2014 Joint Usage and Research Report
Title of Research Project

Analysis of the molecular mechanisms underlying the mutualinteractions between normal cells and transformed cells inepithelia in vitro and in vivo

Institution
ApplicantJob titleand name

Visiting
researcher

Name

University College London
Dr. Masazumi Tada
Dr. Katarzyna Anton

Purpose of the Research Project

The aim of this project is to investigate mutual interactions at(approx. 250 words)

the interface between normal cells and transformed cells at theinitiation of carcinogenesis in the two different model systems:
Madin_Darby Canine Kidney (MDCK) cells and the zebrafish
embryonic epithelium. Fujita and Tada labs found out theconserved mechanisms, by which transformed cells send
signal(s) to neighboring normal cells and in turn the normal cells
respond to the transformed cells, resulting in transformed cell
extrusion from the epithelium. The initial research purpose
upon this grant application was to uncover novel mechanisms
underlying transformed cell extrusion by comparing thedifferences in outputs between the two model systems. However,
we decided to refocus to further identify the novel conservedmechanisms, since our collaborative paper (Kajita et al., 2014)
was published in Nature Communications.
Tada lab found out a potentially novel mechanism in zebrafish
but impinged upon difficulties to demonstrate this based on invivo time-lapse analysis because of the limited availability ofreagents. To complement this, Fujita lab offered to use MDCK
cells to override the difficulties. Based on several lines ofexperimental evidence, we hypothesized that transformed cell
extrusion is regulated by cell cycle progression. To test thishypothesis, Dr Anton from Tada lab visited Fujita lab, andcarried out experiments in MDCK cells using the reagent thatallows us to visualize cell cycle transition in live imaging.
(227 words)

Development of the Research

Real time imaging analysis of transformed Src cell extrusion in

Project and Results

the zebrafish embryonic epithelium revealed that an extruding(approx.. 850 words)

Src cell is highly associated with the presence of a small cell orpart of the cell on the opposite side of the extruding Src cell fromthe epithelium. Importantly, this cell-like structure rarely
contains the nucleus and aster microtubules. This is not in anauthentic manner but reminiscent of cell division in theepithelial plane perpendicular to the orthogonal axis, as opposed
to normal cells divide in the same plane. Thus, Src cells might beextruded from the epithelium through atypical or asymmetriccell division. This prompted us to test the hypothesis that Src
cells might be extruded in the timing of cell division from thehost epithelium. To support this hypothesis, we demonstrated
that inhibition of G2/M transition, but not of G1/S transition,effectively suppressed Src cell extrusion from the zebrafish
epithelium. However, it was technically challenging to capture
images of extruding Src cells in the phase of cell cycle (G1, S orG2) in the embryonic epithelium, due to rapid cell cycle
progression, although we tested several different reagents inzebrafish.
During her visit to Fujita lab, Dr Anton further investigated therelationship between the cell cycle progression and Src cell
extrusion in MDCK cells. To consolidate this idea, first Dr Anton
was able to inhibit Src cell extrusion using an inhibitor specificfor G2/M transition, but not for G1/S transition. Along with Dr
Kajita in Fujita lab, next Dr Anton has established a stable
MDCK cell line harboring the live imaging marker for cell cycletransition called Fucci in which we can visualize changes incolors in the different phases (red in G1 and green in S, G2 orM). This fluorescent reporter line allowed us to examine if thecell cycle progression occurs in relation to Src cell extrusion. The
time-lapse analysis using the Fucci reporter line revealed thatSrc cells become extruded predominantly in the phase, G2/Mtransition. Finally, using the FACS cell sorter Dr Anton assessed
if DNA contents in both normal and transformed cells arecorrelated with their state in cell cycle progression. Because of
the limited time (for only 5 weeks), Dr Anton could not have aconclusive result from the last experiment. Together with thedata in zebrafish, Dr Anton demonstrated that the timing of Src

cell extrusion is highly correlated with the cell cycle progression,in particular G2/M transition.
This collaborative project enabled us to validate our hypothesisin the two different systems in vitro and in vivo, and the datafrom this work will be contributed to an important part ofpublication in the near future. Currently, we are planning toprepare a manuscript aiming at a highly profiled journal,
provisionally entitled “G2/M transition is a morphogenetic
check-point that determines elimination of transformed cells
from host epithelia”.
Other than the main part of the achievement by Dr Anton,
during my visit (for 5 days), Fujita and Tada labs bothexchanged the data and idea in several on-going projects. Of
those, a project related to transformed cell extrusion andautophagy was particularly interesting to both of us. Dr Kon inFujita lab found that the molecular/genetic pathways activating
autophagy are involved in the regulation of transformed cell
extrusion in MDCK cells. On the other hand, Tada lab hasexperimental evidence that an oncogenic form of the tumor
suppressor gene Adenopolyposis coli (Apc), which has been foundin 80% of colorectal cancer cases in humans, is capable ofactivating the autophagy pathway during cell extrusion and acts
independently of canonical Wnt signaling. Despite the fact thatthe extrusion rate of Apc cells is much lower than that of Srccells, that of Apc cells is much more facilitated in other genetic
backgrounds. This increased extrusion rate is highly correlatedwith more aggressive behavior of Apc cells than in wild typebackground.
These data prompted us to raise the hypothesis that themolecular/genetic pathways upstream of autophagy might play apivotal role in the regulation of transformed cell extrusion inparticular in the Apc mutant background. We will explore thisproject further in the two systems, in addition to each project ineither lab. Thus, we will aim to uncover a new aspect of cancer
progression in a multiple-hit model using the two model
systems: in vitro and in vivo.

Overall, this joint grant has led us to fruitful collaborations,through which we will be able to publish several good papers in
the coming year or two. We would like to keep this collaboration
in a longer term, so that we are planning to apply for anothercollaboration grants such as one from the Royal Society, UK.
(784 words)
Publication【Conference, symposium, workshop etc.】*Enter the names of conference

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or journal and its vol. No. where
the above work was presented.
【Journals】
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