Elucidating the signaling pathways regulating syndromic and nonsyndromic rhabdomyosarcoma
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Costello Syndrome (CS) is a rare autosomal dominant disorder which is
estimated to affect rough 350 patients worldwide. CS patients are born with failure-tothrive,
coarse facial features, short stature, skeletal and skin abnormalities, and a widerange
of intellectual disabilities. CS is the result of heterozygous germline mutations
in the proto-oncogene HRAS. These activating germline mutations result in
constitutive activation of the RAS/MAPK signaling pathway and leads to increased
cell proliferation, cell differentiation, and migration. CS patients also have a higher
predisposition for developing malignances including neuroblastoma, early onset
bladder carcinoma, and rhabdomyosarcoma. The most common malignancy seen in
CS patients is embryonal rhabdomyosarcoma (ERMS). In our CS cohort 14 of our 141
CS patients have developed ERMS. In this study we were able to analyze eleven
ERMS tumors from eight of the fourteen unrelated patients.
ERMS is the most common pediatric soft tissue sarcoma and accounts for
roughly 4.5% of all pediatric cancers (Ries et al., 1999). It is thought to be derived
from the skeletal muscle lineage. ERMS has been classically described by LOH at the
11p15.5 (Visser et al., 1997, Kratz et al., 2007; Menke et al., 2015) with or without the
presence of a mutation in any of the RAS genes. Very few studies had been conducted
to better understand tumorigenesis in the context of syndromic and nonsyndromic
ERMS, therefore we aimed to take a closer look into the cytogenetic, molecular, and
functional levels of ERMS in both populations. For the first time, this research showed
that ERMS tumors, both syndromic and nonsyndromic, have complete uniparental
disomy of chromosome (UPD11). We showed that ERMS tumors display either
UPD11 or segmental LOH, which includes but is not limited to the 11p15.5 locus as
previously thought. Though it has been speculated throughout the literature that lossof-
imprinting (LOI) occurs at the 11p15.5 imprinted gene cluster there have been no
previous studies with ERMS to show this. However, the work in this dissertation
suggests that both CS and sporadic ERMS tumors, may display LOI by misexpression
of the maternally expressed genes H19 and CDKN1C from the paternal allele. Though
it is extremely tempting to claim our experiments prove LOI at this locus in ERMS, it
could prove to be a technology artifact. It was not only beneficial to take a closer look
into the cytogenetic, molecular and functional levels to understand ERMS
tumorigenesis, but it was also critical to understand important crosstalk between the
RAS signaling pathway and other critical pathways involved in ERMS.
While there have been some previous ERMS gene expression studies to
identify top players involved in tumorigenesis there have been no studies that included
a CS ERMS cohort. Therefore in this study, microarray gene expression analysis was
used to identify possible differences between the ERMS group consisting of both CS
and sporadic ERMS and the control group consisting of CS and control fibroblasts.
This study enabled for us to identify genes that were ERMS specific in both
syndromic and nonsyndromic ERMS models. The analysis from this study also
validated previous studies that reported the RAS/MAPK signaling as a key player in
ERMS and enabled the discovery of novel genes that interact with the RAS/MAPK
pathways and new ERMS specific genes.
In our CS cohort alone, 3 out of the 14 patients have unfortunately succumbed
to this disease thus demonstrating the need to identify new therapeutic agents that will
help control ERMS cell proliferation and increase the survival rate in CS patients. In
this study, high-throughput screening (HTS) was used as a tool to identify cytotoxic
compounds in order to study tumor resistance and further determine its mechanism of
action. From this study, Zardaverine was identified as a novel compound that
specifically inhibited CS ERMS cell proliferation and cell invasion.
This dissertation will focus on demonstrating the effectiveness of using a rare
disorder such as CS as a syndromic model for ERMS, for further advancement of our
current understanding of this type of solid tumors. In addition, this study revealed the
importance of using HTS as a tool that may help to develop potential novel
therapeutics for CS ERMS.