The Role And Mechanism Of HOXA9Gene In The TMZ-resistance And RT-resistance Of Human Glioma

Gliomas are the most common adult primary central nervous system tumours, whichaccount for approximately40~50%of brain tumours. Glioblastoma multiforme (GBMWHO IV) is the most common gliomas and accounts for approximately60~70%ofmalignant gliomas with the worst prognosis. GBM is characterised by a short course andhigh mortality of disease, which prognosis is far worse than that of other tumours becauseof its rapid growth and invasion of the central nervous system and resistance toconventional therapies. Imaging-guided neurosurgery, stereotactic radiation therapy andcytotoxic chemotherapy have been unable to fundamentally cure GBM. GBM patientsusually die from tumour recurrence within six months of the initial surgery. Difficulties ontreatment are closely associated with the malignant biology phenotype of gliomas.Although temozolomide (TMZ)-based chemotherapy effectively prolongs overall survivaland enhances the quality of life of patients, however, not all GBM patients can be benefitfrom TMZ-based chemotherapy because of the active GSTs system and abundant MGMTcontent of GBM cells. Therefore, it is important to find new ways to treat and curegliomas. Molecular targeted therapy is one of the most promising potential treatments. New markers to predict clinical outcome, tumour recurrence and resistance to therapiesoften determine the diagnosis and therapy of some cancer subtypes.Homeobox genes encode transcription factors important for anteroposteriorpatterning during embryogenesis and are divided into two classes: the class I clusteredhomeobox genes (HOX), and the class II dispersed non-HOX genes (PAX, EMX, MSX,and so on). In humans, there are39class I homeobox (HOX) genes found in four genomicclusters (HOXA at7p15.3, HOXB at17q21.3, HOXC at12q13.3, and HOXD at2q31).Their spatial and temporal expression patterns are critical for body patterning duringnormal development, and HOX genes also have critical postdevelopmental regulatoryfunctions. Aberrant HOX gene expression initiates different types of human diseases andcancers including leukaemia, breast cancer and brain tumours. Multiple HOX genes havebeen shown to be overexpressed in primary astrocytomas and GBM cell lines, whichsuggests an important role for these genes in gliomagenesis, glioma recurrence and drugresistance. However, the potential mechanisms underlying HOX activation, in addition totheir functional relevance in GBM cells, have not been determined. Of the39HOX genesin humans, the HOXA9genes encode critical transcriptional regulators of embryonicdevelopment and postdevelopmental stages that have been implicated in gliomas.However, the potential mechanisms with HOXA9activation and gliomas’ recurrent,TMZ-resistance and radiotherary (RT)-resistance also have not been explored.Therefore, to further understand the relationship between HOXA9and brain gliomasand the role of HOXA9in malignant gliomas’ proliferation and invasion, gliomas’recurrent, TMZ-resistance and RT-resistance, we performed four experiments as follows.1. The significance of HOXA9expression in human gliomasThe expression of HOXA9mRNA and protein were first investigated in five humanglioma cell lines (U87MG、U251、A172、T98G and BT325) by quantitative real time PCR(qRT-PCR) and western blot respectively. The results showed that the HOXA9was widelyexpressed in five human glioma cell lines, and the most were T98G and BT325cells. Andthen, we performed immunohistochemistry (IHC), qRT-PCR and western blot to detect theexpression of HOXA9on158primary glioma specimens,50recurrent glioma specimens and6non-neoplastic brain parenchyma specimens. In the158primary glioma specimens,50recurrent glioma specimens and6non-neoplastic brain parenchyma specimens by IHC,we found that HOXA9expression was significantly higher in gliomas than in normal brainparenchyma and was much more lower in more malignant gliomas than in less malignantgliomas (P<0.01). HOXA9expression was inversely correlated with survivin expression(r=0.906, P<0.01), large tumor diameter (r=0.507, P<0.01), pathological grade (r=0.683,P<0.01), extent of resection (r=0.152, P=0.029) and type of adjuvant treatment (r=0.366,P<0.01) in gliomas. Spearman’s rank correlation analysis did not show a statisticallysignificant correlation between HOXA9and patients’ gender (P=0.624), age (P=0.415)and frequent intra-tumorous necrosis (P=0.351). Based on IRS results, we divided theglioma specimens into two categories for the Kaplan-Meier survival analysis. HOXA9expression was associated with OS and PFS in all the glioma patients. Patients whosetumors high expressed HOXA9had a significantly shorter overall survival [OS; median,8months;95%confidence interval (CI),4-10months] compared with