Neuroblastoma is a disease of the sympathoadrenal lineage of the neural crest, and these tumours can develop anywhere in the sympathetic nervous system. At least three key pathways regulate the formation, migration and differentiation of the neural crest cells: the bone morphogenetic protein (BMP) pathway (Barembaum & Bronner-Fraser, 2005), the fibroblast growth factor pathway (Sauka-Spengler & Bronner-Fraser, 2008) and the Wnt signalling pathway (Crane & Trainor, 2006). Wnt signalling plays an important role in neural crest cells. For example, mouse embryos in which Wnt1 and Wnt3 were knocked out displayed severe loss of neural crest cells (Varga & Wrana, 2005). In addition, conditioned medium of cells overexpressing wingless (*Drosophila* homolog of Wnt1) could induce avian neural crest cells (Garcia-Castro *et al.*, 2002). Aberrant regulation of the embryonic developmental Wnt pathway has been implicated in many solid childhood tumours (reviewed in Koesters & Doeberitz, 2003), but a clear role for Wnt signalling in

Many genes perform their functions by interaction of their encoded proteins with other proteins. The search for NBPF-interacting proteins revealed two hits so far: chibby and clusterin, which together can form a tri-molecular complex with NBPF1 (Vandepoele *et al.*, 2010). Interestingly, both NBPF1 and clusterin are candidate tumour suppressors for neuroblastoma (Vandepoele *et al.*, 2008; Chayka *et al.*, 2009). This suggests that the

Chibby (Cby) is a 15-kDa protein that has been strongly conserved throughout evolution from fly to human. It was originally identified as an inhibitor of the canonical Wnt-β-catenin pathway (Takemaru *et al.*, 2003). Cby interacts with the C-terminal activation domain of βcatenin and blocks its transcriptional activation potential by two mechanisms. First, it competes with T cell factor/lymphoid-enhancer factor (TCF/LEF) transcription factors for binding with β-catenin (Takemaru *et al.*, 2003). Second, in cooperation with 14-3-3 adaptor proteins, it facilitates nuclear export of β-catenin (Li *et al.*, 2008). Inhibition of the Wnt-βcatenin pathway is necessary for cardiomyocyte differentiation, proliferation and repair, and Cby has been implicated in facilitating cardiomyocyte differentiation (Singh *et al.*, 2007). Additionally, several molecules of the Wnt-β-catenin pathway play important roles in the inhibition of adipogenesis, and different findings indicate that Cby is required for adipocyte differentiation (Li *et al.*, 2007). Cby also plays a role in other processes. Reports on the binding of Cby to polycystin-2 (Hidaka *et al.*, 2004) and on the Cby knockout mouse (Voronina *et al.*, 2009; Love *et al.*, 2010) implicate Cby in the development of motile airway cilia. Germline inactivation of *Cby* resulted in complete absence of mucociliary transport due to scarcity of motile cilia in the nasal epithelium (Voronina *et al.*, 2009). In a follow-up study, characterisation of the lung morphology of Cby knockout mice showed that Cby-/- lungs were normal at birth, but alveolar airspace became enlarged, proliferation became reduced, and lung epithelial cells differentiated abnormally, which together affected pulmonary function (Love *et al.*, 2010). Both the lungs and nasal epithelia of Cby knockout mice showed increased expression of two direct β-catenin target genes, namely *Axin2* and *CyclinD1*, but it is not clear whether the ciliary phenotypes are related to the modest elevation of these Wnt related genes, since no change in the localisation of certain components of the Wnt-β-catenin pathway was observed. Presence of endogenous Cby at the base of cilia and the phenotypes related to the loss of Cby show that Cby is important in ciliogenesis (Voronina *et al.*, 2009;

Neuroblastoma is a disease of the sympathoadrenal lineage of the neural crest, and these tumours can develop anywhere in the sympathetic nervous system. At least three key pathways regulate the formation, migration and differentiation of the neural crest cells: the bone morphogenetic protein (BMP) pathway (Barembaum & Bronner-Fraser, 2005), the fibroblast growth factor pathway (Sauka-Spengler & Bronner-Fraser, 2008) and the Wnt signalling pathway (Crane & Trainor, 2006). Wnt signalling plays an important role in neural crest cells. For example, mouse embryos in which Wnt1 and Wnt3 were knocked out displayed severe loss of neural crest cells (Varga & Wrana, 2005). In addition, conditioned medium of cells overexpressing wingless (*Drosophila* homolog of Wnt1) could induce avian neural crest cells (Garcia-Castro *et al.*, 2002). Aberrant regulation of the embryonic developmental Wnt pathway has been implicated in many solid childhood tumours (reviewed in Koesters & Doeberitz, 2003), but a clear role for Wnt signalling in

interactions between these three proteins are functionally important.

**5. NBPF-interacting proteins**

**5.1 Chibby**

Love *et al.*, 2010).

neuroblastoma has remained elusive. Nevertheless, there is mounting evidence that the Wnt pathway is involved in neuroblastoma (Table 1).

The MSX1 homeobox transcription factor functions as an intermediate between the BMP and Wnt signalling pathways to ensure proper differentiation of the neural crest cells. Elevated expression of BMP in non-neuronal ectoderm switches on the neural crest differentiation program and induces the expression of MSX1, which leads to induction of the expression of BMP and Wnt in neural crest cells (Ramos & Robert, 2005). Msx genes might regulate *Wnt1* expression at the dorsal midline of the neural tube (Bach *et al.*, 2003). Additionally, MSX1 overexpression in the *MYCN*-amplified neuroblastoma cell line SJNB8 induced the expression of four Wnt inhibitor genes: Dickkopf 1 (DKK1), Dickkopf 2 (DKK2) and Dickkopf 3 (DKK3) and secreted frizzled-related protein 1 (SFRP-1) (Revet *et al.*, 2010). Analysis of the expression profiles of MSX1, DKK1, -2, -3 and SFRP-1 in a series of neuroblastic tumours showed a significant and positive correlation between MSX1 and DKK2 expression and between MSX1 and DKK3 expression. Additionally, stronger DKK2 and DKK3 expression correlated with a significantly better prognosis. Expression profiling of neuroblastic tumours and cell lines for the different Wnt family genes showed that both the canonical Wnt3a and the non-canonical Wnt5 were strongly expressed and that they could activate upstream Wnt signalling in neuroblastoma cells by phosphorylating the dishevelled co-receptor DVL3 (Revet *et al.*, 2010). The importance of DKK3 in neuroblastoma was already shown in previous reports, in which DKK3 was implicated as a marker for neuroblastic tumour maturation and was shown to be down-regulated by *MYCN* (Bell *et al.*, 2007; Koppen *et al.*, 2008).

Additionally, β-catenin has been shown to be strongly expressed and aberrantly localised in the nucleus in high-risk neuroblastoma cells without *MYCN* amplification (Liu *et al.*, 2008). Expression profiling of primary neuroblastoma tumours demonstrated increased expression of *WNT* ligands (*WNT1*, *WNT6*, *WNT7A*, *WNT10B*), *DVL1* and *TCF7* in high-risk neuroblastoma tumours without *MYCN* amplification. Also several β-catenin target genes,

Table 1. Wnt signalling is important in neuroblastoma tumourigenesis and chemoresistance.Schematic representation of different Wnt signalling components implicated in neuroblastoma. NB: neuroblastoma; MNA: *MYCN* amplification. See text for references.

The *NBPF* Gene Family 201

Clusterin knockout mice are more prone to autoimmune myocarditis (McLaughlin *et al.*, 2000) and to transformation of the prostate epithelium (Bettuzzi *et al.*, 2009). In homozygous knockout mice, the normal portion of prostate tissue is characterised by stronger expression of the proliferation marker Ki67 and shows activated NF-κB signalling, but this was not observed in wild type mice (Bettuzzi *et al.*, 2009). Additionally, in TRAMP mice, a transgenic animal model of prostate cancer (Greenberg *et al.*, 1995), the *CLU* gene is dramatically downregulated during onset and progression of prostate cancer (Caporali *et al.*, 2004). Crossing of these TRAMP mice with the clusterin knockout mice led to development of a more advanced invasive disease in the progeny, confirming the role of *CLU* as a negative

On the contrary, overexpression of clusterin in LNCaP prostate cancer cells conferred resistance to both androgen ablation *in vivo* and cytotoxic chemotherapy (Miyake *et al.*, 2000c). Additionally, the administration of clusterin antisense oligonucleotides in prostate cancer xenograft models delayed progression to androgen independence and enhanced chemosensitivity (Miyake *et al.*, 2000b). A human clusterin antisense oligonucleotide (OGX-011) synergistically enhanced the cytotoxic effects of paclitaxel in human xenografts of prostate cancer (Miyake *et al.*, 2000a). This antisense oligonucleotide is currently undergoing clinical trials for tumour therapy. The phase I trial demonstrated that OGX-011 is well tolerated and that it inhibits clusterin expression in prostate cancer (Chi *et al.*, 2005). The phase II trial assessed the effects of combined therapy with androgen ablation and OGX-011 given prior to radical prostatectomy in patients with metastatic castration-resistant prostate cancer. This study showed that the combined therapy was associated with improved

The biological role of clusterin is controversial in both prostate cancer and neuroblastoma. Indeed, also in neuroblastoma it has been shown that clusterin can function both as a tumour suppressor gene and as an oncogene. Overexpression of clusterin impaired the invasion of neuroblastoma cell lines by inhibiting the NF-κB activity by stabilising IκBs, the inhibitors of NF-κB signalling (Santilli *et al.*, 2003). Additionally, Oncomine expression analysis (Rhodes *et al.*, 2007) showed a lower expression level of the *CLU* gene in primary human neuroblastomas with *MYCN* amplification, and further investigation demonstrated that MYCN acts as a negative regulator of clusterin expression by inducing transcription of the 17-5p~92 microRNA cluster (Chayka *et al.*, 2009). This polycistronic cluster encodes six microRNAs (miR-17-5p, -18a, -19a, -19b, -20a and -92) and is homologous to the miRNA 106a~92 cluster (Mestdagh *et al.*, 2010b). Most miRNAs encoded by this cluster are expressed at higher levels in neuroblastoma cell lines overexpressing *MYCN* or carrying *MYCN* amplification. MYCN transcriptionally induces the expression of the miRNA 17- 5p~92 cluster by directly binding to its promoter. The therapeutic potential of this miRNA cluster was demonstrated by injecting a miR-17-5p-specific antagomir into nude mice that had been injected with a neuroblastoma cell line with *MYCN* amplification. Tumour growth *in vivo* was reduced substantially, and this reduction was associated with down-modulation of miR-17-5p activity and with a reciprocal increase of both p21 and BIM protein levels. Nevertheless, the expression pattern of miR-17-5p in primary tumours is complex, because high levels of miR-17-5p expression were detected in both *MYCN*-amplified and non *MYCN*-amplified tumours (Fontana *et al.*, 2008). Additionally, it has been demonstrated that miR-17-5p~92 dampens TGF-β signalling by acting both upstream and downstream of pSMAD2/SMAD4 (Mestdagh *et al.*, 2010a). This finding further underscores its ability to

tumour modulator in prostate cancer (Bettuzzi *et al.*, 2009).

regulate multiple components of the same pathway.

survival (Chi *et al.*, 2010).

