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Class III phosphatidylinositol-3-OH kinase controls epithelial integrity through endosomal LKB1 regulation

Abstract

The molecular mechanisms underlying the interdependence between intracellular trafficking and epithelial cell polarity are poorly understood. Here we show that inactivation of class III phosphatidylinositol-3-OH kinase (CIII-PI3K), which produces phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes, disrupts epithelial organization. This is caused by dysregulation of endosomally localized Liver Kinase B1 (LKB1, also known as STK11), which shows delocalized and increased activity accompanied by dysplasia-like growth and invasive behaviour of cells provoked by JNK pathway activation. CIII-PI3K inactivation cooperates with RasV12 to promote tumour growth in vivo in an LKB1-dependent manner. Strikingly, co-depletion of LKB1 reverts these phenotypes and restores epithelial integrity. The endosomal, but not autophagic, function of CIII-PI3K controls polarity. We identify the CIII-PI3K effector, WD repeat and FYVE domain-containing 2 (WDFY2), as an LKB1 regulator in Drosophila tissues and human organoids. Thus, we define a CIII-PI3K-regulated endosomal signalling platform from which LKB1 directs epithelial polarity, the dysregulation of which endows LKB1 with tumour-promoting properties.

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Figure 1: Disruption of endosomal transport mediated by CIII-PI3K causes epithelial integrity defects in Drosophila and Caco-2 organoids.
Figure 2: CIII-PI3K restriction of LKB1 signalling is required for epithelial integrity.
Figure 3: Interaction of LKB1 and the endosomal transport machinery.
Figure 4: Endosomal control of LKB1 transport and signalling.
Figure 5: Screening for PtdIns3P effectors regulating the LKB1 pathway identifies the conserved FYVE-domain protein WDFY2.
Figure 6: The endosomal protein WDFY2 restricts LKB1 activity.
Figure 7: Restriction of LKB1 activity by WDFY2 is crucial for epithelial integrity.
Figure 8: Reduction of LKB1 activity suppresses oncogenic characteristics of CIII-PI3K loss in vivo.

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Acknowledgements

We thank T. Håve, A. Engen, E. Rønning, A.-G. Bergersen, C. Bassols and the Advanced Light and Electron Microscopy core facility (OUS) for technical assistance, and the Stenmark laboratory for discussions. We thank J. Poulton for initial characterization of CIII-PI3K mutant phenotypes and D. St. Johnston and D. T. Bergstrahl for discussions. We thank the Bloomington Stock Center, DSHB and Addgene for reagents. This work was partly supported by the Research Council of Norway, Centre of Excellence scheme (179571), by grants from the Norwegian Cancer Society to F.O’F. (5768696), T.E.R. (PK01-2009-0386) and H.S. (605009), and the South-Eastern Norwegian Health Authority to T.E.R. (39717), E.M.W. (2015014), A.B. (35424) and V.H.L. (2014039).

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Contributions

Conceived and designed by T.E.R. with F.O’F., V.H.L. and H.S. contributing to the design and interpretation of experiments. Experiments in Drosophila were performed by F.O’F. and N.S.K. Caco-2 cyst protocols were developed by V.H.L. and implemented by V.H.L. and M.S. (WDFY2 rescue experiments with F.O’F.). A.J. and V.H.L. performed co-immunoprecipitation experiments. Stable cell lines generated by M.S. aided by A.J. and K.W.T. Live-cell imaging experiments were performed by M.S., E.M.W. and F.O’F. Confocal imaging performed by F.O’F., V.H.L. and M.S. Additional cell culture experiments were performed by F.O’F. and A.J. while western blotting was performed by F.O’F., V.H.L., A.J. and N.S.K. S.W.S. and A.B. performed correlative light and electron microscopy experiments. F.O’F. drafted the manuscript with subsequent contributions from V.H.L., H.S. and T.E.R.

