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MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination

Nature Cancer volume 1, pages 222–234 (2020)Cite this article

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Abstract

Tumor-initiating cells (TICs) are considered the culprits of cancer development and progression. Dysregulation of metastasis suppressor protein 1 (MTSS1) has been widely observed in tumor metastasis, but its functional contribution and mechanism in cancer is poorly understood. Here we report a role of MTSS1 in suppressing TICs in breast cancer. Mtss1 knockout (KO) enhances the mammary epithelial TIC subpopulation in both luminal and basal-like breast cancer mouse models. MTSS1 also suppresses tumorsphere formation in breast cancer cells. Mechanistically, MTSS1 interacts with the E3 ligase RanBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) to facilitate RBCK1-mediated p65 ubiquitination and degradation, thus suppressing the NF-κB signaling pathway and tumorigenesis. In addition, actin beta-like 2 (ACTBL2) competes with RBCK1 for MTSS1 binding, leading to p65 stabilization. Importantly, MTSS1 silencing promotes patient-derived organoid formation and xenograft growth. MTSS1 downregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal a new paradigm of NF-κB regulation and may have important implications in therapeutics targeting TICs.

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Fig. 1: Mtss1 KO accelerates mammary tumor formation and lung metastasis in MMTV-PyMT and MMTV-Wnt1 mice.
Fig. 2: Mtss1 KO enhances the expansion and activities of TICs in the mammary glands of MMTV-PyMT and MMTV-Wnt1 mice.
Fig. 3: MTSS1 inhibits the stem-like properties of breast cancer cells.
Fig. 4: ACTBL2 interacts with MTSS1 and inhibits the suppressing role of MTSS1 in tumorigenicity.
Fig. 5: MTSS1 suppresses TICs by inhibiting p65 protein level.
Fig. 6: MTSS1 promotes RBCK1-mediated p65 ubiquitination.
Fig. 7: The role and expression of MTSS1 in clinical human breast tumors.

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Data availability

The RNA sequencing data of Mtss1-overexpressing cells have been deposited with the Gene Expression Omnibus (GSE126606) and National Omics Data Encyclopedia (https://www.biosino.org/node, under ID no. OEP000280). Previously published breast cancer proteomics data that were reanalyzed in this study were generated by the National Cancer Institute’s CPTAC and downloaded from the cBioPortal database (http://www.cbioportal.org/)64,65. The TCGA RNA expression dataset of breast cancer used in the GSEA was obtained from the TCGA data portal (https://portal.gdc.cancer.gov). The CSC and MaSC-related gene sets used for the GSEA analyses were from previous publications66,67. Specifically, these sets are the genes upregulated in normal CD44+ versus CD24+ MECs (N_CD44_UP), upregulated in metastatic CD44+ tumor cells versus metastatic CD24+ tumor cells (MET_CD44_UP)66 and the genes upregulated in the MaSC-enriched population versus the luminal population of murine mammary glands (MaSC_UP)67. The NF-κB target gene sets used for the GSEA analyses were obtained from the GSEA MSigDB Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp)41, including HINATA_NFKB_TARGETS_KERATINOCYTE_UP and HALLMARK_TNFA_SIGNALING_VIA_NFKB, or previous studies6 (NFKB-UP-FEUERHAKE). Source data for Figs. 1–7 and Extended Data Figs. 1–9 have been provided with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank P. Lappalainen (University of Helsinki) for providing the Mtss1 KO mice, and X. Miao, S. Yan, Y. Zhai, Z. Weng, K. Wang, Y. Yuan and Y. Bu at the Shanghai Institute of Nutrition and Health core facilities for their technical support. The study was funded by the National Natural Science Foundation of China (grant nos. 81661148048, 81725017, 81872367, 81430070 to G.H. and 91853128 to R.H.), the Chinese Academy of Sciences (grant no. QYZDB-SSW-SMC013 to G.H. and XDA12040323 to R.H.) and the Ministry of Science and Technology of China (grant no. 2017YFA0103502 to G.H.).

