LLGL2 rescues nutrient stress by promoting leucine uptake in ER+ breast cancer

Abstract

Drosophila Lgl and its mammalian homologues, LLGL1 and LLGL2, are scaffolding proteins that regulate the establishment of apical–basal polarity in epithelial cells1,2. Whereas Lgl functions as a tumour suppressor in Drosophila1, the roles of mammalian LLGL1 and LLGL2 in cancer are unclear. The majority (about 75%) of breast cancers express oestrogen receptors (ERs)3, and patients with these tumours receive endocrine treatment4. However, the development of resistance to endocrine therapy and metastatic progression are leading causes of death for patients with ER+ disease4. Here we report that, unlike LLGL1, LLGL2 is overexpressed in ER+ breast cancer and promotes cell proliferation under nutrient stress. LLGL2 regulates cell surface levels of a leucine transporter, SLC7A5, by forming a trimeric complex with SLC7A5 and a regulator of membrane fusion, YKT6, to promote leucine uptake and cell proliferation. The oestrogen receptor targets LLGL2 expression. Resistance to endocrine treatment in breast cancer cells was associated with SLC7A5- and LLGL2-dependent adaption to nutrient stress. SLC7A5 was necessary and sufficient to confer resistance to tamoxifen treatment, identifying SLC7A5 as a potential therapeutic target for overcoming resistance to endocrine treatments in breast cancer. Thus, LLGL2 functions as a promoter of tumour growth and not as a tumour suppressor in ER+ breast cancer. Beyond breast cancer, adaptation to nutrient stress is critically important5, and our findings identify an unexpected role for LLGL2 in this process.

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Fig. 1: LLGL2 promotes proliferation in ER+ breast cancer cells by regulating leucine uptake.
Fig. 2: SLC7A5 interacts with LLGL2 and regulates proliferation in ER+ breast cancer cells.
Fig. 3: LLGL2 and YKT6 regulate cell surface levels of SLC7A5 in ER+ breast cancer cells.
Fig. 4: LLGL2 is an ER target and is required for E2-induced proliferation under leucine stress.

Data availability

The Kaplan–Meier plot data that support the findings of this study are available in Kaplan–Meier Plotter (http://kmplot.com/analysis/) with the identifier 10.18632/oncotarget.1033735. The ChIP–seq data GSM153472236 and GSM166901437 that support the findings of this study are available from Cistrome Data Browser (http://cistrome.org/db/#/). The ChIP–seq data from breast cancer patients (GSE32222) have been previously published26. Source data are provided. All other relevant data are available from the corresponding author on reasonable request.

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Acknowledgements

We thank members of the Muthuswamy laboratory for discussions, R. Schiff for TamR cells, T. Xiao for advice on CRISPR–Cas9, J. Zoeller for advice on mouse experiments, F. Au-Yeung for assistance with BioID experiments, and M. Yuan for assistance with mass spectrometry. The work was supported by funding from the National Institutes of Health (NIH) (grant 5P01CA120964; J.M.A.), a long term postdoctoral fellowship (LT000091/2014) from the Human Frontier Science Program and research funds from the Yamagata prefectural government and the City of Tsuruoka (Y.S.), and the Breast Cancer Research Foundation (S.K.M.).

Reviewer information

Nature thanks Jason Carroll, Zach Schafer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors

Contributions

Y.S. and S.K.M. designed, performed, and interpreted experiments, and co-wrote the paper. L.L. performed ChIP–seq analysis from the breast cancer patient database. E.C. and B.R. performed BioID analysis. J.M.A. performed metabolomics analysis. A.L. and C.S. contributed to K–M plot analysis from the breast cancer patient database. M.B. contributed to the design of experiments. M.B. and J.M.A. contributed to editing.

