Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 2: SAMe and MTA induce the expression of miR-34a and miR-34b and all four treatments induce apoptosis and inhibit growth of colon cancer cells. (A) RKO (left) and SW620 (right) cells were treated with 250 μM SAMe or MTA for 24 hours. The mRNA levels of miR-34a and miR-34b were compared to vehicle treated controls using real-time PCR. Results represent mean ± SEM from 4-5 experiments, *p<0.001 vs. control. (B) MAT2A 3’UTR reporter activity in RKO and SW620 cells treated with miR-34a, miR-34b or empty vector (EV). Results represent mean ± SEM from 3 experiments, *p<0.01 vs. EV. RKO (C) and SW620 (D) cells were treated with 250 μM SAMe or MTA, overexpression of miR-34a or miR-34b as described in Methods for 24 hours and were processed for apoptosis, growth by BrdU, miR-34 and miR-34b transfection efficiency measurements. Results represent mean ± SEM from 3 experiments, *p<0.01 vs. EV.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Activity Assay, Plasmid Preparation, Over Expression, Transfection

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 3: Effects of SAMe, MTA, miR-34a and miR-34b on MAT2A and MAT2B expression. RKO (A) and SW620 (B) cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A mRNA levels, MAT2A and MAT2B protein levels (MATα2 and MATβ) were measured by real-time PCR and Western blotting, respectively. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 to 4 experiments, *p<0.05 (real-time PCR) and *p<0.01 (Western blotting) vs. empty vector (EV) control. (C) RKO cells were treated with 10 nM siRNA against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression was measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.001 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 4: Effects of MAT2A, MAT2B, miR-34a and miR-34b in colon cancer cell migration, invasion and growth. RKO cells were transfected with empty vector (EV), miR-34a, miR-34b, MAT2A or MAT2B expression vectors alone or in combination for 24 hours for measurement of cell migration (A), invasion (B) and growth (C) as described in Methods. Results represent mean ± SEM from 3 to 4 experiments, *p<0.05 vs. EV. †p<0.05 vs. MAT2A overexpression. (D) shows transfection efficiency. (E) RKO cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.04 vs. EV.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Migration, Transfection, Plasmid Preparation, Expressing, Over Expression, Western Blot, Control

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 6: MAT2A and MAT2B expression is upregulated in human prostate and pancreatic cancers. (A) Representative IHC of MATα2 and MATβ is shown from normal (n = 8) and prostate cancer patient tissues (n = 40). (B) IHC of MATα2 and MATβ from 4 separate normal pancreatic tissues and PDACs are shown, scale = 100 μm. Magnification x 20.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 8: MAT2A and MAT2B expression is down-regulated by SAMe, MTA, miR-34a and miR-34b in prostate cancer cell line. CWR22Rv1 cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A and MAT2B mRNA (A) and protein (B) levels were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 to 4 experiments, *p<0.04 vs. empty vector (EV) control. (C) Effect of SAMe and MTA treatment (250 μM for 24 hours) on miR-34a and miR-34b expression in CWR22Rv-1 cells. Results represent mean ± SEM from 5 experiments, *p<0.05 vs. control. (D) CWR22Rv-1 cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.02 vs. EV. (E) CWR22Rv-1 cells were transfected with EV, MAT2A or MAT2B overexpression vector and cell migration was measured as described in Methods. Results represent mean ± SEM from 3 experiments, *p<0.002 vs. EV control.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control, Transfection, Migration

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 9: MAT2A and MAT2B expression is down-regulated by SAMe, MTA, miR-34a and miR-34b in pancreatic cancer cell line. MIA PaCa-2 cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A and MAT2B mRNA (A) and protein (B) levels were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.02 vs. empty vector (EV) control. (C) Effect of SAMe and MTA treatment (250 μM for 24 hours) on miR-34a and miR-34b expression in MIA PaCa-2 cells. Results represent mean ± SEM from 4 experiments, *p<0.05 vs. control. (D) MIA PaCa-2 cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.05 vs. EV. (E) MIA PaCa- 2 cells were transfected with EV, MAT2A or MAT2B overexpression vector and cell migration was measured as described in Methods. Results represent mean ± SEM from 3 experiments, *p<0.03 vs. EV control.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control, Transfection, Migration