*e.g. MYC* and *CD44*, were found to be coordinately upregulated in high-risk neuroblastomas without *MYCN* amplification in comparison to high-risk *MYCN*-amplified or intermediaterisk neuroblastomas (Liu *et al.*, 2008).

Another study implicated the Wnt-β-catenin pathway in neuroblastoma chemoresistance (Flahaut *et al.*, 2009). Gene expression profiling of two doxorubicin-resistant neuroblastoma cell lines identified the frizzled1 Wnt receptor (*FZD1*) gene as the most strongly overexpressed transcript in both cell lines in comparison with their sensitive parental cell lines. FZD1 silencing resulted in significant restoration of drug sensitivity and induced a parallel strong decrease in the expression of MDR1 (Flahaut *et al.*, 2009), another β-catenin target gene frequently associated with the resistance of neuroblastoma tumours to chemotherapeutic drugs (Haber *et al.*, 1997). Moreover, FZD1 and/or MDR1 expression was significantly enhanced in the group of relapsed patients after chemotherapy, whereas no significant increase in expression was measured in the group of non-relapsed patients (Flahaut *et al.*, 2009).

These data indicate that Wnt signalling is important in neuroblastoma tumourigenesis and chemoresistance (overview in Table 1). Therefore, we hypothesise that important players in the Wnt-β-catenin pathway might play important roles in neuroblastoma. But nothing is known yet about the role of Cby in neuroblastoma tumourigenesis or chemoresistance, and further investigation is needed.

#### **5.2 Clusterin**

Clusterin (CLU), also known as apolipoprotein J, is a heterodimeric sulfated glycoprotein that is expressed in most human tissues and body fluids and is associated with many biological activities, including regulation of apoptosis and cancer (Trougakos & Gonos, 2002). Research on *CLU* function and its relation to tumourigenesis has been intensive, but many contradictory data have been reported. These contradictions can be explained mainly by the existence of three protein isoforms with different sub-cellular localisations and biological functions (Rizzi & Bettuzzi, 2010).

The most extensively studied form of CLU is the secreted form (sCLU), which is a glycosylated heterodimer present in almost all physiological fluids. sCLU is exported from the cell and released in secretions, in which it acts as an extracellular chaperone (Humphreys *et al.*, 1999). Cytoplasmic clusterin has been shown to inhibit apoptosis by interfering with Bax activation in the mitochondria (Zhang *et al.*, 2005). Clusterin binds and stabilises the Ku70-Bax protein complex and serves as a cytosolic retention factor for Bax. Depletion of clusterin disrupts the Ku70-Bax complex and triggers Bax activation and its relocalisation to the mitochondria (Trougakos *et al.*, 2009). Cytoplasmic clusterin also promotes tumour cell survival by cooperating with c-Myc during transformation (Zhang *et al.*, 2005). Additionally, a large amount of CLU confers resistance to cytotoxic agents on different cancer cell types (Lourda *et al.*, 2006). However, a nuclear form of clusterin originating from an alternative translation start site has been shown to be proapoptotic in prostate cancer cells (Moretti *et al.*, 2007), breast cancer cells (Leskov *et al.*, 2003) and neuroblastoma (Yang *et al.*, 2000).

The main question is whether clusterin is a positive or negative modulator of mammalian tumourigenesis. It is becoming clear that *CLU* can act either as a tumour suppressor (Caporali *et al.*, 2004; Chayka *et al.*, 2009) or as a tumour promoter (Lourda *et al.*, 2006), but the time-course of the disease and the selection pressures imposed on the cancer by chemotherapy or other treatments have to be taken into account.

*e.g. MYC* and *CD44*, were found to be coordinately upregulated in high-risk neuroblastomas without *MYCN* amplification in comparison to high-risk *MYCN*-amplified or intermediate-

Another study implicated the Wnt-β-catenin pathway in neuroblastoma chemoresistance (Flahaut *et al.*, 2009). Gene expression profiling of two doxorubicin-resistant neuroblastoma cell lines identified the frizzled1 Wnt receptor (*FZD1*) gene as the most strongly overexpressed transcript in both cell lines in comparison with their sensitive parental cell lines. FZD1 silencing resulted in significant restoration of drug sensitivity and induced a parallel strong decrease in the expression of MDR1 (Flahaut *et al.*, 2009), another β-catenin target gene frequently associated with the resistance of neuroblastoma tumours to chemotherapeutic drugs (Haber *et al.*, 1997). Moreover, FZD1 and/or MDR1 expression was significantly enhanced in the group of relapsed patients after chemotherapy, whereas no significant increase in expression was measured in the group of non-relapsed patients

These data indicate that Wnt signalling is important in neuroblastoma tumourigenesis and chemoresistance (overview in Table 1). Therefore, we hypothesise that important players in the Wnt-β-catenin pathway might play important roles in neuroblastoma. But nothing is known yet about the role of Cby in neuroblastoma tumourigenesis or chemoresistance, and

Clusterin (CLU), also known as apolipoprotein J, is a heterodimeric sulfated glycoprotein that is expressed in most human tissues and body fluids and is associated with many biological activities, including regulation of apoptosis and cancer (Trougakos & Gonos, 2002). Research on *CLU* function and its relation to tumourigenesis has been intensive, but many contradictory data have been reported. These contradictions can be explained mainly by the existence of three protein isoforms with different sub-cellular localisations and

The most extensively studied form of CLU is the secreted form (sCLU), which is a glycosylated heterodimer present in almost all physiological fluids. sCLU is exported from the cell and released in secretions, in which it acts as an extracellular chaperone (Humphreys *et al.*, 1999). Cytoplasmic clusterin has been shown to inhibit apoptosis by interfering with Bax activation in the mitochondria (Zhang *et al.*, 2005). Clusterin binds and stabilises the Ku70-Bax protein complex and serves as a cytosolic retention factor for Bax. Depletion of clusterin disrupts the Ku70-Bax complex and triggers Bax activation and its relocalisation to the mitochondria (Trougakos *et al.*, 2009). Cytoplasmic clusterin also promotes tumour cell survival by cooperating with c-Myc during transformation (Zhang *et al.*, 2005). Additionally, a large amount of CLU confers resistance to cytotoxic agents on different cancer cell types (Lourda *et al.*, 2006). However, a nuclear form of clusterin originating from an alternative translation start site has been shown to be proapoptotic in prostate cancer cells (Moretti *et al.*, 2007), breast cancer cells (Leskov *et al.*, 2003) and

The main question is whether clusterin is a positive or negative modulator of mammalian tumourigenesis. It is becoming clear that *CLU* can act either as a tumour suppressor (Caporali *et al.*, 2004; Chayka *et al.*, 2009) or as a tumour promoter (Lourda *et al.*, 2006), but the time-course of the disease and the selection pressures imposed on the cancer by

chemotherapy or other treatments have to be taken into account.

risk neuroblastomas (Liu *et al.*, 2008).

(Flahaut *et al.*, 2009).

**5.2 Clusterin**

further investigation is needed.

biological functions (Rizzi & Bettuzzi, 2010).

neuroblastoma (Yang *et al.*, 2000).

Clusterin knockout mice are more prone to autoimmune myocarditis (McLaughlin *et al.*, 2000) and to transformation of the prostate epithelium (Bettuzzi *et al.*, 2009). In homozygous knockout mice, the normal portion of prostate tissue is characterised by stronger expression of the proliferation marker Ki67 and shows activated NF-κB signalling, but this was not observed in wild type mice (Bettuzzi *et al.*, 2009). Additionally, in TRAMP mice, a transgenic animal model of prostate cancer (Greenberg *et al.*, 1995), the *CLU* gene is dramatically downregulated during onset and progression of prostate cancer (Caporali *et al.*, 2004). Crossing of these TRAMP mice with the clusterin knockout mice led to development of a more advanced invasive disease in the progeny, confirming the role of *CLU* as a negative tumour modulator in prostate cancer (Bettuzzi *et al.*, 2009).

On the contrary, overexpression of clusterin in LNCaP prostate cancer cells conferred resistance to both androgen ablation *in vivo* and cytotoxic chemotherapy (Miyake *et al.*, 2000c). Additionally, the administration of clusterin antisense oligonucleotides in prostate cancer xenograft models delayed progression to androgen independence and enhanced chemosensitivity (Miyake *et al.*, 2000b). A human clusterin antisense oligonucleotide (OGX-011) synergistically enhanced the cytotoxic effects of paclitaxel in human xenografts of prostate cancer (Miyake *et al.*, 2000a). This antisense oligonucleotide is currently undergoing clinical trials for tumour therapy. The phase I trial demonstrated that OGX-011 is well tolerated and that it inhibits clusterin expression in prostate cancer (Chi *et al.*, 2005). The phase II trial assessed the effects of combined therapy with androgen ablation and OGX-011 given prior to radical prostatectomy in patients with metastatic castration-resistant prostate cancer. This study showed that the combined therapy was associated with improved survival (Chi *et al.*, 2010).

