Molecular Target Therapy against Neuroblastoma

*Hidemi Toyoda, Dong-Qing Xu, Lei Qi and Masahiro Hirayama*

### **Abstract**

Neuroblastoma, originated from neural crest cells, is the most common extracranial solid tumor in childhood. Treatment is of limited utility for high-risk neuroblastoma and prognosis is poor. The high incidence of resistance of advancedstage neuroblastoma to conventional therapies has prompt investigators to search for novel therapeutic approaches. Activation of IGF-R/PI3K/Akt/mTOR signaling pathway correlates with oncogenesis, poor prognosis, and chemotherapy resistance in neuroblastoma. Therefore, we investigated the effect of IGF-R/PI3K/Akt/mTOR signaling inhibitors in neuroblastoma. Significantly, IGF-R/PI3K/Akt/mTOR signaling inhibitors effectively inhibited cell growth and induced cell cycle arrest, autophagy, and apoptosis in neuroblastoma cells. Moreover, IGF-R/PI3K/Akt/ mTOR signaling inhibitors significantly reduced tumor growth in mice xenograft model without apparent toxicity. Therefore, these results highlight the potential of IGF-R/PI3K/Akt/mTOR signaling pathway as a promising target for neuroblastoma treatment. Therefore, IGF-1R/PI3K/Akt/mTOR signaling inhibitors should be further investigated for treatment in clinical trials for high-risk neuroblastoma.

**Keywords:** neuroblastoma, insulin-like growth factor (IGF), phosphatidylinositol 3-kinase (PI3K), protein kinase-B (Akt), mammalian target of rapamycin (mTOR)

### **1. Introduction**

Neuroblastoma (NB) is one of the most common extracranial solid tumors of early childhood [1, 2]. Prognosis of patients with NB depends on tumor stage, patient's age, and biologic feature of the tumor cells [3]. In patients under 1 year of age, NB is curable and sometimes spontaneously regress [4]. However, in older children with advanced stage, often the tumor is very aggressive, and patients have poor prognosis despite treatment with high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation. Although immunotherapeutic therapy such as anti-GD2 monoclonal antibody has improved outcomes of advanced stage of NB, a number of patients still relapse and eventually die of disease [5]. The high incidence of resistance of high-risk NB to conventional therapies has prompted us to search for novel therapeutic approaches.

It was reported that phosphorylated protein kinase-B (Akt) correlates with poor patients' prognosis in NB [6], and the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) pathway has subsequently been linked to augmented cell survival [7] and increased resistance to chemotherapy in NB [8].

Therefore, targeting the PI3K/Akt/mTOR signaling pathway by appropriate inhibitors appears to be a promising strategy for overcoming therapy resistance [9].

Here, we demonstrate that NB cell lines are heterogeneous in their insulin growth factor-1 (IGF-1) receptor-mediated signaling [10]. The pattern of IGF-1 receptor/PI3K/Akt/mTOR pathway-mediated proliferation is an important determinant of the response to IGF-1 receptor antagonistic therapy in human NB [10]. Furthermore, our results highlight the potential of IGF-1 receptor/PI3K/Akt/mTOR signaling pathway as a promising target for NB treatment [10–13].

