1. Serotonin induces migration of congenital melanocytic
nevus cells via upregulation of Twist1/2 expression.
Joie Zabec,1 Sarah Koury,1 Cláudia Salgado,2 Dipanjan Basu2 and Miguel Reyes-Múgica2 k
1 First Experiences in Research, Dietrich School of Arts & Sciences, University of Pittsburgh
2 Department of Pathology, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center
Results Conclusion
• The scratch assay showed that cells treated with serotonin have
more migration into the scratched area than the control.
• Quantitative PCR analysis of Twist 1 and Twist 2 shows
upregulation of these EMT genes in cells when treated with
serotonin.
• The research performed here indicates Serotonin may promote
the migration of nevus cells which leads to progression and
spread of nevus cells and that could increase the risk of
malignant transformation.
Introduction
• Large/giant congenital melanocytic nevi
(L/GCMN) are characterized by abnormal
large patches of dark colored skin.
• Occurrence of L/GCMN is approximately 1 in
20,000 to 1 in 500,000 births).
• An aggressive subset of L/GCMN is called
Neurocutaneous melanocytosis (NCM),
which has a very poor prognosis and high
mortality rate.
• Previous research has found oncogenic
mutations in NRAS1 or BRAF4 in these lesions.
• However, because it is so rare, there is little
research and treatment options.
Methods
• The nevomelanocytic cells were derived from patient’s lesions of
L/GCMN from the Gavin Bailey Tissue Repository at the Reyes-Mύgica
lab.
Scratch Assay
• Cells were treated with increased dosage of serotonin and observed over
72 hours.
• A scratch assay was preformed by creating a scratch in the monolayer
culture migration of cells into the scratch was monitored over a time.
Quantitative Real Time PCR
• Total RNA was extracted from the cells using Qiagen Rneasy mini kit and
cDNA were prepared using Superscript-II (Invitrogen) reverse
transcriptase.
• PCR was preformed using SYBR green qPCR master mix containing ROX
dye (Thermo Scientific).
• GAPDH was used as the endogenous housekeeping control.
• Primers were chosen for genes known to affect epithelial to
mesenchymal transition (EMT) and were from realtimeprimers.com.
Future Direction
• In the future, more genes related to the EMT pathway could be
tested to understand the molecular mechanism by which
serotonin induces the EMT.
• It remains to be determined whether a serotonin receptor
antagonist will rescue the migratory phenotype of
nevomelanocytes.
References1. Basu, D., Salgado, C. M., Bauer, B. S., Johnson, D., Rundell, V., Nikiforova, M., … Reyes-Mugica, M. (2015)
Nevospheres from neurocutaneous melanocytosis cells shows reduced viability when treated with specific
inhibitors of NRAS signaling pathway. Neuro-Oncology, 1-10.
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of epithelial-mesenchymal transition regulators SNAI2 and TWIST1 in thyroid carcinomas. Mod Pathol 26(1),
54-61.
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4. Salgado, C., Basu, D., Nikiforova, M., Bauer, B. S., Johnson, D., … Reyes-Mugica, M. (2015) BRAF mutations are
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Figure 2. Nevus Cell Migration Scratch Assay. Cells cultured from
L/GCMN patient P08 showed increased migration upon serotonin
treatment in an in vitro “wound-healing assay” or “scratch assay.”
Figure 2. Quantitative PCR shows upregulation of EMT
genes, related to cell migration. Quantitative PCR analysis of
relative Twist 1 and Twist 2 expression compared to the
control GAPDH from patient C76N.
Figure 1. Image of patient C76N’s
massive garment-like giant nevus.
Basu et al.: Tumor spheroids of
NCM respond to NRAS inhibitors
Nevus Cell Migration
• Nevocytes and mastocytes
share the stem cell factor
receptor CD1175.
• Serotonin, a biogenic amine,
is a secreted factor of mast
cells.
• We hypothesize that
serotonin induces the EMT
pathway, which leads to the
proliferation and migration
of nevus cells.
Figure 1. Epithelial to mesenchymal transition (EMT
pathway) is influenced by several genes including
Twist, Snail and Zeb.
http://flipper.diff.org/app/items/info/6934
Acknowledgements
We would like to acknowledge Rangos Research Center at Children’s Hospital of Pittsburgh of UPMC and
the Gavin Bailey tissue repository at the Reyes-Mugica Lab, Division of Pediatric Pathology, University of
Pittsburgh.