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 k1 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.

2. Buehler, D., Hardin, H., Shan, W., Montemayor-Garcia, C., Rush, R. S., Asioli, S., … Lloyd, R. V. (2013). Expression of epithelial-mesenchymal transition regulators SNAI2 and TWIST1 in thyroid carcinomas. Mod Pathol 26(1), 54-61.

3. Qin, Q., Xu, Y., He, T., Qin, C., Xu, J. (2012). Normal and disease-related biological functions of Twist 1 and underlying molecular mechanisms. Cell Research, 22(1), 90-106. Retrieved from www.nature.com/cr

4. Salgado, C., Basu, D., Nikiforova, M., Bauer, B. S., Johnson, D., … Reyes-Mugica, M. (2015) BRAF mutations are also associated with neurocutaneous melanocytosis and large/giant congenital melanocytic nevi. Pediatric and Development Pathology 18, 1-9.

5. Salgado, C., Silver, R. B., Bauer, B. S., Basu, D., Schmitt, L., … Reyes-Mugica, M. (2014) Skin of patients with large/giant congenital melanocytic nevi shows increased mast cells. Pediatric and Developmental Pathology 17, 198-203.

6. Zhong, J., Ogura, K., Wang, Z., Inuzuka,, H. (2013). Degration of the transcription factors twist, an oncoprotein that promotes cancer metastasis. Discovery of Medicine, 15(80), 7-15. Retrieved from http://www.discoverymedicine.com/Jiateng-Zhong/2013/01/24/degradation-of-the-transcription-factor-twist-an-oncoprotein-that-promotes-cancer-metastasis/

7. Zhuo, X., Chang, A., Huang, C., Yang, L., Xiang, Z., Zhou, Y. (2014). Expression of TWIST, an inducer of epithelial-mesenchymal transition, in nasopharyngeal carcinoma and its clinical significance. International Journal of Clinical and Experimental Pathology 7(12), 8862-8868. Retrieved from ww.ijcep.com

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

AcknowledgementsWe 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.