Corresponding authors

Correspondence to Fergal O’Farrell or Tor Erik Rusten.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Disruption of CIII-PI3K, but not atg14, results in loss of epithelial integrity.

a, Egg chamber with follicle epithelial cells harbouring GFPvps34−/− mutant clones (indicated by hatched line) labelled with anti-Armadillo (Arm). b, Egg chamber with follicle epithelial cells harbouring GFPvps34 mutant clones (hatched line) labelled with anti-E-Cadherin (E-Cad). c, Egg chamber with follicle cells harbouring GFP+ (MARCM) vps34−/− mutant clones. d, Egg chamber with follicle epithelial cells harbouring GFP+ vps34−/− mutant clones that express the vps34 rescue construct. e, Egg chamber with follicle epithelial cells harbouring GFPatg14−/− mutant clones (marked with hatched line) labelled with anti-E-Cad. Scale bars, 25 μm. All experiments were performed 3 times with similar results.

Supplementary Figure 2 Increased p-AMPK levels induced by CIII-PI3K loss, is not observed in other polarity mutant phenotype or by loss of atg14, and can be suppressed by lkb1 co-disruption.

a, Egg chambers harbouring GFP+ clones of lkb1X5−/− in the follicular epithelium labelled with anti-E-Cad and-aPKC. White line traces the expanse of the clones. b, Egg chambers harbouring GFP+ clones of lkb1X5−/− in the follicular epithelia labelled with anti-p-AMPK and anti-E-Cad. c, Wing epithelium in which GFP and lkb1RNAi were co-expressed, labelled with anti-dLkb1. Hatched line marks the Dorsal (D)/Ventral (V) boundary. d, Wing epithelium in which GFP-tagged Lkb1 was expressed in the dorsal region, D, labelled with anti-dLkb1. Hatched line indicates the D/V boundary. e, Caco-2 cell lysate following indicated treatment for 4 h was subject to western blot analysis with anti-p-AMPK, -AMPK and-Tubulin. f,g, Egg chamber in which Actin FLP-out Gal4 clones expressing GFP and interfering RNA directed against the apical polarity regulating protein crumbsRNAi and basolateral component and tumour suppressor lethal giant larvae locus lglRNAi were generated and labelled with anti-p-AMPK and anti-Arm. Hatched lines mark the clones. h, Egg chambers harbouring GFP clones of atg14−/− mutant (atg14d5.2) labelled with anti-p-AMPK and anti-Arm. Hatched line marks the clone area. i, Egg chambers harbouring GFP+ clones of vps15−/−lkb1X5−/− in the follicular epithelium labelled with anti-E-cad. j, Egg chambers harbouring GFP+ clones of vps15−/−lkb1X5−/− in the follicular epithelium labelled with anti-p-AMPK. Scale bars, 25 μm, except in (i), 10 μm). All experiments were performed 3 times with similar results.

Supplementary Figure 3 Disruption of endosomal transport leads to increased p-AMPK levels.

a, Wing epithelia expressing interfering RNA directed against the vps15 locus (vps15RNAi) in the dorsal region (D) labelled with anti-p-AMPK. Hatched line indicates Dorsal/Ventral boundary. Scale bar, 25 μm.XZ cross-section shown to right. b, vps34RNAi was expressed in the dorsal domain of the larval wing epithelia, labelled with anti-Rab7. D/V boundaries indicated with hatched line. Scale bar, 25 μm. c,d, Wing discs expressing GFP-tagged Rab5 alone or together with and vps34RNAi (d) were labelled with anti-Rab7. Merged channel on right indicates regions of overlap (yellow). Scale bar, 5 μm. e, ap driven expression of GFP::Rab7 and mCherry-tagged AMPKα (Ch::AMPKα) show overlap in the wing as indicated by arrows. Scale bar, 10 μm. f, Wing cells expressing GFP-tagged Rab7 were labelled with anti-p-AMPK. Arrows indicate anti-p-AMPK signal overlapping Rab7 vesicles. Scale bar, 2 μm. g, Wing disc expressing GFP::Rab5CA (constitutive active) in the dorsal compartment cells were labelled with anti-p-AMPK. Hatched line denotes the D/V boundary. Scale bar, 5 μm. h, Structured illumination microscopy captured image of GFP::Rab5CA vesicle from wing disc labelled with anti-p-AMPK. Scale bar 1 μm. All experiments were performed 3 times with similar results.