## **2. Heterogeneity of IGF-1 receptor/Akt pathway-mediated proliferation in NB**

IGF-1, IGF-2, and insulin belong to a family of mitogenic growth factors and are involved in normal growth and differentiation of most tissues. The biological actions of both IGFs and insulin can be mediated by the IGF-1 receptor which is involved in mitogenic, anti-apoptotic, and oncogenic transforming responses [14, 15]. The IGF-1 receptor has two extracellular α-subunits and two intracellular β-subunits that form a heterotetrameric complex. Ligand interaction with α-subunits triggers the autophosphorylation of tyrosine kinase domains within the β-subunit [16–18]. The tyrosine kinase domains are connected to several intracellular pathways such as PI3K/Akt [19, 20]. Dysregulation of the IGF-1 receptor pathway is involved in promoting oncogenic transformation, cell proliferation, metastasis, angiogenesis, and resistance in numerous malignant diseases, such as multiple myeloma [21], carcinomas [22], and NB [23]. Since high cellular heterogeneity is a hallmark of NB, which may account for the wide range of clinical presentations and nonuniform response to treatment, we hypothesized that NB cells are heterogeneous in their IGF-1 receptor signaling-mediated cell proliferation. Thirty-one NB cell lines were cultured in three different conditions, insulincontaining serum-free medium (SFM), RPMI1640 without FBS (serum starvation medium), and RPMI1640 with 10% FBS (serum-containing medium). Based on the response patterns, 31 cell lines were subdivided into three groups [10]. Group 1, which consisted of three NB cell lines, could proliferate for more than 3 days in SFM, RPMI1640 without FBS, and RPMI1640 with 10% FBS [10]. Group 2, which consisted of 10 cell lines, could proliferate in SFM and RPMI1640 with 10% FBS but not in RPMI1640 [10]. Group 3, which consisted of 18 NB cell lines, proliferated only in RPMI1640 with 10% FBS [10]. NB cell proliferation in RPMI1640 in the presence of exogenous IGF (IGF-1, IGF-2) and insulin was examined. These IGF and insulin accelerated cell proliferation in group 1 and group 2 NB cell lines but not in group 3 NB cell lines [10]. Group 1 NB cell lines were able to proliferate in RPMI1640 in the absence of exogenous IGF and insulin [10].

## **3. Impairment of Akt activation and cell proliferation in NB by IGF-1 receptor inhibitor, picropodophyllin (PPP)**

The IGF-1 receptor inhibitors, such as IGF-1 receptor-neutralizing antibodies and IGF-1 receptor antisense/siRNA, have been shown to block cancer cell proliferation [24]. Selective IGF-1 receptor inhibitor, picropodophyllin (PPP), lacks inhibitory activity on tyrosine phosphorylation of insulin receptor tyrosine kinase (RTK) [25]. Inhibition of the IGF-1 RTK with PPP is noncompetitive with respect to ATP, suggesting interference with the IGF-1 receptor at substrate level [26]. It is reported that PPP specifically blocks phosphorylation of the Tyr1136 residue in the activation loop

**65**

**inhibitor, MK2006**

*Molecular Target Therapy against Neuroblastoma DOI: http://dx.doi.org/10.5772/intechopen.81706*

of IGF-1 receptor kinase [27]. Inhibition of IGF-1 receptor with PPP has been demonstrated in a lot of cancers such as multiple myeloma [26], melanoma [28], breast cancer [29], and glioblastoma cells [30]. PPP inhibited Akt activation and suppressed cell proliferation in group 1 and group 2 NB cell lines but less in group 3 NB cell lines [10]. Elevation of ERK phosphorylation was only observed in group 1 NB cell lines [10]. U0126 is a MEK inhibitor effectively suppressed ERK phosphorylation, U0126 (2.5 μM) did not suppress cell proliferation induced by IGF-1, IGF-2, or insulin in group 1 and group 2 NB cell lines [10]. In groups 1 and 2 NB cell lines, IGF-1 receptor/PI3K/Akt pathway is critical for cell proliferation. Although IGF-1, IGF-2, and insulin activated Akt in group 3 NB cell line, cell proliferation was not increased in RPMI1640. This suggests that the activation of IGF-1R/Akt pathway is insufficient for cell proliferation [10]. Since IGF-1 and IGF-2 regulate apoptosis [31] and cell cycle progression [32], activation of caspase 3 and PARP was examined in NB cell lines. In group 1 NB cell lines, caspase 3 and PARP were not cleaved in RPMI1640 [10]. In group 2 NB cell lines, caspase 3 and PARP were cleaved in RPMI1640, and the cleavage of caspase 3 was suppressed by addition of IGF-1, IGF-2, and insulin [10]. In group 3 NB cell lines, cleavages of caspase 3 and PARP were observed in RPMI1640 [10]. However, they were not suppressed by IGF-1, IGF-2, and insulin, even though Akt activation was upregulated by IGF-1, IGF-2, and insulin [10]. Since PPP induced G2/M arrest and apoptosis by inhibiting IGF-1 receptor [26, 33], cell cycle phase distribution in NB cells was examined in the presence of PPP. PPP treatment (2.5 μM) for 12 hours increased the G2/M fraction and shifted cell cycle profile from G0-G1 dominant to G2/M dominant in group 1 and group 2 NB cell lines [10]. However, group 3 NB cell lines did not show G2/M arrest and the G0/G1 fraction was not affected [10]. Furthermore, cyclin B1, a marker protein of G2/M phase of cell cycle, was upregulated, and cyclin D1, a marker protein of G0/G1, synchronously declined in group 1 and group 2 NB cell lines [10]. However, accumulation of cyclin B1 and decline of cyclin D1 were not observed in group 3 NB cell lines [10]. This may be explained by the insensitiveness of group 3 cell lines to PPP-induced G2/M arrest.

