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Tumor and Stem Cell Biology

Arginine Methylation of SREBP1a via PRMT5 Promotes De Novo Lipogenesis and Tumor Growth

Liu Liu, Xiaoping Zhao, Li Zhao, Jiajin Li, Hao Yang, Zongping Zhu, Jianjun Liu and Gang Huang
DOI: 10.1158/0008-5472.CAN-15-1766 Published March 2016

Abstract

Dysregulation of the sterol regulatory element-binding transcription factors sterol regulatory element-binding protein (SREBP) and SREBF activates de novo lipogenesis to high levels in cancer cells, a critical event in driving malignant growth. In this study, we identified an important posttranslational mechanism by which SREBP1a is regulated during metabolic reprogramming in cancer cells. Mass spectrometry revealed protein arginine methyltransferase 5 (PRMT5) as a binding partner of SREBP1a that symmetrically dimethylated it on R321, thereby promoting transcriptional activity. Furthermore, PRMT5-induced methylation prevented phosphorylation of SREBP1a on S430 by GSK3β, leading to its disassociation from Fbw7 (FBXW7) and its evasion from degradation through the ubiquitin–proteasome pathway. Consequently, methylation-stabilized SREBP1a increased de novo lipogenesis and accelerated the growth of cancer cells in vivo and in vitro. Clinically, R321 symmetric dimethylation status was associated with malignant progression of human hepatocellular carcinoma, where it served as an independent risk factor of poor prognosis. By showing how PRMT5-induced methylation of SREBP1a triggers hyperactivation of lipid biosynthesis, a key event in tumorigenesis, our findings suggest a new generalized strategy to selectively attack tumor metabolism. Cancer Res; 76(5); 1260–72. ©2016 AACR.

Introduction

De novo lipogenesis is highly activated in many types of malignant tumors, resulting in intracellular unusual lipids accumulation, which is involved in tumor progression (1–3). In contrast to normal cells, in malignant tumors, lipids are mainly derived from new synthesis instead of extracellular lipids uptake. Lipids contribute to multiple aspects of tumor biology because of their diverse biologic roles. However, the underlying mechanisms of the reprogramming of de novo lipogenesis remain unclear.

At the molecular level, excessive activation of sterol regulatory element-binding proteins (SREBP) transcription factors directly upregulates lipogenic enzymes to trigger de novo lipogenesis in tumors (4). In mammals, three isoforms of SREBP proteins have been identified. SREBP1a and SREBP1c are produced from the SREBF1 gene, whereas the SREBF2 gene encodes SREBP2 (5). SREBP1 primarily regulates genes involved in the synthesis of fatty acids, triglycerides, and phospholipids, whereas SREBP2 preferentially activates genes for cholesterol synthesis (5–7). SREBP is essential to maintain the balance between protein and lipid biosynthesis, and in turn, it is involved in the progression, migration, or poor prognosis of cancer (8–10). Drugs targeting SREBP1-triggered upregulation of lipogenesis have been proven as anticancer agents in glioblastoma therapy (11). Recent evidence indicates that posttranslational modifications (PTM) of SREBP1 are essential for de novo lipogenesis. PTMs influence the function of SREBP in protein degradation or DNA binding (12–14). However, it is unknown whether SREBP is a methylated protein.

Protein arginine methylation, catalyzed by members of the protein arginine methyltransferase (PRMT) family, exists universally in the nucleus and cytoplasm (15). In mammals, according to the methylation products, PRMTs are classified into three types (16, 17). As the only well-characterized type II PRMT that generates symmetric dimethyl arginine (SDMA) modification, PRMT5 is involved in tumor progression via both epigenetic silencing and organelle biogenesis (18, 19). PRMT5 overexpression was found in various types of cancer, and PRMT5 was considered a target for cancer therapy (20–22). However, little is known about the effect of PRMT5 on tumor metabolic regulation.

In this study, we identified new molecular mechanisms of lipid reprogramming in cancer cells. As a binding partner of SREBP1a, PRMT5 modulates de novo lipogenesis in an SREBP1a-dependent manner. The underlying mechanism involves arginine methylation modification and subsequently inhibition of proteasome degradation of SREBP1a. Our in vivo and in vitro data indicated that specific arginine methylation of SREBP1a by PRMT5 was essential for cancer development. Moreover, the arginine methylation status of SREBP1a in tumors correlated with the malignant progression of human hepatocellular carcinoma (HCC). These data provided a rationale for using the PRMT5–SREBP1a pathway as a potential target for therapeutic intervention in carcinomas with abnormal intercellular lipid accumulation.

Materials and Methods

Cell culture and reagents

The cells used in our research were purchased through ATCC. All cell lines were passaged fewer than 6 months after purchase, and authenticated by short tandem repeat analysis. HepG2, HEK293T, MCF-7, and HeLa cells were cultured in DMEM (Gibco). SW480 and A549 cells were maintained in RPMI1640 (Gibco). Routinely, media were supplemented with 10% FBS (Hyclone), 100 mg/mL penicillin (Gibco), and 100 mg/mL streptomycin sulfate (Gibco). In low lipid situations, charcoal-stripped FBS (CSFBS; Gibco) was used instead of FBS. Lipofectamine 2000 reagent (Invitrogen) was used for transient transfection. The siRNA sequences used are shown in Supplementary Table S1.