those whose tumorslow expression of HOXA9(median,44months; CI,36-47months; P<0.01). Similarly,patients whose tumors high expressed HOXA9had a significantly shorter progression-freesurvival (PFS; median,7months; CI,3-8months) compared with those whose tumors lowexpression of HOXA9(median,43months; CI,34-46months; P<0.01). Cox stepwiseproportional hazards model involving the IRS score of HOXA9and survivin expressionand eight clinical parameters (age, gender, pathologic grade, Kamofsky performance sore,largest tumor diameter, extent of resection, type of adjuvant treatment, intra-tumornecrosis) identified five prognostic variables including pathologic grade (P=0.000), KPS(P=0.000), extent of resection (P=0.015), largest tumor diameter (P=0.000) and HOXA9expression (P=0.007). Therefore, HOXA9activation is a novel, independent, and negativeprognostic marker in human gliomas. The results showed that the HOXA9locates innucleus and cytoplasm of glioma cells, especially in nucleus; A weak to strong range ofHOXA9staining with increasing pathologic grade of gliomas at the mRNA and proteinlevels, especially recurrent GBM.2. Construct HOXA9RNAi expression lentivirus to infect T98G cells, and effects of HOXA9down-regulation on T98G cells proliferation, apoptosis and invasion in vitroAccording to HOXA9cDNA sequence, the specific RNAi fragments targetingHOXA9gene were designed and synthesized, which were cloned into pLKO.1-HOXA9vector of HOXA9shRNA was constructed. After lentiviral packaging, HOXA9RNAiexpression lentivirus infected T98G cells. pLKO.1-HOXA9shRNA-1, pLKO.1-HOXA9shRNA-2and pLKO.1-NC were transfected into T98G cells. After puromycin selection,stably transfected cell lines including T98G-S1(T98G shRNA-1), T98G-S2(T98GshRNA-2) and T98G-NC (negative control group) were established. To detect expressionof HOXA9mRNA and protein in T98G-S1, T98G-S2and T98G-NC using qRT-PCR andWestern blot. The result showed that expressions of HOXA9in T98G cells weresignificantly inhibited. Based on T98G-S1, T98G-S2, and T98G-NC cells, we found thatthe downregulation of HOXA9gene expression significantly inhibited proliferation,migration and invasion of glioma cells.3. Overexpression of HOXA9is partially associated with MGMT-independentTMZ-resistance in glioblastoma cells via phosphoinositide-3-kinase pathways andNF-kappaB pathwaysIn the158primary glioma specimens,50recurrent glioma specimens and5gliomas’cells (U87MG, U251, A172, T98G and BT325) by qRT-PCR and western blot, we foundthat the expressions of MGMT mRNA and protein were very lower or hardly detected insome gliomas’ specimens and BT325cell, however, they were TMZ resistanced. Giventhat HOXA9plays an important role on BT325cells with TMZ-resistance through PI3Kpathways, we investigated whether knockdown of HOXA9or block of PI3K pathways canenhance BT325cells to TMZ-sensitivity and subsequently reverse TMZ-resistancethrough repressed HOXA9expression in T98G and BT325cells. The results showed thatknockdown of HOXA9was done with siRNA that resulted in at least70%inhibition ofHOXA9mRNA and protein expression72h posttransfection in T98G and BT325cells asassessed by PI3K signaling specific inhibitor LY294002, and LY294002also enhanced theprotein expression of NF-kappaB p65. Therefore, overexpression of HOXA9is associatedwith MGMT-independent TMZ-resistance in glioblastoma cells via PI3K pathways and NF-kappaB pathways.4. Overexpression of HOXA9is associated with RT-resistance in glioblastoma cells viaNF-kappaB pathwaysIn the158primary glioma and50recurrent glioma patients’ clinical follow-upparameters, we found that the patients with high-grade gliomas were higher RT-resistance.Studies showed that the NF-kappaB signaling pathway plays an important role in tumordevelopment and progression, and results in unsatisfactory treatment outcome. Inhibitionof the NF-kappaB signaling cascade may sensitize the resistant cancer cells toradiotherapy. Given it was an important role to RT-resistance in GBM by NF-kappaBsignaling pathways, knockdown of HOXA9by siRNA could be enhanced radiosensitive.When glioblastoma T98G and BT325cells upon radiated with4Gy treatment, we foundthat the mRNA and protein expression of NF-kappaB p65increased as well andknockdown of HOXA9was done with siRNA that resulted in at least70%inhibition ofNF-kappaB p65and I B mRNA and protein expression48h posttransfection in T98Gand BT325cells upon the same radiated treatment. These results support thatoverexpression of HOXA9is partially associated with RT-resistance in glioblastoma cellsvia NF-kappaB pathways.In sum, HOXA9gene may play an important role in the pathogenesis andproliferation of human gliomas. HOXA9is not only a negative prognostic biomarker ofbrain gliomas, but also a potential candidate target for gene therapy in malignant gliomas.