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Regulation of a PRMT5/NF-κB Axis by Phosphorylation of PRMT5 at Serine 15 in Colorectal Cancer - PubMed

  • ️Wed Jan 01 2020

Regulation of a PRMT5/NF-κB Axis by Phosphorylation of PRMT5 at Serine 15 in Colorectal Cancer

Antja-Voy Hartley et al. Int J Mol Sci. 2020.

Abstract

The overexpression of PRMT5 is highly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1β-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1β-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics.

Keywords: NF-κB; PRMT5; colorectal cancer; phosphorylation; serine.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1

Identification of phosphorylation of Serine 15 (S15) on PRMT5. (A) Top panel, mass spectrometry (MS) experiment identifies S15 as a phosphorylated residue in response to IL-1β treatment. A mass shift of 80 Da was observed, indicating the existence of the phosphorylation modification. Bottom panel, Gel-code blue stained MS gel indicates a purified strong FLAG-PRMT5 protein band (left). Western blot analysis confirmation of the identity of the purified band as PRMT5 (right). (B) Establishment of wild type (WT) or Serine 15 to Alanine (S15A) mutant FLAG-PRMT5 overexpressing stable cells. Western blot images, showing overexpression of FLAG-PRMT5 constructs probed with anti-PRMT5, or FLAG, or β-actin respectively, in HEK293 cells or HT29, DLD1, and HCT116 colon cancer cells. (C) Confirmation of phosphorylation of PRMT5 at S15 using co-immunoprecipitation and Western blot analysis. Either HEK293 (top panel) or HT29 cells (bottom panel) were treated with 10 ng/mL of IL-1β or left untreated for 1 h. Samples were collected and the WT-PRMT5 (Flag-WT) or S15A (Flag-S15A) protein was further immunoprecipitated with anti-FLAG beads and subjected to Western analysis using an anti-phospho-serine motif antibody (pSER). The inputs were probed with anti-PRMT5 antibody. (D) Cross-species alignment of amino acid sequences from PRMT5 proteins (residues 1–60). The conserved S15 residue is indicated on the top by the red asterisk (

www.uniprot.com

).

Figure 2
Figure 2

Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation and differentially regulates a subset of NF-κB target genes in response to IL-1β. (A) Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation. NF-κB luciferase assay, conducted for vector control (Ctrl), or with the overexpression of WT-PRMT5 (WT) or S15A mutant in the presence or absence of 10 ng/mL of IL-1β treatment in HEK293, and HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT + IL-1β group. (B) Phosphorylation of PRMT5 at S15 differentially regulates a subset of NF-κB target genes. Top panel: pie-chart (left, yellow and orange), representing data from human Illumina array assay. Data indicates that upon overexpression of WT-PRMT5, the expression of ≈48% of NF-ĸB target genes were further augmented by >= 1.5-fold following 10 ng/mL IL-1β stimulation. Among these genes, ≈39% of genes (pie-chart, right, gray and blue) could be downregulated by 2-fold or more (S15A + IL-1β/WT + IL-1β ≤ 0.5) by the S15A mutation. Bottom panel: table, showing a short list of typical NF-ĸB target genes that were upregulated by WT-PRMT5 (WT) but not by the S15A mutant. (C) Confirmation of Illumina Array data with qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in HEK293 and HT29, DLD1, HCT116 colon cancer cells. Ctrl: vector control cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. † p < 0.05 vs. Ctrl group; * p < 0.05 vs. Ctrl+IL-1β group; # p < 0.05 vs. WT+IL-1β group. (D) Ingenuity Pathway Analysis (IPA): Subsets of genes upregulated by WT-PRMT5 overexpression but downregulated by S15A were used to conduct the IPA. Enrichment results indicating top biological functions, disease networks, and upstream regulators are shown as dots scaled by –log(p). The size of the dot shows the significant level of enrichment. (E) IPA representative network, showing genes regulated by S15A with NF-κB as one of the critical nodes in this network.