GST pull-down assay

GST-tagged PRMT5 and mSREBP1a derivatives were expressed in Escherichia coli (E. coli) BL21-DE3 cells (Sangon Biotech) by induction with 0.4 mmol/L IPTG (Ameresco). GST-tagged proteins were purified using Glutathione Sepharose 4B (GE Healthcare). Different GST recombinant protein beads were incubated with appropriate cell lysates for 1 hour at 4°C before immunoblotting.

Mass spectrometry

GST-PRMT5–bound proteins were resolved by SDS–PAGE and stained with Coomassie blue R250 (Dingguo). We excised approximately 72 kDa protein bands from the SDS-PAGE gels and subjected them to liquid chromatography coupled with electrospray ionization and a hybrid quadrupole linear ion-trap mass spectrometry (LC-ESI-LTQ-MS; Thermo Fisher Scientific).

For intracellular lipids assays, cells were lysed in cell lysis buffer P0013 (Beyotime) at 4°C. Lipid molecules in lysates were tested by gas chromatography-mass spectrometry (GC-MS; Agilent Technologies). The quantity of the lipids was normalized to the total protein levels.

In vitro methylation assay

Enzymes GST-PRMT5 and substrates GST-mSREBP1a were expressed in E. coli BL21-DE3 (Sangon Biotech) and purified by Glutathione Sepharose 4B (GE Healthcare). GST fusion proteins were eluted using 10 mmol/L reduced glutathione (Sigma-Aldrich). Subsequently, enzymes and substrates were mixed in methylation reaction buffer (50 mmol/L Tris, 0.1 mmol/L EDTA, 50 mmol/L NaCl; Sangon Biotech), with or without 16 μmol/L methyl donor S-adenosyl-methionine (SAM; New England Biolabs), to a total volume of 40 μL and incubated at 30°C for 90 minutes. The reactions were stopped by the addition of sample loading buffer for immunoblotting.

Xenograft study

Different viruses were infected into HepG2 cells to form stable ectopic mSREBP1a-expressing HepG2, with or without PRMT5 knockdown. Different HepG2 cells were injected subcutaneously into the shoulder sides of BALB/c nude mice (20, male, 5-week-old, Shanghai Laboratory Animal Center, Shanghai, China). Mice were sacrificed 21 days after injection, and the subcutaneous tumors were removed, weighed, and their sizes were measured. Mice were handled in accordance with the guidelines published in the Animal Ethics Committee of Renji Hospital Shanghai Jiao Tong University School of Medicine (Shanghai, China).

Statistical analyses

Data were analyzed using GraphPad Prism 6 software (GraphPad Software) or SPSS 19.0 (SPSS). Quantitative data are expressed as the mean ± SD of at least three independent experiments. Statistical differences between groups were assessed by the unpaired two-tailed t test or the ANOVA test. The χ2 test was used for rate comparisons. Kaplan–Meier analysis was used in the survival duration assay. Multivariate Cox regression analysis was performed to identify independent risk factors. Asterisk means a P value < 0.05 and was considered statistically significant.

Results

PRMT5 interacts with mSREBP1a in an enzyme activity–dependent manner

On the basis of the importance of PRMT5 in tumor progression (23–25), we asked which regulatory signaling factors are directly regulated by PRMT5. GST-tagged PRMT5 was used as a bait, followed by MS. Unexpectedly, we identified the mature form of SREBP1a (mSREBP1a, 68 kDa) as a PRMT5-interacting protein (Fig. 1A). The associations between proteins PRMT5 and mSREBP1a at both endogenous and exogenous levels were confirmed by coimmunoprecipitation assays (Fig. 1B–E and Supplementary Fig. S1A). Furthermore, in HepG2 cells, endogenous SREBP1 was overlapped with PRMT5 (Supplementary Fig. S1B). We found that endogenous PRMT5 could be copurified with both two isoforms (1a and 1c) of SREBP1 (Fig. 1F). SREBP1a is the predominant SREBP1 in cultured cancer cells and has much higher transcriptional activity with its extended N-termini (4, 26). Therefore, we focused our research on mSREBP1a.

Figure 1.
Figure 1.

PRMT5 interacts with mSREBP1a. A, HEK293T cell lysates were incubated with purified GST-PRMT5 protein. Bound proteins were eluted and analyzed by SDS-PAGE. The silver staining showed the location of GST-PRMT5 and its associated protein mSREBP1a (∼72 kDa). B, HEK293T extracts were immunoprecipitated with anti-PRMT5 or control rabbit IgG and immunoblotted by anti-SREBP1. Ten percent input of extracts is shown. C, extracts of HEK293T cotransfected with Flag-mSREBP1a and HA-PRMT5 for 48 hours were immunoprecipitated with anti-Flag or mouse IgG and immunoblotted by anti-HA. Ten percent input of extracts is shown. D, extracts of HEK293T transfected with Flag-PRMT5 for 48 hours were immunoprecipitated with anti-Flag or mouse IgG and immunoblotted by anti-SREBP1. Ten percent input of extracts is shown. E, lysates of HEK293T transfected with Flag-mSREBP1a for 48 hours were immunoprecipitated with anti-Flag or mouse IgG and immunoblotted by anti-PRMT5. Ten percent input of lysates is shown. F, lysates of HEK293T transfected with Flag-mSREBP1a or Flag-mSREBP1c for 48 hours were immunoprecipitated with Flag M2 affinity gel and immunoblotted with anti-PRMT5. Ten percent input of respective proteins is shown. G, lysates of HEK293T cotransfected with Flag-mSREBP1a and HA-PRMT5 WT or ΔM for 48 hours were immunoprecipitated with Flag M2 affinity gel and immunoblotted with anti-HA. Ten percent input of respective proteins is shown. H, lysates from HEK293T transfected with Flag-mSREBP1a for 48 hours were incubated with either purified protein GST or GST-PRMT5 derivatives. Bound Flag-mSREBP1a proteins were immunoblotted by anti-Flag. Same amount of the proteins were resolved in SDS–PAGE for Coomassie blue staining. I and J, after 48 hours of transfection, the colocation (yellow) of exogenous Cherry-mSREBP1a (red) and EGFP-PRMT5 WT or ΔM (green) in HepG2 cells were analyzed using a fluorescence microscope (scale bar, 20 μm).

