TRIM24Promotes Glioma Growth And Enhances Chemoresistance

Gliomas include astrocytoma, oligodendroglioma, ependymoma, mixed glioma, etc.The most common primary malignant tumors in the central nervous system are malignantgliomas, which include anaplastic astrocytoma (AA, WHO grade III), anaplasticoligodendroglioma (AO, WHO grade III) and glioblastoma multiforme (GBM, WHOgrade IV astrocytoma). GBM accounts for the majority of malignant gliomas. Despitegreat advances in surgical techniques, radiotherapy strategies and chemotherapymodalities recently, the median survival is only2to3years for patients with AA,3to5years for patients with AO, and12to15months for patients with GBM. These dismaloutcomes render malignant gliomas urgent subjects of cancer research. Identification oftumor biomarkers may improve the accuracy of prognostic prediction, increase theefficacy of conventional treatment and pave the way for molecular therapeutic approaches.Tripartite motif-containing24(TRIM24), formerly known as transcriptionintermediary factor1(TIF1), is the founding member of the TIF family, which ischaracterized by a conserved N-terminal tripartite motif consisting of a RING domain, twoB-box zinc fingers and a coiled-coil region. The C-terminal tandem plant homeodomain(PHD) and Bromodomain (Bromo) distinguish the TIF family members from other TRIM proteins. TRIM24targets p53degradation via its RING domain, participates in chromatinremodeling via its PxVxL motif, interacts with nuclear receptors via its LxxLL motif andrecognizes a specific histone signature via its PHD-Bromo domains. Due to its versatility,close associations of TRIM24with several cancers have been unveiled in recent years.Nevertheless, it acts as a tumor suppressor in hepatocellular carcinoma while promotescarcinogenesis in myeloid leukemia and breast cancer.The array-based comparative genomic hybridization had detected TRIM24geneamplification in about1/3GBM cases, especially for patients older than50years,indicating that TRIM24might participate in glioma development and progression. Toaddress the possible correlation between TRIM24and gliomas, we collected surgicalsamples to measure TRIM24expression levels therein, conducted a series of in vitro andin vivo analysis to find out its cellular and molecular mechanisms in gliomas, and exploredits potential clinical significance as well.1. TRIM24is overexpressed in gliomas in a grade-dependent pattern andparticipates in GBM recurrenceImmunohistochemistry was used to evaluate TRIM24expression levels in normalbrain tissue (n=15), pilocytic astrocytoma (PA, WHO grade I)(n=24), diffuse astrocytoma(DA, WHO grade II)(n=57), AA (n=63), newly diagnosed GBM (n=297), recurrent GBM(n=48) and oligodendroglioma (n=44). Normal brain glia and oligodendrogliomapresented negative TRIM24immunological staining or weak staining in few cells,whereas TRIM24was overexpressed in astrocytomas. Moreover, as the tumor pathologicalgrade escalated, TRIM24levels were elevated, except that no significant differenceexisted between PA and DA. By using proteins extracted from frozen preserved tissuesamples, this specific expression profile was further validated by Western blot. Pairedcomparison between newly diagnosed GBM and recurrent GBM samples obtained fromthe same patients showed that TRIM24expression generally increased at the time of GBMrecurrence, especially for those that presented relatively low levels previously. Theseresults suggest that TRIM24may be involved in glioma development and its expressionlevels are positively associated with glioma malignancy. 2. TRIM24expression levels are inversely correlated with prognosis of patients withne wly diagnosed GBMsFollow-up began in2009and ended in2013, and during which time, new cases wereadded intermittently. A total of297newly diagnosed GBM patients were included andtheir age, sex, extent of resection, KPS, chemotherapy status, overall survival (OS) andprogression-free survival (PFS) time were recorded. The Kaplan-Meier method wasemployed to build survival curves for patients whose tumors presented low TRIM24expression and those whose tumors presented high TRIM24expression. The median OSfor patients with low TRIM24expression was13.9months (95%CI,11.5to16.3), ascompared with11.0months (95%CI,10.6to11.4) for those with high express ion. Themedian PFS for patients with low and high TRIM24expression was6.5months (95%CI,5.5to7.5) and5.3months (95%CI,4.8to5.8), respectively. The log-rank testdemonstrated significant differences in both OS and PFS between subjects with low andhigh TRIM24expression (P<0.001). The multivariate Cox proportional hazards regressionmodels found that KPS, age and TRIM24level emerged as significant independentprognostic factors for OS, whilst KPS and TRIM24level also possessed independentpredictive value for PFS. These results suggest that detection of TRIM24levels mayimprove the predictive strength of currently available prognostic clinical indicators and itslevels may be further positively associated with tumor malignancy within one