TRIM22 acts as a transcription factor to regulate the transcription of SPHK2 in the nucleus
We collected glioma samples of different grades from Xijing Hospital for detection (NBT; n = 10, WHO II; n=92, WHO III; n=65, WHO IV; n=70). We found that TRIM22 was highly expressed in GBM (Fig. 1a) and mainly located in the nucleus (Fig. 1b). In addition, cell lines U251MG, A172, TJ905, H4 with high expression of TRIM22 and primary GBM cell lines P1 and P2 were selected. Western blot (Fig. 1c) and immunofluorescence (Fig. 1d) showed that the expression of TRIM22 was mainly located in the nucleus. To study the role of TRIM22 in the nucleus, we used ChIP-Seq and enrichment analysis to find that TRIM22 binds to the DNA fragment of VEGF pathway related genes (MAPKAPK3, NFATC2, PP3CC, PRKCA, SPHK1, SPHK2), which may play the role of transcription factors (Fig. 1e, Additional file 1: Fig. S1a). In addition, ChIP- qPCR (Fig. 1f) in 293T and Yeast one hybrid (Fig. 1g) in vitro showed that TRIM22 could bind to the gene fragment of SPHK2. These results show that TRIM22 acts as a transcription factor to regulate the transcription of SPHK2 in the nucleus.
TRIM22 binds to the transcriptional region of SPHK2
To study the detailed binding sites of TRIM22 and SPHK2, according to the information of multiple transcripts of SPHK2, the transcription start site (TSS) and the position of peak in the sequencing results, four pairs of different primers were designed to explore potential binding sites (Additional file 1: S Table 3, Additional file 1: Fig. S1b). In 293T cells, ChIP-qPCR showed that TRIM22 bound most closely to the site interval of peak (Additional file 1: Fig. S1c). In addition, different TRIM22 truncates (Flag-TRIM22-ΔRING, Flag-TRIM22-ΔB-Box, Flag-TRIM22-ΔCC and Flag-TRIM22-ΔSPRY) were transfected into U251MG, A172, TJ905, H4 and two primary cells P1 and P2 for CUT&Tag. We were surprised to find that both TRIM22 (Fig. 2a) and its truncate (Additional file 1: Fig. S2a-b, Additional file 1: Fig. S3a-b) bind to SPHK2-4. In Yeast one hybrid, three repeated SPHK2-4 were used as bait sequence, and we also obtained the results consistent with those in vivo (Fig. 2b). To further explore how TRIM22 regulates the expression of SPHK2, Using CRISPR/Cas9 technology, we constructed TRIM22-knockout (KO) cell line (Additional file 1: Fig. S1d). qPCR results showed that knockout of TRIM22 reduced the mRNA expression of SPHK2, while overexpression of TRIM22 could promote its expression in four cell lines and two primary cells (Fig. 2c). These results suggest that TRIM22 protein positively regulates the expression of SPHK2.
TRIM22 positively regulates SPHK2/MAPK signaling pathway through K48-linked ubiquitination of Raf-1
Our results show that TRIM22 binds transcripts of SPHK2 and positively regulates SPHK2 expression. The enrichment results of ChIP-Seq also showed that TRIM22 may affect the VEGF pathway, and although both MAKP and PI3K/AKT pathways are in the VEGF pathway, only the MAPK pathway is downstream of SPHK2. (Additional file 1: Fig. S1a). To explore the effect of TRIM22 on SPHK2/MAPK pathway, using an ERK1/2 dependent transcriptional reporter containing three copies of an ERK1/2 response element located upstream of luciferase. Dual luciferase reporter assay showed that knockout of TRIM22 inhibited the activity of MAPK signal (Fig. 3a). Knockout of TRIM22 inhibited the expression of SPHK2/MAPK pathway proteins, including SPHK2, Ras, P-Raf-1Ser338, P-MEK1/2Ser217/221 and P-ERK1/2Thr202/Tyr204. Although the expression of phosphorylated protein was down regulated after knockout of TRIM22, there was no significant change in the expression of MEK1/2 and ERK1/2 (Fig. 3b, Additional file 1: Fig. S4a). Interestingly, the expression of Raf-1 was up-regulated but the mRNA level did not change (Fig. 3c). In addition, knockout of TRIM22 attenuated the degradation of Raf-1 (Fig. 3d, Additional file 1: Fig. S4b) and reduces K48-linked ubiquitination of Raf-1 (Fig. 3e, Additional file 1: Fig. S4c). These results suggest that TRIM22 regulates SPHK2/MAPK pathway and TRIM22 deletion attenuates K48-linked ubiquitination of Raf-1.