Notably, the exogenous mSREBP1a could interact with wild-type (WT) PRMT5 WT, but not ΔM (27), a mutant of PRMT5 that lacked the sequence GAGRG at 365–369 aa, which is essential for its enzymatic activity (Fig. 1G). In the GST pull-down assay, we further found that amino acids 1–369 of PRMT5 were required for the interaction with mSREBP1a, yet 1–364 aa truncated PRMT5 or 365–369 aa (GAGRG) deleted PRMT5 did not interact with mSREBP1a in vitro (Fig. 1H). Again, Cherry-tagged mSREBP1a was colocalized with EGFP-tagged PRMT5 WT, but not ΔM (Fig. 1I and J).

Taken together, we proved that PRMT5 interacts physically with mSREBP1a, and that the enzyme activity of PRMT5 is essential for the association with mSREBP1a.

PRMT5 symmetrically dimethylates mSREBP1a on arginine 321

The requirement for the PRMT5 enzymatic region in the interaction with mSREBP1a led us to examine whether PRMT5 is responsible for direct arginine methylation of mSREBP1a. Our results showed that in the presence of adenosine-2′,3′-dialdehyde (Adox), a specific inhibitor of PRMTs, the SDMA modification of mSREBP1a was dramatically reduced (Fig. 2A). In the mSREBP1a protein sequence, we found three arginines (Arg321, Arg446, Arg480) flanked by glycines and resembling canonical methylation sites (Fig. 2B). To check for SDMA modification of these residues, we created a series of mSREBP1a mutants carrying a single lysine mutation at each of these sites separately, namely, R321K, R446K, and R480K. Mutation at Arg321, but not at Arg446 or Arg480, resulted in decreased SDMA modification of mSREBP1a (Fig. 2C). Furthermore, PRMT5 knockdown caused reductions in the amount of symmetric dimethylated arginine in WT, R446K, and R480K mutants of mSREBP1a, whereas had no effect on the SDMA level of R321K (Fig. 2D). We further demonstrated that purified WT mSREBP1a but not R321K protein was symmetrically dimethylated by PRMT5 WT, while PRMT5 ΔM could not dimethylate mSREBP1a in vitro (Fig. 2E). There are two type II PRMTs (PRMT5 and PRMT9) that could catalyze the SDMA modification of proteins (17). Our research proved that PRMT5 was the only type II PRMT promoting SDMA modification of mSREBP1a as well as interacting with mSREBP1a (Supplementary Fig. S1C and S1D). Thus, we demonstrated that mSREBP1a was symmetrically dimethylated by PRMT5 on R321.

Figure 2.
Figure 2.

Arginine methylation of mSREBP1a on 321R by PRMT5 promotes its transcriptional activity. A, HepG2 cells were treated with or without Adox (0.5 mmol/L) for 8 hours after transfection with Flag-mSREBP1a for 36 hours. Cell lysates were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-SDMA, which was used to analyze SDMA marks on mSREBP1a. B, the location of three RG motifs in mSREBP1a. The lysines (K) replacing arginine (R) sites are indicated in gray. C, extracts of HepG2 transfected with Flag-mSREBP1a derivatives for 48 hours were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-SDMA. D, lysates of HepG2 transfected with Flag-mSREBP1a derivatives in the absence or presence of PRMT5 for 72 hours were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-SDMA. E, either GST-mSREBP1a WT or R321K were incubated with GST-PRMT5 WT or ΔM in methylation assays with or without SAM before immunoblotting with antibodies against SDMA, SREBP1, or PRMT5. F, the mRNA levels of the indicated genes in HepG2 transfected with siRNA-PRMT5-2 or siRNA-negative control (NC) for 36 hours were analyzed by real-time PCR. G, HepG2 cells were transfected with siRNA-PRMT5-1 or siRNA-PRMT5-2 (or siRNA-NC) for 36 hours before luciferase reporter assay. H, HepG2 cells were cotransfected with Flag-mSREBP1a WT or R321K (or empty vector as the control) plus siRNA-PRMT5 or siRNA-NC for 36 hours before luciferase reporter assay.

However, both mSREBP1a WT and R321K could interact with endogenous PRMT5 (Supplementary Fig. S1E). Using three truncated fragments of mSREBP1a (1–209 aa, 61–373 aa, and 323–487 aa), we further demonstrated that the 323–487 aa region of mSREBP1a (the common domain of the two SREBP1 isoforms) was required for PRMT5 interaction in vitro (Supplementary Fig. S1F).

