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Cancer Research

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

Published: September 13th, 2018

DOI:

10.3791/58416

1Department of Pathology and Molecular Medicine, Queen's University
* These authors contributed equally

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

The Hippo signaling pathway was first identified in Drosophila as a novel regulator of cell growth and animal size1,2. Since its initial discovery, mounting evidence has convincingly shown that the Hippo pathway plays critical roles in the development (e.g., early embryonic development, organ size control, and three-dimensional [3-D] morphology), tumorigenesis (e.g., tumor development, metastasis, angiogenesis, immune evasion, genomic instability, stress response, and drug resistance), and tissue homeostasis (e.g., stem cell renewal and differentiation and tissue regeneratio....

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1. Investigation of a Putative Regulator of Hippo Signaling Using the LATS-BS

  1. Plating and transfection of cells
    1. Preparation of cell culture
      1. Warm 1x PBS, DMEM containing 10% FBS and 1% penicillin/streptomycin, and 0.25% trypsin-EDTA to 37 °C in a water bath for approximately 30 min.
      2. Thoroughly clean a biosafety cabinet with 70% ethanol.
      3. Place tissue culture dishes, Pasteur pipettes, serological pipettes, and pipette tips into the tissue cu.......

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The LATS-BS was cotransfected with different genes to evaluate their effect on LATS activity (Figure 3). In this experiment, Renilla was used as an internal control. The transient expression of YAP-5SA, a constitutive active form of YAP, causes increasing levels of YAP transcriptional targets and a subsequent increase in LATS kinase activity through an established feedback pathway25. MST, the upstream activator of LATS in the .......

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While the Hippo pathway plays critical roles in various biological processes, and dysregulation of the Hippo pathway leads to cancer6, how the Hippo pathway is regulated in response to various stimuli is not completely understood. In addition, there has been no quantitative and real-time system to assess the activity of core Hippo components. Recently, we developed and validated a novel biosensor for measuring LATS kinase activity in the Hippo pathway24. This biosensor can .......

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This work was supported by grants from the Canadian Institute of Health Research (CIHR#119325, 148629) and the Canadian Breast Cancer Foundation (CBCF) to XY. TA is supported by the Vanier Canada Graduate Scholarship and the Ontario International Graduate Scholarship. HJJVR is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology.

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Name Company Catalog Number Comments
Trypsin-EDTA  Life Technologies 2520056 0.25%
Fetal Bovine Serum (FBS) Sigma F1051
DMEM Sigma D6429-500ml DMEM with high glucose 
Penicillin Streptomycin Life Technologies 15140122
PolyJet In Vitro DNA Transfection Reagent  Signagen SL100688.5
5x Passive lysis buffer Promega E194A 30 ml
Dual-Glo® Luciferase Assay System Promega E2940 100 ml kit
20/20 luminometer Turner Biosystems 998-2036 Single tube reader luminometer
GloMax®Navigator with dual injectors Promega GM2010 96-well plates reader luminometer

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