Journal: Cancer Communications
Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling
doi: 10.1002/cac2.70072
Figure Lengend Snippet: Methionine metabolism promotes H3K4me3 methylation modification to upregulate PDGFB expression in CTC clusters. (A) Western blot analysis of PERK, MAT2A, and H3K4me3 levels in control and PERK‐KO MDA‐MB‐231/LM2‐CTC clusters treated with or without PF9366 or SAM. (B) The volcano plot depicting differentially expressed genes affected by methionine ( n = 3). (C) Metaplot comparing H3K4me3 enrichment profiles in MDA‐MB‐231/LM2‐CTC clusters cultured in CM or MRM. The plot is centered on TSS (±3.0 Kb) ( n = 3). (D) Bioinformatics analysis filtered out 10 survival‐related genes as downstream targets of H3K4me3. (E) Heatmap showing survival‐associated gene expression in MDA‐MB‐231/LM2‐CTC clusters under CM or MRM conditions ( n = 3). (F) Genome browser tracks of H3K4me3 at the PDGFB gene locus by ChIP‐seq ( n = 3). (G) TEM images of MDA‐MB‐231/LM2‐CTC cluster. (H‐J) Representative immunofluorescence images (left) and quantification (right) of PDGFB in CTC clusters from NCG‐MDA‐MB‐231/LM2 mouse model (H, n = 20); or in the complete or dispersed CTC clusters (I, n = 15); or in control or PDGFB‐knockdown CTC clusters (J, n = 15). Arrows indicate cell‐cell border; circle indicates intracellular regions. (K) Representative images of flow cytometry (left) and quantification of AnnexinV + MDA‐MB‐231/LM2‐CTC clusters (right) in control (shNC) and PDGFB‐knockdown (shP#1/2/3) groups ( n = 5). (L) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters (shNC and shPDGFB #3) were injected into NCG mice via the tail vein, and metastatic burden was assessed by bioluminescence imaging at 24 h. (M‐N) Bioluminescence images (left) and quantification of fluorescence intensity (right) showing lung metastasis after tail vein injection at 24 h ( n = 5). Results represent mean ± SD. One‐way ANOVA test in A and K; student's t‐test in H, I, J, M, and N. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: MDA‐MB‐231/LM2, MDA‐MB‐231 lung metastasis 2; CTCs, circulating tumor cells; FC, fold change; PERK, PRKR‐like endoplasmic reticulum kinase; ERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; MAT2A, methionine adenosyltransferase 2A; H3K4me3, trimethylation of histone H3 at lysine 4; SAM, S‐adenosylmethionine; CM, control medium; MRM, methionine‐restricted medium; No., number; TSS, transcription start site; ChIP‐seq, chromatin immunoprecipitation sequencing; RNA‐seq, RNA sequencing; PDGFB, platelet‐derived growth factor subunit B; IGF2BP3, insulin‐like growth factor 2 mRNA‐binding protein 3; PHGDH, phosphoglycerate dehydrogenase; ANXA11, annexin A11; ITGA6, integrin subunit alpha 6; ITGB4, integrin subunit beta 4; LOX, lysyl oxidase; PIK3C2B, phosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 beta; IRF9, interferon regulatory factor 9; ATP2A3, ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 3; TEM, transmission electron microscopy; DAPI, 4′,6‐diamidino‐2‐phenylindole; shNC, negative control short hairpin RNA; shP#, short hairpin RNA targeting PDGFB; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; BLI, lung Bioluminescence imaging; ANOVA, analysis of variance.