PRMT5-mediated symmetric dimethylation induces SREBP1a transcriptional activity

To determine whether the PRMT5-mediated SDMA modification regulates SREBP1a function, we silenced PRMT5 using two specific siRNAs (siRNA-PRMT5-1, 2) and analyzed the transcript levels of SREBF1 and its target genes in HepG2 cells (Fig. 2F and Supplementary Fig. S2A). The target lipogenic genes [ATP citrate lyase (ACLY), fatty acid synthase (FASN), elongation of very long chain fatty acids protein 6 (ELOVL6), stearoyl-CoA desaturase (SCD1), and glycerol-3-phosphate acyltransferase (GPAT)] of SREBP1 were downregulated by PRMT5 knockdown to different degrees. In contrast, the level of SREBF1 mRNA was not regulated by PRMT5. We then used an SREBP1 reporter plasmid, which contained a SRE from the FASN promoter, to analyze the effect of PRMT5 on SREBP1 transcriptional activity. As shown in Fig. 2G, PRMT5 knockdown decreased the activity of SREBP1. Conversely, SREBP1 transcriptional activity was dramatically increased upon PRMT5 WT overexpression, whereas ΔM had no effect on FASN luciferase activity (Supplementary Fig. S2B). In addition, R321K mutation completely blocked the transcriptional activation of FASN mediated by mSREBP1a (Fig. 2H). Moreover, either WT or R321K mSREBP1a-meditated FASN transcription was compromised by PRMT5 knockdown (Fig. 2H). Similarly, the mRNA levels of lipogenic genes tested were induced by mSREBP1a WT but not R321K (Supplementary Fig. S2C–S2G). When PRMT5 was knocked down, mSREBP1a-mediated transcription of FASN, ELOVL6, SCD1, and GPAT was almost completely ablated by R321K mutation, whereas the transcription of ACLY was only partially blocked (Supplementary Fig. S2C–S2G). These data demonstrated that SREBP1a transcriptional activity was regulated by PRMT5 via SDMA modification on R321.

PRMT5 regulates mSREBP1a protein stability via the ubiquitin–proteasome pathway

On the basis of the result that PRMT5-induced methylation of mSREBP1a obviously upregulated its target genes transcription, whereas its own mRNA showed no change, we hypothesized that PRMT5 might regulate the protein mSREBP1a at posttranscriptional level via SDMA modification. As expected, PRMTs inhibitor Adox, as well as PRMT5 knockdown, apparently decreased the protein level of mSREBP1a, whereas the SREBP1a precursor (preSREBP1a) level showed no change (Fig. 3A and B). PRMT5 knockdown-mediated reduction of mSREBP1a was observed in several cancer cell lines (Supplementary Fig. S3A), which process could be inhibited by the proteasome inhibitor MG132 (Fig. 3C). In addition, PRMT5 WT, rather than ΔM overexpression, increased the mSREBP1a protein level (Supplementary Fig. S3B). Protein synthesis inhibitor cycloheximide was also used to observe the degradation of mSREBP1a. The results showed PRMT5 knockdown caused faster degradation of mSREBP1a than in control cells (Fig. 3D). In accordance with this fast degradation, the ubiquitination of mSREBP1a was significantly increased by PRMT5 knockdown (Fig. 3E).

Figure 3.
Figure 3.

PRMT5 regulates mSREBP1a protein stability. A, lysates of HepG2 treated with or without Adox (0.5 mmol/L) for 8 hours were immunoblotted with anti-SREBP1. β-Tublin provided the loading control. B, lysates of MCF-7 cells transfected with siRNA-PRMT5-1 (or siRNA-PRMT5-2) or siRNA-NC for 72 hours were immunoblotted with anti-SREBP1 or anti-PRMT5. β-Actin provided the loading control. C, HepG2 cells were transfected with siRNA-PRMT5 or siRNA-NC for 48 hours. MG132 (100 μmol/L) was then added for 6 hours, whereas DMSO worked as the control. Lysates were immunoblotted with anti-SREBP1 or PRMT5. β-Actin provided the loading control. D, HepG2 cells were transfected with siRNA-NC or siRNA-PRMT5 for 48 hours, followed by 0, 1/2, 1, 2, and 3 hours cycloheximide (CHX; 100 μg/mL) treatment. Lysates were immunoblotted with anti-SREBP1 or anti-PRMT5. mSREBP1a band intensity was normalized to β-actin, then normalized to the time = 0 controls. E, HepG2 cells were cotransfected with Flag-mSREBP1a, HA-ubiquitin (UB), and siRNA-PRMT5 (or siRNA-NC) for 66 hours, followed by 6 hours MG132 (100 μmol/L) treatment. Lysates were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-HA. F, lysates of HEK293T cotransfected with WT Flag-mSREBP1a or R321K and HA-PRMT5 (or empty vector) for 48 hours were immunoblotted with anti-Flag or anti-PRMT5. β-Tubulin provided the loading control. G, lysates of HepG2 cotransfected with HA-UB and WT Flag-mSREBP1a or R321K for 66 hours followed by 6 hours MG132 (100 μmol/L) treatment were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-HA. H, HEK293T cells were transfected with WT Flag-mSREBP1a or R321K for 36 hours. Cycloheximide (100 μg/mL) was then added and cells were harvested at 0, 1/2, 1, 2, 3, and 4 hours. Lysates were immunoblotted with anti-Flag. Flag-mSREBP1a levels was normalized to β-actin, then normalized to the time = 0 controls.