Figure 2
Figure 2

Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation and differentially regulates a subset of NF-κB target genes in response to IL-1β. (A) Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation. NF-κB luciferase assay, conducted for vector control (Ctrl), or with the overexpression of WT-PRMT5 (WT) or S15A mutant in the presence or absence of 10 ng/mL of IL-1β treatment in HEK293, and HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT + IL-1β group. (B) Phosphorylation of PRMT5 at S15 differentially regulates a subset of NF-κB target genes. Top panel: pie-chart (left, yellow and orange), representing data from human Illumina array assay. Data indicates that upon overexpression of WT-PRMT5, the expression of ≈48% of NF-ĸB target genes were further augmented by >= 1.5-fold following 10 ng/mL IL-1β stimulation. Among these genes, ≈39% of genes (pie-chart, right, gray and blue) could be downregulated by 2-fold or more (S15A + IL-1β/WT + IL-1β ≤ 0.5) by the S15A mutation. Bottom panel: table, showing a short list of typical NF-ĸB target genes that were upregulated by WT-PRMT5 (WT) but not by the S15A mutant. (C) Confirmation of Illumina Array data with qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in HEK293 and HT29, DLD1, HCT116 colon cancer cells. Ctrl: vector control cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. † p < 0.05 vs. Ctrl group; * p < 0.05 vs. Ctrl+IL-1β group; # p < 0.05 vs. WT+IL-1β group. (D) Ingenuity Pathway Analysis (IPA): Subsets of genes upregulated by WT-PRMT5 overexpression but downregulated by S15A were used to conduct the IPA. Enrichment results indicating top biological functions, disease networks, and upstream regulators are shown as dots scaled by –log(p). The size of the dot shows the significant level of enrichment. (E) IPA representative network, showing genes regulated by S15A with NF-κB as one of the critical nodes in this network.

Figure 2
Figure 2

Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation and differentially regulates a subset of NF-κB target genes in response to IL-1β. (A) Phosphorylation of PRMT5 at S15 is critical for NF-ĸB activation. NF-κB luciferase assay, conducted for vector control (Ctrl), or with the overexpression of WT-PRMT5 (WT) or S15A mutant in the presence or absence of 10 ng/mL of IL-1β treatment in HEK293, and HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT + IL-1β group. (B) Phosphorylation of PRMT5 at S15 differentially regulates a subset of NF-κB target genes. Top panel: pie-chart (left, yellow and orange), representing data from human Illumina array assay. Data indicates that upon overexpression of WT-PRMT5, the expression of ≈48% of NF-ĸB target genes were further augmented by >= 1.5-fold following 10 ng/mL IL-1β stimulation. Among these genes, ≈39% of genes (pie-chart, right, gray and blue) could be downregulated by 2-fold or more (S15A + IL-1β/WT + IL-1β ≤ 0.5) by the S15A mutation. Bottom panel: table, showing a short list of typical NF-ĸB target genes that were upregulated by WT-PRMT5 (WT) but not by the S15A mutant. (C) Confirmation of Illumina Array data with qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in HEK293 and HT29, DLD1, HCT116 colon cancer cells. Ctrl: vector control cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. † p < 0.05 vs. Ctrl group; * p < 0.05 vs. Ctrl+IL-1β group; # p < 0.05 vs. WT+IL-1β group. (D) Ingenuity Pathway Analysis (IPA): Subsets of genes upregulated by WT-PRMT5 overexpression but downregulated by S15A were used to conduct the IPA. Enrichment results indicating top biological functions, disease networks, and upstream regulators are shown as dots scaled by –log(p). The size of the dot shows the significant level of enrichment. (E) IPA representative network, showing genes regulated by S15A with NF-κB as one of the critical nodes in this network.

Figure 3
Figure 3

S15A mutant of PRMT5 disrupts its interaction with p65 and attenuates occupancy of p65 at NF-κB target gene. (A) Co-immunoprecipitation (IP) experiments, HEK293 and HT29 cells were treated or left untreated with 10 ng/mL of IL-1β for 1 h, WT-PRMT5 (Flag-WT) or S15A (Flag-S15A) was immunoprecipitated with anti-FLAG beads. Samples were then subjected to Western blot analysis (WB) and probed with anti-p65 antibody. Inputs were probed with anti-p65 and anti-Flag antibodies. (B) Architecture of the IL8 promoter showing the location of the NF-ĸB binding site. (C) Chromatin immunoprecipitation (ChIP) assay in HEK29 and HT29 cells to detect occupancy of p65 at the typical NF-κB target gene, IL8′s promoter upon IL-1β stimulation.

