| Summary: | MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult
tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a
restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the
latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene
transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the
MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in
Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four
ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric
soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)].
ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS
and ARMS, but the MYCN amplification and expression levels shows a significant correlation and
are greater in ARMS, in which they are associated with adverse outcome.
We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28
and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent
inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was
followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN
expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated-
PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix
microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced
or repressed, with both genes previously described as targets of N-myc or Myc, and new genes
undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility,
metastasis, angiogenesis and muscle development). The changes in the expression of the most
relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the
MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS
xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic
tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity),
whereas treatment with the mutated-PNA had no effect.
Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS,
particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.
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