To elucidate the biological function of PRMT6 in colorectal cancer, we first analyzed its expression using data from the TCGA and GEO databases. The results indicated that PRMT6 is significantly upregulated in colorectal cancer tissues compared to adjacent normal tissues (Fig. 1A-C). Additionally, ROC curve analysis demonstrated that PRMT6 served as a promising marker for distinguishing between tumor and non-tumor colorectal tissues (Fig. 1D). The GEPIA database further confirmed that PRMT6 was overexpressed in multiple types of cancer (Figure S1A). To further validate the role of PRMT6 in colorectal cancer, we examined its expression levels in colorectal cancer tissues and adjacent normal tissues using western blot (7 pairs) and IHC assays (69 pairs). The results showed that intratumoral PRMT6 expression was significantly higher compared to normal tissues (Fig. 1E-G). Furthermore, patients with higher T and clinical stages exhibited elevated PRMT6 expression (Fig. 1H-J). PRMT6 expression showed no correlation with patients’ age or gender (Figure S2B and S2C). We then categorized colorectal cancer patients into two groups based on PRMT6 expression levels. Analysis of data from GSE36864 and our own clinical cohort revealed that patients with higher PRMT6 expression had worse overall survival (Fig. 1K, L). Interestingly, similar trends were observed in multiple types of cancers (Figure S1D). Overall, these findings suggested that PRMT6 might function as an oncogene in colorectal cancer.
Fig. 1PRMT6 functions as an oncogene in colorectal cancer. A-C. Expression levels of PRMT6 were compared between colorectal cancer and adjacent normal tissues using the TCGA and GSE24550 datasets. D. ROC curve analysis was used to evaluate the potential of PRMT6 as a diagnostic marker for distinguishing colorectal tumor tissues from normal tissues. E. Western blot analysis was performed to evaluate PRMT6 expression levels in colorectal cancer tissues compared to adjacent normal tissues. F, H. Representative images of immunohistochemistry (IHC) staining showing PRMT6 expression in colorectal cancer tissues and adjacent normal tissues.G. Statistical analysis comparing PRMT6 expression levels between colorectal cancer tissues and adjacent normal tissues. I, J. Statistical analysis of PRMT6 expression levels in patients categorized by T stage and clinical stage. K, L. Kaplan-Meier survival analysis of patients from the GSE36864 dataset, stratified by PRMT6 expression levels. Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
PRMT6 positively regulated the proliferation, migration and invasion ability of colorectal cancer cell in vitroTo test our hypothesis, we constructed SW48 and RKO cell lines stably expressing either an empty vector or flag-tagged PRMT6 using lentiviral transduction. Western blot analysis confirmed the successful expression of PRMT6 (Fig. 2A). Next, we assessed the proliferation ability of vector and PRMT6-overexpressing cancer cells. CCK8 assays showed that overexpression of PRMT6 significantly promoted cancer cell proliferation (Fig. 2B). Cells overexpressing PRMT6 exhibited higher cellular ATP levels than vector controls (Fig. 2C) and demonstrated more active DNA replication (Fig. 2D, E). Metastasis was a hallmark of advanced malignant tumors, so we also evaluated the effect of PRMT6 on cancer cell metastasis. Transwell assays demonstrated that PRMT6-overexpressing cells had enhanced migration and invasion capabilities compared to vector controls (Fig. 2F, G).
Fig. 2PRMT6 overexpression promotes cancer cell proliferation, migration, and invasion. (A) Western blot analysis was performed to confirm the successful construction of colorectal cancer cells stably expressing either vector or PRMT6. (B) CCK8 assays were conducted to evaluate the proliferation ability of colorectal cancer cells stably expressing either vector or PRMT6. (C) Cell viability assays were used to assess the intracellular ATP levels in colorectal cancer cells stably expressing either vector or PRMT6. D, E. BrdU assays were performed to evaluate the DNA replication ability of colorectal cancer cells stably expressing either vector or PRMT6. F, G. Transwell assays were conducted to assess the migration and invasion abilities of colorectal cancer cells stably expressing either vector or PRMT6.Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
Subsequently, we knocked down PRMT6 expression in SW48 and RKO cancer cells. Western blot analysis confirmed effective knockdown (Fig. 3A). Our data consistently demonstrated that silencing PRMT6 significantly inhibited cancer cell proliferation, DNA replication, and cellular ATP levels (Fig. 3B-E). Transwell assays further showed that cells with reduced PRMT6 expression exhibited lower migration and invasion abilities than control shNC cells (Fig. 3F, G). In summary, these findings demonstrated that PRMT6 positively regulated the proliferation, migration, and invasion capabilities of colorectal cancer cells.
