SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer

Abstract

Background: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC).

Methods: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC.

Findings: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors.

Interpretation: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. FUND: This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017-87301-R and SAF2014-52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015-71017/BMC MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovacion 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007-2009 and 2011-2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971-16 (P.A.), MINECO:SAF2015-64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (M.S.R.), MINECO - BFU2016-76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Keywords: HCC; LKB1; SIRT1; STRADα; SUMO.

Fig. 1

Liver Kinase B1 (LKB1) offers survival and invasiveness advantage to human hepatoma cells during hypoxic stress. LKB1 overexpression was induced in human hepatoma Huh-7 cell line by using the pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1) and compared to control overnight transfection with the pcDNA™3.3-TOPO® plasmid (Ctrl), followed by 24 h treatment under control conditions of normoxia and complete media (21%oxygen, 10% serum), serum deprivation (SD) and hypoxia (1% oxygen, 10% serum). a. Representative Western blot of LKB1, its downstream target AMP-activated protein (AMPK) and phosphorylated AMPK at Thr172 and hypoxia inducible factor (HIF1α), a hypoxic marker, are shown. [β-actin was used as loading control]. Quantifications are shown in Suppl. Fig. 1b; b. Cell viability as detected by staining of attached cells with crystal violet dye; c. Time-course of cell viability and cell migration using a wound-healing scratch assay after LKB1 overexpression under hypoxia; d. Representative immunofluorescence staining for LKB1 (FLAG) in Huh-7 hepatoma cells and quantification of the percentage of LKB1 nuclear positive staining cells. Scale bar corresponds to 50 μm; and e. Western blot of LKB1 levels in cytoplasmic and nuclear fractions [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control for cytoplasmic fractions and Histone H3 for nuclear fractions]. Quantifications are shown in Suppl. Fig. 1b. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p < 0·05 and **p < 0·01 are indicated (Mann-Whitney U test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Liver Kinase B1 (LKB1) is SUMOylated by SUMO-2 in human hepatoma cells. a. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-1, 2, or 3, with the pcDNA3-FLAG-LKB1 Wild type plasmid (LKB1 WT) in the presence and absence of ubiquitin conjugating enzyme 9 (UBC9). b. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 with the LKB1 WT plasmid in the presence of the different SUMO E3 ligases PIAS 1, 2α, 2β, and 4. c. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and with the LKB1 WT plasmid in the presence of the different SUMO-specific proteases, SENPs 1–7. d. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and with the LKB1 WT plasmid or the pcDNA3-FLAG-LKB1 Kinase Dead K78I plasmid. e. Immunoprecipitation assay between LKB1 and the STe20-Related ADaptor (STRADα) cofactor after STRADα overexpression in Huh-7 human hepatoma cells. Normalized quantifications relative to inputs are shown below the panel.

Fig. 3

Liver Kinase B1 (LKB1) is SUMOylated by SUMO-2 at Lys178 in human hepatoma cells. a. Schematic representation of LKB1 showing the Nuclear (NLS) localization and the Kinase domain (KDN). SUMOylation LKB1 mutants used are also shown and described. b. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and the LKB1 SUMO mutants, LKB1 K96R, LKB1 K97R, LKB1 K178R and LKB1 K235R. Normalized quantifications relative to inputs are shown below each panel.

