The Role of CDKN3 in Neuroblastoma Differentiation

Abstract

BackgroundNeuroblastoma (NB) is a pediatric cancer that accounts for 15% of cancer related deaths of children and 7% of new childhood cancer cases in the United States. NB arises from neural crest cells that fail to differentiate. The defected neural crest cells grow indefinitely into tumors that can be surgically removed, however residual NB cells are left behind leading to relapse. The undifferentiated cells can be treated with differentiation therapy which is to force NB cells to differentiate into mature cells leading to growth arrest and apoptosis. The problem with current differentiation treatment is that 13‐cis‐retionic acid (retinoic acid) is the only agent used and it has not significantly improved survival rates.The reason that only one differentiating agent is used to treat NB is because the cause of differentiation loss is unclear. One mechanism that may lead to differentiation loss involves cell cycle regulation, because cell cycle arrest has been linked to differentiation. To identify cell cycle regulators involved in NB differentiation, a screen was conducted by treating cells on 96‐well plates with siRNA. The screen used neurite outgrowth as a marker of differentiation since differentiated NB cells have neurites and undifferentiated cells lack them. This screen identified that CDKN3 knock down leads to significant neurite outgrowth which suggests that CDKN3 is involved in differentiation. CDKN3 is a cell cycle regulator that is shown to be overexpressed in some cancers however it has no known role in NB.AimsThe goal of our study is to (1) Confirm that CDKN3 is involved in NB differentiation; (2) Demonstrate the function of CDKN3 in cell survival and proliferation regulation; (3) Determine if CDKN3 plays a role in mediating the differentiation‐inducing effects of retinoic acid and MYCN knockdown.MethodsTo measure neurite outgrowth NB cells are grown on 96‐well plates then treated using CDKN3 siRNA. Differentiation is confirmed using western blot for NB differentiation markers: growth associated protein 43 (GAP 43), neuron specific enolase (NSE), and B‐Tubulin III. Cell survival is measured using MTT assays and cell proliferation is measured using colony formation assays. To measure CDKN3 levels following retinoic acid treatment and MYCN knockdown western blot is used.ResultsWe have found that siCDKN3 NB cells show increased expression of differentiation markers; siCDKN3 cells also have increased neurite outgrowth and are less viable than control cells.ConclusionsIn conclusion it appears that CDKN3 is involved in differentiation in NB because knockdown of CDKN3 resulted in increased differentiation markers, neurite outgrowth, and a decrease in cell viability.Future workWe will examine the effect of siCDKN3 on proliferation. We will also examine CDKN3 levels following retinoic acid treatment and MYCN knockdown.Support or Funding InformationDoD PRMRP Discovery Award (PR151532) 09/01/2013‐02/28/2017 Goal: To comprehensively identify neuroblastoma differentiation‐inducing microRNAs by combining a functional high‐content screening approach and a library of microRNA mimics used to raise the intracellular microRNAs levels. Startup funds from Texas State University. 09/01/2016‐present. Goal: To investigate the mechanisms of neuroblastoma cell differentiation.