Fig. 3Silencing PRMT6 significantly inhibits cancer cell proliferation, migration, and invasion. (A) Western blot analysis was performed to confirm the successful construction of colorectal cancer cells stably expressing either shNC or shPRMT6. (B) CCK8 assays were conducted to evaluate the proliferation ability of colorectal cancer cells stably expressing either shNC or shPRMT6. (C) Cell viability assays were used to assess the intracellular ATP levels in colorectal cancer cells stably expressing either shNC or shPRMT6. D, E. BrdU assays were performed to evaluate the DNA replication ability of colorectal cancer cells stably expressing either shNC or shPRMT6. F, G. Transwell assays were conducted to assess the migration and invasion abilities of colorectal cancer cells stably expressing either shNC or shPRMT6. Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
PRMT6 positively activated MYC signaling via stabilizing c-MYCTo elucidate the mechanism underlying PRMT6-mediated colorectal cancer progression, we first conducted Gene Set Enrichment Analysis (GSEA) using 2 TCGA and 10 GEO datasets. FDR < 0.25 and P < 0.05 were considered to indicate statistical significance. Finally, analysis from all datasets indicated that MYC signaling was more active in tumors with higher PRMT6 expression, which inspired us to investigate whether PRMT6 could regulate MYC signaling (Fig. 4A and Figure S2A). Western blot analysis showed that overexpression of PRMT6 significantly increased c-MYC expression (Figure S2B), while knockdown of PRMT6 led to a significant decrease in the expression of c-MYC and the downstream genes expression of MYC signaling (CDK4 and GLUT1) (Fig. 4B). Additionally, we transfected SW48 cells with either wild-type PRMT6 (WT) or an enzyme activity-deficient mutant (PRMT6 KLA) and treated the cells with DMSO or the PRMT6 inhibitor PRMT6-IN-3 (Fig. 4C and Figure S2C). We then ectopically expressed either PRMT6 WT or the KLA mutant in SW48 cells stably expressing shPRMT6-3’UTR. Western blot analysis and CCK8 assays demonstrated that overexpression of PRMT6 WT, but not the KLA mutant, restored c-MYC signaling activation and cancer cell proliferation that had been inhibited by PRMT6 silencing (Figure S2D and 2E). The results demonstrated that PRMT6 enzyme activity was essential for PRMT6-mediated activation of MYC signaling. However, RT-PCR analysis revealed that PRMT6 did not affect the mRNA expression of c-MYC (Figures S3A and S3B). We further investigated the post-transcriptional regulation of c-MYC by PRMT6 using cycloheximide (CHX) to inhibit endogenous c-MYC synthesis. Western blot analysis showed that knockdown of PRMT6 accelerated the degradation rate of c-MYC (Fig. 4D and E). These findings suggested that PRMT6 regulated c-MYC expression at the post-transcriptional level. Since the autophagy-lysosome pathway and the ubiquitin-proteasome pathway were the two main mechanisms for protein degradation, we treated vector and PRMT6-overexpressing cells with chloroquine (CQ), an autophagy-lysosome pathway inhibitor, and MG132, a ubiquitin-proteasome pathway inhibitor. The results indicated that MG132, but not CQ, abrogated PRMT6-induced c-MYC expression (Fig. 4F). Subsequently, we assessed the polyubiquitin levels of endogenous c-MYC in shNC and shPRMT6 cells. Silencing PRMT6 significantly increased the polyubiquitin level of c-MYC (Fig. 4G). In a cohort of 69 colorectal tumor cases, we evaluated the levels of intratumoral PRMT6, c-MYC, and the percentage of intratumoral cancer cells positive for Ki-67. Statistical analysis showed a positive correlation between PRMT6 expression and both c-MYC expression and the number of Ki-67-positive cancer cells (Fig. 4H and I, and Figure S3C). c-MYC expression showed no correlation with patients’ age or gender (Figure S3D and 3E). Patients with higher T and clinical stages exhibited elevated c-MYC expression (Figure S3F and 3G). Analysis of GEPIA database revealed that c-MYC was significantly upregulated in colorectal cancer tissues compared to adjacent normal tissues (Figure S3H). Collectively, our data revealed that PRMT6 acted as an activator of MYC signaling by blocking the polyubiquitination of c-MYC.