SPHK2 and MAPK inhibitors attenuated TRIM22-promoted proliferation of GBM
It has been reported that TRIM22 is highly expressed in GBM and promotes the proliferation of GBM[21]. To explore whether TRIM22 promotes GBM proliferation through SPHK2/MAPK signaling pathway, K145, SPHK2 inhibitor and Selumetinib, a highly potent non-ATP competitive MEK1/2 inhibitor, were used for in vivo and in vitro experiments. Overexpression of TRIM22 activated MAPK signal, while the fluorescence intensity decreased significantly after treatment with K145 and Selumetinib for 24 hours (Fig. 4a, Additional file 1: Fig. S5a). K145 and Selumetinib also inhibited the growth rate of cells promoted by TRIM22 (Fig. 4b, Additional file 1: Fig. S5b) and the expression rate of Ki67 (Fig. 4c, Additional file 1: Fig. S5c). In vivo, consistent with previous reports, A172 cell line and primary cell line P1 overexpressing TRIM22 accelerated the proliferation of GBM 15 days after tumor implantation. The tumor growth rate of NOD/SCID mice injected with K145 and Selumetinib daily intraperitoneally was slower (Fig. 4d). In addition, injection of K145 and Selumetinib improved the survival rate of mice (A172: Flag-EV vs. Flag-TRIM22; 44 days vs. 32.5 days; P<0.05; Flag-TRIM22 vs. Flag-TRIM22+K145 or Flag-TRIM22+Selumetinib; 32.5 days vs. 45.5 or 49 days; P<0.05; P1: Flag-EV vs. Flag-TRIM22; 45.5 days vs. 35 days; P<0.05; Flag-TRIM22 vs. Flag-TRIM22+K145 or Flag-TRIM22+Selumetinib; 35 days vs. 48 or 52 days; P<0.05; Fig. 4e). These results illustrate TRIM22 promotes proliferation of GBM through SPHK2/MAPK signaling pathway.
TRIM22 ubiquitinates Raf-1
TRIM22 contains four structures: RING, B-Box, CC and SPRY. To explore which structure is critical for TRIM22-mediated activation of MAPK signaling, we tested TRIM22-ΔRING, TRIM22-ΔB-Box, TRIM22-ΔCC and TRIM22-ΔSPRY in ERK1/2 luciferase reporter assays compared with TRIM22-FL and EV groups. The results showed that compared with TRIM22-FL group, only TRIM22-ΔRING group significantly reduced luciferase activity (Fig. 5a, Additional file 1: Fig. S6a). Furthermore, overexpression of TRIM22 increased the expression of key proteins in SPHK2/MAPK pathway, while the lack of RING domain inhibited this change. The change trend of Raf-1 protein was opposite to that of other proteins (Fig. 5b, Additional file 1: Fig. S6b). In CHX assay, overexpression of TRIM22 accelerated the degradation of Raf-1 and the results were similar in TRIM22-ΔRING group and Flag-EV group (Fig. 5c, Additional file 1: Fig. S6c). Both in vitro (Fig. 5d) and in vivo (Fig. 5e, Additional file 1: Fig. S6d), overexpression of TRIM22 induced endogenous K48-linked ubiquitination of Raf-1, while the RING domain deletion had no effect. These results illustrate that the change of Raf-1 is caused by ubiquitin degradation pathway induced by TRIM22.
