The Role Of The Transcriptional Factor HOXC9 In Glioblastoma And The Mechanism Investigation Of HOXC9 In Autophagy Regulation

Glioblastoma,which is classified as a grade IV glioma,is the most common and deadly type of brain tumor.It exhibits a high rate of cellular proliferation and highly aggressive and infiltrative metastasis.Despite advances in surgical and pharmacological therapies,glioblastoma remains incurable and has a low survival rate.Therefore,identifying alternative therapeutic approaches is critical but cannot be achieved without understanding the molecular mechanisms underlying the initiation and progression of glioblastoma.HOX genes belong to the homeobox family,which encodes evolutionarily conserved transcription factors that provide positional information during differentiation and morphogenesis along the anteroposterior axis.They also control other cellular processes,such as cell proliferation,cell migration,the determination of cell morphology,and apoptosis,in cancer cells.The dysregulation of HOX genes has been reported to cause abnormal development and malignancy,for example,HOXA9 dysregulation in leukemia,HOXB13 dysregulation in ovarian cancer,and HOXB5 dysregulation in breast cancer.In some cases,HOX genes act as tumor suppressors,including HOXC6 in serous ovarian cancer and HOXC9 in neuroblastoma.As a member of the HOX family,HOXC9 has been reported to induce cell differentiation and reduce self-renewal capabilities in neuroblastoma.However,it has been reported that HOXC9 is highly expressed in CD133(+)astrocytomas and that it promotes cell proliferation.The abnormal expression of HOXC9 has also been observed in non-small cell lung cancer,breast cancer,and canine mammary tumors,but the role HOXC9 played in these cancers has not been identified.The role of HOXC9 in tumorigenesis and tumor progression is therefore complicated and needs to be further clarified.In this study,we demonstrate that HOXC9 plays an oncogenic role in glioblastoma.Knockdown of HOXC9 significantly reduced cell proliferation and induced autophagy,which indicates that HOXC9 may be a potential therapeutic target for glioblastoma treatment.The main results are as follows:(1)High HOXC9 expression is an indicator of a poor prognosis in patients with glioblastomaHOXC9 gene expression dysregulation has been reported in many types of cancer,however,it has not yet been well defined in glioblastoma.To investigate whether HOXC9 is a prognostic marker for glioblastoma,we conducted a microarray-based search using two databases,and these two databases showed that high HOXC9 expression was strongly associated with a poor outcome,whereas low HOXC9 expression was correlated with good overall survival.Moreover,increased HOXC9 expression was significantly correlated with advanced tumor stages and was a meaningful prognostic indicator for the different stages of glioma that were analyzed(grade II-IV).To further confirm our results,we examined the expression of HOXC9 in several glioblastoma cell lines and primary tumor specimens and their corresponding peritumoral tissues.HOXC9 was commonly expressed in all four cell lines.The A172 cell line,which is a relatively benign cell line with no tumor formation ability,had a relatively low level of HOXC9 expression.Moreover,the expression levels of HOXC9 in primary tumor specimens were much higher than those in their corresponding peritumoral tissues,where HOXC9 was barely expressed.H&E staining and immunohistochemistry(IHC)assays that were performed on the representative primary specimen and the associated peritumoral tissue confirmed the results of the Western blot analysis.Our data suggest that a higher expression of HOXC9 predicts a poor prognosis in patients with glioblastoma,indicating that HOXC9 may play an oncogenic role in tumor development.(2)HOXC9 is essential for sustaining glioblastoma cell proliferation,migration,and invasionHOXC9 was knocked down in three glioblastoma cell lines.Using microscopy,we found that sh HOXC9 cells showed a clear decrease in cell numbers and severe morphological changes compared with the corresponding control cells,suggesting an inhibitory effect on cell growth.Accordingly,we analyzed cell growth and proliferation in these cells using MTT and Brd U assays,and the results showed that silencing HOXC9 led to sharp declines in growth curves and the proportion of Brd U-positive cells.These results indicate that HOXC9 accelerates glioblastoma cell growth and proliferation.To determine whether cell migration and invasion were affected by HOXC9 silencing,wound healing and Transwell assays were performed.The results of the wound healing assays showed that gap widths were much wider in the sh HOXC9 group than in the control group after 24 h.Consistent with these results,sh HOXC9 cells showed a much weaker ability to migrate across the membrane of Transwell chambers,either with or without Matrigel,demonstrating that the loss of HOXC9 led to a significant reduction in migration and invasion.These results suggest that HOXC9 is essential to sustaining cell migration and invasion.