Abstract
Malignant peripheral nerve sheath tumors (MPNST) are rare, highly aggressive sarcomas that can occur spontaneously or from pre-existing plexiform neurofibromas in neurofibromatosis type1 (NF1) patients. MPNSTs have high local recurrence rates, metastasize easily, are generally resistant to therapeutic intervention and frequently fatal for the patient. Novel targeted therapeutic strategies are urgently needed. Standard treatment for patients presenting with advanced disease is doxorubicin based chemotherapy which inhibits the actions of the enzyme topoisomerase IIα (TOP2A). Recent molecular studies using murine models and cell lines identified the bromodomain containing protein 4 (BRD4) and enhancer of zeste homolog 2 (EZH2) as novel targets for MPNST treatment. We investigated the expression and potential use of BRD4, EZH2 and TOP2A as therapeutic targets in human NF1-derived MPNSTs. The transcript levels of BRD4, EZH2 and TOP2A were determined in paired formalin-fixed paraffin-embedded (FFPE) neurofibroma/MPNST samples derived from the same NF1 patient and in a set of plexiform neurofibromas, atypical neurofibromas and MPNST. We further examined the effect on cell viability of genetic or pharmacological inhibition of BRD4, EZH2 and TOP2A in an MPNST cell line panel. Our results indicated that in MPNST samples BRD4 mRNA levels were not upregulated and that MPNST cell lines were relatively insensitive to the bromodomain inhibitor JQ1. We corroborated that EZH2 mRNA expression is increased in MPNST but failed to confirm its reported pivotal role in MPNST pathogenesis as EZH2 knockdown by siRNA did not interfere with cellular proliferation and viability. Finally, the relation between TOP2A levels and sensitivity for doxorubicin was examined, confirming reports that TOP2A mRNA levels were overexpressed in MPNST and showing that MPNST cell lines exhibited relatively high TOP2A protein levels and sensitivity to doxorubicin. We tentatively conclude that the potential for effective therapeutic intervention in MPNST by targeting BRD4, EZH2 and TOP2A individually, may be limited. Clinical studies are necessary to ultimately prove the relevance of BRD4 and EZH2 inhibition as novel therapeutic strategies for MPNST.
Highlights
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder which has a de novo incidence of one in 3000 individuals [1,2,3]
We examined bromodomain containing protein 4 (BRD4) mRNA expression by qRT-PCR in a series of nine paired human malignant peripheral nerve sheath tumors (MPNST) and plexiform neurofibroma formalin-fixed paraffin-embedded (FFPE) samples, each pair derived from the same patient (Fig 1A)
The paired sample analyses indicated significantly higher BRD4 mRNA levels in 6 of the neurofibromas compared to their corresponding MPNST whereas in most fresh frozen samples there was no significant difference in BRD4 mRNA levels between plexiform neurofibromas and MPNSTs
Summary
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder which has a de novo incidence of one in 3000 individuals [1,2,3]. Despite the high expression of TOP2A, advanced MPNST patients do not respond well to doxorubicin given a 2 year overall survival rate of approximately 20%, which is roughly equivalent to the outcome of patients with metastatic STS other than MPNST [7]. This poor outcome clearly underscores the need to get better insight into the exact relationship between TOP2A expression and doxorubicin sensitivity in MPNST and the necessity to reveal new leads for treatment
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