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 10: MAT2A and MAT2B proteins stabilize each other in CWR22Rv-1 and MIA PaCa-2 cells. CWR22Rv-1 (A) and MIA PaCa-2 (B) cells were treated with 10 nM siRNA against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.03 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 11: MAT2A and MAT2B proteins stabilize each other in RKO, CWR22Rv-1 and MIA PaCa-2 cells. RKO (A), CWR22Rv-1 (B) and MIA PaCa-2 (C) cells were treated with a second siRNA (10 nM) against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.05 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 12: Summary diagram of miR-34a/b-MAT2A/MAT2B axis and treatment with SAMe or MTA. In normal non- hepatic tissues, miR-34a and miR-34b negatively regulate MAT2A expression mainly by suppressing its protein translation. In cancer cells miRNA-34a and miR-34b are often down-regulated, releasing the inhibition on MAT2A expression. This indirectly raises MAT2B expression as the two proteins interact and stabilize each other. Higher MAT2A and MAT2B expression leads to cancer cell growth, migration and invasion. Treatment with SAMe or MTA in cancer cells increases the expression of miR-34a and miR-34b. Potential mechanisms involved include via lowering DNA methylation of miR-34a/b promoters, and inhibition of IL-6/STAT3 signaling pathway. SAMe and MTA could also suppress MAT2A and MAT2B by other mechanisms not shown. Collectively this results in lower MAT2A and MAT2B expression, increased apoptosis, decreased cell growth, migration and metastasis.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Inhibition, Migration, DNA Methylation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: MATα2 is secreted by CRC cells in extracellular vesicles. A Exopred and Exocarta software predicts MATα2 is secreted via exosomes ( B ) CRC cells transfected to overexpress DDK-MAT2A (M2A) secreted more MATα2 when compared to empty vector (EV). C NanoSight analysis of extracellular vesicles isolated from culture media of CRC cells transfected to overexpress MAT2A (MAT2A OE) and empty vector (EVec) with peak corresponding to size range of exosomes. D Images from NanoSight analysis of extracellular vesicles. E Immunofluorescence microscopy of human hepatocytes treated with exosomes isolated from culture media from CRC transfected to overexpress DDK-MAT2A and empty vector (EVec) shows internalization of EV-MATα2 with localizing to the nuclues via DAPI staining

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Software, Transfection, Plasmid Preparation, Isolation, Immunofluorescence, Microscopy, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 is internalized by hepatocytes and alters MAT1A and MAT2A expression A mRNA levels of MAT1A and MAT2A isolated from human hepatocytes treated with exosomes from CRC cells. B Western blot analysis of endogenous nuclear and cytosolic MATα2 in human hepatocytes treated with exosomes from CRC cells. Lamin B1 and tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Mean ± SEM from n = 3, * p < 0.05 vs. EVec exo. C-D Confocal microscopy of human liver spheroids treated with exosomes from CRC cells transfected with empty vector (EVec) or MAT2A-His vector at day 7 for 24 h showing effect on MATα1 and MATα2-His expression. E mRNA levels of MAT1A and MAT2A isolated from human liver spheroids after the exosome treatment. Mean ± SEM from n = 3, * p < 0.02 vs.control and † p < 0.05 vs. EVec exo

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Expressing, Isolation, Western Blot, Confocal Microscopy, Transfection, Plasmid Preparation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Integrated analysis of MATα2 genomic binding profiles in CRC cells. A Heatmap showing the read density distribution of MATα2 ChIP-seq peaks across all human chromosomes. Read intensities are color-coded from low (purple) to high (yellow) density. B Genomic annotation of MATα2 binding peaks. C Top five DNA-binding motifs enriched within MATα2-bound peaks ranked by motif enrichment score. D Pathway enrichment analysis of MATα2-associated genes. E Predicted consensus sequences logos representing the top three de novo motifs identified from MATα2 binding sites by Jaspar software. F ChIP-seq tracks showing MATα2 (blue), RNA polymerase II (POL II; red) and input control (black) signals across the MAT1A (lower) or MAT2A (upper) locus. The Y-axis represents normalized read enrichment. The bottom track shows the gene structure with exons (black boxes) and introns (dashed lines)

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Binding Assay, ChIP-sequencing, Software, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 acts as a transcription factor to alter MAT1A and MAT2A expression. A Promoter activities in human hepatocytes transfected with human MAT1A or MAT2A promoter constructs and then treated with exosomes from RKO cells expressing empty vector (EVec exo) or MAT2A (EV-MATα2) as described in Methods. B ChIP analysis of the human MAT1A and ( C ) human MAT2A promoters showing binding of MATα2-His to different regions of the promoters. Mean ± SEM from n = 8, * p < 0.05 and ** p < 0.01 vs. EVec exo for MAT1A promoter; n = 7, * p < 0.04 vs. EVec exo for MAT2A promoter