R321 of mSREBP1a was demonstrated as the major SDMA site modified by PRMT5. Our data showed that an R321K mutation blocked the increase of mSREBP1a protein level in response to PRMT5 overexpression (Fig. 3F). Conversely, R321K blocked the decrease of mSREBP1a induced by PRMT5 knockdown (Supplementary Fig. S3C). Besides, R321K showed higher level of ubiquitination than WT while degraded faster (Fig. 3G and H). To exclude the possibility that Lys321 ubiquitination induced the following degradation of mSREBP1a R321K (28), we mutated 321 arginine of mSREBP1a to alanine (R321A), and observed no difference of ubiquitin level between R321K and R321A (Supplementary Fig. S3D). Thus, we demonstrated that R321 SDMA modification by PRMT5 regulated the turnover of mSREBP1a via the ubiquitin–proteasome pathway.

The phosphorylation status of S430 modulates PRMT5-mediated degradation of mSREBP1a

As described above, PRMT5-induced SDMA modification on R321 affected the degradation of mSREBP1a. Previous studies indicate that ubiquitin ligase F-box and WD repeat domain-containing 7 (Fbw7) interacts with mSREBP1a and promotes its degradation via ubiquitin–proteasome pathway, which is dependent on the phosphorylation of T426 and S430 (28). Reasonably, we asked whether there was a cross-talk between PRMT5-induced methylation and T426 or S430 phosphorylation in mSREBP1a. Indeed, PRMT5 knockdown induced the serine phosphorylation of mSREBP1a, whereas decreased the SDMA level of mSREBP1a (Fig. 4A). However, threonine phosphorylation of mSREBP1a was unchanged (Fig. 4A). Consistently, mSREBP1a WT and its R321K/R446K/R480K mutants showed similar levels of threonine phosphorylation, whereas R321K had the highest serine phosphorylation level (Fig. 4B). To further analyze the role of serine phosphorylation in PRMT5 regulation of mSREBP1a, we generated a double mutant containing both S430A and R321K mutations. Interestingly, the R321K mutant showed a strong association with Fbw7, which was essential for the GSK3β-regulated mSREBP1a degradation, whereas the R321K/S430A mutation completely blocked their interaction (Fig. 4C). Meanwhile, the process that R321K accumulated much more ubiquitin than WT mSREBP1a was inhibited in R321K/S430A (Fig. 4D). Collectively, these data suggested that methylation deficiency of mSREBP1a might initiate subsequent posttranscriptional events, as indicated by S430 phosphorylation. Thus, the S430A mutation could rescue the decrease of mSREBP1a protein induced by R321K mutation (Fig. 4E). Surprisingly, although S430 phosphorylation deficiency rescued the decrease of FASN promoter activity induced by R321K mutation, the more stable mSREBP1a R321K/S430A did not induce stronger activation of FASN promoter than WT (Fig. 4F), which led us to compare the activity of WT mSREBP1a with R321K. MG132 was used to inhibit the degradation of mSREBP1a, and mSREBP1a R321K was proved to have a lower transcriptional activity than WT (Fig. 4G and Supplementary Fig. S3E).

Figure 4.
Figure 4.

The cross-talk between methylation and phosphorylation of mSREBP1a. A, lysates of HepG2 transfected with Flag-mSREBP1a and siRNA-PRMT5 (or siRNA-NC) for 72 hours were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with the indicated antibodies. P-Ser, phosphorylated serine; P-Thr, phosphorylated threonine. B, lysates of HepG2 transfected with indicated Flag-mSREBP1a derivatives for 48 hours were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with indicated antibodies (the same sample as Fig. 2C). C, lysates of HEK293T transfected with HA-Fbw7 and indicated Flag-mSREBP1a derivatives for 48 hours were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-HA. D, HEK293T cells were cotransfected with indicated Flag-mSREBP1a derivatives and HA-UB for 66 hours followed by 6 hours MG132 (100 μmol/L) treatment. Lysates were immunoprecipitated with Flag M2 affinity gel and normalized before immunoblotting with anti-HA. E, lysates of HEK293T transfected with indicated Flag-mSREBP1a derivatives for 48 hours were immunoblotted with anti-Flag. β-Actin provided the loading control. F, HepG2 cells were cotransfected with indicated Flag-mSREBP1a derivatives for 24 hours before luciferase reporter assay. G, HepG2 cells were transfected with Flag-mSREBP1a WT or R321K for 48 hours followed by 6 hours MG132 (100 μmol/L) treatment before luciferase reporter assay. Western blot analysis showed the protein level of Flag-mSREBP1a WT and R321K in HepG2. H and I, HepG2 cells were transfected with Flag-mSREBP1a WT or R321K for 30 hours before 6 hours 20 mmol/L LiCl (NaCl as the control) or 3 hours TWS119 (DMSO as the control) treatment. Lysates were immunoblotted with anti-Flag. β-Actin or β-tubulin provided the loading control.

As reported, GSK3β regulates the stability of protein mSREBP1a via S430 phosphorylation (28). Here, we confirmed that lithium chloride (LiCl) and TWS119, inhibitors of GSK3β, could stabilize both WT and R321K mutant of mSREBP1a (Fig. 4H and I). More importantly, the protein level was comparable between two mSREBP1a during GSK3β inhibition, whereas R321K degraded quicker than WT in control groups (Fig. 4H and I). Thus, the results showed that there is cross-talk between PRMT5-mediated SDMA modification at R321 and GSK3β-induced S430 phosphorylation in relation to mSREBP1a protein stability.

