学位論文内容の要旨（ Summary of dissertation）
博士の専攻分野の名称

博士（医 学）（Degree conferred: Doctor of Philosophy）

氏 名

艾力江 卡德尔（Name of recipient: Ailijiang Kadeer ）学 位 論 文 題 名（Title of dissertation）
Plectin is a novel regulator for apical extrusion of RasV12-transformed cells（プレクチンは Ras 変異細胞の頂端側への逸脱に関わる新規制御因子である）【Background and Objectives】
In multicellular organisms, oncogenic mutations occur within the epithelial tissues at
the initial stage of carcinogenesis, though the fate of the transformed cells remained
enigmatic. Recent studies have revealed that the newly emerging transformed cells areoften eliminated from the epithelium. During this process, normal and transformed
epithelial cells compete with each other for survival, a process called cell competition.
However, underlying mechanisms of this process still remain to be explored. Plectin is aversatile cytoskeleton cross-linker. Here, I aim to clarify a role of plectin in cellcompetition between normal and RasV12-transformed cells.
【Methods and Results】
In this study, normal Madin-Darby canine kidney (MDCK) cells and MDCK-pTR
GFP-RasV12-transfromed (hereafter RasV12) cells were mainly used for theexperimental cell culture system. Under three different cell culture conditions (normalMDCK cells alone, RasV12 cells alone and their mixture at 1:1 ratio) I first usedanti-phospho-tyrosine antibody and performed immunoprecipitation. I found thatamount of precipitated plectin increased under the mix culture condition, comparedwith normal or RasV12 cells alone. By western blotting, I demonstrated that plectin was
not tyrosine-phosphorylated. Next, I examined the subcellular localization of plectin byimmunofluorescence analysis. I mixed RasV12 cells with normal MDCK cells at a ratioof 1:50. I found that plectin was non-cell-autonomously accumulated in RasV12 cells
surrounded by normal cells. To understand the functional role of plectin in apicalextrusion, I knocked down plectin in RasV12 cells. Plectin knockdown significantly
suppressed apical extrusion of RasV12 cells, indicating that plectin is a positive
regulator for apical extrusion.
Plectin is a cytoskeletons organizer. I thus examined the correlation between

plectinandmicrotubules

or keratin5+8. Microtubules

andkeratin5+8

wereaccumulated in RasV12-transformed cells and partially co-localized with plectin. And
disruption of microtubules by nocodazole suppressed accumulation of plectin andkeratin5+8. In addition, nocodazole suppressed apical extrusion of RasV12 cells.
Conversely,

plectin-knockdown

suppressedaccumulationofmicrotubules

andkeratin5+8. These findings indicate that plectin and microtubules were mutuallyaccumulated to co-regulate apical extrusion of RasV12 cells.
In our previous studies, EPLIN was identified as a crucial regulator in RasV12cells for apical extrusion. I performed immunoprecipitation with anti-EPLIN antibody
to examine the relevance between EPLIN and plectin. I found the increased interaction
between EPLIN and plectin under the mix culture condition. In addition, knockdown ofplectin or knockdown of EPLIN diminished EPLIN accumulation or plectin
accumulation in RasV12 cells, respectively. Similarly, EPLIN-knockdown suppressed
microtubule accumulation, and nocodazole treatment diminished EPLIN accumulation.
These findings indicate that plectin, EEPLIN, and microtubules mutually affect theiraccumulation in a complex in RasV12 cells surrounded by normal cells.
In the previous study, caveolin-1 (Cav-1) and PKA were identified as downstreamof EPLIN in RasV12 cells surrounded by normal cells. I found that plectin-knockdown
diminished Cav-1 accumulation, whereas knockdown of Cav-1 in RasV12 cells did notaffect accumulation of plectin. Furthermore, plectin-knockdown profoundly suppressed
the non-cell-autonomous activation of PKA. These data suggest that in concert withEPLIN and microtubules, plectin regulates these downstream regulators for apical
extrusion of RasV12-transformed cells.
【Discussion】
In this study, we have revealed that plectin is a new player in apical elimination ofRasV12-transformed cells from the epithelium. Plectin is co-accumulated with EPLIN
and microtubules in RasV12 cells surrounded by normal cells to regulate downstream
signaling molecules Cav-1 and PKA, thereby promoting apical extrusion. In thismultiple-molecular complex, plectin seems to act as a key adaptor that interlinks thesecytoskeletal proteins. There still remains a question to be addressed: what are theupstream and downstream regulators of the plectin-EPLIN-microtubules complex?
【Conclusion】
Apical extrusion of transformed cells can be regarded as a cancer preventive process,which is supposed to occur at the initial stage of carcinogenesis. Thus, theplectin-EPLIN-microtubules complex could be a potential drug target; the activation of
the plectin complex is expected to enhance the eradication of newly emerging and/orremaining transformed cells from epithelia. Further development of this study would
open a new avenue for cancer preventive medicine.