Corresponding author

Correspondence to Senthil K. Muthuswamy.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Relationship between ER status and LLGL1 or LLGL2 expression in breast cancer.

a, Protein expression of LLGL1 and LLGL2 in breast cancer cell lines. The relative ratios of signal intensities are shown. b, c, Immunohistological staining for LLGL2 in ER+ and ER breast cancer tissues. d, Quantification of LLGL2 signal intensity and its relationship to ER status. e, Correlation between copy number variation of LLGL genes and mRNA expression in breast cancer data from The Cancer Genome Atlas (TCGA). f, Kaplan–Meier plot of survival for patients with ER/PR breast cancer. b, c, Images are representative of three biological replicates. e, n = 967 for LLGL1, n = 963 for LLGL2; f, n = 298; statistical analysis conducted by log-rank test (f). Source data

Extended Data Fig. 2 LLGL2, but not LLGL1, promotes cell proliferation in ER+ breast cancer cells.

a, Overexpression of LLGL in T47D cells. b, 2D cell proliferation of LLGL-overexpressing MCF-7 (left) and T47D (right) cells in 10% FBS medium. c, Sphere formation efficiency of LLGL-overexpressing T47D cells in serum-free medium. d, 2D cell proliferation of LLGL-overexpressing MCF-7 (left) and T47D (right) cells in serum-free medium. e, Knockdown of LLGL2 in T47D cells. f, Sphere formation efficiency of LLGL2-KD T47D cells in serum-free medium. g, 2D cell proliferation of LLGL2-KD MCF-7 (left) and T47D (right) cells in serum-free medium. h, MTT assay of LLGL2-KD MCF-7 cells under 10% FBS or serum-free culture conditions. i, Rescue of LLGL2 expression in LLGL2-KD MCF-7 cells. j, k, 2D proliferation (j) and sphere formation efficiency (k) of LLGL2-rescued MCF-7 cells. l, Knockdown of LLGL2 using short hairpin RNA (shRNA) targeting different LLGL2 sequences from the shRNA used in Extended Data Fig. 2i and rescue of LLGL2-KD using RNAi-resistant Flag–LLGL2 in MCF-7 cells. m, 2D cell proliferation of LLGL2-KD and LLGL2-rescued MCF-7 cells in serum-free medium. n, Sphere formation ability of LLGL2-KD and LLGL2-rescued MCF-7 cells in serum-free medium. o, LLGL2-knockdown in T47D cells and rescue of LLGL2-KD using RNAi-resistant Flag–LLGL2 in T47D cells. p, Sphere formation ability of LLGL2-KD and LLGL2-rescued T47D cells. q, Changes in live (left) and dead (right) cell numbers of MCF-7 cells growing in 2D in serum-free medium. bd, fh, j, k, m, n, p, q, Mean ± s.e.m.; a, e, i, l, o, Images are representative of three biological replicates. b, d, g, j, m, q, n = 3; h, n = 6; c, f, k, n, p, n = 9 measurements from three biological replicates performed in triplicate. Statistical analysis was conducted by two-tailed t-test (f, h), one-way ANOVA followed by Tukey’s post-test (c, k, n, p) and two-way ANOVA followed by Tukey’s post-test (b, d, g, j, m, q). Source data

Extended Data Fig. 3 Changes in intracellular metabolites and amino acid levels.

a, 2D proliferation of LLGL2-KD cells in culture medium supplemented with 10% dialysed FBS. b, Heatmap of down- or upregulated metabolites in cultured cells (left) and in tumours (right) generated from LC–MS/MS. The top 50 metabolites are shown. c, Relative intracellular levels of essential amino acids in vivo. d, Sphere formation efficiency of LLGL2-KD MCF-7 (top) and T47D (bottom) cells in nutrient stress conditions supplemented with 10× LQ. e, Sphere formation efficiency of LLGL2-OE MCF-7 cells in 10× LQ medium. f, Leu-Ile concentration in culture medium in which control (shGFP), LLGL2-KD or LLGL2-OE MCF-7 cells were cultured. g, Relative intracellular amount of amino acids in LLGL2-OE MCF-7 cells. h, Relative intracellular amount of amino acids in LLGL2-KD MCF-7 cells with or without supplementation with 10× LQ. i, Leu-Ile concentration in culture media used in this study. j, Heatmap of whole metabolites in culture media used in this study. a, df, i, Mean ± s.e.m.; c, g, h, mean ± s.d.; a, c, fi, n = 3; d, e, n = 9 measurements from three biological replicates performed in triplicate. Statistical analysis was conducted by two-tailed t-test (c, g), one-way ANOVA followed by Tukey’s post-test (d, e, h, i), and two-way ANOVA followed by Tukey’s post-test (a, f). Source data