The biological role of clusterin is controversial in both prostate cancer and neuroblastoma. Indeed, also in neuroblastoma it has been shown that clusterin can function both as a tumour suppressor gene and as an oncogene. Overexpression of clusterin impaired the invasion of neuroblastoma cell lines by inhibiting the NF-κB activity by stabilising IκBs, the inhibitors of NF-κB signalling (Santilli *et al.*, 2003). Additionally, Oncomine expression analysis (Rhodes *et al.*, 2007) showed a lower expression level of the *CLU* gene in primary human neuroblastomas with *MYCN* amplification, and further investigation demonstrated that MYCN acts as a negative regulator of clusterin expression by inducing transcription of the 17-5p~92 microRNA cluster (Chayka *et al.*, 2009). This polycistronic cluster encodes six microRNAs (miR-17-5p, -18a, -19a, -19b, -20a and -92) and is homologous to the miRNA 106a~92 cluster (Mestdagh *et al.*, 2010b). Most miRNAs encoded by this cluster are expressed at higher levels in neuroblastoma cell lines overexpressing *MYCN* or carrying *MYCN* amplification. MYCN transcriptionally induces the expression of the miRNA 17- 5p~92 cluster by directly binding to its promoter. The therapeutic potential of this miRNA cluster was demonstrated by injecting a miR-17-5p-specific antagomir into nude mice that had been injected with a neuroblastoma cell line with *MYCN* amplification. Tumour growth *in vivo* was reduced substantially, and this reduction was associated with down-modulation of miR-17-5p activity and with a reciprocal increase of both p21 and BIM protein levels. Nevertheless, the expression pattern of miR-17-5p in primary tumours is complex, because high levels of miR-17-5p expression were detected in both *MYCN*-amplified and non *MYCN*-amplified tumours (Fontana *et al.*, 2008). Additionally, it has been demonstrated that miR-17-5p~92 dampens TGF-β signalling by acting both upstream and downstream of pSMAD2/SMAD4 (Mestdagh *et al.*, 2010a). This finding further underscores its ability to regulate multiple components of the same pathway.

The *NBPF* Gene Family 203

Based on these findings, which were observed in both prostate cancer and neuroblastoma, a role for clusterin in tumourigenesis has been proposed (Figure 6). This model proposes that clusterin acts as a tumour suppressor in the early stages of cancer but can become a tumour promoter in the more malignant and advanced stages, namely when tumours become resistant to therapy (Rizzi & Bettuzzi, 2010). Under physiological conditions, the basal expression level of *CLU* in benign cells is low and confined to the secreted form of CLU (sCLU). Several apoptotic stimuli lead to the production of nuclear CLU (nCLU), which in turn induces extensive cell death. At the onset of cancer, early genetic lesions disrupt or inactivate apoptotic pathways or lead to the activation of other survival pathways. Under these conditions of clonal selection, a key step in the transformation process would be the prevention of CLU from entering the nucleus in order to prevent induction of apoptosis. At this point, cancer cells are characterised by strong expression of cytoplasmic CLU (cCLU) and are therefore resistant to death by cytotoxic therapeutic agents. This resistant phenotype can promote cell transformation that leads to late cancer stages associated with impaired

A yeast two-hybrid screening with an N-terminal fragment of the NBPF11 protein identified Cby as a binding partner of NBPF11 (Vandepoele *et al.*, 2010). This interaction is not exclusive to NBPF11 but is common to other NBPF members. For instance, Cby can also interact with NBPF1. The interaction was confirmed by other techniques, such as coimmunoprecipitation and mitochondrial recruitment (Vandepoele *et al.*, 2010). It was shown that the coiled-coil region in the N-terminal domain of NBPF interacts with the coiled-coil region in the C-terminal domain of Cby. This C-terminal portion of Cby harbours not only the binding site for NBPF, but also the binding site for most other known interaction partners of Cby, such as β-catenin (Takemaru *et al.*, 2003), polycystin-2 (Hidaka *et al.*, 2004)

Functional analysis of the interaction showed that NBPF did not influence Cby-mediated Wnt signalling, nor did it compete with β-catenin binding to Cby. This indicates the presence of two different pools of Cby, one binding to β-catenin and the other binding to NBPF, or alternatively, a dimeric Cby complex that can bind simultaneously to β-catenin and NBPF. Besides its repressor function in the Wnt pathway, Cby can also play an important role in ciliogenesis, as shown in the Cby knockout mouse (see section 5.1). The role of NBPF in the formation of cilia remains uncertain, as NBPF1 overexpression did not interfere with the formation of primary cilia (Vandepoele *et al.*, 2010). An additional yeast two-hybrid screen identified clusterin as a new binding partner for Cby and showed that the C-terminus of CLU, which contains a coiled-coil domain, interacts with both the N-terminal and C-terminal domains of Cby. Additionally, co-immunoprecipitation experiments in HEK293T cells demonstrated that NBPF1, Cby and clusterin can form a tri-molecular

Since intracellular CLU can be found both in the nucleus and in the cytoplasm (see section 5.2), the effect of Cby or NBPF or their combination on the subcellular localisation of CLU

The identification of two proteins that interact with NBPF is a clue to the functions of NBPF, but further investigation is needed. Anyway, the identification within the complex of two proteins (NBPF1 and CLU) as candidate tumour suppressor genes linked to neuroblastoma (Vandepoele *et al.*, 2008; Chayka *et al.*, 2009) is intriguing and warrants

complex (Figure 7), because all three proteins were co-purified in every setting.

was further investigated, but no difference was observed (Vandepoele *et al.*, 2010).

mitosis, which causes further genomic instability (Rizzi & Bettuzzi, 2010).

**5.3 NBPF1, Chibby and clusterin can form a tri-molecular complex**

and TC1 (Jung *et al.*, 2006).

further scrutiny.

When *MYCN*-transgenic mice were crossed with clusterin knockout mice, the progeny showed reduced tumour-free survival, active NF-κB signalling, and epithelial to mesenchymal transition (Chayka *et al.*, 2009); this resembles the phenotype of TRAMP mice in which CLU was homozygously deleted (Bettuzzi *et al.*, 2009). These data indicate that clusterin functions as a TSG for neuroblastoma, even though several reports indicate that it can promote neuroblastoma. Treatment with histone deacetylase inhibitors (HDACIs) resulted in upregulation of clusterin in different cancer cell lines, including neuroblastoma (Liu *et al.*, 2009; Subramanian *et al.*, 2011). Suppression of clusterin in combination with high-dose HDACIs synergistically enhanced HDACI-induced cell death through cytochome-c-mediated apoptosis in HDACI-resistant cancer cells. Additionally, combining OGX-011 with low-dose HDACIs enhanced HDACI-induced growth arrest in both HDACIsensitive and -resistant cancer cell lines. Moreover, in mice xenografted with neuroblastoma cells, combining OGX-011 and valproate (the only HDACI proven to be safe in young children) synergistically inhibited tumour growth *in vivo*, whereas neither OGX-011 nor valproate alone had an effect on tumour progression (Liu *et al.*, 2009).

Fig. 6. Proposed model for CLU in tumourigenesis.

In normal cells and under physiological conditions the basal expression level of *CLU* in benign cells is low and confined to the secreted form of CLU (sCLU) (1). Several apoptotic stimuli (2) lead to the production of nuclear CLU (nCLU) (3), which in turn induces extensive cell death (4). In the early phases of tumourigenesis, genetic lesions lead to acquisition of survival advantages and consequent clonal selection (5). A key step in the transformation process is prevention of CLU from entering the nucleus. Cells with high levels of cytoplasmic CLU are then selected for, which results in acquisition of a deathresistant phenotype (6). This phenotype is associated with impaired mitosis (7), which leads eventually to genomic instability (8) (Modified after Rizzi & Bettuzzi, 2010, Society for Endocrinology (2010), reproduced by permission).

When *MYCN*-transgenic mice were crossed with clusterin knockout mice, the progeny showed reduced tumour-free survival, active NF-κB signalling, and epithelial to mesenchymal transition (Chayka *et al.*, 2009); this resembles the phenotype of TRAMP mice in which CLU was homozygously deleted (Bettuzzi *et al.*, 2009). These data indicate that clusterin functions as a TSG for neuroblastoma, even though several reports indicate that it can promote neuroblastoma. Treatment with histone deacetylase inhibitors (HDACIs) resulted in upregulation of clusterin in different cancer cell lines, including neuroblastoma (Liu *et al.*, 2009; Subramanian *et al.*, 2011). Suppression of clusterin in combination with high-dose HDACIs synergistically enhanced HDACI-induced cell death through cytochome-c-mediated apoptosis in HDACI-resistant cancer cells. Additionally, combining OGX-011 with low-dose HDACIs enhanced HDACI-induced growth arrest in both HDACIsensitive and -resistant cancer cell lines. Moreover, in mice xenografted with neuroblastoma cells, combining OGX-011 and valproate (the only HDACI proven to be safe in young children) synergistically inhibited tumour growth *in vivo*, whereas neither OGX-011 nor

valproate alone had an effect on tumour progression (Liu *et al.*, 2009).

Fig. 6. Proposed model for CLU in tumourigenesis.

Endocrinology (2010), reproduced by permission).

In normal cells and under physiological conditions the basal expression level of *CLU* in benign cells is low and confined to the secreted form of CLU (sCLU) (1). Several apoptotic stimuli (2) lead to the production of nuclear CLU (nCLU) (3), which in turn induces extensive cell death (4). In the early phases of tumourigenesis, genetic lesions lead to acquisition of survival advantages and consequent clonal selection (5). A key step in the transformation process is prevention of CLU from entering the nucleus. Cells with high levels of cytoplasmic CLU are then selected for, which results in acquisition of a deathresistant phenotype (6). This phenotype is associated with impaired mitosis (7), which leads eventually to genomic instability (8) (Modified after Rizzi & Bettuzzi, 2010, Society for

Based on these findings, which were observed in both prostate cancer and neuroblastoma, a role for clusterin in tumourigenesis has been proposed (Figure 6). This model proposes that clusterin acts as a tumour suppressor in the early stages of cancer but can become a tumour promoter in the more malignant and advanced stages, namely when tumours become resistant to therapy (Rizzi & Bettuzzi, 2010). Under physiological conditions, the basal expression level of *CLU* in benign cells is low and confined to the secreted form of CLU (sCLU). Several apoptotic stimuli lead to the production of nuclear CLU (nCLU), which in turn induces extensive cell death. At the onset of cancer, early genetic lesions disrupt or inactivate apoptotic pathways or lead to the activation of other survival pathways. Under these conditions of clonal selection, a key step in the transformation process would be the prevention of CLU from entering the nucleus in order to prevent induction of apoptosis. At this point, cancer cells are characterised by strong expression of cytoplasmic CLU (cCLU) and are therefore resistant to death by cytotoxic therapeutic agents. This resistant phenotype can promote cell transformation that leads to late cancer stages associated with impaired mitosis, which causes further genomic instability (Rizzi & Bettuzzi, 2010).