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Authors and Affiliations

  1. CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Min Cong, Yuan Wang, Cheng Lian, Xueqian Zhuang, Xiaoxun Li, Peiyuan Zhang, Yingjie Liu, Xue Zhang & Guohong Hu

  2. State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Shanghai, China

    Yang Yang & Ronggui Hu

  3. State Key Laboratory of Oncology in South China; Department of Breast Oncology, Sun Yat-Sen University, Guangzhou, China

    Jun Tang

  4. Department of Breast Surgery, Qilu Hospital of Shandong University, Ji’nan, China

    Qifeng Yang

  5. Department of Oncology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China

    Hua Xiong

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  1. Min Cong
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Contributions

G.H. supervised the study. M.C. and G.H. designed the experiments and drafted the manuscript. M.C., Y.W., Y.Y., C.L., X. Zhuang., X.L., P.Z., Y.L. and X. Zhang. performed the experiments. J.T., Q.Y. and H.X. contributed to clinical sample collection and analysis. H.X. and R.H. helped design the experiments. All authors discussed the results and commented on the manuscript.

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Extended data

Extended Data Fig. 1 Confirmation of Mtss1 deficiency and analysis of mammary gland development in the KO mice.

(a) Mtss1 mRNA expressing profile in female mice in indicated Mtss1 genotype. (n = 3 mice per group). (b-c) MTSS1 protein expression in female mice in indicated Mtss1 genotype. (d) Representative whole mounts and H&E-stained sections of normal mammary glands of C57BL/6 and FVB mice of the indicated genotypes at week 8. Scale bars, 100 μm. (e) Schematic of breeding scheme used to generate PyMT;Mtss1+/+, PyMT;Mtss1+/− and PyMT;Mtss1-/- mice. The experiments in B-D were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 1.

Source Data Extended Data Fig. 1

Extended Data Fig. 2 Mtss1 KO enhances the expansion and activities of TICs in mammary glands.

(a) Representative flow cytometry of luminal subset cells of normal glands (NG) and Wnt1-driven preneoplastic glands of 6-week-old females of the indicated genotypes. (b) Quantification of flow cytometry analyses in (A). (n = 6 mice per group). (c) Tumorsphere formation assays with WT or KO MECs dissociated from preneoplastic glands of MMTV-PyMT mice (n = 6 mice per group for tumorsphere numbers and n = 6 tumorspheres measured per group for tumorsphere sizes). Scale bars, 100 μm. (d)Tumorsphere numbers and representative images of tumorsphere with WT or KO MECs dissociated from preneoplastic glands of MMTV-Wnt1 mice (n = 6 mice per group). Scale bars, 100 μm. (e)Tumorsphere sizes with WT or KO MECs dissociated from preneoplastic glands of MMTV-Wnt1 mice (n = 6 tumorspheres measured per group for tumorsphere sizes). Scale bars, 100 μm. (f) Quantitation and representative images of tumorsphere formation of WT or KO Lin-CD24+CD29low luminal cells dissociated from preneoplastic glands of MMTV-PyMT (n = 6 mice per group) mice. (g and h) Mammary tumor incidence (G) and volumes (H) 4 weeks after orthotopic transplantations of Lin-CD24+CD29low luminal cells from preneoplastic glands of PyMT;Mtss1+/+ and PyMT;Mtss1-/- mice. (n = 10 mice per group). (i) Percentages with or without lung metastasis 10 weeks after orthotopic transplantations of Lin-CD24+CD29low luminal cells dissociated from preneoplastic glands of PyMT;Mtss1+/+ and PyMT;Mtss1-/- mice. (n = 10 mice per group). (j and k) Mammary tumor incidence (J) and volumes (K) 5 weeks after orthotopic transplantations of Lin-ALDH+ cells from established tumors of PyMT;Mtss1+/+ and PyMT;Mtss1-/- mice. (n = 10 mice per group). Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (B-F), limiting dilution analysis (G, J), Mann-Whitney U-test (H, K) or Chi-squared test (I). The experiments in A and C-F were repeated three times independently with similar results, and the results of one representative experiment are shown.

Source Data

Extended Data Fig. 3 MTSS1 and ACTBL2 regulate tumorsphere formation of breast cancer cells.