Extended Data Fig. 4 Detailed analysis of BioID candidates.

a, Overexpression of Flag–BirA* (F-B)–LLGL2 in MCF-7 cells. b, Sphere formation ability of MCF-7 cells expressing F-B–LLGL2. c, List of cell polarity and cell adhesion molecules identified by BioID analysis. d, Kaplan–Meier plots of survival of patients with ER+/PR+ breast cancer to examine the effects of SLC4A7, SLC38A1, SLC7A2 and SLC1A5. b, Mean ± s.e.m.; n = 9 measurements from three biological replicates performed in triplicate. d, n = 479 (SLC7A2 and SLC38A1), n = 577 (SLC4A7 and SLC1A5). a, Images are representative of three biological replicates. Statistical analysis was conducted by two-tailed t-test (b) and log-rank test (d). Source data

Extended Data Fig. 5 Localization and function of SLC7A5 in ER+ breast cancer.

a, b, Immunohistological images of SLC7A5 in ER+ or ER breast cancer (top) and table summarizing the relationship between SLC7A5 expression and ER status (b, bottom). c, Total cell lysates (Fig. 2c). d, Immunoprecipitation of LLGL2 in LLGL2-OE MCF-7 cells. e, Immunoprecipitation of endogenous SLC7A5 in T47D cells. f, Immunostaining of SLC7A5 and LLGL2 in LLGL2-OE MCF-7 cells. Scale bars, 10 µm. g, 2D growth of SLC7A5-KD MCF-7 cells. h, Knockdown of SLC7A5 in MCF-7 cells. i, Sphere formation ability of SLC7A5-KD and SLC7A5-rescued MCF-7 cells. j, Knockdown of SLC7A5 in T47D cells. k, Sphere formation ability of SLC7A5-KD and SLC7A5-rescued T47D cells. l, Treatment of parental MCF-7 xenograft tumours with JPH203; the graph shows changes in total body mass. Arrows point to days of JPH203 administration. m, Overexpression of Flag-tagged SLC7A5 in T47D cells. n, 2D proliferation of SLC7A5-OE T47D cells. g, i, k, l, n, Mean ± s.e.m.; af, h, j, m, images are representative of three biological replicates. g, n, n = 3; l, n = 5; i, k, n = 9 measurements from three biological replicates performed in triplicate. Statistical analysis was conducted by one-way ANOVA followed by Tukey’s post-test (i, k) and two-way ANOVA followed by Tukey’s post-test (g, l, n). Source data

Extended Data Fig. 6 Cell junction localization of LLGL2 and SLC7A5.

a, Immunostaining for SLC7A5 (green) in control (shGFP) and LLGL2-KD cells. The percentage of cell–cell junctions with SLC7A5 is indicated. Scale bar, 10 µm. b, Total protein levels of SLC7A5 in LLGL2-KD T47D cells. c, Percentage of cell–cell junctions that were positive for SLC7A5. d, Total cell lysates (Fig. 2m). e, Surface protein levels of SLC7A5 in T47D cells. f, Immunohistological images of LLGL2 and SLC7A5 in ER+ and ER breast cancer. Numbers indicate cell–cell junctions that show localization of SLC7A5. g, Total protein levels of SLC7A5 in breast cancer cell lines. Immunoblots (left) and the relative ratio of signal intensity (right) are shown. h, 2D cell proliferation of breast cancer cells cultured in nutrient stress condition, 1/10 LQ medium or nutrient stress conditions in the presence of SLC7A5 inhibitor (BCH). i, 2D growth of MDA-MB 231 cells in nutrient stress (control), Leu-depleted (Leu–) and Leu/Gln-depleted (Leu/Gln–) conditions. j, Normalized intensity of surface protein levels of SLC7A5 in ER+ or ER breast cancer cells (Fig. 2o). k, Overexpression of LLGL2 in MDA-MB 231 cells. l, Surface protein levels of SLC7A5 in LLGL2-OE MDA-MB 231 cells (image shown is long exposure to detect SLC7A5 band). h, i, Mean ± s.e.m.; a, b, dg, k, l, images are representative of three biological replicates. h, i, n = 3. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s post-test (h, i). Source data