#### **5.3 NBPF1, Chibby and clusterin can form a tri-molecular complex**

A yeast two-hybrid screening with an N-terminal fragment of the NBPF11 protein identified Cby as a binding partner of NBPF11 (Vandepoele *et al.*, 2010). This interaction is not exclusive to NBPF11 but is common to other NBPF members. For instance, Cby can also interact with NBPF1. The interaction was confirmed by other techniques, such as coimmunoprecipitation and mitochondrial recruitment (Vandepoele *et al.*, 2010). It was shown that the coiled-coil region in the N-terminal domain of NBPF interacts with the coiled-coil region in the C-terminal domain of Cby. This C-terminal portion of Cby harbours not only the binding site for NBPF, but also the binding site for most other known interaction partners of Cby, such as β-catenin (Takemaru *et al.*, 2003), polycystin-2 (Hidaka *et al.*, 2004) and TC1 (Jung *et al.*, 2006).

Functional analysis of the interaction showed that NBPF did not influence Cby-mediated Wnt signalling, nor did it compete with β-catenin binding to Cby. This indicates the presence of two different pools of Cby, one binding to β-catenin and the other binding to NBPF, or alternatively, a dimeric Cby complex that can bind simultaneously to β-catenin and NBPF. Besides its repressor function in the Wnt pathway, Cby can also play an important role in ciliogenesis, as shown in the Cby knockout mouse (see section 5.1). The role of NBPF in the formation of cilia remains uncertain, as NBPF1 overexpression did not interfere with the formation of primary cilia (Vandepoele *et al.*, 2010). An additional yeast two-hybrid screen identified clusterin as a new binding partner for Cby and showed that the C-terminus of CLU, which contains a coiled-coil domain, interacts with both the N-terminal and C-terminal domains of Cby. Additionally, co-immunoprecipitation experiments in HEK293T cells demonstrated that NBPF1, Cby and clusterin can form a tri-molecular complex (Figure 7), because all three proteins were co-purified in every setting.

Since intracellular CLU can be found both in the nucleus and in the cytoplasm (see section 5.2), the effect of Cby or NBPF or their combination on the subcellular localisation of CLU was further investigated, but no difference was observed (Vandepoele *et al.*, 2010).

The identification of two proteins that interact with NBPF is a clue to the functions of NBPF, but further investigation is needed. Anyway, the identification within the complex of two proteins (NBPF1 and CLU) as candidate tumour suppressor genes linked to neuroblastoma (Vandepoele *et al.*, 2008; Chayka *et al.*, 2009) is intriguing and warrants further scrutiny.

The *NBPF* Gene Family 205

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Barembaum, M. & Bronner-Fraser, M. (2005). Early steps in neural crest specification.

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Fig. 7. NBPF1, Cby and CLU form a tri-molecular complex.

An overview of the mutual interactions between NBPF1, Cby and CLU. The coiled-coil region in the N-terminus of NBPF1 interacts with the coiled-coil region in the cytoplasmic domain of Cby. The C-terminus of CLU interacts with the full-length Cby protein, as interactions with both the N-terminus and C-terminus are observed. It is uncertain whether CLU and NBPF1 interact directly (boxed question mark) (Modified after Vandepoele *et al.*, 2010, Elsevier (2010), reproduced by permission).

## **6. Conclusion**

In this chapter we summarise the growing evidence for the recent emergence of the *NBPF* gene family and for its role in human evolution. The NBPF/DUF1220 domains in particular seem to have been important in this context. This evidence is based on the large increase in copy number of the NBPF/DUF1220 domain in humans. On the other hand, *NBPF* genes seem to be involved in cancer and in brain and developmental disorders due to their location in unstable high-identity duplication blocks, which leads to recurrent chromosomal rearrangements. One case of particular interest is neuroblastoma. Evidence for the involvement of *NBPF* genes in this type of cancer comes not only from disruption of *NBPF1* in a neuroblastoma patient, but also from gene expression studies and the association with neuroblastoma of a copy number variation of an *NBPF* paralog. Additionally, the regulatory elements and the identification of NBPF-interacting proteins indicate that the *NBPF* gene family plays a role in neuroblastoma. Involvement of *NBPF* genes in neuroblastoma and possibly as well in other cancers points to the value of studying this gene family further. Every step in the functional understanding of these genes could bring us closer to unravelling the molecular mechanisms underlying neuroblastoma development and could help us to develop more effective therapy. Such studies could also shed light on the impact these novel genes had on recent primate evolution.

## **7. Acknowledgments**

We thank Dr. Amin Bredan for critically editing the manuscript. Research supported by the Fund for Scientific Research, Flanders (FWO), the Foundation against Cancer, Belgium, and the Queen Elisabeth Medical Foundation (G.S.K.E.), Belgium.

## **8. References**

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An overview of the mutual interactions between NBPF1, Cby and CLU. The coiled-coil region in the N-terminus of NBPF1 interacts with the coiled-coil region in the cytoplasmic domain of Cby. The C-terminus of CLU interacts with the full-length Cby protein, as interactions with both the N-terminus and C-terminus are observed. It is uncertain whether CLU and NBPF1 interact directly (boxed question mark) (Modified after Vandepoele *et al.*,

In this chapter we summarise the growing evidence for the recent emergence of the *NBPF* gene family and for its role in human evolution. The NBPF/DUF1220 domains in particular seem to have been important in this context. This evidence is based on the large increase in copy number of the NBPF/DUF1220 domain in humans. On the other hand, *NBPF* genes seem to be involved in cancer and in brain and developmental disorders due to their location in unstable high-identity duplication blocks, which leads to recurrent chromosomal rearrangements. One case of particular interest is neuroblastoma. Evidence for the involvement of *NBPF* genes in this type of cancer comes not only from disruption of *NBPF1* in a neuroblastoma patient, but also from gene expression studies and the association with neuroblastoma of a copy number variation of an *NBPF* paralog. Additionally, the regulatory elements and the identification of NBPF-interacting proteins indicate that the *NBPF* gene family plays a role in neuroblastoma. Involvement of *NBPF* genes in neuroblastoma and possibly as well in other cancers points to the value of studying this gene family further. Every step in the functional understanding of these genes could bring us closer to unravelling the molecular mechanisms underlying neuroblastoma development and could help us to develop more effective therapy. Such studies could also shed light on the impact

Research supported by the Fund for Scientific Research, Flanders (FWO), the Foundation against Cancer, Belgium, and the Queen Elisabeth Medical Foundation (G.S.K.E.), Belgium.

Aitman, T.J., Dong, R., Vyse, T.J., Norsworthy, P.J., Johnson, M.D., Smith, J., Mangion, J.,

Roberton-Lowe, C., Marshall, A.J., Petretto, E., Hodges, M.D., Bhangal, G., Patel, S.G., Sheehan-Rooney, K., Duda, M., Cook, P.R., Evans, D.J., Domin, J., Flint, J.,

Fig. 7. NBPF1, Cby and CLU form a tri-molecular complex.

2010, Elsevier (2010), reproduced by permission).

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**Part 3**

**Neuroblastoma,**

 **Therapeutic Targets - Present and Future**

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## **Part 3**

**Neuroblastoma, Therapeutic Targets - Present and Future**

214 Neuroblastoma – Present and Future

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**10**

*Spain*

**Retinoic-Acid-Induced Downregulation**

Juan M. Escamilla1, Christine Bäuerl1, Carlos M. R. López1, Satu P. Pekkala1, Samuel Navarro2 and Domingo Barettino1

*2Dept. of Pathology, School of Medicine, University of Valencia, Valencia*

Neuroblastoma is a common tumor of the childhood arising from embryonal sympathetic neural cell precursors. Despite of the improved therapeutic strategies, the survival rate of high-risk neuroblastoma patients is poor. Although complete clinical remissions can be achieved, relapse is relatively frequent, indicating a role for the persistence of the minimal residual disease (for review, Maris, 2010). Treatments with derivatives of retinoic acid (RA), the biologically active form of vitamin A, produce significant improvements on the therapy of high-risk neuroblastoma patients, when used together with intensive multimodal therapies (Reynolds et al., 2003, for review). Despite some controversy on dosage and treatment schedules (Matthay & Reynolds, 2000; Reynolds et al., 2003), treatment with 13 *cis*-RA (isotretinoin) has been reported to produce a modest but significant increase in the event-free survival of high-risk neuroblastoma patients (Matthay et al., 2009; Matthay et al., 1999). RA and its derivatives, the retinoids, are differentiating agents that induce growth arrest, differentiation and/or apoptosis of neuroblastoma cells *in vitro* (Lovat et al., 1997; Pahlman et al., 1984; Sidell, 1982; Thiele et al., 1985). However the molecular bases of RA

With the aim of identifying possible molecular targets for RA treatment, we have analyzed RA-induced gene expression changes in SH-SY5Y cultured human neuroblastoma cells (Lopez-Carballo et al., 2002), by means of Ordered Differential Display RT-PCR (Matz et al., 1997). Among more than 60 genes identified, we could found *LAMR1* (also called *RPSA*), encoding the precursor for the 67-KDa Laminin Receptor (67LR) (Wewer et al., 1986; Yow et al., 1988), whose mRNA levels were remarkably reduced after RA treatment. The 67LR is a multifunctional protein which is involved in cell adhesion, required for maintaining of cell viability (Scheiman et al., 2010; Susantad & Smith, 2008), acts as receptor for infectious agents like viruses and prions (Vana et al., 2009) and mediates the actions of the green tea polyphenol epigallocatechin-3-gallate (Umeda et al., 2008). Expression of 67LR has been

therapeutic actions in neuroblastoma have not been established yet.