PRMT5 promotes de novo lipogenesis through mSREBP1a methylation

The main function of SREBP1a is triggering de novo lipogenesis, which led us to check the possible regulation of PRMT5 to lipid metabolism. Intracellular fatty acids were analyzed by MS. As shown in Fig. 5A, major fatty acids were decreased by PRMT5 knockdown. In addition, total triglycerides level was also decreased by PRMT5 knockdown (Fig. 5B). Conversely, WT PRMT5 overexpression increased the level of intracellular triglycerides, whereas the inactive mutant had no effect on triglycerides level (Fig. 5C). Furthermore, Oil red O staining showed that the number of intracellular lipids droplets was significantly decreased when PRMT5 silencing (Fig. 5D). Again, the enzyme motif was required for PRMT5-mediated accumulation of lipids droplets (Fig. 5E). Compared with WT, mSREBP1a R321K decreased the levels of fatty acids in HepG2 cells (Fig. 5F). In addition, R321K mutation caused mSREBP1a to have no effect on cellular triglyceride synthesis (Fig. 5G). Consistently, there were more lipid droplets in cells overexpressing WT mSREBP1a than in cells expressing R321K (Fig. 5H). These data suggested that PRMT5 increased lipogenesis of cancer cells via R321 methylation of mSREBP1a.

Figure 5.
Figure 5.

PRMT5 induces lipids synthesis via mSREBP1a methylation. A, lipids metabolites of HepG2 transfected with siRNA-PRMT5 or siRNA-NC for 96 hours were analyzed by GC-MS. B, triglycerides levels in HepG2 transfected with siRNA-PRMT5 or siRNA-NC for 96 hours were quantitated by colorimetric method and normalized by total protein levels. C, effects of Flag-PRMT5 WT or ΔM overexpression for 72 hours (empty vector worked as the control) on triglycerides level in HepG2. D, representative Oil red O staining images in HepG2 transfected with siRNA-PRMT5 or siRNA-NC for 72 hours (scale bar, 10 μm). E, representative Oil red O staining images in HepG2 transfected with Flag-PRMT5 WT or ΔM (or empty vector) for 48 hours (scale bar, 10 μm). F, lipids metabolites of HepG2 transfected with Flag-mSREBP1a WT or R321K for 72 hours were analyzed by GC-MS. G, triglyceride levels in HepG2 transfected with Flag-mSREBP1a WT or R321K for 72 hours were quantitated by a colorimetric method and normalized by total protein levels. H, representative Oil red O staining images in HepG2 transfected with Flag-mSREBP1a WT or R321K for 48 hours (scale bar, 10 μm).

Methylation of mSREBP1a by PRMT5 promotes the growth of cancer cells

Several types of cancer have shown higher levels of SREBP1, which correlated with malignant progression and poor prognosis. Interestingly, in lipid-deficient conditions, R321K mutation completely blocked the growth promotion effect of WT mSREBP1a to control cells (Fig. 6A). The colony formation assay also revealed that WT mSREBP1a, rather than R321K, significantly promoted the anchorage-independent growth of cancer cells (Fig. 6B). However, in the absence of PRMT5, the growth advantage induced by WT mSREBP1a was blocked, whereas the growth of cells expressing R321K was comparable with the control group, regardless the protein level of PRMT5 (Fig. 6A and B).

Figure 6.
Figure 6.

PRMT5 regulates cancer cells growth in vitro and in vivo via mSREBP1a methylation. A, after transfection with Flag-mSREBP1a WT or R321K (empty vector worked as a control) plus siRNA-PRMT5 or siRNA-NC in low-lipid situation (1% CSFBS), the numbers of HepG2 cells were counted at 48 and 96 hours. B, after 21 days of transfection with mSREBP1a WT or R321K (or empty vector) and siRNA-PRMT5 or siRNA-NC, colony formation assays of HepG2 cells were performed. C–F, nude mice were injected with 1 × 107 HepG2 cells stable expressing LV-mSREBP1a WT or R321K and LV-shRNA-PRMT5 or shRNA-NC: C, representative image of mice with xenograft tumors (L, left; R, right); D, image of tumors isolated from nude mice. E, the weight of tumors when mice were sacrificed. F, representative images of hematoxylin and eosin (H&E) staining and Ki67 staining of tumor samples (scale bar, 20 μm).

To determine whether the PRMT5-mediated methylation of mSREBP1a provided a growth advantage to cancer cells in vivo, we performed xenograft studies. Compared with the R321K group, tumors in mice injected with cells stably expressing WT mSREBP1a were larger and heavier (Fig. 6C–E). When PRMT5 was stably silenced, ectopic expression of either WT or R321K mSREBP1a resulted in comparable tumor volumes and weights (Fig. 6C–E). Furthermore, xenograft tumors were stained with an antibody against Ki67 to observe the proliferation status of tumors (29). Unsurprisingly, tumors derived from the R321K were less proliferative than those derived from cells expressing WT SREBP1a, whereas PRMT5 knockdown completely blocked the growth advantage conferred by SREBP1a in vivo (Fig. 6F).

Together, these in vivo and in vitro results indicated that the 321R methylation of mSREBP1a by PRMT5 promoted cancer cell proliferation and tumor growth.