Figure 4
Figure 4

Phosphorylation of PRMT5 at S15 regulates cell growth, anchorage-independent growth and migration of colon cancer cells. (A) Cell growth assay compares cell numbers of vector control (Ctrl), WT-PRMT5 (WT), or S15A-PRMT5 (S15A) mutant-overexpressing cells in HT29, DLD1, and HCT116 colon cancer cells. A total of 2 × 104 cells were seeded and counted using a cell counting chamber at days 3, 5, 7, and 9. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group. (B) Top panel: anchorage-independent growth assay with colon cancer cells overexpressing WT or S15A mutant compared to Ctrl. Representative images of colonies for HT29, DLD1, and HCT116 are shown. Bottom panel: quantification of the average colony size and number is shown below the corresponding cell type. The data represent the means ± standard error of mean (SEM) for three independent experiments. Scale bar = 25 µm. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group. (C) Top panel: Boyden Chamber Transwell assay, showing migration of colon cancer cells overexpressing WT or S15A mutant compared to Ctrl. Representative photos of crystal violet stained cells are shown with 20× magnification. Bottom panel: quantification of the average number of migrated cells is shown. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group.

Figure 4
Figure 4

Phosphorylation of PRMT5 at S15 regulates cell growth, anchorage-independent growth and migration of colon cancer cells. (A) Cell growth assay compares cell numbers of vector control (Ctrl), WT-PRMT5 (WT), or S15A-PRMT5 (S15A) mutant-overexpressing cells in HT29, DLD1, and HCT116 colon cancer cells. A total of 2 × 104 cells were seeded and counted using a cell counting chamber at days 3, 5, 7, and 9. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group. (B) Top panel: anchorage-independent growth assay with colon cancer cells overexpressing WT or S15A mutant compared to Ctrl. Representative images of colonies for HT29, DLD1, and HCT116 are shown. Bottom panel: quantification of the average colony size and number is shown below the corresponding cell type. The data represent the means ± standard error of mean (SEM) for three independent experiments. Scale bar = 25 µm. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group. (C) Top panel: Boyden Chamber Transwell assay, showing migration of colon cancer cells overexpressing WT or S15A mutant compared to Ctrl. Representative photos of crystal violet stained cells are shown with 20× magnification. Bottom panel: quantification of the average number of migrated cells is shown. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. Ctrl group; # p < 0.05 vs. WT group.

Figure 5
Figure 5

S15 phosphorylation is located in the triose phosphate isomerase (TIM) barrel domain of PRMT5 and regulates IL-1β-inducible PRMT5 methyltransferase activity. (A) Overview of the domain architecture of human PRMT5. The S15 residue is located in the unique N-terminal TIM-Barrel, which might be critical for PRMT5 substrate binding. (B) S15 phosphorylation is critical for the methyltransferase activity of PRMT5. AlphaLISA assay was conducted by using biotinylated histone H4 as a PRMT5 substrate. Graph shows detection of specific methyltransferase activity of WT-PRMT5 (WT) or S15A-PRMT5 (S15A) mutant enzymes purified from HEK293 cells in the presence or absence of 10 ng/mL of IL-1β. E444D-PRMT5 (E444D) was used as an enzymatic dead mutant control. S-adenosyl methionine (SAM) was used as the methyl donor for the reaction. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. WT group; # p < 0.05 vs. WT + IL-1β group.

Figure 5
Figure 5

S15 phosphorylation is located in the triose phosphate isomerase (TIM) barrel domain of PRMT5 and regulates IL-1β-inducible PRMT5 methyltransferase activity. (A) Overview of the domain architecture of human PRMT5. The S15 residue is located in the unique N-terminal TIM-Barrel, which might be critical for PRMT5 substrate binding. (B) S15 phosphorylation is critical for the methyltransferase activity of PRMT5. AlphaLISA assay was conducted by using biotinylated histone H4 as a PRMT5 substrate. Graph shows detection of specific methyltransferase activity of WT-PRMT5 (WT) or S15A-PRMT5 (S15A) mutant enzymes purified from HEK293 cells in the presence or absence of 10 ng/mL of IL-1β. E444D-PRMT5 (E444D) was used as an enzymatic dead mutant control. S-adenosyl methionine (SAM) was used as the methyl donor for the reaction. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. WT group; # p < 0.05 vs. WT + IL-1β group.