**1. Introduction**

**of Human Neuroblastoma Cells**

*Consejo Superior de Investigaciones Científicas,Valencia,*

*1Instituto de Biomedicina de Valencia,*

**of the 67 KDa Laminin Receptor Correlates**

**with Reduced Biological Aggressiveness**

## **Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells**

Juan M. Escamilla1, Christine Bäuerl1, Carlos M. R. López1, Satu P. Pekkala1, Samuel Navarro2 and Domingo Barettino1 *1Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas,Valencia, 2Dept. of Pathology, School of Medicine, University of Valencia, Valencia Spain*

### **1. Introduction**

Neuroblastoma is a common tumor of the childhood arising from embryonal sympathetic neural cell precursors. Despite of the improved therapeutic strategies, the survival rate of high-risk neuroblastoma patients is poor. Although complete clinical remissions can be achieved, relapse is relatively frequent, indicating a role for the persistence of the minimal residual disease (for review, Maris, 2010). Treatments with derivatives of retinoic acid (RA), the biologically active form of vitamin A, produce significant improvements on the therapy of high-risk neuroblastoma patients, when used together with intensive multimodal therapies (Reynolds et al., 2003, for review). Despite some controversy on dosage and treatment schedules (Matthay & Reynolds, 2000; Reynolds et al., 2003), treatment with 13 *cis*-RA (isotretinoin) has been reported to produce a modest but significant increase in the event-free survival of high-risk neuroblastoma patients (Matthay et al., 2009; Matthay et al., 1999). RA and its derivatives, the retinoids, are differentiating agents that induce growth arrest, differentiation and/or apoptosis of neuroblastoma cells *in vitro* (Lovat et al., 1997; Pahlman et al., 1984; Sidell, 1982; Thiele et al., 1985). However the molecular bases of RA therapeutic actions in neuroblastoma have not been established yet.

With the aim of identifying possible molecular targets for RA treatment, we have analyzed RA-induced gene expression changes in SH-SY5Y cultured human neuroblastoma cells (Lopez-Carballo et al., 2002), by means of Ordered Differential Display RT-PCR (Matz et al., 1997). Among more than 60 genes identified, we could found *LAMR1* (also called *RPSA*), encoding the precursor for the 67-KDa Laminin Receptor (67LR) (Wewer et al., 1986; Yow et al., 1988), whose mRNA levels were remarkably reduced after RA treatment. The 67LR is a multifunctional protein which is involved in cell adhesion, required for maintaining of cell viability (Scheiman et al., 2010; Susantad & Smith, 2008), acts as receptor for infectious agents like viruses and prions (Vana et al., 2009) and mediates the actions of the green tea polyphenol epigallocatechin-3-gallate (Umeda et al., 2008). Expression of 67LR has been

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor

Chemiluminiscent signals were developed with ECL (GE Healthcare).

for *ACTB.*

**2.3 Western blot**

**2.4 Immunofluorescence**

excitation filters.

**2.5 Migration and invasion assays**

Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells 219

quantification of the *LAMR1* expression levels, duplicate Northern Blots were exposed to Fuji IP capture plates, measured using a Fuji FLA-5000 laser scanner and analyzed using Fuji ImageQuant software. *LAMR1* absolute values were normalized to the values obtained

Whole cell extracts were obtained by lysis of the cells in RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% Glycerol) containing 0.5% Nonidet P-40, 0.1% SDS, protease inhibitors (1 mM PMSF, 40 g/ml aprotinin and 40 g/ml leupeptin) and phosphatase inhibitors (1 mM sodium ortovanadate, 1 mM NaF). After 10 min incubation on ice, the lysate was cleared by centrifugation (16,100xg, 10 min, 4ºC), and protein concentration determined. Extracts were diluted with 2x sample buffer containing freshly added 50 mM Dithiothreitol. Western blot analysis of proteins from whole cell extracts was performed as described (Lopez-Carballo et al., 2002). Antibodies against the 67- KDa Laminin Receptor (Wewer et al., 1987), were obtained from Abcam. Monoclonal antibodies against the 37-KDa Laminin Receptor precursor (MPLR, Buto et al., 1997) were a generous gift of Dr. S. Menard (Istituto Nazionale Tumori, Milan, Italy). Antibodies against beta-actin were purchased from Sigma. Horseradish-peroxidase-conjugated secondary antibodies were obtained from GE Healthcare, and Jackson ImmunoResearch.

SH-SY5Y neuroblastoma cells were treated with RA or vehicle for 96 h, scraped from the plate and washed in PBS. A drop of the cell suspension was applied onto poly-lysine-coated slides and let to settle for 10 min at room temperature. The slides were fixed with 4% paraformaldehyde in PBS for 20 min, and washed three times with PBS. After blocking with 3% Bovine Serum Albumin in Phosphate Buffered Saline (PBS) for 40 min, slides were sequentially incubated with Anti-67-KDa Laminin Receptor (Abcam, diluted 1:50 in the same buffer) and Fluorescein-isothiocyanate (FITC)-conjugated anti-rabbit IgG (Jackson ImmunoResearch, diluted 1:100 in the same buffer). After washing with PBS, the slides were counter-stained with Hoechst 33258 (Scharlab, 50 mg/ml) for 5 min, rinsed with PBS and mounted with anti-fade mounting medium (Dako-Cytomation). Slides were sequentially photographed in a Leica fluorescence microscope equipped with FITC- and UV-specific

The effects of RA treatments, and Laminin 1-derived peptide P11 on the migration and invasion capacities of SH-SY5Y neuroblastoma cells were tested according to published protocols (Albini et al., 1987; McCarthy et al., 1983), with slight modifications. A modified, light-opaque Boyden chamber (Falcon HTS FluoroBlok, 8 m pore size; Becton-Dickinson) was used in both migration and invasion assays. Cells were treated with 1 M RA or vehicle in culture medium during the indicated times, and labeled in the plate with Calcein AM (5 M; Molecular Probes-Invitrogen), during 30 min at 37ºC. Labeled cells were detached from the plate by scraping and counted. Migration/haptotaxis assays (see Fig. 3A) were used to measure cell movement toward an immobilized laminin-rich extracellular matrix protein gradient. For that purpose, the lower chamber of the plate was coated with BD *Matrigel*

correlated with the biological aggressiveness, and the invasive and metastatic capacities of tumors from diverse origin. Many examples in which the expression of 67LR is increased in a tumor in respect to the corresponding normal tissue have been reported, and elevated expression of 67LR correlates with tumor progression and has been considered as adverse prognostic factor in several human cancers (Castronovo, 1993; Menard et al., 1997; Menard et al., 1998; Viacava et al., 1997, for review). *LAMR1* encodes a 37 KDa precursor protein (Wewer et al., 1986; Yow et al., 1988), that is acylated and glycosylated to generate the mature 67LR (Buto et al., 1998; Katagiri et al., 2005; Landowski et al., 1995) A role of 67LR in stabilizing the binding of integrins to laminin has been postulated (Magnifico et al., 1996), and it has been proposed that binding of 67LR contributes to laminin remodeling, thereby facilitating laminin degradation by proteolytic enzymes and increasing the release of motility fragments (Ardini et al., 2002; Berno et al., 2005; Vande Broek et al., 2001). Therefore, 67LR could play a role in the degradation of basal membrane whose major component is laminin, a critical step of the metastasis process and a prerequisite for tumor vascularization, involving the invasion of tumor mass by endothelial cells.

We report here that RA treatment of human neuroblastoma cells induces a reduction on the mRNA and protein levels of 67LR. In parallel, RA treatment impaired neuroblastoma cell migration towards laminin in haptotaxis assays *in vitro* and reduced invasiveness through *Matrigel* in *in vitro* invasion assays, processes in which 67LR played a major role. In addition, we show by immunohistochemical methods that more than two thirds of the human highrisk neuroblastic tumors assayed expressed 67LR to certain extent. The results showed here support an important role for 67LR in migration towards and invasion through the basement membrane, critical steps on the metastasis process. RA-induced downregulation of 67LR correlates with a reduction in the biological aggressiveness *in vitro* of neuroblastoma cells, and supports the idea of 67LR as an important molecular target for the therapeutic actions of RA in neuroblastoma.

## **2. Experimental procedures**

## **2.1 Cell culture and treatments**

SH-SY5Y cells (ATCC no. CRL-2266) were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin. LA-N-1 cells (ECACC no. 06041201) were grown in a 1:1 mixture of EMEM and Ham's F-12 medium containing 10% heatinactivated FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 1% non-essential amino acids. Cell cultures were kept in a humidified incubator at 37ºC with 5% CO2. The medium was replaced every 2 days and the cells were split before they reached confluence. Cycloheximide (CHX), all*-trans*-retinoic acid (RA), and LY294002 (LY) were purchased from Sigma. The different compounds were dissolved in Dimethyl-sulfoxide and added to the culture medium at the indicated concentrations. Peptide P11 (Ac-CDPGYIGSR-NH2, (Graf et al., 1987b) and its Scrambled control (Ac-YCIPGDRGS-NH2) were synthesized and purified by HPLC to >90%. Peptides were dissolved in serum-free culture medium at 60 g/ml (60 M).

#### **2.2 RNA analysis**

Northern Blot analysis of total RNA from SH-SY5Y cells with [32P]-labeled probes were performed as previously described (Lopez-Carballo et al., 2002). *LAMR1* probe consisted of a 1 Kb fragment obtained from the EST clone IMAGE 1271323. Probes for Cyclophilin A (*PPIA)* and beta-actin (*ACTB*) consisted in cDNA fragments generated by RT-PCR. For

quantification of the *LAMR1* expression levels, duplicate Northern Blots were exposed to Fuji IP capture plates, measured using a Fuji FLA-5000 laser scanner and analyzed using Fuji ImageQuant software. *LAMR1* absolute values were normalized to the values obtained for *ACTB.*

## **2.3 Western blot**

218 Neuroblastoma – Present and Future

correlated with the biological aggressiveness, and the invasive and metastatic capacities of tumors from diverse origin. Many examples in which the expression of 67LR is increased in a tumor in respect to the corresponding normal tissue have been reported, and elevated expression of 67LR correlates with tumor progression and has been considered as adverse prognostic factor in several human cancers (Castronovo, 1993; Menard et al., 1997; Menard et al., 1998; Viacava et al., 1997, for review). *LAMR1* encodes a 37 KDa precursor protein (Wewer et al., 1986; Yow et al., 1988), that is acylated and glycosylated to generate the mature 67LR (Buto et al., 1998; Katagiri et al., 2005; Landowski et al., 1995) A role of 67LR in stabilizing the binding of integrins to laminin has been postulated (Magnifico et al., 1996), and it has been proposed that binding of 67LR contributes to laminin remodeling, thereby facilitating laminin degradation by proteolytic enzymes and increasing the release of motility fragments (Ardini et al., 2002; Berno et al., 2005; Vande Broek et al., 2001). Therefore, 67LR could play a role in the degradation of basal membrane whose major component is laminin, a critical step of the metastasis process and a prerequisite for tumor

