Arginine is an epigenetic regulator targeting TEAD4 to modulate OXPHOS in prostate cancer cells

Abstract

Arginine plays diverse roles in cellular physiology. As a semi-essential amino acid, arginine deprivation has been used to target cancers with arginine synthesis deficiency. Arginine-deprived cancer cells exhibit mitochondrial dysfunction, transcriptional reprogramming and eventual cell death. In this study, we show in prostate cancer cells that arginine acts as an epigenetic regulator to modulate histone acetylation, leading to global upregulation of nuclear-encoded oxidative phosphorylation (OXPHOS) genes. TEAD4 is retained in the nucleus by arginine, enhancing its recruitment to the promoter/enhancer regions of OXPHOS genes and mediating coordinated upregulation in a YAP1-independent but mTOR-dependent manner. Arginine also activates the expression of lysine acetyl-transferases and increases overall levels of acetylated histones and acetyl-CoA, facilitating TEAD4 recruitment. Silencing of TEAD4 suppresses OXPHOS functions and prostate cancer cell growth in vitro and in vivo. Given the strong correlation of TEAD4 expression and prostate carcinogenesis, targeting TEAD4 may be beneficially used to enhance arginine-deprivation therapy and prostate cancer therapy.

Fig. 1. Arginine globally modulates metabolic gene expression via histone acetylation.

a Heat map of Affymetrix microarray analysis shows arginine (Arg+) globally induced metabolic pathways, including oxidative phosphorylation pathway, glucose, fatty acid, and DNA metabolic pathways in CWR22Rv1 prostate cancer cells. The color key indicates the fold change to control. b After starvation overnight, cells were treated with arginine and then harvested at different time points for total acetyl-histone H3 ELISA assay. This data shows that arginine regulated the total level of histone H3 acetylation at different time points. c After starvation overnight, cells were treated with arginine and then harvested at different time points for total acetyl-CoA assay. ELISA data show that arginine regulated the total level of acetyl-CoA at different time points. d Heat map of acetylated histone H3 ChIP-seq peaks showing that the distribution of peaks are close to transcription star sites (TSS). e Overlap of arginine stimulation Arg (+) and arginine deprivation Arg (−) ChIP-seq peaks. f Acetylated Histone H3 ChIP-sequencing peaks classified by human genomic annotation (hg38), showing that the peaks of histone acetylation are mainly enriched in the promoter region of genes. Data are presented as mean values ± SEM of independent experiments (n = 3 in b, c). **p < 0.01, ***p < 0.001, ****p < 0.0001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 2. Arginine epigenetically modulates mitochondrial OXPHOS genes.

a Ingenuity pathway analysis on metabolic pathways shows arginine stimulation epigenetically modulates key metabolic pathways, including plasma membrane biosynthesis, DNA synthesis, and OXPHOS pathways. b Ingenuity pathway analysis on cell signaling pathways shows arginine stimulation mainly induced mTOR pathway (highlighted in blue). c ChIP-seq distribution for acetylated histone H3 at representative OXPHOS genes loci. d The summary list of OXPHOS genes enriched in the arginine-stimulation group based on ChIP-seq data. The upregulated genes in microarray data are marked with an asterisk (*). e ChIP-qPCR of histone H3 acetylation confirmed arginine stimulation induced histone acetylation on the promoter region of OXPHOS genes. The value indicated the fold enrichment after arginine stimulation. f After starvation, cells were treated with arginine for 24 h and then harvested. Both total lysate and mitochondrial fraction (Mito) were used for immunoblotting. These data show that arginine induced OXPHOS protein expression. g After starvation, cells were treated with arginine for 24 h and then harvested for individual complex activity assay. These data show arginine stimulation increased mitochondrial complex activities. Data are presented as mean values ± SEM of independent experiments (n = 3 in e, g). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 3. Arginine targets TEAD4 to modulate the mitochondrial functions.

a The recognition motifs were enriched in ChIP-seq data after arginine stimulation. Top consensus sequences classified by the program DREME, clustered by similarity and order by p value. b Each candidate was first knocked down by lentivirus transduction for 24 h, following an antibiotic selection for 1 week. Cells were then harvested for real-time PCR analysis of OXPHOS gene expression. The color key indicates the relative gene expression to scramble control. c Seahorse assay for mitochondrial respiration activities after silencing each TF candidates via shRNA. Arginine deprivation (−) serves as a negative control. O oligomycin, F FCCP, R/A rotenone/antimycin. The individual shRNA plot is shown in Supplementary Fig. 2c. d Each TEAD was first knocked down by lentivirus transduction for 24 h, following an antibiotic selection for 1 week. Seahorse assay for mitochondrial respiration activities. O oligomycin, F FCCP, R/A rotenone/antimycin. e ChIP-qPCR data show the binding activity of TEAD4 with (Arg+) or without (Arg−) arginine stimulation. The value indicates the fold enrichment to negative control (Arg−). Lane A is the recruitment profile of TEAD4 onto OXPHOS promoters after 24 h of arginine re-stimulation, whereas lane B is that from cultures grown in arginine-supplemented media for a long time. TEAD4 binding sites (BS) of OXPHOS promoters are shown in Supplementary Fig. 2b. f Advance image cytometer analysis shows that silencing of TEAD4 increased cellular ROS production after arginine deprivation for 48 h. g Scramble or shTEAD4 cells were first incubated with arginine-free media for 48 h and then incubating with MitoSOX for 15 min as described by the manufacturer. Immunofluorescent staining shows that silencing of TEAD4 increased mitochondrial ROS (MitoSOX) after arginine deprivation (all groups p value < 0.0001) (scale bar = 10 μm). Data are presented as mean values ± SEM of independent experiments (n = 3 in b–e, n = 4 in f, g). *p < 0.05, **p < 0.01, ***p < 0.001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 4. Arginine mediated nuclear retention of TEAD4.

