),one of the top 20 DEGs,is significantly downregulated in all Cr(?)-transformed cells.Given it is an important component of hedgehog(Hh)signaling pathway,downregulation of HHIP suggested the possible participation of HHIP and Hh signaling pathway in malignant transformation induced by Cr(?).Hh signaling is closely related to many fundamental cellular processes,including growth,differentiation,migration,tissue polarity,and self-renewal during embryonic development.Activation of Hh signaling is initiated by binding of Hh ligands to their receptors,Patched-homolog 1 and 2(PTCH 1&2).There are three Hh ligands in mammals,including sonic hedgehog(SHH),indian hedgehog(IHH)and desert hedgehog(DHH),which can elicit a similar response after binding to the same receptor.In the absence of Hh ligands,PTCH interacts with G protein-coupled receptor Smoothened homolog(SMO)and inhibits the activity of SMO.Binding of Hh ligands to PTCH leads to the activation of SMO,which subsequently results in nuclear translocation of glioma-associated oncogene homolog(GLI)as well as transcriptional activation of Hh target genes.HHIP,one of Hh downstream target genes,is able to bind to Hh ligands with an affinity similar to PTCH1.However,binding of HHIP to Hh ligand does not activate SMO due to the lack of signal transduction capacity.Hence,HHIP plays an important role in establishing a negative regulatory feedback loop during the activation of Hh signaling pathway.In the adult lung,Hh signaling only exists in airway epithelial progenitor cells,a kind of stem cells.Aberrant activation of Hh signaling has been reported in both SCLC and NSCLC and crucial for the survival and progression of lung cancer cells.Reduced expression of HHIP in Cr(?)-transformed cells suggested the important role of HHIP and Hh signaling in Cr(?)-induced malignant transformation and lung carcinogenesis.Therefore,the main purpose of the current study was to elucidate the functions of HHIP in cell malignant transformation induced by Cr(?)and to explore potential mechanisms underlying the modulation of Cr(?)on HHIP expression.Objectives1.To determine the expression level of HHIP in Cr(?)-transformed cells and human NSCLC samples;2.To explore the epigenetic mechanisms underlying downregulated HHIP expression in Cr(?)-transformed cells;3.To investigate the roles of HHIP on Hh signaling pathway in Cr(?)-transformed cells;4.To study the effects of ectopic HHIP expression on cell proliferation and anchorage-independent growth in Cr(?)-transformed cells.Methods1.Analysis of the expression levels of HHIP and main genes in Hh signaling pathwaysHHIP protein and mRNA expression in Cr(?)-transformed cells were analyzed by western blot and RT-qPCR.The mRNA expression of the major components in Hh signaling pathway,including SHH,PTCH1,SMO,and GLI1-3,were analyzed by RT-qPCR.Immunofluorescence staining was used to detect the subcellular localization of exogenous HHIP protein in transfected cells.The mRNA sequencing data and associated clinical information of lung cancer patients were obtained from the Cancer Genome Atlas(TCGA)database.Student's t test was used to verify the statistical significance of the difference in HHIP mRNA expression between lung cancer tumor tissue and adjacent normal lung tissue.Kala-Meier method and log-rank test were used to verify the statistical significance of differences in overall survival(OS)and disease-specific survival(DSS)between lung cancer patients with high HHIP mRNA expression and those with low HHIP mRNA expression.2.Analysis of the epigenetic mechanism about HHIP downregulation in Cr(?)-transformed cellsThe involvement of DNA methylation and histone deacetylation in HHIP downregulation was identified by treating Cr(?)-transformed cells with Trisostatin A(TSA),5-aza-2-deoxycytidine(5-AZA)and combination of TSA and 5-AZA.Chromatin immunoprecipitation conjugated quantitative PCR(ChIP-qPCR)was used to measure the enrichment of histone modifications in the HHIP promoter region,including H3K4me3,H3K9me2,H3K27me3 and H3K9ac.The methylation-specific polymerase chain reaction(MSP)was used to analyze the methylation level of CpG islands in the HHIP promoter region.3.Analysis of growth properties in Cr(?)-transformed cells with ectopic HHIPexpressionCell lines stably expressing ectopic HHIP were established by transfection of pCMV6-HHIP plasmid into Cr(?)-transformed cells and selected with Geneticin(G418).Cell proliferation was assessed by MTS cell viability assay.Soft-agar assay was used to detect the anchorage-independent growth.4.Statistical analysisResults from at least three independent experiments were expressed as mean ± SD.Significant difference between any two groups was determined by Student's t-test.P-values<0.05 were considered to be statistically significant.The data were analyzed using GraphPad Prism 7.Results1.HHIP expression is reduced in Cr(?)-transformed cell and NSCLC tissues.The results from Western blot and RT-qPCR showed that the levels of HHIP protein and mRNA were significantly reduced in all four Cr(?)-transformed cells compared to those in BEAS-2B cells,suggesting that down-regulation of HHIP gene is a common phenomenon in Cr(?)-transformed cells.Concordantly,the number of colonies in Cr(?)-transformed cells was significantly increased in soft-agar assay.Thus,our results indicate that Cr(?)