Supplementary Figure 4 LKB1 localises to endosomal membranes where RAB7 localisation is CIII-PI3 K dependent.

a, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged RAB5 were imaged over time. Double positive punctae were tracked and their movement traced (right panels). Arrowheads indicate the appearance and final positions of individual tracks. b, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged RAB7 were imaged over time. Double positive punctae were tracked and their movement traced (right panels). Arrowheads indicate the appearance and final positions of individual tracks. c, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged RAB7 were treated with SAR405 or DMSO (not shown) and imaged over time. d, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged RAB5 were treated with SAR405 and imaged over time. Arrowheads indicate newly formed LKB1 punctae. Asterisks indicate landmark large vesicles for orientation. Scale bar, 5 μm in all. e, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged RAB5 were treated with SAR405 (45 min) prior to the addition of Alexa-647 labelled Transferrin (15 min). Arrowheads indicate triple positive punctae. Scale bar, 5 μm. All experiments were performed 3 times with similar results.

Supplementary Figure 5 LKB1 localises to Rab7-positive endosomes.

a, Widefield fluorescent microscope image of a 110 nm thick plastic section containing Caco-2 cells expressing mCherry-tagged RAB7. An electron micrograph of the same section (a′) was used to correlate the fluorescent signal with the underlying ultra-structure. b, Distribution of mCherry-tagged RAB7 and GFP-tagged LKB1 (inverted LUT in b′) within the section. c, Enlarged insets as indicated in b. d, Intensity line profile of mCherry-tagged RAB7 (as indicated by red arrow in c) and GFP::LKB1 (d′, white arrow in c). e, Correlation of the fluorescent widefield image with the electron micrograph of the same section at high magnification (enlarged inset as indicated in a′ and c) reveals the presence of endosomes at sites where both mCherry-tagged RAB7 and GFP-tagged LKB1 fluorescent signal can be detected. All experiments were performed 3 times with similar results.

Supplementary Figure 6 Endosomal, physical and genetic interaction of Lkb1 and WDFY2.

a, Follicular epithelial cells of developing egg chambers expressing GFP-tagged 2XFYVE (PI3P binding probe) and mCherry-tagged WDFY2. Arrowheads indicate some of the double positive punctae. Scale bar, 10 μm. b, Caco-2 cells stable expressing GFP-tagged WDFY2 and mCherry-tagged LKB1 labelled with phalloidin 647 (Actin). Scale bar, 10 μm. c, Caco-2 cells stably expressing GFP-tagged WDFY2RA and mCherry-tagged LKB1. Scale bar, 10 μm. d, Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged WDFY2 were imaged over time and double positive punctae were tracked and traced (right panel). Arrowheads indicate start and stop points of individual punctae. Scale bar, 5 μm. e, In vitro pull-down experiments using Drosophila and human proteins, where 35S-labelled LKB1 was produced in the rabbit reticulocyte lysate and GST-tagged WDFY2 (or WDFY2RA) was produced in bacteria. f, Wild type, WDFY2 and WDFY2RA expressing Caco-2 cells were either control or SAR405 treated for 4 h prior to lysis and western blotting for p-AMPK and AMPK. From image intensity analysis the ratio of p-AMPK to AMPK was determined and average values from n, number of samples, were plotted, normalising control samples to 1; n values are indicated in the graph. Standard deviation error bars are shown. P-values were determined by paired two-tailed Student’s t-test. n.s., non-significant difference. g, Caco-2 cells cultured in matrigel and treated with siRNA were labelled as indicated. Scale bar, 10 μm. Experiments were performed 3 times, unless indicated otherwise, with similar results.

