Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells

doi:10.7150/thno.44688

. 2020 Jul 25;10(21):9458-9476.

doi: 10.7150/thno.44688. eCollection 2020.

Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells

Lutao Du¹, Xiaoyan Liu¹, Yidan Ren¹, Juan Li¹, Peilong Li¹, Qinlian Jiao^{1

2}, Peng Meng³, Fang Wang⁴, Yuli Wang¹, Yun-Shan Wang¹, Chuanxin Wang¹

Affiliations

¹ Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China.
² International Biotechnology R&D Center, Shandong University School of Ocean, 180 Wenhua Xi Road, Weihai, Shandong 264209, China.
³ The Medical Department of IVD Division, 3D Medicines, Inc., Pujiang Hi‑tech Park, Shanghai 201114, China.
⁴ Institute of basic medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China.

PMID: 32863939
PMCID: PMC7449925
DOI: 10.7150/thno.44688

Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells

Lutao Du et al. Theranostics. 2020.

. 2020 Jul 25;10(21):9458-9476.

doi: 10.7150/thno.44688. eCollection 2020.

Authors

Lutao Du¹, Xiaoyan Liu¹, Yidan Ren¹, Juan Li¹, Peilong Li¹, Qinlian Jiao^{1

2}, Peng Meng³, Fang Wang⁴, Yuli Wang¹, Yun-Shan Wang¹, Chuanxin Wang¹

Affiliations

¹ Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China.
² International Biotechnology R&D Center, Shandong University School of Ocean, 180 Wenhua Xi Road, Weihai, Shandong 264209, China.
³ The Medical Department of IVD Division, 3D Medicines, Inc., Pujiang Hi‑tech Park, Shanghai 201114, China.
⁴ Institute of basic medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China.

PMID: 32863939
PMCID: PMC7449925
DOI: 10.7150/thno.44688

Abstract

Rationale: It has been proposed that cancer stem/progenitor cells (or tumor-initiating cells, TICs) account for breast cancer initiation and progression. Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent class-III histone deacetylases and mediate various basic biological processes, including metabolic homeostasis. However, interplay and cross-regulation among the sirtuin family are not fully understood. As one of the least studied sirtuin family members, the mitochondrial sirtuin SIRT4 is a tumor suppressor gene in various cancers. However, its role in cancer stemness, as well as initiation and progression of breast cancer, remains unknown. Methods: The expression of SIRT4 in breast cancer was analyzed using the TCGA breast cancer database and 3 GSEA data. Normal breast epithelial cells MCF10A and breast cancer cell lines MCF-7, MDA-MB-231, BT549, MDA-MB-468 were used to establish SIRT4 gene knockdown and corresponding overexpression cells. Identified MTT cytotoxicity assays, cell invasion and motility assay, sorting of SP, confocal immunofluorescence microscopy, mouse mammary stem cell analysis, glutamine and glucose production, clonogenic and sphere-formation assay, mass spectrometric metabolomics analysis and ChIP-seq to further explore SIRT4 biological role in breast cancer. Results: We elucidated a novel role for SIRT4 in the negative regulation of mammary gland development and stemness, which is related to the mammary tumorigenesis. We also uncovered an inverse correlation between SIRT4 and SIRT1. Most importantly, SIRT4 negatively regulates SIRT1 expression via repressing glutamine metabolism. Besides, we identified H4K16ac and BRCA1 as new prime targets of SIRT4 in breast cancer. Conclusions: These results demonstrate that SIRT4 exerts its tumor-suppressive activity via modulating SIRT1 expression in breast cancer and provide a novel cross-talk between mitochondrial and nuclear sirtuins.