Figure 6
Figure 6

Knockdown of PKCι attenuates phosphorylation of PRMT5 and disrupts PRMT5-mediated NF-ĸB signaling. (A) Human Protein Reference Database (HPRD) predicted PKC and PKA phospho-motif in position 13-15 of PRMT5. (B) Co-immunoprecipitation (IP) experiments, HEK293 and HT29 cells were treated or left untreated with 10 ng/mL of IL-1β for 1 h, Flag-WT-PRMT5 (Flag-WT) was immunoprecipitated with anti-FLAG beads. Samples were then subjected to Western blot analysis and probed with the indicated antibodies. Inputs were probed with anti-PKCι and anti-Flag antibodies. (C) Establishment of vector control, WT, or S15A Flag-PRMT5 overexpressing HEK293 stable cells with or without co-expression of shscramble or shPKCι constructs. Western blot analysis, probed with anti-PKCι, PRMT5, or β-actin, respectively. Image J quantification of PKCι to loading control (β-actin) ratios for three independent Western blots is shown below. * p < 0.05 vs. shscramble counterparts. (D) Phosphorylation of PRMT5 using co-immunoprecipitation and Western blot analysis. Either HEK293 cells with vector control and shscramble, Flag-WT and shscramble, or Flag-WT and shPKCι were treated with 10 ng/mL of IL-1β or left untreated for 1 h. Samples were collected and Flag-WT was further immunoprecipitated with anti-FLAG beads and subjected to Western analysis using an anti-phospho-serine motif antibody (pSer). The inputs were probed with anti-PRMT5 or Flag antibody. (E) Co-immunoprecipitation (IP) experiment, HEK293 cells were treated or left untreated with 10 ng/mL of IL-1β for 1 h, WT-PRMT5 (Flag-WT) or S15A (Flag-S15A) was immunoprecipitated with anti-FLAG beads. Samples were then subjected to Western blot analysis and probed with anti-p65 antibody. Inputs were probed with anti-p65 and anti-Flag antibodies. (F) NF-κB activity was determined by luciferase assay, in established cells shown in Figure 6C. The data represent the mean ± SD from three independent experiments. # p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT+IL-1β group; n.s: not significant. (G) NF-κB activity luciferase assay in control or cells overexpressing WT-PRMT5 (WT) or S15A treated with or without PKCι inhibitor CRT0066854 in the presence or absence of 10 ng/mL of IL-1β stimulation in both HEK293 and HT29 cell systems. The data represent the mean ± SD from three independent experiments. # p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT+IL-1β group; n.s: not significant. (H) Top panel: box-whisker plots showing gene expression (transcript levels) of PKCι across colorectal adenocarcinoma (COAD) tumors and normal tissue based on individual cancer stages. Bottom panel: log-rank test was used to indicate statistical significance between normal and each cancer stage. Individual cancer stages are based on AJCC (American Joint Committee on Cancer) pathologic tumor stage information (

http://ualcan.path.uab.edu

).

