PgmNr Z6001: Investigating the role of tetraploid intermediates in melanoma progression.

Authors:
R. Darp 1,2 ; S. Gujja 1 ; Y. Edwards 1 ; D. Pellman 3 ; L. Zon 4 ; N. Ganem 5 ; C. Ceol 1,2


Institutes
1) University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA, USA; 2) University of Massachusetts Medical School, Department of Molecular, Cellular and Cancer Biology, Worcester, MA, USA; 3) Howard Hughes Medical Institute, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Children’s Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA, USA; 4) Howard Hughes Medical Institute, Stem Cell Program and Division of Hematology/Oncology of Boston Children’s Hospital and Dana Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA; 5) Departments of Pharmacology and Experimental Therapeutics and Medicine, Division of Hematology and Oncology, Boston University School of Medicine, Boston, MA, USA.


Abstract:

Tetraploid cells and their aneuploidy progeny have been proposed as intermediates in tumor development. Support for this hypothesis comes from deep sequencing studies that suggest at least one third of solid tumors have undergone a genome doubling event during their progression and additional studies that have implicated genome doubling as an important step early in tumor progression. These studies propose that nascent tetraploid tumor cells can more easily sample genetic configurations that are advantageous for growth because they, as compared to diploid cells, are buffered against deleterious effects of mutations and changes in gene dosage. Studies using a melanoma-prone Tg(mitfa:BRAFV600E);p53(lf) strain suggest a role for tetraploidy and genome doubling in the genesis of tumors in these fish. We have found that, unlike wild-type melanocytes, melanocytes in Tg(mitfa:BRAFV600E) animals are binucleate and tetraploid. In tissue culture, newly-formed tetraploid cells undergo a p53-dependent G1 cell cycle arrest. A similar arrest likely occurs in zebrafish melanocytes, as melanocytes in Tg(mitfa:BRAFV600E); p53(lf) animals exhibit 8N and greater DNA content, suggesting bypass of an arrest that halts progression of tetraploid Tg(mitfa:BRAFV600E) melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as a contributor to melanoma initiation. To gain insight into the mechanism by which BRAFV600E generates binucleate cells, we have established a lentiviral-based doxycycline inducible in vitro model system to induce BRAFV600E expression. We are currently using live-cell imaging approaches to determine the mechanism by which these binucleates arise in culture. We are also conducting retrospective bioinformatic analyses to determine whether melanomas in Tg(mitfa:BRAFV600E);p53(lf) zebrafish progress through tetraploid intermediates, supporting the notion that the combination of oncogenic BRAFV600E and mutated p53 causes the generation and progression of nascent tetraploid melanoma cells.