Keywords: SIRT1; SIRT4; breast cancer; cancer stemness; glutamine metabolism.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

**Figure 1**
**SIRT4 expression is downregulated in breast cancer and related to mammary gland development and stemness.** (A) SIRT4 expression levels in breast cancers compared to healthy tissues from the TCGA data set of 1247 samples. (B) Kaplan-Meier analysis indicating the overall survival of breast cancer patients with high (red) (n = 1944) or low (black) (n = 2007) SIRT4 expression. (C) SIRT4 KO mice were obtained from The Jackson Laboratory. SIRT4 knockout efficiency was measured by immunoblotting (left panel) and qRT- PCR (right panel). (D) Whole-mount analyses were conducted on 8-week-old SIRT4 wild-type (SIRT4^WT) (n = 15), and SIRT4^-/- (n = 18) mice and representative images of mammary gland side-branches are shown. (E) Percent of growth. (F) The number of mammary gland side-branches was quantified. (G) Distribution of Linneg mouse mammary cells according to their expression of CD24 and CD49f was analyzed on 8-week-old SIRT4^-/- mice and littermate controls (left). Mammary stem cells (MSCs), according to their expression of CD24hiCD49fhi in Linneg (right), were quantified by a flow cytometric analysis. (**H, I and J**) Schematic representation of limiting dilution transplantation experiments with LinnegCD24hiCD49fhi MSCs (H). A total of 300, 1000, or 1 x 10⁴ LinnegCD24hiCD49fhi MSCs isolated from 8-week-old SIRT4^-/- mice and littermate controls were injected into the cleared fat pad of 3-week-old FVB/NJ female mice. Whole-mount analyses were then conducted at 6 weeks after injection (I, J). Representative images of mammary gland side-branches are shown (I). The resulting data were analyzed by the Chi-square test (p < 0.001) (J). Scale bars, 100 µm (D) and 50 µm (I).

**Figure 2**
**SIRT4 deletion promotes mammary tumorigenesis.** (A) SIRT4^-/- mice were crossed with MMTV-Neu mice to generate MMTV-Neu transgenic mice in wild-type and SIRT4-null genetic backgrounds. The mammary tumor was developed at an average age of 240 days. Representative images are shown. Using these mice, we monitored (B) tumor weight, (C) tumor incidence, (D) overall survival time, (E) Ki67 and SIRT4 stained tumor sections, (F) SIRT4 protein expression in tumors isolated from MMTV-neu and MMTV-neu; SIRT4^-/- mice. Data are means ± SEM. p < 0.01; t-test (B and C). Scale bars, 50 µm (E).

**Figure 3**
**SIRT4 inhibits self-renewal and expansion of breast tumor-initiating cells (BTICs).** (A) Heatmap summarizing genes differentially expressed in SIRT4^-/- compared to SIRT4^WT mice. (B) Volcano plot displaying differentially expressed genes. Up-regulated genes (3123) are highlighted in red. Down-regulated genes (2477) are highlighted in green. Black dots represent genes not differentially expressed. (C) Enrichment of a stem cell signaling in GSEA analysis of genes altered as described above. (**D, E, F**) Immunoblotting (upper panel) and mRNA expression (bottom panel) of SIRT4 in CSC-enriched mammospheres (D), side population (SP) cells (E), and EPCAM+ cells (F) as well as their corresponding controls, i.e., adherent cells (A), non-SP cells (C), and EPCAM- cells (E). (G) Quantification of CD44⁺/CD24^- subpopulations (right panel) and immunoblotting of SIRT4 expression (left panel) in MDA-MB-231, MCF-7, and BT549 cells transfected with sh-SIRT4 or negative control (NC). (H) Quantification of CD44⁺/CD24^- subpopulations (right panel) and immunoblotting of SIRT4 expression (left panel) in MDA-MB-468, MCF-10A, and SK-BR-3 cells transfected with SIRT4 or control vector (Vec). (**I, J**) Sphere formation efficiency of cells described in G (I) and H (J), respectively. (**K, L**) Hoechst SP assay of cells described in G (K) and H (L), respectively. (M) Tumor formation ability of MDA-MB-231 cells expressing control (Vector) or SIRT4 vector. The transfected MDA-MB-231 cells were assayed for the ability to form tumors by subaxillary injection of 1 × 10⁶, 1 × 10⁵, 10,000, 1,000, and 100 cells into nude mice. The numbers of tumors formed and the number of injections that were performed is listed for each population. Data are means ±SEM. ^**p < 0.01; t-test. Scale bars, 100 µm (I and J).