Article Snippet: The following antibodies were used for western blotting: PERK (1:1, 000, ab229912, Abcam), pPERK (1:1, 000, DF7576, Affinity Biosciences), eukaryotic translation initiation factor 2 alpha (EIF2α, 1:1, 000, ET7111‐34, HUABIO), phosphorylated EIF2α (pEIF2α, 1:1, 000, #3398, Cell Signaling Technology, Danvers, MA, USA), IRE1α (1:1, 000, ab37073, Abcam), phosphorylated IRE1α (pIRE1α, 1:1, 000, ab48187, Abcam), the spliced form of X‐box binding protein 1 (XBP1s, 1:1, 000, 24868‐1‐AP, Proteintech), activating transcription factor 6 (ATF6, 1:1, 000, ER1706‐34, HUABIO), phosphoserine aminotransferase 1 (PSAT1, 1:1, 000, 10501‐1‐AP, Proteintech), phosphoglycerate dehydrogenase (PHGDH, 1:1, 000, 14719‐1‐AP, Proteintech), methylenetetrahydrofolate reductase (MTHFR, 1:1, 000, ab203786, Abcam), MAT2A (1:1, 000, #84478, Cell Signaling Technology), ATF4 (1:1, 000, #11815, Cell Signaling Technology), PI3K (1:1, 000, #4249, Cell Signaling Technology), p‐AKT (1:1, 000, #4060, Cell Signaling Technology), t‐AKT (1:1, 000, #9272, Cell Signaling Technology), β‐actin (1:10, 000, EM21002, HUABIO).
Techniques: Methylation, Modification, Expressing, Western Blot, Control, Cell Culture, Gene Expression, ChIP-sequencing, Immunofluorescence, Knockdown, Flow Cytometry, Injection, Imaging, Fluorescence, Knock-Out, RNA Sequencing, Derivative Assay, Binding Assay, Transmission Assay, Electron Microscopy, Negative Control, shRNA
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![a. L-[ 3 H(G)]-serine transport rate (cpm/10 6 cells, resulting from 15 min uptake, left panel or 3 hours uptake, right panel) in Eµ- Myc (#688) cells following 24 hours incubation in serine and glycine-containing medium, with (+) or without (-) Asn (0.37 mM) and ASNase (0.003 IU/ml). Data are expressed as mean ± SD (n≥ 3 experiments). P-value from t-test. b. c. Relative abundance (peak area) of total intracellular serine ( b. ) and glycine ( c. ) levels in Eµ -Myc (#688) cells cultivated for 24 hours in Asn, L-serine and glycine-free medium, (+) or not (-) with serine, glycine (Ser/Gly; 0.4 mM/0.4 mM), Asn (0.37 mM) and ASNase (0.003 IU/ml) (n=3 biological replicates). P-value from 2-way Anova followed by Tukey’s test. d. Total protein extracts prepared from whole live Eµ- Myc #506 (left panel) and #688 (right panel) cells cultivated for 24 hours in glutamine (Gln) and Asn-free medium supplemented (+) or not (-) with Gln (2 mM), Asn (0.37 mM) or ASNase (0.003 IU/ml), were immunoblotted for the indicated proteins. ERK2, loading control. Immunoblots shown are representative of 2 independent experiments for each Eµ- Myc line. e. Relative quantification of the expression levels of indicated proteins in Eµ- Myc #506 (square, n=2 independent experiments) and in Eµ- Myc #688 (triangle, n=2 independent experiments) cells, following 24 hours of incubation as in d. Data are represented as mean ± SD. P-value from P-value from t-test. f. g. Relative abundance (peak area) of 13 C-labelled serine m+3 ( f. ) and 13 C-labelled glycine m+2 ( g. ) isotopologues in Eµ- Myc (#688) cells cultivated for 24 hours in glucose and asparagine-free medium supplemented with 25 mM [U- 13 C]-glucose and with (+) or without (-) Asn (0.37 mM) and ASNase (0.003 IU/ml). Data are expressed as mean ± SD (n=4 biological replicates and two technical replicates). P-value from t-test. h. Total protein extracts prepared from BCL harvested in C57BL/6 mice bearing Eµ- Myc (#506) lymphoma, treated with Vehicle or ASNase every 48 hours till disease endpoint, were immunoblotted for the indicated proteins (Vehicle, n=4 mice; ASNase, n=10 mice). ERK2, loading control. i. Percentage of <t>PHGDH</t> activity in BCL harvested from Eµ- Myc cells-bearing C57BL/6 mice treated as in g. Data are expressed as mean ± SD (n=9 mice/group). P-value from t-test. j. Schematic representation of in vivo [U- 13 C]-glucose consecutive bolus protocol following Vehicle or ASNase treatment in C57BL/6 mice bearing