Figure 6
Figure 6

Knockdown of PKCι attenuates phosphorylation of PRMT5 and disrupts PRMT5-mediated NF-ĸB signaling. (A) Human Protein Reference Database (HPRD) predicted PKC and PKA phospho-motif in position 13-15 of PRMT5. (B) Co-immunoprecipitation (IP) experiments, HEK293 and HT29 cells were treated or left untreated with 10 ng/mL of IL-1β for 1 h, Flag-WT-PRMT5 (Flag-WT) was immunoprecipitated with anti-FLAG beads. Samples were then subjected to Western blot analysis and probed with the indicated antibodies. Inputs were probed with anti-PKCι and anti-Flag antibodies. (C) Establishment of vector control, WT, or S15A Flag-PRMT5 overexpressing HEK293 stable cells with or without co-expression of shscramble or shPKCι constructs. Western blot analysis, probed with anti-PKCι, PRMT5, or β-actin, respectively. Image J quantification of PKCι to loading control (β-actin) ratios for three independent Western blots is shown below. * p < 0.05 vs. shscramble counterparts. (D) Phosphorylation of PRMT5 using co-immunoprecipitation and Western blot analysis. Either HEK293 cells with vector control and shscramble, Flag-WT and shscramble, or Flag-WT and shPKCι were treated with 10 ng/mL of IL-1β or left untreated for 1 h. Samples were collected and Flag-WT was further immunoprecipitated with anti-FLAG beads and subjected to Western analysis using an anti-phospho-serine motif antibody (pSer). The inputs were probed with anti-PRMT5 or Flag antibody. (E) Co-immunoprecipitation (IP) experiment, HEK293 cells were treated or left untreated with 10 ng/mL of IL-1β for 1 h, WT-PRMT5 (Flag-WT) or S15A (Flag-S15A) was immunoprecipitated with anti-FLAG beads. Samples were then subjected to Western blot analysis and probed with anti-p65 antibody. Inputs were probed with anti-p65 and anti-Flag antibodies. (F) NF-κB activity was determined by luciferase assay, in established cells shown in Figure 6C. The data represent the mean ± SD from three independent experiments. # p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT+IL-1β group; n.s: not significant. (G) NF-κB activity luciferase assay in control or cells overexpressing WT-PRMT5 (WT) or S15A treated with or without PKCι inhibitor CRT0066854 in the presence or absence of 10 ng/mL of IL-1β stimulation in both HEK293 and HT29 cell systems. The data represent the mean ± SD from three independent experiments. # p < 0.05 vs. Ctrl+IL-1β group; ** p < 0.05 vs. WT+IL-1β group; n.s: not significant. (H) Top panel: box-whisker plots showing gene expression (transcript levels) of PKCι across colorectal adenocarcinoma (COAD) tumors and normal tissue based on individual cancer stages. Bottom panel: log-rank test was used to indicate statistical significance between normal and each cancer stage. Individual cancer stages are based on AJCC (American Joint Committee on Cancer) pathologic tumor stage information (

http://ualcan.path.uab.edu

).

Figure 7
Figure 7

Inhibition of PKCι attenuates NF-ĸB activation and target gene expression in response to IL-1β. (A) Establishment of shscramble and shPKCι HT29, DLD1, HCT116 colon cancer cell lines. Western blot analysis, probed with anti-PKCι or β-actin (loading control), respectively. (B) Knockdown of PKCι attenuates NF-ĸB activation. NF-κB luciferase assay, conducted for shscramble and shPKCι in the presence or absence of 10 ng/mL of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. #p < 0.05 vs. shscramble+IL-1β group. (C) Small-molecule inhibition of PKCι attenuates NF-ĸB activation. NF-κB luciferase assay, conducted for vehicle control (Ctrl) or increasing µM concentrations of PKCι inhibitor CRT0066854 in the presence or absence of 10 ng/mL of IL-1β treatment in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. *p < 0.05 vs. Ctrl+IL-1β group. (D) qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in shscramble and shPKCι colon cancer cells in the absence and presence of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble group; # p < 0.05 vs. shscramble+IL-1β group. (E) qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in vehicle and PKCι inhibitor CRT0066854 treated colon cancer cells in the absence and presence of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. vehicle group; #p < 0.05 vs. vehicle+IL-1β group.