**Figure 4**
**Proteome-wide analysis of SIRT4 deficiency-induced expression in mouse mammary gland cells.** (A) Heatmap depicting proteins differentially expressed in mammary epithelial cells from SIRT4^WT and SIRT4^-/- mice. Up-regulated proteins are highlighted in pink. Down-regulated proteins are highlighted in purple. (B) Volcano plot displaying differentially expressed proteins in SIRT4^-/- compared to SIRT4^WT mice. (C) Venn diagram showing the overlap of genes (purple) and proteins (yellow) differentially expressed in mammary epithelial cells from SIRT4^WT and SIRT4^-/- mice (upper); Protein-Protein-Interaction Network including the 14 overlapped proteins in Venn diagram (bottom). (D) Representative IHC staining images of SIRT4 and SIRT1 in tumor sections isolated from SIRT4^WT and SIRT4^-/- mice. (**E, F**) Immunoblotting (upper panel) and mRNA expression (bottom panel) of SIRT1 in tumors isolated from SIRT4^-/- and SIRT4^WT mice (E), and in MDA-MB-231 and BT549 cells transfected with control (Vector), SIRT4 or mutated SIRT4 (H161Y) vector (F). Data are means ±SEM. ^**p < 0.01; t-test (E and F). Scale bars, 100 µm (D).

**Figure 5**
**SIRT4 deficiency down-regulates BRCA1 expression.** (**A, B**) Immunoblotting of acetyl-histone H4 at lys16 (H4K16ac), acetyl-histone H3 at lys9 (H3K9ac), Oct4, SOX2, and Nanog in mammary cells isolated from SIRT4^-/- as well as SIRT4^WT mice (A), and in MDA-MB-231 (left panel) as well as BT549 cells (right panel) transfected with control (Vector), SIRT4 or mutated SIRT4 (H161Y) vector (B). (C) Heatmap summarizing chromatin immunoprecipitation (ChIP)-seq data for H4K16ac, comparing mammary cells from SIRT4^WT and SIRT4^-/- mice (left panel), as well as MDA-MB-231 cells transfected with control (Vector) or SIRT4 vector (right panel). Profiles are centered on H4K16ac binding peaks and depict signal intensity (relative fold enrichment) with green color. (D) ChIP signal of IgG (left panel, red peaks), H3K9ac (middle, blue peaks), and H4K16ac (right panel, green peaks) in the indicated genomic regions of BRCA1 in cells described in C. (**E, F**) Mammary cells from SIRT4^WT as well as SIRT4^-/- mice (E) and transformed MDA-MB-468 cells (F) as described in C and Fig.3G were chromatin immunoprecipitated for IgG and H4K16ac. Pull-down at the putative H4K16ac binding sites was assessed by qRT- PCR and calculated as the percentage of IgG input. Error bars are SEM for 3 technical replicates. (G) Luciferase reporter assays showing the impact of SIRT4 deletion (left), as well as overexpression of SIRT4 and its mutation (H161Y) on BRCA1 promoters (right panel) in SIRT4^-/- as well as SIRT4^WT mammary cells, and MDA-MB-231 as well as BT549 cells, respectively. (**H, I**) Immunoblotting (H) and mRNA expression (I) of BRCA1 in cells described above. Data are means ±SEM. ^**p < 0.01; t-test.