-transformed cells exhibit reduced HHIP expression at both mRNA and protein levels,accompanied by enhanced anchorage-independent growth.To investigate the expression levels of HHIP in human lung cancer,we obtained RNA-seq data of lung cancer samples and normal lung tissues from the TCGA database.Analyzing RNA sequencing data of LUAD(tumor=514,normal=59)and LUSC(tumor=502,normal=51)revealed significantly reduced HHIP expression in primary tumors of both LUAD and LUSC compared with normal tissues.However,HHIP gene expression level is not significantly correlated with OS and DSS in lung cancer patients.Our results suggested that HHIP expression level may serve as a biomarker for early diagnosis of lung cancer,however,it can not be used as a relevant indicator for evaluating the prognosis of lung cancer patients.2.Promoter methylation and histone modification contribute to downregulated expression of HHIP in Cr(?)-transformed cells.To explore whether DNA methylation or histone modification is involved in the down-regulation of HHIP in Cr(?)-transformed cells,we treat two Cr(?)transformed cell lines with trichostatin A(TSA,a histone deacetylase inhibitor),or 5-aza-2-deoxycytidine(5-AZA,a DNA methyltransferase inhibitor),or a combination of TSA and 5-AZA.RT-qPCR results showed that,despite 5-AZA or TSA alone significantly induce HHIP mRNA levels,co-treatment of 5-AZA and TSA produced a greater effect(20-to 30-fold)on re-activation of HHIP transcription,suggesting that DNA methylation and histone deacetylation play important roles in silencing HHIP in Cr(?)-transformed cells.Analyzing the methylation status of the HHIP promoter using methylation-specific PCR(MSP)further confirmed the partially methylated HHIP promoter in two Cr(?)-transformed cell lines but not in parental BEAS-2B cells.Moreover,ChIP-qPCR analysis showed that silencing histone marks H3K27 trimethylation was enriched in HHIP promoter region,accompanied by reduced levels of active histone marks(H3K9 acetylation and H3K4 trimethylation).In summary,these results indicate that the epigenetic mechanism is involved in Cr(?)-induced HHIP gene expression.3.Hh signaling pathway is aberrantly activated in Cr(?)-transformed cells.HHIP is not only a downstream target gene but also a natural inhibitor of Hh signaling pathway.Down-regulation of HHIP gene may imply either the inactivation of the Hh signaling pathway(as Hh target gene),or the activation of the pathway(as Hh inhibitor).To test the activity of the Hh signaling in Cr(?)-transformed cells,we analyzed the mRNA expression levels of major components of Hh signaling,including SHH,SMO,GL11-3 and PTCH1.The results showed that the expression levels of GLI1-3 and PTCH1 mRNA in Cr(?)transformed cells were significantly higher than those in BEAS-2B cells,suggesting an enhanced activity of Hh signaling.It has been reported that culturing cells under serum starvation can increase the length of cell primary cilia and activate Hh signaling pathway.Therefore,we explored the effect of serum starvation culture on Hh signaling pathway in Cr(?)-transformed cells.Consistently,serum starvation induced a greater increase of Hh target genes in Cr(?)-transformed cells compared to BEAS-2B cells,suggesting that Hh signaling is less inhibited and therefore more easily activated in Cr(?)-transformed cells.In addition,compared with normal serum culture(10%FBS),Cr(?)-transformed cells under serum starvation culture(0.1%FBS)significantly increased their SHH mRNA expression levels.To rule out the possibility that Cr(?)directly activates the Hh signaling path,we exposed BEAS-2B and normal human bronchial epithelial(NHBE)cells to Cr(?)for 48 hours and then detected expression levels of GLI1,PTCH1,and HHIP mRNA.The expression levels of GLI1,PTCH1,and HHIP mRNA decreased significantly in BEAS-2B cells when treated with higher doses of Cr(?)(2 ?M).However,only a significant reduction in PITCH1 mRNA was detected in BEAS-2B cells after treated with a lower dose of Cr(?)(1 ?M).And GLI1 mRNA expression levels decreased significantly in NHBE cells when treated with higher doses of Cr(?)(5 ?M).The above results show that acute Cr(?)exposure inhibits the activity of the Hh signaling path in BEAS-2B cells.In summary,the down-regulation of HHIP gene expression in Cr(?)transformed cells is not the outcome of Hh inhibition but rather the cause for the aberrant activation of Hh signaling pathway in these cells.4.Ectopic HHIP expression inhibited aberrantly activated Hh signaling and reduced cell proliferation and anchorage-independent growth in Cr(?)-transformed cellsTo determine whether downregulation of HHIP and subsequent dysregulation of Hh pathway contribute to Cr(?)-induced cell transformation,we ectopically expressed HHIP in Cr(?)-transformed cells.Overexpressing of HHIP mRNA and protein were confirmed by RT-qPCR,western blot,and immunofluorescence staining.Our results showed that the levels of GLI1 and GLI2 mRNA in HHIP overexpressing cells were significantly lower than those control cells,suggesting that aberrantly activated Hh signaling in Cr(?)-transformed cells was reversed by ectopic expression of HHIP.Consistent with reduced Hh signaling,Cr(?)-transformed cells with HHIP expression exhibited a reduced cell proliferation and anchorage-independent growth.In summary,the expression of foreign aid HHIP not only restores the abnormal activation of the Hh signaling pathway in Cr(?)transformed cells,but also inhibits the proliferation and anchorage-independent growth of Cr(?)transformed cells.ConclusionsOur study uncovered a new mechanism underlying Cr(?)-induced lung carcinogenesis.Cr(?)exposure induced DNA hypermethylation and histone modification in the HHIP promoter region,leading to downregulation of HHIP and aberrant activation of Hh signaling.This facilitated cell proliferation and anchorage-independent growth,eventually contributing to Cr(?)-induced cell transformation. |
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