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 induces MAT2A expression and oncogenic activity in RKO cells. A RKO cells were treated with exosomes from RKO cells expressing empty vector (EVec exo) or MAT2A-His-Tag vector (EV-MATα2) as described in Methods and cell entry was visualized under fluorescent microscopy using His-tag antibody. B Real-time PCR shows the effect at the MAT2A mRNA level. Mean ± SEM from n = 3, * p < 0.02 vs. EVec exo. C Western blotting was done in total cell lysate, cytoplasmic and nuclear fractions showing increased MATα2 levels. Densitometry were measured by ImageJ. Mean ± SEM from n = 3, * p < 0.002 for total lysate, * p < 0.02 for cytoplasmic, * p < 0.03 for nuclear fractions vs. EVec exo. D ChIP analysis of the human MAT2A promoter showing binding of MATα2-His to different predicted motifs. Mean ± SEM from n = 3, * p < 0.05 vs. EVec exo. Effects of the same treatments on EdU ( E ) ( n = 3, * p < 0.05 and ** p < 0.01 vs. control), migration ( F ) ( n = 3, * p < 0.004 and ** p < 0.0001 vs. 0 h EVec exo, † p < 0.003 vs. 24 h EV-MATα2), and invasion ( G ) ( n = 3, * p < 0.008 vs. EVec exo)

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Expressing, Activity Assay, Plasmid Preparation, Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Control, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Cancer cells secrete more truncated MATα2, which is required for survival. A Medium from RKO cells overexpressing MAT2A-His or empty vector (EVec) was separated into exosomes and EV-free media that only has truncated MATα2 (MATα2-t). Note MATα2-His has higher MW than full length endogenous MATα2, which has the same MW as MATα2-t-His. Endogenous MATα2-t has the lowest MW. B MATα2 protein sequence and predicted cleavage site based on PrediSI is at proline 30. C RKO (CRC), MiaPACA (pancreatic adenocarcinoma) and RV1 (prostate adenocarcinoma) cells secrete more MATα2-t as compared to the respective non-malignant cells (HCoEpC, HPDE, RWPE1). Mean ± SEM from n = 3, * p < 0.01 vs. HCoEpC cells; * p < 0.03 and † p < 0.01 vs. HPDE cells; * p < 0.03 and † p < 0.001 vs. RWPE1 cells. D RKO and HT29 cells treated with anti-MATα2 (20 µg/ml) for 48 h and TUNEL staining shows CRC cells underwent apoptosis. E MTT assay in RKO and HT29 cells treated with anti-MATα2 shows a fall in viability. Mean ± SEM from n = 3, * p < 0.03 and † p < 0.04 vs. control. F RKO cells were transfected with MATα2-DDK for 48 h and increasing amount of anti- MATα2 Ab was added, followed by pull-down of MATα2 Ab using beads, then western blotted the pull-down with anti-DDK Ab. The MATα2 antibody was able to bring down freely secreted MATα2 in a dose-dependent manner. This correlated with a dose-dependent increase in active caspase 3

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Plasmid Preparation, Sequencing, TUNEL Assay, Staining, MTT Assay, Control, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Secreted MATα2-t activates FAK and is required to maintain MAT2A expression. A RKO cells were treated with EV-free media containing MATα2-t as described in Methods and western blotted for pFAK and total FAK. Mean ± SEM from n = 3, * p < 0.04 vs. EVec ( B ) RKO cells were treated with anti-MATα2 Ab for 48 h and western blotted for pFAK, total FAK, pro-caspase 3 and active caspase 3. Mean ± SEM from n = 3, * p < 0.03 vs. control. C FAK and MAT2A mRNA levels in RKO cells from the above treatments were measured by real-time PCR. Mean ± SEM from n = 3, * p < 0.002 vs. EVec; * p < 0.002 vs. control. D RKO cells were CRISPR/Cas9 gene edited (HDR) to mutate proline to leucine at position 30 (canonical motif: PDLD) and at positions 131 and 133 glycine to leucine (non-canonical motif: GXGD); western blotted for pFAK and FAK. Mean ± SEM from n = 5–6, * p < 0.01 vs. wild-type (WT) for PDLD. E Immunoblotting of secreated MATα2 (MATα2-t) in culture media from RKO cells gene edited PDLD motif. Mean ± SEM from n = 3, * p < 0.04 vs. WT. F PDLD gene edited RKO and HT29 cells exhibited increased apoptosis on TUNEL staining (blue for RKO and brown for HT29 depending to pH culture media)

Article Snippet: Furthermore, MATα2 is reported to be present in circulation on the Human Protein Atlas ( MAT2A protein expression summary - The Human Protein Atlas ).

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, CRISPR, TUNEL Assay, Staining