**Figure 6**
**SIRT1 is required for the SIRT4 deficiency-induced BCSC phenotype.** (A) Sphere formation efficiency of MDA-MB-468 (left) and MCF-7 cells (right panel) transfected with different combinations of control vector, sh-SIRT4, and sh-SIRT1. (B) Quantification of CD44⁺/CD24^- subpopulations (C) SIRT4, SIRT1, H4K16ac, BRCA1, SOX2, Oct4, and Nanog in MDA-MB-468 (left) and MCF-7 cells (right panel) described above. (D) Immunofluorescence images of cells described in (A) stained with antibodies against BRCA1/DAPI. (E) Representative ventral view images of bioluminescence from mice described above. (F) Tumor volume. (G) Quantification of E. Data are means ±SEM. ^**p < 0.01; t-test. Scale bars, 100 µm (A) and 20 µm (D).

**Figure 7**
**Glutamine metabolism disorder mediates SIRT4-induced SIRT1 inhibition in breast cancer cells.** (A) Heatmap showing the changes in metabolites levels between SIRT4^WT and SIRT4^-/- mice. Up-regulated metabolites are highlighted in red. Down-regulated metabolites are highlighted in purple. (B) Measurement of Glutamine uptake (upper left), NH4+ production (upper right), Glucose uptake (bottom left), and lactate production (bottom right panel) in mammary epithelial cells from SIRT4^WT and SIRT4^-/- mice. (**C, D**) Immunoblotting of indicated proteins isolated from SIRT4^WT and SIRT4^-/-mammary cells with or without BPTES (10 µM) (C) and 968 (10 µM) (D) treatment. (E, F, G) Quantification of Hoechst SP assay (E), sphere formation efficiency (F), and CD44+/CD24- subpopulations (G) in SIRT4^WT and SIRT4^-/-mammary cells with or without BPTES (left) and 968 (right panel) treatment. (**H, I**) Representative ventral view images of bioluminescence from mice with injections of cells described above (H) and its quantification (I). (J) Representative IHC staining images of BRCA1 in tumor sections isolated from mice described in H. Data are means ± SEM. ^**p < 0.01; t-test. Scale bars, 100 µm (J).

**Figure 8**
**EX-527 significantly eliminates SIRT4 depletion-induced BTICs and xenograft formation.** (**A, B, C**) Quantification of Hoechst SP assay (A), sphere formation efficiency (B), and CD44⁺/CD24^- subpopulations (C) in transformed MDA-MB-468 (left) and MCF-7 cells (right panel) described in Fig. 3G with or without treatment of EX-527, a highly potent and selective inhibitor of SIRT1. (D) Immunoblotting of indicated proteins isolated from transformed MDA-MB-468 (left) and MCF-7 cells (right panel) described above with or without EX-527 treatment. (**E, F**) Representative ventral view images of bioluminescence from mice with injections of cells described above (E) and its quantification (F). (G) A schematic model was illustrating the biological processes regulated by SIRT4 in breast cancer. Data are means ± SEM. p < 0.01; t-test.

See this image and copyright information in PMC

Cited by

Progress of Breast Cancer basic research in China.
Wang X, Wang C, Guan J, Chen B, Xu L, Chen C. Wang X, et al. Int J Biol Sci. 2021 May 11;17(8):2069-2079. doi: 10.7150/ijbs.60631. eCollection 2021. Int J Biol Sci. 2021. PMID: 34131406 Free PMC article. Review.
Research Progress of Sirtuin4 in Cancer.
Bai Y, Yang J, Cui Y, Yao Y, Wu F, Liu C, Fan X, Zhang Y. Bai Y, et al. Front Oncol. 2021 Jan 8;10:562950. doi: 10.3389/fonc.2020.562950. eCollection 2020. Front Oncol. 2021. PMID: 33585187 Free PMC article. Review.
CCL16 maintains stem cell-like properties in breast cancer by activating CCR2/GSK3β/β-catenin/OCT4 axis.
Shen W, Zhang X, Tang J, Zhang Z, Du R, Luo D, Liu X, Xia Y, Li Y, Wang S, Yan S, Yang W, Xiang R, Luo N, Luo Y, Li J. Shen W, et al. Theranostics. 2021 Jan 1;11(5):2297-2317. doi: 10.7150/thno.51000. eCollection 2021. Theranostics. 2021. PMID: 33500726 Free PMC article.
Mitochondrial Sirtuins in Chronic Degenerative Diseases: New Metabolic Targets in Colorectal Cancer.
Colloca A, Balestrieri A, Anastasio C, Balestrieri ML, D'Onofrio N. Colloca A, et al. Int J Mol Sci. 2022 Mar 16;23(6):3212. doi: 10.3390/ijms23063212. Int J Mol Sci. 2022. PMID: 35328633 Free PMC article. Review.
CDC45 promotes the stemness and metastasis in lung adenocarcinoma by affecting the cell cycle.
Liu Y, Han T, Xu Z, Wu J, Zhou J, Guo J, Miao R, Xing Y, Ge D, Bai Y, Hu D. Liu Y, et al. J Transl Med. 2024 Apr 8;22(1):335. doi: 10.1186/s12967-024-05038-5. J Transl Med. 2024. PMID: 38589907 Free PMC article.