To investigate whether TRIM22 binds directly to Raf-1. We performed Co-IP experiments using 293T cells transfected with Flag-TRIM22 and HA-Raf-1 and the results show that Flag and HA bring down each other (Fig. 5f). Furthermore, the physical binding of endogenous TRIM22 and Raf-1 was also confirmed in four cell lines and two primary cells (Fig. 5g, Additional file 1: Fig. S6e). To further study which domain of the two proteins plays a crucial role in binding, a series of deletion mutants of TRIM22 and Raf-1 were constructed and transfected into 293T cells. The CC and SPRY domains of TRIM22 are the necessary conditions for bringing down HA-Raf-1, while the C1d domain of Raf-1 is the necessary condition for bringing down f Flag-TRIM22 (Fig. 5h). Thus, TRIM22 promotes proteasomal-mediated degradation of Raf-1 via its RING domain, a negative regulator of MAPK signaling.
Raf-1 mediates the proliferation of GBM promoted by TRIM22
Phosphorylation at S338 site activates Raf-1 protein[22]. To further study the role of Raf-1 in TRIM22 promoted proliferation of GBM, we constructed a mutant of phosphorylated Raf-1, and the serine at site 338 was mutated to alanine (Raf-1S338A). The plasmid was packaged with lentivirus and transfected into cells for stable expression. Luciferase Report showed that Raf-1S338A inhibited the activation of MAPK pathway caused by overexpression of TRIM22 (Fig. 6a, Additional file 1: Fig. S7a). The growth of GBM cells (Fig. 6b, Additional file 1: Fig. S7b) and positive rate of Ki67 (Fig. 6c, Additional file 1: Fig. S7c) promoted by overexpression of TRIM22 was also inhibited by Raf-1S338A. Moreover, in vivo, compared with Flag-TRIM22 group, Raf-1S338A group showed significant inhibitory effect at 15 days after intracranial tumor implantation (Fig. 6d). The Flag-TRIM22 group had shorter survival, while there was no difference between Flag-EV and Raf-1S338A group (A172; Flag-EV vs. Flag-TRIM22+ Raf-1S338A; 41 days vs. 42 days; P>0.05; Flag-TRIM22 vs. Flag-TRIM22+ Raf-1S338A; 30.5 days vs. 42 days; P<0.05; P1; Flag-EV vs. Flag-TRIM22+ Raf-1S338A; 47 days vs. 42 days; P>0.05; Flag-TRIM22 vs. Flag-TRIM22+ Raf-1S338A; 29.5 days vs. 42 days; P<0.05; Fig. 6e). These results indicated that Raf-1 is a key effector in TRIM22-promoted growth of GBM cell populations in vitro and in vivo.
TRIM22 promotes K63-linked ubiquitination of ERK1/2
TRIM22 knockout decreased the expression of key proteins of SPHK2/MAPK pathway, including SPHK2, Ras and phosphorylated MEK1/2 and ERK1/2 (Fig. 3b, Additional file 1: Fig. S4a), while overexpression of TRIM22 promoted its up regulation (Fig. 5b, Additional file 1: Fig. S6b). The parallel change trend of these proteins and TRIM22 makes us speculate whether TRIM22 may directly regulate the activation of these proteins. To explore whether TRIM22 directly regulates these key proteins, anti-TRIM22 antibody was used for Co-IP experiment. We found that ERK1/2 was associated with TRIM22 in four GBM cell lines and two primary GBM cells (Fig. 7a, Additional file 1: Fig. S8a). The same results were obtained by Co-IP with anti-ERK1/2 antibody (Fig. 7b, Additional file 1: Fig. S8b). Furthermore, we found that K63 ubiquitination levels of ERK1/2 paralleled TRIM22 protein levels. TRIM22 knockout reduced K63-linked ubiquitination levels of ERK1/2 (Fig. 7c, Additional file 1: Fig. S8c) and overexpression TRIM22 promotes it. The specific mechanism may also be related to the RING domain of TRIM22(Fig. 7d, Additional file 1: Fig. S8d). These results suggested that TRIM22 might regulate activation of MAPK through ERK1/2 activation.