R321 SDMA of mSREBP1a is increased in tumors and correlates with poor prognosis of HCC

The finding that the methylation of mSREBP1a by PRMT5 promotes cancer cell de novo lipogenesis and proliferation led us to examine R321 symmetric dimethylation in human carcinoma tissues. Ninety pairs of HCC samples with adjacent normal liver tissues were examined by immunostaining using an antibody against R321(SDMA)-mSREBP1a. The antibody was designed to specifically recognize the 321R-SDMA–modified SR(methylated)GEKRTAHNC motif of mSREBP1a, and its validity was verified (Supplementary Fig. S4A–S4D). The signal of R321(SDMA)-mSREBP1a was strongly displayed in the nuclei of HCC tissues, while weak and diffused in normal hepatic tissues (Fig. 7A). R321(SDMA)-mSREBP1a staining showed a significant increase of methylated mSREBP1a in tumors compared with normal tissues (Fig. 7B). R321(SDMA)-mSREBP1a expression in HCC tissues significantly correlated with tumor size (P = 0.002), histologic grade (P = 0.034), and tumor–node–metastasis (TNM) stage (P = 0.005; Supplementary Table S2). However, other clinicopathologic factors had no significant correlation with mSREBP1a methylation (Supplementary Table S2). The survival duration of 90 patients with low or moderate versus high (Fig. 7C–E) R321(SDMA)-mSREBP1a staining of tumors were compared. Patients whose tumors had low or moderate R321(SDMA)-mSREBP1a staining had a median survival of 54 and 28.5 months, respectively. However, the median survival of patients decreased to only 5.5 months if their tumors showed high levels of R321(SDMA)-mSREBP1a. The levels of R321(SDMA)-mSREBP1a were negatively correlated with survival duration of HCC patients (Fig. 7F). Furthermore, the IHC score of R321(SDMA)-mSREBP1a was recognized as an independent predictor for HCC patient survival (moderate vs. low, HR = 2.932, P = 0.034; high vs. low, HR = 9.857, P = 0.001). These results revealed a close relationship between 321R symmetric dimethylation of mSREBP1a and the aggressive clinical behavior of HCC.

Figure 7.
Figure 7.

R321 symmetric dimethylation of mSREBP1a is related with malignant progression of HCC. A, the intracellular accumulation of 321R(SDMA)-mSREBP1a antibody in HCC and normal hepatic tissues via immunohistochemical staining (scale bar, 40 μm). B, semiquantitative analyses of IHC data of 321R(SDMA)-mSREBP1a staining in HCC and normal hepatic tissues. C–E, according to the staining score, tumor samples were categorized into three groups: low (C), moderate (D), and high staining (E; scale bar, 40 μm) of 321R(SDMA)-mSREBP1a. H&E, hematoxylin and eosin. F, Kaplan–Meier analysis of overall survival depending on the 321R(SDMA)-mSREBP1a level in HCC tumor tissues. G, model of the effect of PRMT5 in de novo lipogenesis. In tumorous cells, PRMT5 directly interacts with mSREBP1a via motif GAGRG of the PRMT5 methyltransferase domain, subsequently symmetrically dimethylating mSREBP1a. The PRMT5-mediated symmetrical dimethylation on 321R inhibits GSK3β-mediated 430S-phosphorylation ubiquitin–proteasome degradation of mSREBP1a. Finally, stabled mSREBP1a overactivates lipogenic gene transcription, resulting in abnormal lipids accumulation in cancer cells, as well as malignant progression of carcinoma.

Discussion

Tumorigenesis involves not only uncontrolled cell proliferation, but also corresponding adjustments to energy metabolism to fuel cancer cell growth and division. The most prominent metabolic characteristic of tumors is the “aerobic glycolysis,” or the Warburg effect (30). Another characteristic metabolic change in cancer cells is the highly activated de novo lipogenesis (31). Most normal cells and tissues preferentially consume circulating lipids, whereas cancer cells largely rely on de novo lipogenesis, because of poor vascularization, hypoxia, or other limits that exist in the tumor microenvironment (32). As essential constituent of biologic membrane lipids, the excessive accumulation of intracellular fatty acids is essential for malignant progression of cancer.

During tumor development, target genes of transcriptional factor SREBP1 are commonly upregulated to trigger de novo lipogenesis (33). The PI3K/Akt signaling pathway, AMPK pathway, and P53 regulate the activity of SREBP1 during tumorigenesis (34–37). SREBP1 is synthesized as a precursor protein, which is further processed into a mature form in the Golgi apparatus (5). Ultimately, the phosphorylation-marked SREBP1 is ubiquitinated and then degraded in the proteasome. PTMs regulate the function of SREBP1 to influence cellular biochemical metabolism and biologic status. As far as we know, PTMs of SREBP1 involve phosphorylation, acetylation, and sumoylation (12, 14, 38). Interestingly, our data proved that mSREBP1a is also a methylated protein, and verified that Arg321 of mSREBP1a is the major residue modified by PRMT5 (Fig. 2).

As the major PRMT catalyzing the SDMA modification of proteins, PRMT5 is involved in tumorigenesis via histone methylation and transcriptional gene silencing (39, 40). Some nonhistone proteins are also directly methylated by PRMT5, resulting in tumor progression (41–43). In our study, when we used GST-PRMT5 as the bait to find its possible partners (∼72 kDa), mSREBP1a had the highest number of identified peptides via MS assay. We further found that PRMT5 regulates lipid metabolism and proliferation of cancer cells via its association with SREBP1a (Figs. 1, 2, 5, and 6). The symmetric dimethylation of SREBP1a by PRMT5 enhanced its stability, in which the ubiquitin–proteasome pathway is involved, thus powerfully promoting its transcriptional activity (Figs. 2 and 3).