Fig. 4

Liver Kinase B1 (LKB1) SUMOylation at Lys178 by SUMO-2 regulates human hepatoma cell survival by hampering LKB1 nucleocytoplasmic shuttling. LKB1 overexpression was induced in Huh-7 cells with pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1 WT) or the pcDNA3-FLAG-LKB1 K178R plasmid (LKB1 K178R) in the presence of His-SUMO-2. a. Western blot analysis of LKB1, [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control]. Quantifications are shown in Suppl. Fig. 4a; b and c. Cell viability as detected by staining of attached cells with crystal violet dye and number of cells; d. Immunoprecipitation assay between LKB1 WT and LKB1 K178R and the STe20-Related ADaptor (STRADα) cofactor after STRADα overexpression in Huh-7 human hepatoma cells. Normalized quantifications relative to inputs are shown below the panel; e. Representative immunofluorescence staining for LKB1 (FLAG) in Huh-7 cells and quantification of the percentage of LKB1 nuclear positive staining cells. Scale bar corresponds to 50 μm. f. LKB1 levels in cytoplasmic (Cyto) and nuclear fractions (Nuc) [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control for cytoplasmic fractions and Histone H3 for nuclear fractions]. Quantifications are shown in Suppl. Fig. 4b. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p < 0·05 is indicated (Mann-Whitney U test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Liver Kinase B1 (LKB1) is modified by SUMO-2 in Lys178 after its acetylation at Lys48 in human hepatoma cells. a. Schematic representation of LKB1 showing the Nuclear (NLS) localization, acetylation domain (AD) and the Kinase domain (KDN). Acetylation LKB1 mutant used is shown. b. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with the pcDNA3-FLAG-LKB1 Wild type plasmid (LKB1 WT), His-SUMO-2 and treatment with sirtuin 1 (SIRT1). c. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection the LKB1 WT, LKB1 acetylation mutant, LKB1 K48R or the LKB1 SUMOylation mutant, LKB1 K178R, with His-SUMO-2, in the presence and absence of the Ex-527, the SIRT1 inhibitor. Normalized quantifications relative to inputs are shown below each panel; d. Representative immunofluorescence staining for FLAG and quantifications in Huh-7 hepatoma cells after transfection with the LKB1 WT or the LKB1 SUMOylation mutant LKB1 K178R and SUMO-2 in the presence and absence of Ex-527. Scale bar corresponds to 50 μm. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p < 0·05 and **p < 0·01 are indicated (Mann-Whitney U test).

Fig. 6

Model of structural and dynamic changes on Liver Kinase B1 (LKB1) upon post-translational changes. a. Structure of LKB1 in the context of its ternary complex with STRADα and MO25 [80] (pdb code: 2WTJ). Residues of LKB1 closer within 6 Å of either STRADα or Mo25 are in yellow, those closer than 4 Å are in orange. MO25 interacts with LKB1 through the A-loop, while STRADα interacts with the β₂-β₃ loop and the CFT_L region. b. Comparison of the structures along the molecular dynamics computations. The Root Mean Square Deviation of trajectory snapshots upon alignment to the initial, energy minimized structure. Upper: data corresponding to unmodified LKB1 is in dark cyan, and that of K178-SUMOylated LKB1 is in blue. Lower: the trajectory of K48-acetylated LKB1 is in ochre, and dark red dots represent that computed for the K48-acetylated and K178-sumoylated. c. Per-residue atomic fluctuations computed along the last 250 ns of the trajectories. Colour attributes in b apply also to this panel. d. Map of changes in atomic fluctuations between unmodified LKB1 and fully modified (ALY at position 48, SUMO at position 178) onto the model obtained by simulated annealing of the mobile parts of LKB1 using the 2WTJ coordinates. Residues in red are more mobile in the unmodified protein, those in blue are more mobile in the fully modified one. e. Overlay of the structures of unmodified (cyan) and fully modified (red; SUMO in yellow) LKB1. Ribbons represent the coordinates closest to averages of the analyzed trajectory intervals. f. Detail of the terminal part of the CFT_L region of the acetylated and SUMOylated STK domain of LKB1, according to the structure closest to the average of the last 250 ns of MD computations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Liver Kinase B1 (LKB1) is modified by SUMO-2 in mouse models of Hepatocellular Carcinoma (HCC). a. Representative immunohistochemical analysis and quantification of LKB1, SUMO-2/3 and HIF1α staining. Scale bar corresponds to 50 μm; At least 5 animals per group were used; b. Western blot analysis and quantification of LKB1 by using SUMO binding entities (SUBEs) to capture endogenous SUMOylated LKB1; and c. Immunoprecipitation assay and quantification for SUMO-2 and LKB1 in tumor and non-tumor livers of wild type and Glycine-N-methyltransferase (Gnmt^−/−) HCC mice. Three animals per group are shown. Data is shown as mean ± SEM. *p < 0·05 is indicated (Mann-Whitney U test).