gliomapathological grade-GBM, in view of its highly heterogeneous characteristics.3. TRIM24promotes glioma growth in vitro and in vivoTo study the mechanisms whereby TRIM24enhances glioma malignancy, weconstructed retroviruses either separately expressing two shRNAs targeting TRIM24orone negative control shRNA, and lentiviruses either expressing human TRIM24gene ornegative control. Thereafter, human GBM cell lines were infected with these viruses toaddress whether the changes of TRIM24expression levels had impact on glioma growthor not. Cell proliferation assay showed that knockdown of TRIM24prolonged thedoubling time of T98and U87cells. Cell cycle assay indicated that downregulation ofTRIM24in T98cells caused a reduction in the proportion of G2/M phase, whereas upregulation of TRIM24in U251cells led to an increase in G2/M proportion. Soft agarcolony formation assay demonstrated that TRIM24knockdown diminished the numberand size of clones formed by T98and U87cells. Xenograft experiments showed that oncestably infected with retroviruses expressing shRNA targeting TRIM24, T98cells failed todevelop subcutaneous xenograft in nude mice, whereas the cells infected with negativecontrol retroviruses possessed the ability of xenograft formation. These results suggest thatTRIM24promotes glioma growth and targeting TRIM24might be utilized to crippleglioma progression.4. TRIM24activates PI3K/Akt signaling to enhance glioma growthSince PI3K/Akt and Raf/MEK/ERK are the major pathways particip ating in gliomagrowth, Western blot was employed to assess the possible changes in the levels ofactivated Akt and ERK when TRIM24was downregulated, and rescue experiments werecarried out. Knockdown of TRIM24resulted in a reduction of p-Akt level, whereas thephosphorylation of ERK increased concomitantly, possibly reflecting that the inhibitioneffect of activated Akt on Raf/MEK/ERK signal transduction was relieved somewhat. Theinfluence of TRIM24knockdown on Akt and ERK activation was rescued byreintroduction of TRIM24into downregulated cells via infecting with lentivirusexpressing human TRIM24gene. MTT assay showed that compared with TRIM24downregulated cells, the proliferation of TRIM24overexpressed cells were more sensitiveto the pan-PI3K inhibitor LY294002and siRNA targeting PIK3CA as well. Real-timeRT-PCR and Western blot demonstrated a decrease in PIK3CA expression upon TRIM24knockdown. ChIP assay demonstrated TRIM24binding to the promoter of PIK3CA genein T98and U87cells. Reintroduction of wild-type TRIM24rescued PIK3CA and p-Aktlevels in TRIM24downregulated cells, whereas mutant TRIM24deleted of PHD-Bromodomains had no rescue effect. These results suggest that the growth promoting effect ofTRIM24can be emanated through the PI3K/Akt pathway.5. TRIM24enhances glioma resistance to chemotherapyApart from tumor growth, chemoresistance is a major cause for the progression andrecurrence of malignant gliomas. To investigate whether TRIM24has impact on cell response to temozolomide (TMZ) or not, GBM cell lines, infected with either retrovirusexpressing shRNA targeting TRIM24or negative control retrovirus, were exposed to TMZ.MTT assay showed that knockdown of TRIM24increased the cytotoxicity of TMZ to T98and U87cells. Plate colony formation assay demonstrated that TRIM24knockdown hadadditive effect to TMZ treatment. Significant cleavage of procaspase-7, apparent TUNELsignal and evident Annexin V staining emerged when TRIM24depleted T98cells wereexposed to TMZ for one week, whereas minimal apoptosis was observed in control cells.Xenograft models showed that TRIM24knockdown increased the therapeutic efficacy ofTMZ administration in vivo. In the clinical context, for GBM patients with low TRIM24expression, those who received chemotherapy had a better prognosis than those who didnot receive chemotherapy. On the other hand, patients with high TRIM24expression gotno significant benefit from chemotherapy. These results suggest that TRIM24enhanceschemoresistance, and combination of TRIM24knockdown and chemotherapy might havegreater therapeutic efficacy for patients with GBMs.6. TRIM24regulates MGMT expression through PI3K/Akt/NF-κB signalingSince DNA repair enzyme MGMT plays a key role in TMZ resistance, the possiblechanges in its expression levels upon TRIM24knockdown were evaluated. Western blotshowed that knockdown of TRIM24led to a reduction of MGMT protein in T98cells,while real-time RT-PCR addressed that TRIM24knockdown reduced MGMT expressionin the transcriptional level. Although ChIP assay did not show TRIM24binding to theMGMT promoter, dual-luciferase reporter assay indicated that NF-κB activity decreasedupon TRIM24downregulation, similar to the PI3K/Akt inhibitor LY294002. The effect ofTRIM24knockdown on MGMT expression was rescued by reintroduction of TRIM24,whereas transfection of siRNA targeting RelA, which encodes the p65subunit of NF-κB,abolished the rescue action of TRIM24. These results suggest that TRIM24regulatesMGMT expression by means of, or at least partly through, PI3K/Akt/NF-κB signaling.