Article

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

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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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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).

Author information

Affiliations

  1. Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway

    • Fergal O’Farrell
    • , Viola Hélène Lobert
    • , Marte Sneeggen
    • , Ashish Jain
    • , Nadja Sandra Katheder
    • , Eva Maria Wenzel
    • , Sebastian Wolfgang Schultz
    • , Kia Wee Tan
    • , Andreas Brech
    • , Harald Stenmark
    •  & Tor Erik Rusten
  2. Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway

    • Fergal O’Farrell
    • , Viola Hélène Lobert
    • , Marte Sneeggen
    • , Ashish Jain
    • , Nadja Sandra Katheder
    • , Eva Maria Wenzel
    • , Sebastian Wolfgang Schultz
    • , Kia Wee Tan
    • , Andreas Brech
    • , Harald Stenmark
    •  & Tor Erik Rusten

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Fergal O’Farrell or Tor Erik Rusten.

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Videos

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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.