Figure 7
Figure 7

Inhibition of PKCι attenuates NF-ĸB activation and target gene expression in response to IL-1β. (A) Establishment of shscramble and shPKCι HT29, DLD1, HCT116 colon cancer cell lines. Western blot analysis, probed with anti-PKCι or β-actin (loading control), respectively. (B) Knockdown of PKCι attenuates NF-ĸB activation. NF-κB luciferase assay, conducted for shscramble and shPKCι in the presence or absence of 10 ng/mL of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. #p < 0.05 vs. shscramble+IL-1β group. (C) Small-molecule inhibition of PKCι attenuates NF-ĸB activation. NF-κB luciferase assay, conducted for vehicle control (Ctrl) or increasing µM concentrations of PKCι inhibitor CRT0066854 in the presence or absence of 10 ng/mL of IL-1β treatment in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. *p < 0.05 vs. Ctrl+IL-1β group. (D) qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in shscramble and shPKCι colon cancer cells in the absence and presence of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble group; # p < 0.05 vs. shscramble+IL-1β group. (E) qPCR analysis, indicating relative mRNA levels of CCL20 and IL8 in vehicle and PKCι inhibitor CRT0066854 treated colon cancer cells in the absence and presence of IL-1β in HT29, DLD1, HCT116 colon cancer cells. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. vehicle group; #p < 0.05 vs. vehicle+IL-1β group.

Figure 8
Figure 8

Inhibition of PKCι attenuates cell growth, anchorage-independent growth and migration of colon cancer cells. (A) Cell growth assay compares cell numbers of shscramble and shPKCι as well as (B) Vehicle control (Ctrl) and HT29, DLD1, and HCT116 colon cancer cells treated with selective PKCι inhibitor CRT0066854. A total of 2 × 104 cells were seeded and counted using a cell counting chamber at days 3, 5, 7, and 9. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble (A) or Ctrl (B) group. (C) Anchorage-independent growth assay with knockdown (top panel) or small molecule inhibition of PKCι (bottom panel) compared to shscramble and vehicle control (Ctrl), respectively, in colon cancer cells. Representative images of colonies for HT29, DLD1, and HCT116 are shown. Quantification of the average colony size is shown on the right for the corresponding cell type. The data represent the means ± standard error of mean (SEM) for three independent experiments; Scale bar = 25 µm. * p < 0.05 vs. shscramble (top panel) or Ctrl (bottom panel) group. (D) Boyden Chamber Transwell assay, showing migration of shPKCι colon cancer cells compared to shscramble controls. Representative photos of crystal violet stained cells are shown with 20× magnification. Right panel: quantification of the average number of migrated cells is shown. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble group.

Figure 8
Figure 8

Inhibition of PKCι attenuates cell growth, anchorage-independent growth and migration of colon cancer cells. (A) Cell growth assay compares cell numbers of shscramble and shPKCι as well as (B) Vehicle control (Ctrl) and HT29, DLD1, and HCT116 colon cancer cells treated with selective PKCι inhibitor CRT0066854. A total of 2 × 104 cells were seeded and counted using a cell counting chamber at days 3, 5, 7, and 9. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble (A) or Ctrl (B) group. (C) Anchorage-independent growth assay with knockdown (top panel) or small molecule inhibition of PKCι (bottom panel) compared to shscramble and vehicle control (Ctrl), respectively, in colon cancer cells. Representative images of colonies for HT29, DLD1, and HCT116 are shown. Quantification of the average colony size is shown on the right for the corresponding cell type. The data represent the means ± standard error of mean (SEM) for three independent experiments; Scale bar = 25 µm. * p < 0.05 vs. shscramble (top panel) or Ctrl (bottom panel) group. (D) Boyden Chamber Transwell assay, showing migration of shPKCι colon cancer cells compared to shscramble controls. Representative photos of crystal violet stained cells are shown with 20× magnification. Right panel: quantification of the average number of migrated cells is shown. The data represent the means ± standard deviation (S.D.) for three independent experiments. * p < 0.05 vs. shscramble group.

Figure 9
Figure 9

Hypothetical model. IL-1β stimulation activates the NF-κB pathway and induces PKCι-mediated phosphorylation of PRMT5. Phosphorylation of PRMT5 at S15 mediates the PRMT5-p65 interaction and proximal promoter occupancy of p65 at target genes and thus constitute pivotal mechanisms by which PRMT5 can fine-tune NF-κB activation and target gene expression. Furthermore, S15 phosphorylation mediates IL-1β-induced PRMT5 activity. Together, these potentially serve to facilitate the tumor-associated functions exerted by PRMT5-mediated NF-κB activation, including the enhanced proliferation, anchorage-independent growth, and migration associated with PRMT5 overexpression.

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