Previously, we demonstrated that cyclin-dependent kinase 8 (CDK8) and its regulatory partner, cyclin C, mediated the phosphorylation on T402 of SREBP1c (the site corresponding to T426 of SREBP1a), resulting in SREBP1c degradation (44). SREBP1a can also be phosphorylated by GSK3β on T426 and S430. GSK3β-phosphorylated SREBP1a is recognized by the ubiquitin ligase Fbw7, which mediates its subsequent ubiquitination and proteasome degradation (28). Here, we revealed a novel posttranscriptional regulation mechanism of SREBP1a. Symmetrical dimethylation of SREBP1a at R321 inhibited GSK3β-mediated S430 phosphorylation (Fig. 4). As a result, methylated SREBP1a was much more stable and resistant to ubiquitination–proteasome degradation. Methylated mSREBP1a also has higher transcriptional activity than nonmethylated mSREBP1a. However, to reveal the detailed regulation mechanism of arginine methylation in the function of SREBP1a, further research is required.

As major lipids in cells, fatty acids are used for membrane biogenesis, whereas the oxygenolysis of triglycerides produces the direct source of energy, ATP, which is required for all life activities. Fatty acid C16:0 is catalyzed by ACLY, FASN, or ELOVL6, whereas the ELOVL6 also catalyzes C16:0, C16:1n7, C18:0, C18:1n9, and C18:1n7 via the elongation of certain fatty acids (45–47). SCD1 converts saturated fatty acids into monounsaturated fatty acids, such as C16:1n7, C18:1n9, and C18:1n7 (48), whereas GPAT catalyzes the esterification of long-chain acyl-CoAs in triglyceride synthesis. In our research, the transcription of lipogenic target genes of SREBP1a mentioned above, as well as the accumulation of intracellular triglycerides, fatty acids, and lipids droplets, were all inhibited by mSREBP1a methylation deficiency or PRMT5 knockdown (Fig. 5). Thus, it was reasonable to believe that PRMT5-methylated mSREBP1a could promote the transcription of lipogenic genes and subsequently intracellular lipids synthesis, which are essential for cancer development.

The upregulation by PRMT5 of de novo lipogenesis via mSREBP1a suggested that they affect cancer growth. Our in vivo and in vitro growth assays showed that cancer cells overexpressing WT mSREBP1a grew quicker than SDMA-deficient cells, whereas the growth advantage induced by mSREBP1a WT was blocked in the absence of PRMT5 (Fig. 6). As reported, HCC is one of the most common and lethal cancers with unusual intracellular lipid accumulation, which has been proven to be involved in tumor progression (49, 50). Herein, we found that 321R(SDMA)-mSREBP1a was expressed at higher levels in HCC tissues than in normal liver tissues (Fig. 7). Interestingly, 321R(SDMA)-mSREBP1a in HCC tumors mainly existed in the nucleus, which correlated with the ability of symmetrical dimethylated mSREBP1a to promote transcription via its endonuclear binding to DNA SREs (4). In addition, the R321K SDMA status of mSREBP1a in tumors positively correlated with large tumor size, high histologic grade, and advanced TNM stage of HCC (Supplementary Table S2). Significantly, our data identified high level of 321R SDMA modification of SREBP1a in tumors might predict short survival time of surgically treated HCC patients (Fig. 7).

Overall, our data identified mSREBP1a as a novel methylated protein, and further revealed the molecular mechanisms of SREBP1a-mediated reprogramming of lipid metabolism, which is essential for carcinoma progression (Fig. 7G). R321 symmetrical dimethylation of SREBP1a could be a sensitive biomarker to predict the prognosis of patients with carcinoma. Intervention in the PRMT5-mediated mSREBP1a arginine methylation might be a feasible and effective strategy in the metabolic treatment of cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: L. Liu, G. Huang

Development of methodology: L. Liu, X. Zhao, L. Zhao, J. Li, H. Yang, Z. Zhu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Liu, L. Zhao, H. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Liu, X. Zhao, L. Zhao, J. Li, G. Huang

Writing, review, and/or revision of the manuscript: L. Liu, X. Zhao, G. Huang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Liu, J. Liu, G. Huang

Study supervision: J. Liu, G. Huang

Grant Support

The study was supported by research grants from “973” Project (No. 2012CB932604), New Drug Discovery Project (No. 2012ZX09506-001-005), Shanghai First-class Discipline (Medical technology), and National Natural Science Foundation of China (No. 81372195, 81530053, 81471685, and 81471687).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

  • Received June 30, 2015.
  • Revision received November 23, 2015.
  • Accepted December 17, 2015.

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Cancer Research: 76 (5)
March 2016
Volume 76, Issue 5

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Arginine Methylation of SREBP1a via PRMT5 Promotes De Novo Lipogenesis and Tumor Growth
Liu Liu, Xiaoping Zhao, Li Zhao, Jiajin Li, Hao Yang, Zongping Zhu, Jianjun Liu and Gang Huang
Cancer Res March 1 2016 (76) (5) 1260-1272; DOI: 10.1158/0008-5472.CAN-15-1766
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