Mechanism of action of trabectedin in desmoplastic small round cell tumor cells.
BACKGROUND
Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera.


METHODS
We characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP).


RESULTS
JN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis.


CONCLUSIONS
The JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease.
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
S
S
Uboldi
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
I
I
Craparotta
Experimental Oncology and Pharmacogenomics, IRCCS Fondazione "Salvatore Maugeri"-Istituto di Pavia, Pavia, Italy.
G
G
Colella
Experimental Oncology and Pharmacogenomics, IRCCS Fondazione "Salvatore Maugeri"-Istituto di Pavia, Pavia, Italy.
E
E
Ronchetti
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
L
L
Beltrame
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
S
S
Vicario
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
S
S
Marchini
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
N
N
Panini
Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
G
G
Dagrada
Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
F
F
Bozzi
Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
S
S
Pilotti
Cell Biology and Pharmacogenomics Department, PharmaMar, Madrid, 28770, Spain.
C M
CM
Galmarini
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy.
M
M
D'Incalci
Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy. raffaella.gatta@guest.marionegri.it.
R
R
Gatta
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D058405:Desmoplastic Small Round Cell Tumor / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D004149:Dioxoles / Q000494:pharmacology* / Q000627:therapeutic use; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000187:drug effects* / Q000235:genetics; D034802:RNA-Binding Protein EWS; D044005:Tetrahydroisoquinolines / Q000494:pharmacology* / Q000627:therapeutic use; D000077606:Trabectedin; D025721:WT1 Proteins
D018906:Antineoplastic Agents, Alkylating; D004149:Dioxoles; C500986:EWSR1 protein, human; D015514:Oncogene Proteins, Fusion; D034802:RNA-Binding Protein EWS; D044005:Tetrahydroisoquinolines; D025721:WT1 Proteins; C583751:WT1 protein, human; D000077606:Trabectedin
DSRCT; JN-DSRCT-1; Trabectedin