We report here that RA treatment of human neuroblastoma cells induces a reduction on the mRNA and protein levels of 67LR. In parallel, RA treatment impaired neuroblastoma cell migration towards laminin in haptotaxis assays *in vitro* and reduced invasiveness through *Matrigel* in *in vitro* invasion assays, processes in which 67LR played a major role. In addition, we show by immunohistochemical methods that more than two thirds of the human highrisk neuroblastic tumors assayed expressed 67LR to certain extent. The results showed here support an important role for 67LR in migration towards and invasion through the basement membrane, critical steps on the metastasis process. RA-induced downregulation of 67LR correlates with a reduction in the biological aggressiveness *in vitro* of neuroblastoma cells, and supports the idea of 67LR as an important molecular target for the therapeutic

SH-SY5Y cells (ATCC no. CRL-2266) were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin. LA-N-1 cells (ECACC no. 06041201) were grown in a 1:1 mixture of EMEM and Ham's F-12 medium containing 10% heatinactivated FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 1% non-essential amino acids. Cell cultures were kept in a humidified incubator at 37ºC with 5% CO2. The medium was replaced every 2 days and the cells were split before they reached confluence. Cycloheximide (CHX), all*-trans*-retinoic acid (RA), and LY294002 (LY) were purchased from Sigma. The different compounds were dissolved in Dimethyl-sulfoxide and added to the culture medium at the indicated concentrations. Peptide P11 (Ac-CDPGYIGSR-NH2, (Graf et al., 1987b) and its Scrambled control (Ac-YCIPGDRGS-NH2) were synthesized and purified by HPLC to >90%. Peptides were dissolved in serum-free culture medium at 60 g/ml (60 M).

Northern Blot analysis of total RNA from SH-SY5Y cells with [32P]-labeled probes were performed as previously described (Lopez-Carballo et al., 2002). *LAMR1* probe consisted of a 1 Kb fragment obtained from the EST clone IMAGE 1271323. Probes for Cyclophilin A (*PPIA)* and beta-actin (*ACTB*) consisted in cDNA fragments generated by RT-PCR. For

vascularization, involving the invasion of tumor mass by endothelial cells.

actions of RA in neuroblastoma.

**2. Experimental procedures 2.1 Cell culture and treatments**

**2.2 RNA analysis**

Whole cell extracts were obtained by lysis of the cells in RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% Glycerol) containing 0.5% Nonidet P-40, 0.1% SDS, protease inhibitors (1 mM PMSF, 40 g/ml aprotinin and 40 g/ml leupeptin) and phosphatase inhibitors (1 mM sodium ortovanadate, 1 mM NaF). After 10 min incubation on ice, the lysate was cleared by centrifugation (16,100xg, 10 min, 4ºC), and protein concentration determined. Extracts were diluted with 2x sample buffer containing freshly added 50 mM Dithiothreitol. Western blot analysis of proteins from whole cell extracts was performed as described (Lopez-Carballo et al., 2002). Antibodies against the 67- KDa Laminin Receptor (Wewer et al., 1987), were obtained from Abcam. Monoclonal antibodies against the 37-KDa Laminin Receptor precursor (MPLR, Buto et al., 1997) were a generous gift of Dr. S. Menard (Istituto Nazionale Tumori, Milan, Italy). Antibodies against beta-actin were purchased from Sigma. Horseradish-peroxidase-conjugated secondary antibodies were obtained from GE Healthcare, and Jackson ImmunoResearch. Chemiluminiscent signals were developed with ECL (GE Healthcare).

## **2.4 Immunofluorescence**

SH-SY5Y neuroblastoma cells were treated with RA or vehicle for 96 h, scraped from the plate and washed in PBS. A drop of the cell suspension was applied onto poly-lysine-coated slides and let to settle for 10 min at room temperature. The slides were fixed with 4% paraformaldehyde in PBS for 20 min, and washed three times with PBS. After blocking with 3% Bovine Serum Albumin in Phosphate Buffered Saline (PBS) for 40 min, slides were sequentially incubated with Anti-67-KDa Laminin Receptor (Abcam, diluted 1:50 in the same buffer) and Fluorescein-isothiocyanate (FITC)-conjugated anti-rabbit IgG (Jackson ImmunoResearch, diluted 1:100 in the same buffer). After washing with PBS, the slides were counter-stained with Hoechst 33258 (Scharlab, 50 mg/ml) for 5 min, rinsed with PBS and mounted with anti-fade mounting medium (Dako-Cytomation). Slides were sequentially photographed in a Leica fluorescence microscope equipped with FITC- and UV-specific excitation filters.

#### **2.5 Migration and invasion assays**

The effects of RA treatments, and Laminin 1-derived peptide P11 on the migration and invasion capacities of SH-SY5Y neuroblastoma cells were tested according to published protocols (Albini et al., 1987; McCarthy et al., 1983), with slight modifications. A modified, light-opaque Boyden chamber (Falcon HTS FluoroBlok, 8 m pore size; Becton-Dickinson) was used in both migration and invasion assays. Cells were treated with 1 M RA or vehicle in culture medium during the indicated times, and labeled in the plate with Calcein AM (5 M; Molecular Probes-Invitrogen), during 30 min at 37ºC. Labeled cells were detached from the plate by scraping and counted. Migration/haptotaxis assays (see Fig. 3A) were used to measure cell movement toward an immobilized laminin-rich extracellular matrix protein gradient. For that purpose, the lower chamber of the plate was coated with BD *Matrigel*

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor

**surface**

Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells 221

newly synthesized proteins (Fig. 1b). RA activates the PI3K/Akt signaling pathway through a non-genomic mechanism (Lopez-Carballo et al., 2002; Masiá et al., 2007). Such activation is also required for the downregulation of the expression of *LAMR1*, since it did not occur in

**3.2 RA treatment reduces the presence of the 67-KDa Laminin receptor at the cell**

be observed in another neuroblastoma cell line, LA-N-1 (Fig.2C). Immunofluorescent

Fig. 1. (A-C) RA-induced downregulation of the expression of *LAMR1.*

of the 67LR (*LAMR1*, 1.4 Kb), and Actin- (*ACTB*, 2 Kb) as internal control.

the average (bars) and standard deviation (error bars) of a duplicate experiments.

A. Northern Blot. Each lane contains 15 µg of total RNA from SH-SY5Y neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially hybridized with [32P]-labeled DNA probes specific for the mRNAs of 67LR (*LAMR1*, 1.3 kb), and cyclophilin A (peptidyl- prolyl-isomerase A, *PPIA*, 1 Kb) as internal loading control. B. Northern Blot. Each lane contains 15 µg of total RNA from SH-SY5Y neuroblastoma cells pre-treated during 30 min with vehicle (-), Cycloheximide (CHX) or 10 µM LY294002 (LY), and then treated with 1 µM RA (+) or vehicle (-) during 24 h in the presence of the inhibitor. The blot was sequentially hybridized with [32P]-labeled DNA probes specific for the mRNAs

C. Quantitative analysis of the experiment shown in (B). The radioactivity present in each band on the blot was determined with a Fuji FLA5000 phosphorimager. The values obtained for *LAMR1* probe were normalized with the corresponding *ACTB* values. The graph shows

67LR is synthesized as a 37-KD precursor (37LRP) subunit, which is processed to the mature form of the protein on the cell surface (Buto et al., 1998; Landowski et al., 1995). Using antibodies recognizing the mature form of this protein in Western Blots (Wewer et al., 1987), we have detected that RA treatment results in a decrease on the levels of 67LR in cellular extracts of SH-SY5Y cells(Fig. 2A). By means of a monoclonal antibody specific for the 37LRP (Buto et al., 1997), we observed a parallel reduction on the expression of the precursor protein (Fig. 2B). A similar RA-induced decrease in 67LR protein expression could

cells treated with RA in the presence of the PI3K inhibitor LY290004 (Fig. 1b).

Matrix (10 g/cm2; Becton Dickinson) as attractant and filled with serum-free medium. 50,000 labeled cells in serum-free medium were added onto the upper chamber. As control, equivalent experiments in which coating with *Matrigel* was omitted were set. After 6h of incubation at 37ºC, the fluorescent light emitted by the cells that migrated through the opaque filter was measured with a Victor2 multilabel counter (PerkinElmer Life and Analytical Sciences). The assays were performed at least in triplicate. For invasion assays (see Fig. 3C) the porous membrane of the Boyden chamber was covered with BD *Matrigel* matrix (5 g/cm2 in serum-free medium; Becton Dickinson) and allowed to gel for 30 min. 50,000 calcein-labeled cells were carefully layered on top of the *Matrigel* layer and culture medium containing 10% FBS as chemoattractant to promote cell invasion was placed into the lower well. The fluorescent light emitted by cells in the lower chamber after 6 h incubation at 37ºC was measured in triplicate assays as above. Peptide blocking of 67LR was performed by incubating 50,000 labeled cells in 500 l of serum-free medium with P11 peptide or Scrambled control peptide (60 g/ml; 60M) for 30 min at 37ºC and added to the upper chamber of migration/haptotaxis or invasion assays prepared as described above. For statistical analysis, one-way ANOVA together with Tukey post-hoc test were employed.

#### **2.6 Immunocytochemistry**

Immunohistochemical study was performed following streptavidin-biotin method with antigen retrieval using citrate buffer 0.1M, pH 6.0 and heating with autoclave at 1.5 atm during 3 minutes. Primary antibody (Anti-67-KDa Laminin Receptor, Abcam) was diluted 1:100. Positive controls include internal epithelial tissues as well as one case of invasive breast carcinoma. Negative controls consisted in substitution of the primary antibody by mouse ascites and/or PBS. Immunohistochemistry was performed on a tissue microarray composed of cylinders of 1.5 mm in diameter of 49 cases of Neuroblastoma that was assembled using a manual tissue arrayer (MTA , Beecher Inc, USA) (Noguera et al., 2009).

