Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell

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In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v+) subpopulation of 4T1 breast cancer cells, but not that of a CD44v subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v+ cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

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Figure 1: Expression of CD44v in metastatic breast cancer cells with lung colonization potential.
Figure 2: Regulation of alternative splicing of CD44 mRNA and promotion of lung metastasis by ESRP1.
Figure 3: Selective expansion of CD44v+ stem-like cancer cells during lung metastasis.
Figure 4: CD44 splicing enhances ROS defence and lung colonization potential.
Figure 5: EMT marker expression and epigenetic status of the ESRP1 locus in CD44v+ or CD44v 4T1 cells.

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We thank I. Ishimatsu, S.Suzuki, S.Hayashi, Y.Hata and K.Hizume for technical assistance; K.Arai for help in preparation of the manuscript. O.N. and H.S. are grateful to A.Izumi-Taguchi, T.Kodama (Lab for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-Ku, Tokyo 153-8904, Japan), H.Ueda, T.Miyazawa, S.Yamamoto, K.Shiina and G.Nagae (Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo) for technical assistance in ChIP-sequencing analysis. Antibodies against Histone H3K4me3, Pol II were kindly provided by H.Kimura (Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan). This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to H.S.) as well as in part by the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan(to O.N.). Imaging mass spectrometry analysis was supported by Grant-in-Aid from the SENTAN Program from JST and Development of the Next-Generation Integrated Simulation of Living Matter, a Part of Development and Use of the Next-Generation Supercomputer Project of MEXT Japan.

Author information

H.S., K. Takahashi, and O.N. conceived the project and designed experiments. T.Y., K. Tsuchihashi, T.I., T. Motohara, M.Y., G. J. Y., T.W., T.O., Y.K., and A.K. performed experiments. T. Masuko, K.M., H.T., T. Minami, H.A., M.O. and M.S. analysed the data. H.S. and O.N. wrote the manuscript.

Correspondence to Hideyuki Saya or Osamu Nagano.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures and Methods

Supplementary Figures S1-S3 and Supplementary Methods (PDF 359 kb)

Supplementary Data 1

Microarray analysis of CD44v+ cell/CD44v– cells, ES and iPS cells (XLS 38 kb)

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