**4. Impairment of Akt activation and cell proliferation in NB by Akt** 

**5. Inhibition of NB cell proliferation by mTOR inhibitor**

MK2206 selectively inhibits AKT and has potency against Akt1 and Akt2 isoforms than Akt3. In pediatric solid tumors, MK2206 is effective in vitro and in vivo [34, 35]. In clinical trials, stable disease was observed in different kinds of cancers [36, 37]. Our results also suggested that MK2206 (2.5 μM) completely inhibited Akt phosphorylation and cell proliferation in NB cell lines [10]. Furthermore, MK2206 also impaired the cell proliferation induced by exogenous IGF and insulin in NB cell lines [10].

The PI3K/Akt/mTOR signaling cascade is one of the most important intracellular pathways, which is frequently activated in diverse cancers [38, 39]. The PI3K/Akt/ mTOR pathway can be activated by transmembrane tyrosine kinase growth factor receptors, such as IGF-1 receptor, fibroblast growth factor receptors, ErbB family receptors, and others [40, 41]. Both mTOR S2448 and mTOR S2481 were extensively phosphorylated in NB cell lines [13]. Cell proliferation of NB cell lines was inhibited by AZD8055, a potent dual mTORC1-mTORC2 inhibitor [13]. According to the IC50, the NB cell lines were divided into two groups, sensitive to AZD8055 group (IC50 < 0.5 μM) and insensitive to AZD8055 group (IC50 > 0.5 μM) [13]. We also found *Molecular Target Therapy against Neuroblastoma DOI: http://dx.doi.org/10.5772/intechopen.81706*

*Biophysical Chemistry - Advance Applications*

**in NB**

Therefore, targeting the PI3K/Akt/mTOR signaling pathway by appropriate inhibitors appears to be a promising strategy for overcoming therapy resistance [9]. Here, we demonstrate that NB cell lines are heterogeneous in their insulin growth factor-1 (IGF-1) receptor-mediated signaling [10]. The pattern of IGF-1 receptor/PI3K/Akt/mTOR pathway-mediated proliferation is an important determinant of the response to IGF-1 receptor antagonistic therapy in human NB [10]. Furthermore, our results highlight the potential of IGF-1 receptor/PI3K/Akt/mTOR

**2. Heterogeneity of IGF-1 receptor/Akt pathway-mediated proliferation** 

IGF-1, IGF-2, and insulin belong to a family of mitogenic growth factors and are involved in normal growth and differentiation of most tissues. The biological actions of both IGFs and insulin can be mediated by the IGF-1 receptor which is involved in mitogenic, anti-apoptotic, and oncogenic transforming responses [14, 15]. The IGF-1 receptor has two extracellular α-subunits and two intracellular β-subunits that form a heterotetrameric complex. Ligand interaction with α-subunits triggers the autophosphorylation of tyrosine kinase domains within the β-subunit [16–18]. The tyrosine kinase domains are connected to several intracellular pathways such as PI3K/Akt [19, 20]. Dysregulation of the IGF-1 receptor pathway is involved in promoting oncogenic transformation, cell proliferation, metastasis, angiogenesis, and resistance in numerous malignant diseases, such as multiple myeloma [21], carcinomas [22], and NB [23]. Since high cellular heterogeneity is a hallmark of NB, which may account for the wide range of clinical presentations and nonuniform response to treatment, we hypothesized that NB cells are heterogeneous in their IGF-1 receptor signaling-mediated cell proliferation. Thirty-one NB cell lines were cultured in three different conditions, insulincontaining serum-free medium (SFM), RPMI1640 without FBS (serum starvation medium), and RPMI1640 with 10% FBS (serum-containing medium). Based on the response patterns, 31 cell lines were subdivided into three groups [10]. Group 1, which consisted of three NB cell lines, could proliferate for more than 3 days in SFM, RPMI1640 without FBS, and RPMI1640 with 10% FBS [10]. Group 2, which consisted of 10 cell lines, could proliferate in SFM and RPMI1640 with 10% FBS but not in RPMI1640 [10]. Group 3, which consisted of 18 NB cell lines, proliferated only in RPMI1640 with 10% FBS [10]. NB cell proliferation in RPMI1640 in the presence of exogenous IGF (IGF-1, IGF-2) and insulin was examined. These IGF and insulin accelerated cell proliferation in group 1 and group 2 NB cell lines but not in group 3 NB cell lines [10]. Group 1 NB cell lines were able to proliferate in