Fig. 4PRMT6 positively regulates MYC signaling by blocking the poly-ubiquitination of c-MYC. (A) GSEA analysis was performed to assess the correlation between PRMT6 expression levels and MYC signaling activation. (B) Western blot analysis was conducted to detect the expression of identified proteins in shNC and shPRMT6 cancer cells. (C) Western blot analysis was performed to detect the expression of identified proteins in cancer cells ectopically expressing vector, PRMT6, or the PRMT6 KLA mutant. D, E. Cycloheximide (CHX) was applied to inhibit the synthesis of endogenous c-MYC, and Western blot analysis was conducted to assess the degradation rate of c-MYC. F. Western blot analysis was performed to detect the expression of identified proteins in different pre-treated cancer cells. G. Western blot and immunoprecipitation assays were conducted to assess the poly-ubiquitination levels of c-MYC in shNC and shPRMT6 cancer cells. H, I. Scatter plots were generated to assess the relationship between PRMT6 expression levels and c-MYC expression levels (H), or between PRMT6 expression levels and the ratio of Ki-67 positive cells (I). Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
PRMT6 mono-methylated c-MYC at arginine 371After browsing the GENEMANIA database, we identified a potential interaction between PRMT6 and c-MYC (Figure S4A). This interaction was confirmed by immunoprecipitation assays (Fig. 5A and Figure S4B). PRMT6 was an arginine methyltransferase that primarily catalyzes the mono-methylation and asymmetric di-methylation of arginine residues. We hypothesized that PRMT6 could methylate c-MYC. Western blot analysis revealed that c-MYC exhibited lower levels of mono-methylation, but not asymmetrical di-methylation, in shPRMT6 cells compared to shNC cells (Fig. 5B). Next, we transfected SW48 cancer cells with either wild-type PRMT6 (WT) or the enzyme activity-deficient mutant PRMT6 KLA. MG132 was used to inhibit the degradation of endogenous c-MYC. Western blot analysis showed that enzyme activity is necessary for PRMT6-mediated c-MYC methylation (Fig. 5C). Consistently, inhibitors of PRMT6 enzyme activity significantly suppressed c-MYC methylation (Fig. 5D). In vitro methylation assays further confirmed that PRMT6 can mono-methylate c-MYC (Fig. 5E). Using GPS-MSP software, we identified four potential mono-methylation sites on c-MYC (R5, R346, R355, and R371). We constructed c-MYC mutants by replacing these arginine residues with lysine. Interestingly, we found that only the R371K mutant abrogated PRMT6-mediated c-MYC methylation (Fig. 5F). We used the CRISPR-Cas9 system and lentivirus to generate colorectal cancer cells stably expressing either c-MYC WT or c-MYC R371K (Figure S4C, S4D). Western blot analysis confirmed that PRMT6 overexpression increased the mono-methylation level of c-MYC in c-MYC WT cells but not in c-MYC R371K mutant cells (Fig. 5G). Notably, the c-MYC R371K mutant exhibited higher polyubiquitin levels than c-MYC WT (Fig. 5H). PRMT6 overexpression inhibited the polyubiquitination of c-MYC in c-MYC WT cells but not in c-MYC R371K cells (Fig. 5H). Of note, the analysis of c-MYC sequences across various species revealed that arginine at position 371 was conserved. In summary, our data demonstrated that PRMT6 negatively regulated the polyubiquitination of c-MYC by mono-methylating c-MYC at R371.
Fig. 5c-MYC Arg371 is mono-methylated by PRMT6. (A) Western blot and immunoprecipitation assays were performed to confirm the binding between PRMT6 and c-MYC. (B) Western blot and immunoprecipitation assays were conducted to assess the levels of mono-methylation (MMA) and asymmetric di-methylation (ADMA) of c-MYC in shNC and shPRMT6 cancer cells. (C) Western blot and immunoprecipitation assays were used to evaluate the MMA level of c-MYC in cancer cells ectopically expressing vector, PRMT6, or the PRMT6 KLA mutant. (D) Western blot and immunoprecipitation assays were performed to assess the MMA level of c-MYC in cancer cells treated with DMSO or PRMT6 inhibitors. (E) In vitro methylation assays confirmed that PRMT6 can directly mono-methylate c-MYC. (F) c-MYC KR mutants were transfected into cancer cells ectopically expressing either vector or PRMT6. Western blot and immunoprecipitation assays were performed to assess the MMA levels of c-MYC mutants. (G) Western blot and immunoprecipitation assays were conducted to evaluate the MMA levels of c-MYC in different cancer cells. (H) Western blot and immunoprecipitation assays were performed to assess the poly-ubiquitination levels of c-MYC WT and c-MYC R371K in cancer cells ectopically expressing either vector or PRMT6. (I) The amino acid sequence of c-MYC was analyzed for conservation across various species. Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