OxPhos-dependent Eµ- Myc #688 BCL. k. Relative abundance (peak area) of 13 C-labelled serine (m+3) and glycine (m+2) isotopologues in BCL harvested in C57BL/6 mice bearing Eµ- Myc #688 lymphoma, treated as in j . Data are expressed as mean ± SD (n=4 mice/group). P-value from t-test. PHGDH, phosphoglycerate dehydrogenase; PSAT1, phosphoserine aminotransferase 1; PSPH, phosphoserine phosphatase; ASNS, asparagine synthetase; GLUL, glutamine synthetase, GOT1, Glutamate-Oxaloacetate Transaminase-1; ATF4, Activating Transcription Factor 4. ns , not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_36/10__1101_slash_2025__10__24__683536/10__1101_slash_2025__10__24__683536___F2.large.jpg)
![PERK signaling regulates MAT2A via ATF4 to support enhanced methionine metabolism. (A) KEGG enrichment analysis of upregulated genes in scramble control tumor cell clusters compared to PERK‐KO clusters ( n = 3). (B) Volcano plot showing PERK‐related metabolites identified through targeted metabolomics ( n = 6). (C) Heatmap showing metabolites involved in the methionine cycle in scramble and PERK‐KO tumor cell clusters ( n = 6). (D) Intracellular metabolites in NC‐oe and PERK‐oe tumor cell clusters ( n = 6). (E) The schematic diagram for the conversion of [ 13 C 5 ]‐methionine into various metabolites (left) and LC‐MS quantification of M+5 methionine, M+5 SAM, and M+4 SAH following a 16 h incubation with [ 13 C 5 ]‐methionine in scramble and PERK‐KO tumor cell clusters (right) ( n = 4). (F) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC cells cultured in CM or medium lacking Ser, Gly, Met, or Cys for 24 h ( n = 5). (G‐I) Western blot analysis of metabolic enzyme expression in MDA‐MB‐231/LM2‐CTC and B16F10 tumor cell clusters with or without PERK (G), ATF4 expression in MDA‐MB‐231/LM2‐CTC with or without PERK (H), and ATF4 and MAT2A expression in MDA‐MB‐231/LM2‐CTC with or without ATF4 (I). (J) Relative MAT2A expression in MDA‐MB‐231/LM2‐CTC transfected with shNC or shATF4 #2 ( n = 3). (K) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC transduced with shNC or shMAT2A #3 and treated with or without SAM for 24 h (left) or pretreated with PF9366 prior to SAM (right) ( n = 3). (L) Schematic of the in vivo experiment design. (M) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). (N) Representative H&E staining images (left) and quantification of pulmonary nodules (right) in NCG mice at 2 weeks after tail vein injection of MDA‐MB‐231/LM2‐CTC clusters (shNC and shMAT2A #3) ( n = 5). (O) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters were cultured in CM or MRM for 24 h before tail vein injection into NCG mice, with lung metastases assessed at 24 h. (P) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). Results represent mean ± SD. Student's t‐test in D, E, J, M, and P; one‐way ANOVA test in F and K. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; FC, fold change; Sc, scramble; PERK, Protein kinase RNA‐like endoplasmic reticulum kinase; PERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; LC‐MS, liquid chromatography‐mass spectrometry; Met, methionine; SAM; S‐adenosylmethionine; SAH, S‐adenosylhomocysteine; CM, control medium; Ser, serine; Gly, glycine; Cys, cysteine; PSAT1, phosphoserine aminotransferase 1; PHGDH, <t>phosphoglycerate</t> <t>dehydrogenase;</t> MTHFR, methylenetetrahydrofolate reductase; MAT2A, methionine adenosyltransferase 2A; ATF4, activating transcription factor 4; shNC, negative control short hairpin RNA; NC‐oe, negative control‐overexpression; PERK‐oe, PERK‐overexpression; shATF4, short hairpin RNA of ATF4; shMAT2A, short hairpin RNA of MAT2A; MRM, methionine‐restricted medium; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2, MDA‐MB‐231 lung metastasis 2; CTCs, circulating tumor cells; BLI, lung Bioluminescence imaging; H&E, hematoxylin and eosin; ANOVA, analysis of variance.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8491/pmc12728491/pmc12728491__CAC2-45-1706-g004.jpg)