a Cells were first incubated in arginine-free media and harvested at different time points as indicated. Fresh arginine was then added into media and cells were harvested at different time points as indicated. Western blotting shows that arginine (Arg) did not affect TEAD4 protein level, but affected the ratio of nucleus and cytosol fractions (b). c Cells were first incubated in arginine-free media for 24 h. Arginine was then added into media for 24 h. Immunofluorescent staining of TEAD4 shows cytosolic translocation after arginine deprivation (scale bar = 20 μm). The quantification of immunofluorescent staining is shown in d. e Cells were incubated in arginine-free media and harvested at different time points as indicated. Western blotting shows that arginine depletion induced p38 phosphorylation in a time-dependent manner, which is consistent with TEAD4 cytosol translocation (Supplementary Fig. 3a). f Cells were incubated in arginine-free media with or without p38 inhibitor (p38i) SB203580 treatment for 24 h. Western blotting shows that inhibition of p38 activation prevented TEAD4 cytosolic translocation upon arginine deprivation. The total TEAD4 expression is shown in Supplementary Fig. 3b. g Immunofluorescent staining of TEAD4 shows that inhibition of p38 activation impeded TEAD4 cytosolic translocation upon arginine deprivation (scale bar = 20 μm). The quantification of immunofluorescent staining is shown in (h). i After starvation overnight, cells were treated with arginine for 24 h and then harvested for immunoblotting. Western blotting shows that arginine did not affect the phosphorylations of YAP1. j YAP1 was knocked down via shRNA lentivirus infection for 24 h, following an antibiotic selection for 1 week. ChIP-qPCR data show that silencing of YAP1 did not significantly affect the binding activity of TEAD4 on OXPHOS promoter region. k Cells were transduced with two individual YAP1 shRNAs for 24 h, following an antibiotic selection for 1 week. Seahorse assay for mitochondrial respiration activities after silencing of YAP1 via two individual shRNA clones. O oligomycin, F FCCP, R/A rotenone/antimycin. l ChIP-qPCR data show the binding activity of TEAD4, YAP1, PGC1a, and NRF1 on OXPHOS promoters. m ChIP-qPCR data show that the presence (+) or absence (−) of arginine regulates the recruitment of TEAD4 and PGC1a on OXPHOS promoters. Data are presented as mean value ± SEM of independent experiments (n = 3 in d, h, j, l, m; n = 4 in k). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 5. Arginine modulates OXPHOS pathway and TEAD4 recruitment is mTOR dependent.

a Gene set enrichment assay (GSEA, v4.1.0) of microarray data was performed using hallmark gene set collection. Normalized enrichment scores (NES) for pathways indicate the significant difference in the arginine-stimulation group. b GSEA shows that the genes associated with oxidative phosphorylation (upper panel) and mTORC1 pathway (lower panel) are enriched in the arginine-stimulation Arg (+) group. c After starvation overnight, cells were treated with arginine and harvested at different time points as indicated. Western blotting shows that arginine stimulation induced the phosphorylation of mTORC1 (T2446 and S2448) and its downstream target, phosphor-p70 S6K. d qPCR analysis of OXPHOS gene expression after mTOR activation by arginine stimulation and mTOR inactivation by rapamycin treatment (24 h) or shRNA knockdown. The color key indicates the relative gene expression to vehicle or scramble control. e–g Seahorse assay shows that activation of mTOR by 24-h arginine stimulation (e) increases the mitochondrial respiration activity. By contrast, inactivation of mTOR by 24-h rapamycin treatment (f) or shRNA knockdown (g) suppressed the mitochondrial respiration activity. O oligomycin, F FCCP, R/A rotenone/antimycin. h ChIP-qPCR of TEAD4 on OXPHOS gene promoter region shows that silencing of mTOR disrupted the TEAD4 recruitment after arginine stimulation. i Immunofluorescent staining of TEAD4 shows that inhibition of mTOR via rapamycin treatment induced TEAD4 cytosolic translocation (scale bar = 20 μm). The quantification of immunofluorescent staining is shown in j. Data are presented as mean value ± SEM of independent experiments (n = 3 in d–h, j). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 6. Arginine activates mTOR pathway to regulate the level of acetyl-CoA and modulate histone acetylation.