signaling pathway as a promising target for NB treatment [10–13].

RPMI1640 in the absence of exogenous IGF and insulin [10].

**receptor inhibitor, picropodophyllin (PPP)**

**3. Impairment of Akt activation and cell proliferation in NB by IGF-1** 

The IGF-1 receptor inhibitors, such as IGF-1 receptor-neutralizing antibodies and IGF-1 receptor antisense/siRNA, have been shown to block cancer cell proliferation [24]. Selective IGF-1 receptor inhibitor, picropodophyllin (PPP), lacks inhibitory activity on tyrosine phosphorylation of insulin receptor tyrosine kinase (RTK) [25]. Inhibition of the IGF-1 RTK with PPP is noncompetitive with respect to ATP, suggesting interference with the IGF-1 receptor at substrate level [26]. It is reported that PPP specifically blocks phosphorylation of the Tyr1136 residue in the activation loop

**64**

of IGF-1 receptor kinase [27]. Inhibition of IGF-1 receptor with PPP has been demonstrated in a lot of cancers such as multiple myeloma [26], melanoma [28], breast cancer [29], and glioblastoma cells [30]. PPP inhibited Akt activation and suppressed cell proliferation in group 1 and group 2 NB cell lines but less in group 3 NB cell lines [10]. Elevation of ERK phosphorylation was only observed in group 1 NB cell lines [10]. U0126 is a MEK inhibitor effectively suppressed ERK phosphorylation, U0126 (2.5 μM) did not suppress cell proliferation induced by IGF-1, IGF-2, or insulin in group 1 and group 2 NB cell lines [10]. In groups 1 and 2 NB cell lines, IGF-1 receptor/PI3K/Akt pathway is critical for cell proliferation. Although IGF-1, IGF-2, and insulin activated Akt in group 3 NB cell line, cell proliferation was not increased in RPMI1640. This suggests that the activation of IGF-1R/Akt pathway is insufficient for cell proliferation [10]. Since IGF-1 and IGF-2 regulate apoptosis [31] and cell cycle progression [32], activation of caspase 3 and PARP was examined in NB cell lines. In group 1 NB cell lines, caspase 3 and PARP were not cleaved in RPMI1640 [10]. In group 2 NB cell lines, caspase 3 and PARP were cleaved in RPMI1640, and the cleavage of caspase 3 was suppressed by addition of IGF-1, IGF-2, and insulin [10]. In group 3 NB cell lines, cleavages of caspase 3 and PARP were observed in RPMI1640 [10]. However, they were not suppressed by IGF-1, IGF-2, and insulin, even though Akt activation was upregulated by IGF-1, IGF-2, and insulin [10]. Since PPP induced G2/M arrest and apoptosis by inhibiting IGF-1 receptor [26, 33], cell cycle phase distribution in NB cells was examined in the presence of PPP. PPP treatment (2.5 μM) for 12 hours increased the G2/M fraction and shifted cell cycle profile from G0-G1 dominant to G2/M dominant in group 1 and group 2 NB cell lines [10]. However, group 3 NB cell lines did not show G2/M arrest and the G0/G1 fraction was not affected [10]. Furthermore, cyclin B1, a marker protein of G2/M phase of cell cycle, was upregulated, and cyclin D1, a marker protein of G0/G1, synchronously declined in group 1 and group 2 NB cell lines [10]. However, accumulation of cyclin B1 and decline of cyclin D1 were not observed in group 3 NB cell lines [10]. This may be explained by the insensitiveness of group 3 cell lines to PPP-induced G2/M arrest.