Fig. 8

LKB1 SUMOylation in clinical HCC is associated with aggressiveness of the tumor. a. Representative immunohistochemical analysis and quantification of LKB1 and SUMO-2/3 staining in clinical HCC patients (n = 22) and healthy controls (n = 5). Scale bar corresponds to 50 μm. b. Western blot analysis and quantification of LKB1 by using SUMO binding entities (SUBEs) to capture endogenous SUMOylated LKB1 in liver biopsies of HCC patients comparing the tumor with the surrounding tissue. 5 paired samples were used. Surrounding tissue (ST) and tumor (T). c. Volcano plot of gene expression of the main genes involved in SUMO pathway, hypoxia and hypoxia responsiveness genes retrieved from a previous published microarray where HCC patients were separated into more aggressive and less aggressive HCC, according to the tumor doubling time and survival [40]; d. Representative LKB1 staining in more aggressive and less aggressive HCC tumors. Scale bar corresponds to 50 μm. e. Western blot analysis and quantification of LKB1 by using SUBEs to capture endogenous SUMOylated LKB1 in liver biopsies of HCC patients classified as more aggressive or less aggressive tumors (3 samples each were used for less aggressive and more aggressive tumors). Data is shown as mean ± SEM. *p < 0·05 is indicated (Mann-Whitney U test).

Fig. 9

SUMOylation regulates Liver Kinase B1 (LKB1) nucleocytoplasmic shuttling and its oncogenic potential in liver cancer. LKB1 acetylation at Lys48 and posterior SUMOylation at Lys178 by SUMO-2 account for the nuclear retention of LKB1 in liver cancer by hampering the binding of LKB1 to STRADα.

Supplemental Fig. 1

Liver Kinase B1 (LKB1) expression in human hepatoma cells a. Western blot analysis of LKB1 in several human hepatoma cells, Huh-7, Hep G2 and PLC/PRF/5. [α-Tubulin was used as loading control]. b. and c. Quantification of the Western blot shown in Fig. 1a and e, respectively. [Glyceraldehyde 3-phosphate (GAPDH), and β-actin were used as loading control for total and cytoplasmic fractions and Histone H3 for nuclear fractions]. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p < 0·05 and **p < 0·01 versus Ctrl are indicated (Mann-Whitney U test).

Supplemental Fig. 2

Liver Kinase B1 (LKB1) SUMOylation of mouse liver progenitor MLP-29 cells offers survival advantage during hypoxic stress. a. LKB1 overexpression was induced in human MLP-29 cell line by using the pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1) and compared to control overnight transfection with the pcDNA™3.3-TOPO® plasmid (Ctrl), followed by 24 h treatment under control conditions of normoxia and complete media (21%oxygen, 10% serum), serum deprivation and hypoxia (1% oxygen, 10% serum). Quantification of cell viability assessed by Crystal violet assay are shown. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. **p < 0·01 is indicated (Mann-Whitney U test).

Supplemental Fig. 3

LKB1 and SUMO in hypoxia. a. Representative immunohistochemical analysis and quantification of LKB1 and SUMO-2/3 staining in liver biopsies of mice after 2 h30 hypoxia (10% O₂ environment) and control mice (21% O₂ environment). Scale bar corresponds to 50 μm. Data is shown as mean ± SEM. *p < 0·05 is indicated (Mann-Whitney U test). b. Immunoprecipitation assay between LKB1 and SUMO-2/3 in Huh-7 human hepatoma cells during normoxia (21%oxygen, 10% serum) and hypoxia (1% oxygen, 10% serum) conditions for 24 h.

Supplemental Fig. 4

Liver Kinase B1 (LKB1) is SUMOylated by SUMO-2 at Lys178 in mouse liver progenitor MLP-29 cells. a. Ni²⁺-NTA agarose bead pulldown in MLP-29 cells after transfection with His-SUMO-1, 2, or 3, with the pcDNA3-FLAG-LKB1 Wild type plasmid (LKB1 WT) in the presence and absence of ubiquitin conjugating enzyme 9 (UBC9). b. Ni²⁺-NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and the LKB1 SUMO mutants, LKB1 K96R, LKB1 K97R, LKB1 K178R and LKB1 K235R. Normalized quantifications relative to inputs are shown below each panel.

Supplemental Fig. 5

Quantifications of the Western blots shown in a. Fig. 4a and b. Fig. 4e. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p < 0·05 and **p < 0·01 are indicated (Mann-Whitney U test).

Supplemental Fig. 6

Cycloheximide Chase Assay. a. Stability of pcDNA3-FLAG-LKB1 wild type (WT) and b. pcDNA3-FLAG-LKB1 K178R or c. pcDNA3-FLAG-LKB1 K48R proteins as percentage of protein levels remaining after treatment with cycloheximide (CHX). Densitometric analysis and quantifications was obtained from Western blot data. LKB1 WT and mutant proteins at different time points between 0 and 6 h after CHX treatment. Vertical bars are indicative of the calculated half-life for each protein. At least triplicates were used per experimental condition. Data is shown as mean ± SEM.