a After starvation overnight, cells were treated with arginine and harvested at different time points as indicated. qPCR analysis shows the gene expression of ACLY and ACSS2, but not of ACSS1, were upregulated after arginine stimulation in a time-dependent manner. b After arginine starvation, cells were treated with arginine for 24 h. Western blotting shows that arginine stimulation (+) induced the protein expression of ACLY and ACSS2. c Cells were grown either in regular media supplemented with rapamycin or in arginine-free media for 24 h. ELISA data show that inhibition of mTOR activity via rapamycin treatment or arginine deprivation decreased the level of acetyl-CoA (blue) and histone H3 acetylation (purple). d Cells were first transduced with mTOR shRNA for 24 h, following an antibiotic selection for 1 week. Western blotting shows that the protein expression of ACLY and ACSS2 were downregulated after silencing of mTOR. e Cells were first transduced with ACLY or ACSS2 shRNA for 24 h, following an antibiotic selection for 1 week. Western blotting data show that the ratio of acetyl-histone H3 was decreased after silencing of ACLY or ACSS2. f After starvation overnight, cells were treated with arginine and harvested at different time points as indicated. qPCR analysis shows that arginine stimulation globally induced the histone acetyltransferase (HAT) expression. The color key indicates the relative gene expression to vehicle control (at time 0 h). The expression of ACLY and ACSS2 serves as an internal positive control. g After arginine starvation, cells were treated with arginine for 24 h. ELISA data show that arginine stimulation induced the histone H3 acetylation, specifically on lysine residues 9, 18, and 27, as well as H4 acetylation on lysine residues 5 and 12. h Cells were grown in regular media treated with KAT2B inhibitor (KAT2Bi), Garcinol, or in arginine-free media for 24 h. Seahorse data show that inhibition of KAT2B significantly suppressed the mitochondrial respiration activities. O oligomycin, F FCCP, R/A rotenone/antimycin. i Cells were first transduced with KAT2B shRNA for 24 h, following an antibiotic selection for 1 week. ChIP-qPCR shows the TEAD4 recruitment on OXPHOS promoter regions after silencing of KAT2B. j Seahorse data show that silencing of KAT7 significantly suppressed the mitochondrial respiration activities. O oligomycin, F FCCP, R/A rotenone/antimycin. k Cells were first transduced with KAT7 shRNA for 24 h, following an antibiotic selection for 1 week. ChIP-qPCR shows the TEAD4 recruitment on OXPHOS promoter regions after silencing of KAT7. l Diagram of hypothetical model. Data are presented as mean value ± SEM of independent experiments (n = 3 in a, c, f–k). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.

Fig. 7. TEAD4 could be a potential target for cancer therapy.

a TEAD4 expression in Oncomine (www.oncomine.org) Taylor prostate cancer database (n for normal = 29, n for cancer = 131, the center line indicates median, ****p < 0.0001). b Association of TEAD4 expression with Gleason score in Oncomine (www.oncomine.org) Taylor prostate cancer database (n for normal = 25, n for Grade 6 = 41, n for Grade 7 = 74, n for Grade 8 = 8, n for Grade 9 = 7, the center line indicates the median, *p < 0.05, **p < 0.01, ***p < 0.001). c Correlation of TEAD4 expression with tumor recurrence in Oncomine (www.oncomine.org) Taylor prostate cancer database (n for no recurrence = 3, n for recurrence at first year = 9, n for recurrence at third year = 11, n for recurrence at fifth year = 3, whiskers = min to max, bounds of box = lower quartile and upper quartile, the center line indicates the median, *p < 0.05, **p < 0.01). d Kaplan–Meier survival plot shows the correlation of TEAD4 expression with survival rate in Oncomine (www.oncomine.org) Setlur prostate cancer database (n for low expression = 243, n for high expression = 119). e TEAD4 expression in immortalized prostate cell line, RWPE1, and prostate cancer cell lines, CWR22Rv1, PC3, DU145, LNCaP, and C4-2B. f 1 × 10⁵ of scrambled shTEAD4 cells were plated in 24-well plate. Cell growth after silencing of TEAD4 by shRNA in CWR22Rv1 cells. g Doubling time after silencing of TEAD4 by shRNA in CWR22Rv1 cells. h Tumor growth curve of silencing of TEAD4 by shRNA in CWR22Rv1 cells and tumor image by IVIS scan at endpoint. i Isolated tumor image and weight at endpoint. j Tumor growth curve of knockout of TEAD4 by CRISPR-Cas9 in CWR22Rv1 cells and tumor image at endpoint. k Isolated tumor image and weight at endpoint. Data are presented as mean value ± SEM of independent experiments (n = 3 in f, n = 6 in h–k). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using unpaired two-tailed Student’s t test. Source data are provided as a Source data file.