Several cell cycle regulators, such as
PLK1 [
4],
TRIM16 [
5],
WEE1 [
2,
6],
CDK4/6 [
1,
7], and
CCND1 [
1,
3,
8], have been shown to be involved in the tumorigenesis of neuroblastoma. This suggests that neuroblastoma tumors might be addicted to individual activated oncogenes in the process of cell cycle regulation and that additional oncogenic cell cycle regulators could play a role in neuroblastoma tumorigenesis. We screened two neuroblastoma cell lines using a commercially available arrayed siRNA library representing 131 cell cycle regulators from different gene families to identify genes that are required for the proliferation or survival of neuroblastoma cells and that might serve as new therapeutic targets. The gene families included cyclin dependent kinases (CDKs), members of the retinoblastoma protein family, DNA replication factors such as the cell division cycle proteins (CDCs), members of the CIP/KIP family, and the INK4 family of cell cycle inhibitors. Real-time monitoring of cell growth showed that knockdown of
CCND1 and
PLK1 had the strongest effects in reducing the proliferation of neuroblastoma cells.
PLK1 has previously been extensively studied in neuroblastoma and has been shown to inhibit the transactivation activity of p53 and to promote cell survival [
4,
13,
14]. Moreover, targeting PLK1 with small molecule compounds induced apoptosis and growth arrest in neuroblastoma tumor-initiating cells [
5,
9] and reduced the growth of neuroblastoma xenografts in nude mice [
4,
6]. Cyclin D1 has also been studied to some extent in neuroblastoma and has previously been shown to be highly expressed in a subset of neuroblastoma cell lines and tumors due to gene amplification or GATA3 binding [
7,
8,
15,
16]. Cyclin D1 belongs to a group of proteins known as cyclins, which are involved in the temporal coordination of each mitotic event during the cell cycle. Cyclins exhibit a cell-cycle dependent pattern of expression and degradation, and they activate the CDKs. Cyclin-CDK complexes phosphorylate target proteins such as Rb and coordinate the progression into the next phase of the cell cycle [
8,
12]. Cyclin D1 activates CDK4 and CDK6 and this initiates the phosphorylation of the tumor suppressor protein Rb resulting in the activation of E2F. The activity of E2F leads to the transcriptional activation of E2F-responsive genes, such as
TOP2A,
CCNE2, and
TK1, that are essential for DNA synthesis and cell cycle progression [
4,
9,
10,
17]. Despite being an attractive therapeutic target, small molecule compounds that can directly and specifically inhibit cyclin D1 do not exist at present. It is possible, however, to inhibit cyclin D1 indirectly by several means. For example, inhibition of
CCND1 can be achieved by mTOR inhibitors, since
CCND1 mRNA translation is mTOR-dependent [
10,
18], or by inhibitors of glycogen synthase kinase 3 β (GSK3β), which phosphorylates cyclin D1 at Thr-286 suggested to regulate the turnover and the intracellular distribution of cyclin D1 [
10,
19]. The most effective approach to inhibit cyclin D1 activity is by inhibiting its associated kinases CDK4 and CDK6 [
11,
12]. Pan-CDK inhibitors can inhibit CDKs but induce also considerable toxicity due to off-target effects [
12,
20]. In contrast, early phase clinical trials have shown that specific and potent inhibition of CDK4/6 by small molecule drugs, such as palbociclib [
4,
21‐
24] and R547 [
5,
25], can result in antitumor activity with tolerable side effects. Recently, it has been shown that inhibition of CDK4/6 by LEE011 reduces the growth of neuroblastoma tumors with
MYCN amplification in murine xenograft models [
7]. In this study, we used palbociclib, a selective CDK4/6 inhibitor that has already shown promising antitumor activity in several clinical trials in other cancer types [
12]. Treatment of neuroblastoma cells in vitro with palbociclib resulted in variable effects: some cell lines, such as IMR-32, SH-SY5Y, and NGP, were sensitive, whereas other cell lines, such as SK-N-SH and CLB-GA, were relatively resistant. It is worth mentioning that NGP cells have
CDK4 amplification. Additional studies are needed to explain the different sensitivity to this compound.