Extended Data Figure 7 Cell junction localization and interaction of LLGL2 and YKT6 under nutrient stress conditions.

a, Total cell lysates (Fig. 3a). b, Total cell lysates (Fig. 3b). c, Cartoon representation of wild-type LLGL2 and LLGL2 mutants used. d, Immunoprecipitation of wild-type or LLGL2 mutants in HEK293T cells overexpressing indicated LLGL2 or SLC7A5. e, Immunostaining images of MCF-7 cells expressing wild-type Flag–LLGL2 or Flag–LLGL2 Pb mutant. Scale bar, 10 µm. f, Total cell lysates (Fig. 3e). g, Immunoprecipitation of endogenous LLGL2 to detect endogenous YKT6 and SLC7A5 in cell lysates from MCF-7 cells. h, Immunoprecipitation of endogenous YKT6 to detect LLGL2 and SLC7A5 in cell lysates from T47D cells. i, Immunostaining to detect LLGL2 and YKT6 localization in LLGL2-OE MCF-7 cells. j, Total cell lysates (Fig. 3f). k, Knockdown of YKT6 in MCF-7 cells. l, 2D proliferation of YKT6-KD MCF-7 cells. m, Knockdown of YKT6 in T47D cells. n, 2D proliferation of YKT6-KD T47D cells. o, Total cell lysates (Fig. 3h). p, Knockdown of YKT6 in MCF-7 cells using different shRNA. q, Surface protein levels of SLC7A5 in YKT6-KD MCF-7 cells. Numbers indicate relative intensity of signal compared with control. r, Immunoprecipitation of LLGL2 in HEK293T cells transfected with LLGL2 and/or YKT6 vectors. s, Immunostaining images of YKT6 in control (shGFP) and LLGL2-KD MCF-7 cells (left). Percentage of cell–cell junctions with YKT6 (right). Scale bar, 10 µm. l, n, Mean ± s.e.m.; a, b, dk, m, os, Images are representative of three biological replicates. l, n, n = 3. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s post-test (l, n). Source data

Extended Data Fig. 8 LLGL2 is a target of ER.

a, Normalized, relative levels of expression in Fig. 4a. b, Protein expression in T47D cells stimulated with E2. Immunoblot (left) and the relative ratio of signal intensity (right) are shown. c, 2D proliferation of T47D cells in the presence or absence of E2. d, 2D proliferation of MCF-7 in the presence or absence of E2 and/or under Leu-reduced nutrient stress conditions. e, Sphere formation ability of T47D cells in the presence or absence of E2 and/or under Leu-reduced nutrient stress conditions. f, Sphere formation of MCF-7 cells in the presence or absence of E2 stimulation under nutrient stress conditions. cf, Mean ± s.e.m.; b, images are representative of three biological replicates. c, d, n = 3; e, f, n = 9 measurements from three biological replicates performed in triplicate. Statistical analysis was conducted by one-way ANOVA followed by Tukey’s post-test (e, f) or two-way ANOVA followed by Tukey’s post-test (c, d). Source data

Extended Data Fig. 9 LLGL2 is a target of ER.

a, ChIP–seq data from breast cancer samples. b, Scheme of targeting regions for CRISPR–Cas9-mediated genomic editing. Highlighted regions indicate targeted regions. The region of chr17:75538782–75540392 is for control. c, Genomic sequence of the chr17:75538782–75540392 region. Primers for PCR and two guide RNAs for CRISPR are described. d, Genomic sequence of the chr17:75549278–75550868 region. Primers for PCR and two guide RNAs for CRISPR are described.

Extended Data Fig. 10 LLGL2, SLC7A5 and tamoxifen resistance.

a, Total cell lysates (Fig. 4m). b, 2D cell proliferation in the presence or absence of BCH with tamoxifen under nutrient stress conditions. c, Surface protein levels of SLC7A5 in SLC7A5-OE MCF-7 cells. b, Mean ± s.e.m.; a, c, images are representative of three biological replicates. b; n = 3. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s post-test (b). Source data

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Saito, Y., Li, L., Coyaud, E. et al. LLGL2 rescues nutrient stress by promoting leucine uptake in ER+ breast cancer. Nature 569, 275–279 (2019). https://doi.org/10.1038/s41586-019-1126-2

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