Supplementary Figure 7 siRNA efficiency assessment via western blotting.

a, Lysate from Caco-2 cells treated with siRNA targeting VPS34 or Control sequence were subjected to western blot analysis with anti-VPS34. b, Lysate from Caco-2 cells treated with siRNA targeting BECN-1 or Control sequence were subjected to western blot analysis with anti-BECN-1. c, Lysate from Caco-2 cells treated with siRNA targeting ATG14 or Control sequence were subjected to western blot analysis with anti-ATG14. d, Lysate from Caco-2 cells treated with shRNA targeting VPS34 or Control sequence were subjected to western blot analysis using anti-VPS34. e, Lysate from Caco-2 cells treated with siRNA targeting LKB1 or Control sequence were subjected to western blot analysis with anti-LKB1. f, Lysate from Caco-2 cells treated with siRNA targeting SNX3 or Control sequence were subjected to western blot analysis with anti-SNX3. Hatched area indicates the lanes of interest and zoomed region to the right. g, Lysate from Caco-2 cells treated with siRNA targeting SNX18 or Control sequence were subjected to western blot analysis with anti-SNX18. h, Lysate from Caco-2 cells stably expressing GFP-tagged WDFY2 treated with siRNA targeting WDFY2 or Control sequence were subjected to western blot analysis with anti-GFP. Reliable antibody to WDFY2 was not available.

Supplementary Figure 8 Models summarising the proposed role of CIII-PI3K in LKB1 regulation.

a, RAB5 recruits CIII-PI3K which generates PI3P, essential for RAB5 to RAB7 maturation. This maturation step is required to attenuate the polarisation capacity of LKB1 which depends on the PI3P effector protein, WDFY2. b, Overview of the conservation of the CIII-PI3K regulatory system underlying epithelial integrity in Drosophila and human cells. Inactivation of CIII-PI3K or WDFY2 causes epithelial disruption, driven by an increase in LKB1 activity and consequent activation of JNK. In vivo, CIII-PI3K inactivation cooperates with RasV 12 to promote tumour growth through LKB1 activity.

Supplementary Figure 9 Expanded western blots.

a, Expanded view of western blot presented in Fig. 2d. Hatched area indicates the cropped region. b, Expanded view of the western blot in Supplementary Fig. 2e. Hatched area indicates the cropped region. Glucose starvation (No GLU) served as a positive control. c, Expanded view of the blot in Fig. 6f indicating the cropped region with hatched box. d, Expanded view of the western blot in Fig. 6d. Hatched region indicating the cropped region. e, Expanded view of the western blot in Fig. 6e. Hatched region indicating the cropped region. f, Expanded views of western blots presented in Supplementary Fig. 6e.

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LKB1 localises to RAB5-positive endosomal vesicles.

Supporting figure 4a. Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged Rab5 were imaged live for 2 min to visualise the movement of double positive vesicles. Timestamp in minutes and seconds. (AVI 695 kb)

LKB1 localises to RAB7-positive endosomal vesicles.

Supporting figure 4b. Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged Rab7 were imaged live for 2 min to visualise the movement of double positive vesicles. Timestamp in minutes and seconds. (AVI 899 kb)

LKB1 localisation on RAB7-positive endosomal vesicles is disrupted by CIII-PI3K inhibition.

Supporting figure 4c. Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged Rab7 were imaged live for 60 min immediately after the addition of SAR405 to monitor the effect of CIII-PI3K inhibition to the localisation of LKB1. Timestamp in minutes and seconds. (AVI 602 kb)

Inhibition of CIII-PI3K activity does not disrupt the localisation of LKB1 to RAB5-positive vesicles.

Supporting figure 4e. Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged Rab5 were imaged live for 60 min immediately after the addition of SAR405 to monitor the effect of CIII-PI3K inhibition to the localisation of LKB1. Timestamp in minutes and seconds. (AVI 1056 kb)

LKB1 localises to WDFY2-positive vesicles.

Supporting figure 6c. Caco-2 cells stably expressing GFP-tagged LKB1 and mCherry-tagged WDFY2 were imaged live for 2 min to visualise the movement of double positive vesicles. Timestamp in minutes and seconds. (AVI 262 kb)

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O’Farrell, F., Lobert, V., Sneeggen, M. et al. Class III phosphatidylinositol-3-OH kinase controls epithelial integrity through endosomal LKB1 regulation. Nat Cell Biol 19, 1412–1423 (2017). https://doi.org/10.1038/ncb3631

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