See all "Cited by" articles

References

1. Kosciuk T, Wang M, Hong JY, Lin H. Updates on the epigenetic roles of sirtuins. Curr Opin Chem Biol. 2019;51:18–29. - PMC - PubMed
1. O'Callaghan C, Vassilopoulos A. Sirtuins at the crossroads of stemness, aging, and cancer. Aging Cell. 2017;16:1208–1218. - PMC - PubMed
1. Zhu S, Dong Z, Ke X, Hou J, Zhao E, Zhang K, The roles of sirtuins family in cell metabolism during tumor development. Semin Cancer Biol. 2018. - PubMed
1. Tanno M, Sakamoto J, Miura T, Shimamoto K, Horio Y. Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1. J Biol Chem. 2007;282:6823–6832. - PubMed
1. Frazzi R. SIRT1 in Secretory Organ Cancer. Front Endocrinol (Lausanne) 2018;9:569. - PMC - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
Medical
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
Research Materials
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal

[1] Kosciuk T, Wang M, Hong JY, Lin H. Updates on the epigenetic roles of sirtuins. Curr Opin Chem Biol. 2019;51:18–29. - PMC - PubMed

[2] Kosciuk T, Wang M, Hong JY, Lin H. Updates on the epigenetic roles of sirtuins. Curr Opin Chem Biol. 2019;51:18–29. - PMC - PubMed

[3] O'Callaghan C, Vassilopoulos A. Sirtuins at the crossroads of stemness, aging, and cancer. Aging Cell. 2017;16:1208–1218. - PMC - PubMed

[4] O'Callaghan C, Vassilopoulos A. Sirtuins at the crossroads of stemness, aging, and cancer. Aging Cell. 2017;16:1208–1218. - PMC - PubMed

[5] Zhu S, Dong Z, Ke X, Hou J, Zhao E, Zhang K, The roles of sirtuins family in cell metabolism during tumor development. Semin Cancer Biol. 2018. - PubMed

[6] Zhu S, Dong Z, Ke X, Hou J, Zhao E, Zhang K, The roles of sirtuins family in cell metabolism during tumor development. Semin Cancer Biol. 2018. - PubMed

[7] Tanno M, Sakamoto J, Miura T, Shimamoto K, Horio Y. Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1. J Biol Chem. 2007;282:6823–6832. - PubMed

[8] Tanno M, Sakamoto J, Miura T, Shimamoto K, Horio Y. Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1. J Biol Chem. 2007;282:6823–6832. - PubMed

[9] Frazzi R. SIRT1 in Secretory Organ Cancer. Front Endocrinol (Lausanne) 2018;9:569. - PMC - PubMed

[10] Frazzi R. SIRT1 in Secretory Organ Cancer. Front Endocrinol (Lausanne) 2018;9:569. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells

Affiliations

Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

MeSH terms

Substances

Related information

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous