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Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases

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Abstract

TGFβ is a known driver of epithelial-mesenchymal transition (EMT) which is associated with tumor aggressiveness and metastasis. However, EMT has not been fully explored in clinical specimens of castration-resistant prostate cancer (CRPC) metastases. To assess EMT in CRPC, gene expression analysis was performed on 149 visceral and bone metastases from 62 CRPC patients and immunohistochemical analysis was performed on 185 CRPC bone and visceral metastases from 42 CRPC patients. In addition, to assess the potential of metastases to seed further metastases the mitochondrial genome was sequenced at different metastatic sites in one patient. TGFβ was increased in bone versus visceral metastases. While primarily cytoplasmic; nuclear and cytoplasmic Twist were significantly higher in bone than in visceral metastases. Slug and Zeb1 were unchanged, with the exception of nuclear Zeb1 being significantly higher in visceral metastases. Importantly, nuclear Twist, Slug, and Zeb1 were only present in a subset of epithelial cells that had an EMT-like phenotype. Underscoring the relevance of EMT-like cells, mitochondrial sequencing revealed that metastases could seed additional metastases in the same patient. In conclusion, while TGFβ expression and EMT-associated protein expression is present in a considerable number of CRPC visceral and bone metastases, nuclear Twist, Slug, and Zeb1 localization and an EMT-like phenotype (elongated nuclei and cytoplasmic compartment) was only present in a small subset of CRPC bone metastases. Mitochondrial sequencing from different metastases in a CRPC patient provided evidence for the seeding of metastases from previously established metastases, highlighting the biological relevance of EMT-like behavior in CRPC metastases.

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Acknowledgments

We would like to thank the patients and their families who were willing to participate in the Prostate Cancer Donor Program, for without them research of this nature would not be possible. Additionally, we would also like to thank Khanhthy Doan, Funda Vakar-Lopez, Maria Tretiakova, Evan Yu, Elahe Mostaghel and the rapid autopsy teams in the Urology Department at the University of Washington. This material is the result of work supported by resources from the VA Puget Sound Health Care System, Seattle, Washington (RLV is a VA Biomedical Laboratory R&D Senior Research Career Scientist, PHL is a Staff Physician), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the PO1 NIH grant (PO1CA085859), the LUCAS Foundation, W81XWH-10-1-0563 from the CDMRP/U.S. Department of Defense, and an Outstanding New Environmental Scientist Award (ONES) (R01) from the National Institute of Environmental Health Sciences (R01 ES019319). CM is a recipient of a Career Development Award from Jim and Cathrine Allchin.

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Authors and Affiliations

  1. Genitourinary Cancer Research Laboratory, Department of Urology, University of Washington, Box 356510, Seattle, WA, 98195, USA

    Maahum Haider, Xiaotun Zhang, Hung-Ming Lam, Lisha G. Brown, Melanie Ketchanji, Belinda Nghiem, Bryce Lakely, Paul H. Lange, Martine Roudier, Celestia S. Higano, Robert L. Vessella & Colm Morrissey

  2. Divison of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Ilsa Coleman, Roger Coleman, Jason H. Bielas & Peter S. Nelson

  3. Divison of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Nolan Ericson & Jason H. Bielas

  4. Department of Pathology, University of Washington, Seattle, WA, USA

    Lawrence D. True & Jason H. Bielas

  5. Department of Medicine, University of Washington, Seattle, WA, USA

    Bruce Montgomery, Celestia S. Higano & Peter S. Nelson

  6. Department of Veterans Affairs Medical Center, Seattle, WA, USA

    Paul H. Lange & Robert L. Vessella

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  1. Maahum Haider
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10585_2015_9773_MOESM1_ESM.tif

Supplementary material 1 (TIFF 16253 kb) Representative IHC images of TGFβ and Pan-cytokeratin expression in 5 different bone metastases. White arrows highlight epithelial cells with elongated nuclei and the black arrows highlight cells with round nuclei. Bar = 40 microns

10585_2015_9773_MOESM2_ESM.tif

Supplementary material 2 (TIFF 16459 kb) Representative IHC images of Twist and Pan-cytokeratin expression in 5 different bone metastases. White arrows highlight epithelial cells with elongated nuclei and the black arrows highlight cells with round nuclei. Bar = 40 microns

10585_2015_9773_MOESM3_ESM.tif

Supplementary material 3 (TIFF 16695 kb) Representative IHC images of Slug and Pan-cytokeratin expression in 5 different bone metastases. White arrows highlight epithelial cells with elongated nuclei and the black arrows highlight cells with round nuclei. Bar = 40 microns

10585_2015_9773_MOESM4_ESM.tif

Supplementary material 4 (TIFF 16509 kb) Representative IHC images of Zeb1 and Pan-cytokeratin expression in 5 different bone metastases. White arrows highlight epithelial cells with elongated nuclei and the black arrows highlight cells with round nuclei. Bar = 40 microns

10585_2015_9773_MOESM5_ESM.tif

Supplementary material 5 (TIFF 16472 kb) Vimentin was not expressed in the cytoplasm or nuclei of epithelial cancer cells, but did stain in the stromal elements. White arrows highlight epithelial cells with elongated nuclei and the black arrows highlight cells with round nuclei. Bar = 40 microns

10585_2015_9773_MOESM6_ESM.tif

Supplementary material 6 (TIFF 5377 kb) A schematic representation of the mitochondrial genome in metastases from a patient with CRPC. The mitochondrial genome from two normal tissues, the primary tumor, and fifteen metastatic sites was sequenced from one patient. Five sites had a mutation in ND1, eight sites had a mutation in ND1 and ND5, with two further sites having an additional mutation in a tRNA and ND2 respectively

10585_2015_9773_MOESM7_ESM.tif

Supplementary material 7 (TIFF 27464 kb) Representative IHC images of the androgen receptor and Pan-cytokeratin expression in 5 different bone metastases. Black arrows highlight epithelial cells with elongated nuclei. Bar = 20 microns

Supplementary material 8 (DOCX 15 kb) Antibody information

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Haider, M., Zhang, X., Coleman, I. et al. Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases. Clin Exp Metastasis 33, 239–248 (2016). https://doi.org/10.1007/s10585-015-9773-7

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