(a) GSEA analyses showing the enrichment of N_CD44_UP and MaSC_UP gene sets in TCGA breast tumors with lower MTSS1 expression. See Online Methods for the sources of the gene sets. NES, normalized enrichment score. (n = 881 patients). (b) Validation of MTSS1 overexpression and knockdown. Shown are western blots of MTSS1 overexpression in SCP28 and knockdown in MCF7. (c) Py8119 cells were pre-labeled with CFSE (green) or CellTrace Blue, and then mixed in a 1:1 ratio for tumorsphere culturing. The numbers of single-colored or chimeric (red arrow) tumorspheres were counted. Representative images of tumorspheres were shown on the top. The majority of tumorspheres were in single color, indicating that they were mostly originated from clone expansion, rather than cell aggregation. Scale bar, 100 μm. (d-g) Representative images of tumorsphere formation, after MTSS1 overexpression in HMLER and SCP28 (D), or MTSS1 knockdown in MCF7 (E), ZR-75-1 (F) and MCF10AT (G). Scale bars, 100 μm. (h) Cell cycle (left) and apoptosis (right) analyses after Mtss1 overexpression in Py8119 cells. (n = 3 independent experiments). (i) ACTBL2 expression in different subtypes of breast tumors in the TCGA dataset. (n = 699 patients). (j) Tumorsphere formation after ACTBL2 overexpression in HMLER. Scale bars, 100 μm. (k) Representative images of tumorsphere formation after Mtss1 and Actbl2 overexpression in Py8119 cells. Scale bar, 100 μm. Data represent the mean ± s.d. Statistical significance was determined by Wald test with Benjamini–Hochberg’s multiple-comparison correction (A), two-tailed unpaired t-test (H, J) or Mann-Whitney U-test (I). The experiments in B, D-G and J-K were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 3.

Source Data Extended Data Fig. 3

Extended Data Fig. 4 MTSS1 and ACTBL2 regulate p65 protein.

(a) Transcription factors enriched for the Mtss1-downregulated genes identified by promoter binding site analysis. (n =710 downregulated genes). (b) GSEA analyses of NF-κB target gene sets in TCGA breast tumors with different MTSS1 expression levels. See Online Methods for information of the NF-κB target gene sets. (n = 881 patients). (c) P65 protein level in total cell lysate of preneoplastic mammary glands, the Lin+ cells of the glands and bone marrow cells of Mtss1 WT and KO mice. (d) P65 protein level after MTSS1 overexpression in different breast cancer cells. Numbers are p65 relative levels normalized to loading control. (e) P65 protein level after MTSS1 knockdown in different breast cancer cells.(f) P65 protein level in SCP28 after Mtss1 and Actbl2 overexpression. (g) Expression of various signaling pathway molecules in Py8119 after Mtss1 and Actbl2 overexpression. Statistical significance was determined by Wald test with Benjamini–Hochberg’s multiple-comparison correction (B). The experiments in C-G were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 4.

Source Data Extended Data Fig. 4

Extended Data Fig. 5 NF-κB inhibition mediates the role of MTSS1 in TICs.

(a) Representative images of tumorsphere formation after Mtss1 and Rela overexpression in Py8119 cells. Scale bar, 100 μm. (b) Representative images of tumorsphere formation after MTSS1 knockdown with or without BAY11-7082 treatment in MCF7 cells. Scale bar, 100 μm. (c) Representative images of tumorsphere formation after MTSS1 knockdown with or without BAY11-7082 treatment in ZR-75-1 cells. Scale bar, 100 μm. (d and e) Representative images (D) and quantitation (E) of tumorsphere formation after MTSS1 knockdown in MCF7 cells with or without JSH-23, PS-1145, BMS-345451 treatment. Scale bar, 100 μm. (f) Mammary tumor initiation time of Mtss1 WT (n =5, 7, 7 mice for control, JSH-23, and PS-1145 groups, respectively) and KO (n =5, 7, 6 mice for control, JSH-23, and PS-1145 groups, respectively) females with or without JSH-23, PS-1145 treatment. (g) Kinetics of mammary tumor onset of Mtss1 WT (n =5, 7, 7 mice for control, JSH-23, and PS-1145 groups, respectively) and KO (n =5, 7, 6 mice for control, JSH-23, and PS-1145 groups, respectively) females with or without JSH-23, PS-1145 treatment in F. Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (E-F) or log-rank test (G). The experiments in A-E were repeated three times independently with similar results, and the results of one representative experiment are shown.

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Extended Data Fig. 6 CCL20 and IL6RA mediates the role of NF-κB in breast cancer TICs.