(3)The mechanism of HOXC9 silenced induced autophagyWe aimed to determine whether the decrease in cell numbers observed after HOXC9 silencing was caused by apoptosis.Flow cytometry analysis and western blot showed that no apoptosis was detected.We transiently transfected glioblastoma cells with GFP-LC3 B plasmid and examined the cells under a fluorescence microscope.sh HOXC9 cells showed a punctate pattern of GFP-LC3 B fluorescence,representing the recruitment of LC3B-II to autophagosomes and the formation of autophagic vacuoles.This result differed from the diffuse LC3B-associated green fluorescence observed in the control cells.To further confirm our results,immunofluorescence and Western blot analysis were performed to detect the conversion of LC3B-I to LC3B-II and the progression of autophagy.As predicted,after HOXC9 was down-regulated,the formation of LC3B-positive puncta markedly increased.These results are in agreement with what biochemically were demonstrated by the increased conversion of LC3 B and the enhancement of autophagy-associated proteins.Moreover,the induction of autophagy was further supported by Western blot analysis of xenograft tumor tissues.Together,our results provide evidence that HOXC9 silencing induces autophagy but not apoptosis in glioblastoma cells.To further explore the mechanism by which HOXC9 controls autophagy,we focused on the expression of downstream genes involved in the regulation of autophagy,in view of the transcriptional role of HOXC9.The expression levels of a panel of eight genes were analyzed using q RT-PCR.DAPK1 showed the most significant increase in its m RNA level and an obvious increase in its protein level after HOXC9 was silenced.We detected the amount of DAPK1 that was phosphorylated at Ser308 using Western blot analysis,and found that the phosphorylation of DAPK1 was not altered.However,the total amount of DAPK1 protein was significantly increased after HOXC9 down-regulation,indicating that DAPK1 was activated.Similar results were also obtained using xenograft tumor tissues.To gain further insight into the role of DAPK1 in HOXC9-silencing-induced autophagy,we knocked down DAPK1 after HOXC9 was silenced to examine whether this would reduce autophagy.The results showed that the formation of LC3B-positive puncta and the conversion of LC3B-I to LC3B-II were clearly reduced after the knockdown of DAPK1.These results suggest that DAPK1 played a vital role in mediating the autophagy that was induced by the down-regulation of HOXC9.It has been reported that Beclin1 is an indispensable downstream effector of DAPK1 during the regulation of autophagy.We knocked down Beclin1 following HOXC9 silencing to assess whether autophagy would consequently be reduced.The level of autophagy,as indicated by the conversion of LC3B-I to LC3B-II,was markedly decreased in the sh Beclin1 group compared with the sh GFP group.Our results collectively demonstrate that HOXC9 suppresses autophagy through its effect on the DAPK1-Beclin1 pathway.Next,a dual-luciferase reporter assay was performed to explore the mechanism by which HOXC9 modulates DAPK1 expression in glioblastoma cells.DAPK1 promoter activity was significantly increased by the deletion of its upstream region from-1121 bp to-368 bp before the transcription start site(TSS),indicating that this region is associated with the inhibition of DAPK1 transcription.To determine whether the suppression of DAPK1 promoter activity was caused by HOXC9 binding,a Ch IP assay was used to map the HOXC9-binding locus on the DAPK1 promoter.We found that HOXC9-binding sites were enriched in the promoter-proximal region p2(-528 bp to-412 bp).It was previously reported that HOXC9,a transcription factor,can recognize and bind to the DNA sequence T/ATTTAT.We identified an ATTTAT sequence in the DAPK1 promoter region from-442 bp to-437 bp.This finding confirmed our previous results showing that HOXC9 inhibits DAPK1 transcription by directly binding to its promoter.(4)HOXC9 is required for tumorigenesis of glioblastoma cellsNext,the roles of HOXC9 in the self-renewal and tumorigenesis of glioblastoma cells were evaluated.In vitro colony formation was first examined using soft agar assays.Two glioblastoma cell lines were investigated,and the results showed that the HOXC9-silenced group formed substantially fewer and smaller colonies than the control group,suggesting that HOXC9 functions to promote clonogenic and self-renewal abilities.Next,a subcutaneous xenograft mouse model was used to examine the effect of HOXC9 knockdown on tumor formation in NOD/SCID mice.The volumes and weights of sh HOXC9 U87-MG tumors were much smaller than those in the corresponding control group.The sh HOXC9 LN-229 group lost its tumorigenic activity during the same time course.Furthermore,to provide more relevant in vivo data,orthotopic implantations were performed.The data showed that the sh HOXC9 group had smaller tumors by volume and longer survival times than the sh GFP control group,which confirms the results of the subcutaneous xenograft experiments.Consistent with these results,HOXC9 expression,detected by IHC,in the orthotopic xenografts was lower in the sh HOXC9 group than in the control group.These results indicate an oncogenic function of HOXC9 in glioblastoma development.