c-MYC R371 mono-methylation was essential for PRMT6-mediated colorectal cancer cell proliferationTo further elucidate the relationship between PRMT6 signaling and MYC signaling, we constructed SW48 cancer cells stably expressing Vector + c-MYC WT, PRMT6 + c-MYC WT, Vector + c-MYC R371K, or PRMT6 + c-MYC R371K. Western blot analysis confirmed successful construction (Fig. 6A). The results demonstrated that PRMT6 overexpression upregulated the expression levels of c-MYC, CDK4, and GLUT1 in c-MYC WT cells, but not in c-MYC R371K cells (Fig. 6A). CCK8 and BrdU assays revealed that the c-MYC R371K mutant abolished PRMT6-mediated cancer cell proliferation (Fig. 6B-D). Additionally, PRMT6 overexpression increased intracellular ATP levels in c-MYC WT cells, but not in c-MYC R371K cells (Fig. 6E). We further generated SW48 cancer cells stably expressing shNC + c-MYC WT, shNC + c-MYC WT, shPRMT6#1 + c-MYC R371K, or shPRMT6#1 + c-MYC R371K using lentivirus (Fig. 6F). Loss of PRMT6 decreased intracellular c-MYC, CDK4, and GLUT1 expression levels in c-MYC WT cells, but not in c-MYC R371K cells (Fig. 6F). Consistently, silencing PRMT6 significantly inhibited cell proliferation and intracellular ATP levels in c-MYC WT cells, but not in c-MYC R371K cells (Fig. 6G-J). Notably, c-MYC R371K cells exhibited lower expression levels of c-MYC, CDK4, and GLUT1, as well as reduced proliferation ability and intracellular ATP levels compared to c-MYC WT cells (Fig. 6A-J). In summary, these findings demonstrate that mono-methylation of c-MYC at R371 is essential for c-MYC or PRMT6-mediated colorectal cancer cell proliferation.
Fig. 6PRMT6-mediated cancer cell proliferation depends on c-MYC R371 mono-methylation. A, F. Western blot analysis was performed to assess the expression levels of c-MYC, CDK4, and GLUT1 in different pre-treated cancer cells. B, C, G, H. BrdU assays were conducted to evaluate DNA replication ability in different pre-treated cancer cells. D, I. CCK8 assays were performed to detect the proliferation ability in different pre-treated cancer cells. E, J. Cell viability assays were carried out to assess intracellular ATP levels in different pre-treated cancer cells. Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
Silencing of PRMT6 significantly suppressed MYC signaling and tumor proliferation in vivoTo further verify the oncogenic function of PRMT6 in colorectal cancer, we conducted an in vivo xenograft experiment. We subcutaneously injected SW48 cells stably expressing either shNC or shPRMT6 into the backs of 10-week-old female mice. Tumor volumes were measured every three days. After 18 days, all mice were sacrificed, and tumors were isolated for further analysis. The results showed that tumors from the shPRMT6 group had a lower growth rate and weight compared to those from the shNC group (Fig. 7A-C). We also recorded the overall survival time of the mice. Consistently, mice with shPRMT6 tumors had longer overall survival times than those with shNC tumors (Fig. 7D). Next, we performed immunohistochemistry to assess the intratumoral expression levels of PRMT6, c-MYC, and Ki-67. The results indicated that knockdown of PRMT6 significantly inhibited the expression of c-MYC and reduced the proportion of Ki-67-positive cells (Fig. 7E). Western blot analysis further confirmed that silencing PRMT6 markedly decreased the expression of c-MYC and Ki-67 (Fig. 7F). Overall, our data suggested that PRMT6 might be an effective target for colorectal cancer treatment.
Fig. 7Knockdown of PRMT6 significantly inhibits colorectal tumor proliferation in vivo. (A) Representative images of tumors from mice injected with SW48 cells stably expressing shNC or shPRMT6. (B) Tumor growth curves showing the growth rates of tumors derived from mice injected with shNC or shPRMT6 cells. (C) Scatter plot of tumor weights from mice injected with shNC or shPRMT6 cells. (D) Kaplan-Meier survival curves for mice bearing tumors from the three experimental groups. (E) Representative images of immunohistochemistry (IHC) assays showing PRMT6, c-MYC, and Ki-67 expression in tumors. (F) Western blot analysis to detect the expression levels of intratumoral PRMT6, c-MYC, and Ki-67. (G) Working model: PRMT6 mono-methylated c-MYC at arginine 371 site, which suppressed the poly-ubiquitin level of c-MYC, and then promoted colorectal cancer progress. Student’s t-test was employed for statistical analysis, and all Western blot experiments were performed in triplicate
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