(a)Relative mRNA levels of multiple NF-κB target gene in Py8119 cells after Mtss1 overexpression. (b)CCL20 protein level after Mtss1 overexpression in conditioned medium of Py8119 cells. (c) Expression of Ccl20 and Il6ra in Py8119 cells after TNFα treatment. (d) Quantitation and representative images of tumorsphere formation in Py8119 cells with Mtss1, Ccl20 and/or Il6ra overexpression. Scale bars, 100 um. Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (C, D). The experiments in A-D were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 MTSS1 interacts with the RBCK1 and regulates p65 ubiquitination.

(a) Rela mRNA level after Mtss1 overexpression in Py8119 cells. Data represent the mean ± s.d. (b) Quantitation of p65 protein level at the indicated time points in control and Mtss1-overexpressing Py8119 cells after treatment with 25 μg ml−1 cycloheximide (CHX). Related to Fig. 6a. (c)P65 ubiquitination analysis under a denaturing condition (8 M urea lysis buffer) for IP in Py8119 cells after Mtss1 overexpression, with or without treatment of 50 μg ml−1 MG132. (d) Yeast two-hybrid screening of MTSS1-interacting E3 ligase. The screening was performed with full-length MTSS1 expressed in the vector pBGKT7 as the bait, in a human E3 ligase library. The yeast clones which grew on SD-4 medium (deficient in Leu, Trp, His and Ade) were subjected to sequencing. (e) Co-IP assays of ectopically expressed MTSS1 and RBCK1 proteins in 293T cells. (f) MTSS1-RBCK1 Co-IP assays in 293T cells transfected with only RBCK1-HA, or with both Flag-MTSS1 and RBCK1-HA. IP was performed using anti-Flag M2 beads. (g)MTSS1 protein level after RBCK1 overexpression in 293T cells. (h) P65 protein level after RBCK1 overexpression in HeLa cells. (i) P65 ubiquitination analysis in 293T cells transfected with MTSS1, Flag-p65, ubiquitin, and/or RBCK1. Note the two bands corresponding to exogenous (arrow head) and endogenous (arrow) p65 in the anti-p65 immunoblots. (j) P65 ubiquitination analysis in 293T cells transfected with MTSS1, Flag-p65, RBCK1 and wild-type or mutated ubiquitin. (k) P65 ubiquitination analysis in 293T cells transfected with MTSS1, p65, ubiquitin, and wild-type or mutated RBCK1. RBCK1 mutations include deletion of the RBR domain (ΔRBR), and substitution of the 4 cysteines in the domain by serine residues (CS). (l) Co-IP assays of MTSS1 and p65 in 293T cells. (m) Co-IP assays of p65-RBCK1 interaction in Mtss1-overexpressing Py8119 cells with endogenous p65 and RBCK1. (n) Sequential IP assay for MTSS1-RBCK1-p65 interaction. The assay was performed after an initial IP with anti-Flag antibody, then the precipitated complex was recovered after a second IP with anti-HA antibody in 293T. (o) Quantitation of p65-RBCK1 co-localization in HeLa cells with or without MTSS1 overexpression by IF analysis. Related to Fig. 6j (n= 10 fields from 3 independent experiments per group). Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (A, O). In A and B, data from a single experiment are shown and are representative of three independent experiments with similar results. The experiments in C-N were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 7.

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Extended Data Fig. 8 RBCK1 regulates p65 protein and tumorigenesis of breast cancer cells.

(a) P65 protein level after Rbck1 knockdown in Py8119 cells. (b) P65 protein level after Mtss1 overexpression and Rbck1 knockdown in Py8119 cells. (c)Representative images and quantitation of tumorsphere formation after Mtss1 overexpression and Rbck1 knockdown in Py8119 cells. Scale bar, 100 μm. (d and e) Mammary tumor incidence (D) and tumor volumes of 1000-cells injection group (E) 5 weeks after orthotopic transplantation of Py8119 cells with Mtss1 overexpression and/or Rbck1 knockdown. (n = 10 mice per group). (f) Quantitation of MTSS1-RBCK1 co-localization in HeLa cells with or without ACTBL2 overexpression by IF analysis. Related to Fig. 6l. (n= 10 fields from 3 independent experiments per group). (g) Schematic of full length and truncated forms of MTSS1 protein. The positions of MTSS1 domains are indicated by numbers of amino acids. IMD, IRSp53/MIM homology domain; SRD, serine-rich domain; PRD, proline-rich domain; WH2, Wiskott-Aldrich syndrome protein homology 2 domain. (h) Co-IP assays for interaction between MTSS1 protein truncations and ACTBL2 in 293T cells. MTSS1 protein truncations were fused with Myc tag. Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (C, F), limiting dilution analysis (D), Mann-Whitney U-test (E). The experiments in A-C and H were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 8.

