Limits...
Loss of the N-terminal methyltransferase NRMT1 increases sensitivity to DNA damage and promotes mammary oncogenesis.

Bonsignore LA, Butler JS, Klinge CM, Schaner Tooley CE - Oncotarget (2015)

Bottom Line: Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery.Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER).We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY, USA.

ABSTRACT
Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery. Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER). Here we test whether NRMT1 is also important for DNA double-strand break (DSB) repair, and given its previously known roles in cell cycle regulation and the DNA damage response, assay if NRMT1 is acting as a tumor suppressor. We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity. Interestingly, this positions NRMT1 as a tumor suppressor protein involved in multiple DNA repair pathways, and indicates, similar to BRCA1 and BRCA2, its loss may result in tumors with enhanced sensitivity to diverse DNA damaging chemotherapeutics.

No MeSH data available.


Related in: MedlinePlus

NRMT1 depletion also promotes sensitivity to tamoxifen that is independent of hormone receptor expression levels and NF-κB signaling(A) Fold change in cell number of MCF-7 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (B) RT-PCR analysis of ERα, ERβ, and PR mRNA expression levels normalized to GAPDH in five MCF-7 lines transduced with the NRMT1 KD virus as compared to corresponding control lines. Fold change in expression was calculated by setting control equal to one. (C) Fold change in cell number of SKBR-3 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (D) Luciferase assay demonstrating that neither basal nor TNFα induced NF-κB signaling is increased after NRMT1 knockdown. Each bar represents the mean ± SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494936&req=5

Figure 7: NRMT1 depletion also promotes sensitivity to tamoxifen that is independent of hormone receptor expression levels and NF-κB signaling(A) Fold change in cell number of MCF-7 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (B) RT-PCR analysis of ERα, ERβ, and PR mRNA expression levels normalized to GAPDH in five MCF-7 lines transduced with the NRMT1 KD virus as compared to corresponding control lines. Fold change in expression was calculated by setting control equal to one. (C) Fold change in cell number of SKBR-3 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (D) Luciferase assay demonstrating that neither basal nor TNFα induced NF-κB signaling is increased after NRMT1 knockdown. Each bar represents the mean ± SEM of three independent experiments.

Mentions: Though tamoxifen is most commonly known for its role as an estrogen receptor antagonist, it can also produce reactive oxygen species (ROS) and DNA breaks [37, 38]. To test if NRMT1 loss subsequently renders MCF-7 cells more sensitive to tamoxifen treatment, lentivirally transduced MCF-7 cells were treated with 10 μM tamoxifen and assayed for viability over five days. As MCF-7 cells treated with NRMT1 KD virus exhibit higher basal growth, we could not directly compare cell viability between control and NRMT1 KD cells treated with tamoxifen. Instead we compared the change in viability for each cell line between untreated and tamoxifen treated. We found that loss of NRMT1 renders MCF-7 cells more sensitive to tamoxifen (Figure 7A), as the difference between untreated and tamoxifen treated NRMT1 KD cells was significantly higher than the difference between untreated and tamoxifen treated control cells. Similar results were seen when treating cells with the tamoxifen metabolite 4-hydroxytamoxifen (Supplementary Figure 1).


Loss of the N-terminal methyltransferase NRMT1 increases sensitivity to DNA damage and promotes mammary oncogenesis.

Bonsignore LA, Butler JS, Klinge CM, Schaner Tooley CE - Oncotarget (2015)

NRMT1 depletion also promotes sensitivity to tamoxifen that is independent of hormone receptor expression levels and NF-κB signaling(A) Fold change in cell number of MCF-7 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (B) RT-PCR analysis of ERα, ERβ, and PR mRNA expression levels normalized to GAPDH in five MCF-7 lines transduced with the NRMT1 KD virus as compared to corresponding control lines. Fold change in expression was calculated by setting control equal to one. (C) Fold change in cell number of SKBR-3 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (D) Luciferase assay demonstrating that neither basal nor TNFα induced NF-κB signaling is increased after NRMT1 knockdown. Each bar represents the mean ± SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494936&req=5

Figure 7: NRMT1 depletion also promotes sensitivity to tamoxifen that is independent of hormone receptor expression levels and NF-κB signaling(A) Fold change in cell number of MCF-7 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (B) RT-PCR analysis of ERα, ERβ, and PR mRNA expression levels normalized to GAPDH in five MCF-7 lines transduced with the NRMT1 KD virus as compared to corresponding control lines. Fold change in expression was calculated by setting control equal to one. (C) Fold change in cell number of SKBR-3 NRMT1 KD and control cells with treatment of 10 μM tamoxifen or vehicle control. Each data point represents the mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test and by comparing the fold change between vehicle treated groups (NRMT1 KD or control) to the corresponding tamoxifen treated groups (NRMT1 KD or control), * denotes p < 0.05. (D) Luciferase assay demonstrating that neither basal nor TNFα induced NF-κB signaling is increased after NRMT1 knockdown. Each bar represents the mean ± SEM of three independent experiments.
Mentions: Though tamoxifen is most commonly known for its role as an estrogen receptor antagonist, it can also produce reactive oxygen species (ROS) and DNA breaks [37, 38]. To test if NRMT1 loss subsequently renders MCF-7 cells more sensitive to tamoxifen treatment, lentivirally transduced MCF-7 cells were treated with 10 μM tamoxifen and assayed for viability over five days. As MCF-7 cells treated with NRMT1 KD virus exhibit higher basal growth, we could not directly compare cell viability between control and NRMT1 KD cells treated with tamoxifen. Instead we compared the change in viability for each cell line between untreated and tamoxifen treated. We found that loss of NRMT1 renders MCF-7 cells more sensitive to tamoxifen (Figure 7A), as the difference between untreated and tamoxifen treated NRMT1 KD cells was significantly higher than the difference between untreated and tamoxifen treated control cells. Similar results were seen when treating cells with the tamoxifen metabolite 4-hydroxytamoxifen (Supplementary Figure 1).

Bottom Line: Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery.Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER).We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY, USA.

ABSTRACT
Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery. Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER). Here we test whether NRMT1 is also important for DNA double-strand break (DSB) repair, and given its previously known roles in cell cycle regulation and the DNA damage response, assay if NRMT1 is acting as a tumor suppressor. We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity. Interestingly, this positions NRMT1 as a tumor suppressor protein involved in multiple DNA repair pathways, and indicates, similar to BRCA1 and BRCA2, its loss may result in tumors with enhanced sensitivity to diverse DNA damaging chemotherapeutics.

No MeSH data available.


Related in: MedlinePlus