### **3. Results**

#### **3.1 RA treatment of SH-SY5Y neuroblastoma cells results in downregulation of the expression of the metastasis-associated 67-KDa Laminin receptor**

To identify differentially expressed genes during RA treatment of human neuroblastoma cells we have used the Ordered Differential Display RT-PCR technique (Matz et al., 1997). One of the displayed fragments, showing strong downregulation after 24 h of RA treatment, was amplified and sequenced. The sequence obtained (data not shown) corresponded to the *LAMR1* gene (also called *RPSA*), encoding the 67 KDa Laminin Receptor (67LR; Wewer et al., 1986; Yow et al., 1988). Because 67LR has been described as a metastasis-associated gene, whose expression levels are related to tumoral progression (Menard et al., 1997; Menard et al., 1998; Sobel, 1993) we decided to study the regulation of *LAMR1* transcription by RA with some detail. As shown on the Northern Blot in Fig. 1a, *LAMR1* expression levels were significantly reduced after a 24 h RA treatment. This delayed downregulation profile resembled that reported for the coordinate repression of the *ID1*, *ID2* and *ID3* HLH genes (Lopez-Carballo et al., 2002).

As was the case for the ID genes, the mechanism by which RA downregulates *LAMR1* expression appears to be complex. The reduction on the levels of *LAMR1* mRNA observed after 24 h of RA treatment did not occur when cells were pre-treated with the protein synthesis inhibitor Cycloheximide (CHX), indicating an indirect regulation that requires

Matrix (10 g/cm2; Becton Dickinson) as attractant and filled with serum-free medium. 50,000 labeled cells in serum-free medium were added onto the upper chamber. As control, equivalent experiments in which coating with *Matrigel* was omitted were set. After 6h of incubation at 37ºC, the fluorescent light emitted by the cells that migrated through the opaque filter was measured with a Victor2 multilabel counter (PerkinElmer Life and Analytical Sciences). The assays were performed at least in triplicate. For invasion assays (see Fig. 3C) the porous membrane of the Boyden chamber was covered with BD *Matrigel* matrix (5 g/cm2 in serum-free medium; Becton Dickinson) and allowed to gel for 30 min. 50,000 calcein-labeled cells were carefully layered on top of the *Matrigel* layer and culture medium containing 10% FBS as chemoattractant to promote cell invasion was placed into the lower well. The fluorescent light emitted by cells in the lower chamber after 6 h incubation at 37ºC was measured in triplicate assays as above. Peptide blocking of 67LR was performed by incubating 50,000 labeled cells in 500 l of serum-free medium with P11 peptide or Scrambled control peptide (60 g/ml; 60M) for 30 min at 37ºC and added to the upper chamber of migration/haptotaxis or invasion assays prepared as described above. For statistical analysis, one-way ANOVA together with Tukey post-hoc test were employed.

Immunohistochemical study was performed following streptavidin-biotin method with antigen retrieval using citrate buffer 0.1M, pH 6.0 and heating with autoclave at 1.5 atm during 3 minutes. Primary antibody (Anti-67-KDa Laminin Receptor, Abcam) was diluted 1:100. Positive controls include internal epithelial tissues as well as one case of invasive breast carcinoma. Negative controls consisted in substitution of the primary antibody by mouse ascites and/or PBS. Immunohistochemistry was performed on a tissue microarray composed of cylinders of 1.5 mm in diameter of 49 cases of Neuroblastoma that was assembled using a manual tissue arrayer (MTA , Beecher Inc, USA) (Noguera et al., 2009).

**3.1 RA treatment of SH-SY5Y neuroblastoma cells results in downregulation of the**

To identify differentially expressed genes during RA treatment of human neuroblastoma cells we have used the Ordered Differential Display RT-PCR technique (Matz et al., 1997). One of the displayed fragments, showing strong downregulation after 24 h of RA treatment, was amplified and sequenced. The sequence obtained (data not shown) corresponded to the *LAMR1* gene (also called *RPSA*), encoding the 67 KDa Laminin Receptor (67LR; Wewer et al., 1986; Yow et al., 1988). Because 67LR has been described as a metastasis-associated gene, whose expression levels are related to tumoral progression (Menard et al., 1997; Menard et al., 1998; Sobel, 1993) we decided to study the regulation of *LAMR1* transcription by RA with some detail. As shown on the Northern Blot in Fig. 1a, *LAMR1* expression levels were significantly reduced after a 24 h RA treatment. This delayed downregulation profile resembled that reported for the coordinate repression of the *ID1*, *ID2* and *ID3* HLH genes

As was the case for the ID genes, the mechanism by which RA downregulates *LAMR1* expression appears to be complex. The reduction on the levels of *LAMR1* mRNA observed after 24 h of RA treatment did not occur when cells were pre-treated with the protein synthesis inhibitor Cycloheximide (CHX), indicating an indirect regulation that requires

**expression of the metastasis-associated 67-KDa Laminin receptor**

**2.6 Immunocytochemistry**

(Lopez-Carballo et al., 2002).

**3. Results**

newly synthesized proteins (Fig. 1b). RA activates the PI3K/Akt signaling pathway through a non-genomic mechanism (Lopez-Carballo et al., 2002; Masiá et al., 2007). Such activation is also required for the downregulation of the expression of *LAMR1*, since it did not occur in cells treated with RA in the presence of the PI3K inhibitor LY290004 (Fig. 1b).

### **3.2 RA treatment reduces the presence of the 67-KDa Laminin receptor at the cell surface**

67LR is synthesized as a 37-KD precursor (37LRP) subunit, which is processed to the mature form of the protein on the cell surface (Buto et al., 1998; Landowski et al., 1995). Using antibodies recognizing the mature form of this protein in Western Blots (Wewer et al., 1987), we have detected that RA treatment results in a decrease on the levels of 67LR in cellular extracts of SH-SY5Y cells(Fig. 2A). By means of a monoclonal antibody specific for the 37LRP (Buto et al., 1997), we observed a parallel reduction on the expression of the precursor protein (Fig. 2B). A similar RA-induced decrease in 67LR protein expression could be observed in another neuroblastoma cell line, LA-N-1 (Fig.2C). Immunofluorescent

Fig. 1. (A-C) RA-induced downregulation of the expression of *LAMR1.*

A. Northern Blot. Each lane contains 15 µg of total RNA from SH-SY5Y neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially hybridized with [32P]-labeled DNA probes specific for the mRNAs of 67LR (*LAMR1*, 1.3 kb), and cyclophilin A (peptidyl- prolyl-isomerase A, *PPIA*, 1 Kb) as internal loading control.

B. Northern Blot. Each lane contains 15 µg of total RNA from SH-SY5Y neuroblastoma cells pre-treated during 30 min with vehicle (-), Cycloheximide (CHX) or 10 µM LY294002 (LY), and then treated with 1 µM RA (+) or vehicle (-) during 24 h in the presence of the inhibitor. The blot was sequentially hybridized with [32P]-labeled DNA probes specific for the mRNAs of the 67LR (*LAMR1*, 1.4 Kb), and Actin- (*ACTB*, 2 Kb) as internal control.

C. Quantitative analysis of the experiment shown in (B). The radioactivity present in each band on the blot was determined with a Fuji FLA5000 phosphorimager. The values obtained for *LAMR1* probe were normalized with the corresponding *ACTB* values. The graph shows the average (bars) and standard deviation (error bars) of a duplicate experiments.

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor

Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells 223

(Joshi et al., 2006; Joshi et al., 2007; Meseguer et al., 2011; Voigt & Zintl, 2003). To look for RA-induced biological effects in neuroblastoma cells that could be related to the observed downregulation of the 67LR, we used assays for migration/haptotaxis towards *Matrigel* and for invasion through *Matrigel*. Migration and invasion assays described here were based on published methods (Albini et al., 1987; McCarthy et al., 1983), and conducted measuring the proportion of calcein-labeled migrating cells in modified Boyden chamber assays, using FluoroBlok light-opaque transwell inserts. Calcein-labeled SH-SY5Y cells (50,000 cells/well) were seeded onto the upper chamber of a FluoroBlok Insert in serum-free DMEM. For migration/haptotaxis assays, the lower chamber was coated with *Matrigel* (10 μg/cm2) and contained serum-free DMEM. (Fig. 3A). Similar experiments without *Matrigel* coating were set as negative controls for these experiments. Haptotaxis assays with neuroblastoma cells have shown increased migration when *Matrigel* is used as attractant. Surprisingly, short treatments with RA (24 h) resulted in a transient increase on cell migration that however occurred independently of the presence of chemoattractant. Treatment of SH-SY5Y cells

Fig. 3. (A-D) RA treatment reduces migration towards and invasion through *Matrigel*.

the sample and its untreated control. (ANOVA test; \* p<0.05).

A. Schematic representation of the Haptotaxis/migration towards *Matrigel* assay. B. Effect of RA on cell migration towards *Matrigel*. Cells treated with 1 M RA as indicated or untreated controls were assayed in the absence (empty bars) or in the presence (grey bars) of *Matrigel* as chemoattractant, and the increase in fluorescence was measured. The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between the corresponding sample and the untreated control without chemoattractant (ANOVA test; \* p<0.05; \*\* p <0.01). C. Schematic representation of the invasion assay. D. Effect of RA on cell invasion through *Matrigel*. Cells treated with RA as indicated (grey bars) and their corresponding untreated controls (empty bars) were assayed, and the increase in fluorescence was measured. The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between

staining with the same antibody used on fig 2A, showed that 67LR is less abundant on the membranes of RA treated SH-SY5Ycells, as compared with untreated cells (Fig. 2D).

#### **3.3 RA treatment of neuroblastoma cells reduces migration towards and invasion through Matrigel**

Laminin is a basal membrane specific glycoprotein. Apart from its structural function, this protein presents a biological activity linked to adhesion, migration, growth and differentiation of certain cell types. Laminin is the major component of the commercial basal membrane preparation *Matrigel* (Kleinman & Martin, 2005, for review), that was used in the following experiments as source of laminin-rich matrix. RA has been reported to reduce the migratory, invasive and metastatic capacity of neuroblastoma cells in *in vitro* assays

Fig. 2. (A-D) RA-induced reduction on the levels of the 67LR protein.