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Extended Data Fig. 9 The expression and functional role of MTSS1 in human clinical tumors.

(a) Representative flow cytometry sorting of CD44+CD24- and CD44-CD24+ cell subpopulations from breast cancer PDXs. (b) Organoid formation of CD44+CD24- and CD44-CD24+ cell subpopulations from breast cancer PDXs. Scale bars, 100 μm; n = 4 independent experiments.(c) MTSS1 expression in CD44+CD24- and CD44-CD24+ cell subpopulations of three breast cancer PDXs (n = 3 tumors). (d) Expression of the indicated proteins in CD44H and CD44L subpopulations of breast cancer cell lines and a PDX tumor (left), and gastric cancer cell lines and a PDX tumor (right). (e) Expression of the indicated proteins in Py8119 cells after Mtss1 overexpression (left) and in a breast cancer PDX tumor LTMBC-2 after MTSS1 knockdown (right). (f) Representative IF images of patient-derived organoids. CK18, cytokeratin 18; CK14, cytokeratin 14. Scale bar, 100 μm. (g) Patient-derived organoid formation of a gastric tumor, LTMGC-3, after MTSS1 knockdown. The results of primary culture (P0) and the first organoid passage (P1) were shown. Scale bar, 100 μm. n = 4 organoid cultures per group for both P0 and P1. (h) Patient-derived organoid formation of a gastric tumor, LTMGC-1, after MTSS1 knockdown. Shown are organoid numbers and organoid sizes (n = 3 organoid cultures per group for organoid numbers. n = 4 organoids measured for organoid sizes per group). Scale bar, 100 μm. (i) Representative images and numbers of invasive leader cells of organoids from breast cancer PDXs, LTMBC-1, LTMBC-2, LTMBC-3, after MTSS1 knockdown. LTMBC-1: n = 7, 11, 9 organoids counted for Ctrl, KD3 and KD4 respectively; LTMBC-2: n = 9, 11, 8 organoids counted for Ctrl, KD3 and KD4 respectively; LTMBC-3: n = 4, 7, 4 organoids counted for Ctrl, KD3 and KD4 respectively. Scale bar, 100 μm. (j) Representative images and numbers of invasive leader cells of organoids from gastric cancer PDXs, LTMGC-1, LTMGC-3, after MTSS1 knockdown. LTMGC-1: n = 4 organoids counted per group; LTMGC-3: n = 8, 11, 13 organoids counted for Ctrl, KD3 and KD4 respectively. (k) H/E images of local invasion of the breast cancer PDX tumor, LTMBC-2, after MTSS1 knockdown at week 8 after orthotopic transplantation into mice. Scale bar, 100 μm. (l) MTSS1 protein levels in different breast cancer subtypes by IHC staining of the breast cancer tissue microarray. Numbers in parenthesis denote sample sizes of the corresponding subtypes; n = 274 patients. (m) Representative images of various MTSS1 and p65 immunostaining levels in the breast cancer tissue microarray. Scale bar, 20 μm. (n) Representative images of various p65 and RBCK1 immunostaining levels in the breast cancer tissue microarray. Scale bar, 20 μm. (o) P65 protein levels in breast cancer tumors of different RBCK1 expression by IHC staining of the breast cancer tissue microarray. RBCK1 and p65 staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), 3 (strong). Numbers in parenthesis denote sample sizes of the corresponding groups; n = 105 patients. Data represent the mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test (B, C, G-J), Chi-squared test (L, O). The experiments in A-B, D-K and M-N were repeated three times independently with similar results, and the results of one representative experiment are shown. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 9.

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Cong, M., Wang, Y., Yang, Y. et al. MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination. Nat Cancer 1, 222–234 (2020). https://doi.org/10.1038/s43018-019-0021-y

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