A. Western Blot. Each lane contains 20 µg of protein extract from SH-SY5Y neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially incubated with specific antibodies raised against 67LR protein, and with -actin antibodies (-ACT), as loading control. B. Western Blot. Each lane contains 20 µg of protein extract from SH-SY5Y neuroblastoma cells treated with 1 µM RA or vehicle for 72 h. The blot was sequentially incubated with a monoclonal antibody against the 37 KDa Laminin Receptor Precursor protein (37LRP), and with -actin antibodies (-ACT), as loading control. C. Western Blot. Each lane contains 20 µg of protein extract from LA-N-1 neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially incubated with specific antibodies raised against 67LR protein, and with -actin antibodies (-ACT), as loading control. D. Immunofluorescence Microscopy. SH-SY5Y cells were treated during 96 h with 1 M RA or vehicle, scraped from the plate, and applied onto Polylysine-coated slides. The slides were fixed with 4% paraformaldehyde in PBS, and sequentially incubated with rabbit Anti-67LR and FITC-conjugated anti-rabbit IgG. After washing with PBS, the slides were counter-stained with Hoechst 33258 and sequentially photographed in a Leica fluorescence microscope equipped with FITC- and UV-specific excitation filters.

staining with the same antibody used on fig 2A, showed that 67LR is less abundant on the

Laminin is a basal membrane specific glycoprotein. Apart from its structural function, this protein presents a biological activity linked to adhesion, migration, growth and differentiation of certain cell types. Laminin is the major component of the commercial basal membrane preparation *Matrigel* (Kleinman & Martin, 2005, for review), that was used in the following experiments as source of laminin-rich matrix. RA has been reported to reduce the

membranes of RA treated SH-SY5Ycells, as compared with untreated cells (Fig. 2D).

migratory, invasive and metastatic capacity of neuroblastoma cells in *in vitro* assays

Fig. 2. (A-D) RA-induced reduction on the levels of the 67LR protein.

A. Western Blot. Each lane contains 20 µg of protein extract from SH-SY5Y neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially incubated with specific antibodies raised against 67LR protein, and with -actin antibodies (-ACT), as loading control. B. Western Blot. Each lane contains 20 µg of protein extract from SH-SY5Y neuroblastoma cells treated with 1 µM RA or vehicle for 72 h. The blot was sequentially incubated with a monoclonal antibody against the 37 KDa Laminin Receptor Precursor protein (37LRP), and with -actin antibodies (-ACT), as loading control. C. Western Blot. Each lane contains 20 µg of protein extract from LA-N-1 neuroblastoma cells treated with 1 µM RA for the times indicated in the figure. The blot was sequentially incubated with specific antibodies raised against 67LR protein, and with -actin antibodies (-ACT), as loading control. D. Immunofluorescence Microscopy. SH-SY5Y cells were treated during 96 h with 1 M RA or vehicle, scraped from the plate, and applied onto Polylysine-coated slides. The slides were fixed with 4% paraformaldehyde in PBS, and sequentially incubated with rabbit Anti-67LR and FITC-conjugated anti-rabbit IgG. After washing with PBS, the slides were counter-stained with Hoechst 33258 and sequentially photographed in a Leica fluorescence microscope equipped with FITC- and UV-specific

**through Matrigel**

excitation filters.

**3.3 RA treatment of neuroblastoma cells reduces migration towards and invasion**

(Joshi et al., 2006; Joshi et al., 2007; Meseguer et al., 2011; Voigt & Zintl, 2003). To look for RA-induced biological effects in neuroblastoma cells that could be related to the observed downregulation of the 67LR, we used assays for migration/haptotaxis towards *Matrigel* and for invasion through *Matrigel*. Migration and invasion assays described here were based on published methods (Albini et al., 1987; McCarthy et al., 1983), and conducted measuring the proportion of calcein-labeled migrating cells in modified Boyden chamber assays, using FluoroBlok light-opaque transwell inserts. Calcein-labeled SH-SY5Y cells (50,000 cells/well) were seeded onto the upper chamber of a FluoroBlok Insert in serum-free DMEM. For migration/haptotaxis assays, the lower chamber was coated with *Matrigel* (10 μg/cm2) and contained serum-free DMEM. (Fig. 3A). Similar experiments without *Matrigel* coating were set as negative controls for these experiments. Haptotaxis assays with neuroblastoma cells have shown increased migration when *Matrigel* is used as attractant. Surprisingly, short treatments with RA (24 h) resulted in a transient increase on cell migration that however occurred independently of the presence of chemoattractant. Treatment of SH-SY5Y cells

Fig. 3. (A-D) RA treatment reduces migration towards and invasion through *Matrigel*.

A. Schematic representation of the Haptotaxis/migration towards *Matrigel* assay. B. Effect of RA on cell migration towards *Matrigel*. Cells treated with 1 M RA as indicated or untreated controls were assayed in the absence (empty bars) or in the presence (grey bars) of *Matrigel* as chemoattractant, and the increase in fluorescence was measured. The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between the corresponding sample and the untreated control without chemoattractant (ANOVA test; \* p<0.05; \*\* p <0.01). C. Schematic representation of the invasion assay. D. Effect of RA on cell invasion through *Matrigel*. Cells treated with RA as indicated (grey bars) and their corresponding untreated controls (empty bars) were assayed, and the increase in fluorescence was measured. The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between the sample and its untreated control. (ANOVA test; \* p<0.05).

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor

equivalent to those reported for SH-SY5Y cells (data not shown).

the scrambled control peptide had no relevant effect. (Fig. 4A).

(hatched sector) or strong signal (black sector).

signal (36.67%), and 21 (35%) showed strong signal (Fig. 5).

**through Matrigel**

treatment.

Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells 225

chambers of a FluoroBlok transwell was covered with *Matrigel* (5μg/cm2), simulating the basal membrane and 5% FBS was added to the lower chamber as chemoattractant (Fig. 3C). Similarly to what occurred with the migration assay, a transient increase on the invasive capacity of neuroblastoma cells was detected on *Matrigel* invasion assays after 24 h of RA treatment. Nevertheless, treatment with RA for 72 h resulted in strong reduction on the invasiveness of SH-SY5Y neuroblastoma cells (Fig. 3D). In the human neuroblastoma cell line LA-N-1 the effects of RA on migration/haptotaxis and in *in vitro* invasion were

**3.4 The 67-KDa Laminin receptor is involved in migration towards and invasion**

The 67LR binds to the sequence CDPGYIGSR from the Laminin 1 chain (Graf et al., 1987b), and a peptide with that sequence (P11 peptide) impaired the activity of 67LR *in vitro* in laminin-mediated cell attachment and haptotaxis experiments (Graf et al., 1987a) and reduced the formation of experimental metastases *in vivo* (Iwamoto et al., 1987), probably by blocking the Laminin binding sites on 67LR. To assess the involvement of 67LR on the haptotaxis and invasiveness capacities of neuroblastoma cells, we have used P11 peptide to specifically block 67LR. Neuroblastoma cells were incubated with P11 peptide (60 g/ml), or a scrambled peptide of the same chemical composition but different amino acid sequence. Afterwards the cells were used either on migration/haptotaxis experiments or in *in vitro* invasion assays as above. The results obtained showed that incubation of SH-SY5Y cells with P11 peptide significatively reduced migration towards *Matrigel*, whereas addition of

Similarly, addition of peptide P11 also reduced the invasion of neuroblastoma cells through *Matrigel*, whereas incubation with the control peptide had no remarkable effect on invasion (Fig. 4B). These results indicate the involvement of 67LR on laminin-induced migration/haptotaxis and in invasion through *Matrigel*-coated filters. Taken together, our results strongly support that RA-induced downregulation of 67LR contributes to the observed reduction of the biological aggressiveness of neuroblastoma cells after RA

**3.5 Expression of the 67-KDa Laminin receptor in human neuroblastoma tumors**  The expression of 67LR in human neuroblastoma primary tumors was tested by immunocytochemistry with the same 67LR antibody used for Western Blots and Expression of 67LR in neuroblastoma tumor samples was analyzed by immunohistochemistry with anti-67LR antibodies. The graph shows the percentage of the analyzed neuroblastoma samples (n=60) showing weak or no signal (empty sector), moderate intensity signal

immunofluorescence. The antibody provided good results with paraffin sections, showing specific staining on epithelial tissues and invasive carcinomas used as positive controls (data not shown). Paraffin sections of a tissue microarray including 49 high-risk primary neuroblastoma tumor samples, including40 cases with *MYCN* amplification (100 to 500 copies per haploid genome) and 9 cases with *MYCN* gain (2 to 4 copies per haploid genome), and complete sections from 11 additional poorly differentiated neuroblastoma tumors were analyzed. From these 60 different human primary neuroblastoma samples 17 showed no or weak signal (28.33%), 22 showed positive staining with moderate intensity

with RA for 72 h reduced their migration rate and ablated the haptotactic attractive effect of *Matrigel* (Fig. 3B). For *in vitro* invasion assays the porous membrane separating both

Fig. 4. (A-B) The 67-KDa laminin receptor is involved in migration towards and invasion through *Matrigel*.

A. Effect of Laminin peptide P11 on cell migration towards Matrigel. Untreated cells (black bar), as well as cells treated with scrambled peptide (grey bar) or P11 peptide (hatched bar) were assayed for migration using *Matrigel* as chemoattractant, together with a negative control experiment where the attractant was omitted (empty bar). The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between the sample and its control. (ANOVA test; \* p<0.05).

B. Effect of laminin peptide P11 on cell invasion through Matrigel. Untreated cells (black bar), as well as cells treated with scrambled peptide (grey bar) or P11 peptide (hatched bar) were used for *in vitro Matrigel* invasion assays. The graphs show the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between the sample and its control. (ANOVA test; \* p<0.05).

with RA for 72 h reduced their migration rate and ablated the haptotactic attractive effect of

*Matrigel* (Fig. 3B). For *in vitro* invasion assays the porous membrane separating both

Fig. 4. (A-B) The 67-KDa laminin receptor is involved in migration towards and invasion

difference between the sample and its control. (ANOVA test; \* p<0.05).

the sample and its control. (ANOVA test; \* p<0.05).

A. Effect of Laminin peptide P11 on cell migration towards Matrigel. Untreated cells (black bar), as well as cells treated with scrambled peptide (grey bar) or P11 peptide (hatched bar) were assayed for migration using *Matrigel* as chemoattractant, together with a negative control experiment where the attractant was omitted (empty bar). The graph shows the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative

B. Effect of laminin peptide P11 on cell invasion through Matrigel. Untreated cells (black bar), as well as cells treated with scrambled peptide (grey bar) or P11 peptide (hatched bar) were used for *in vitro Matrigel* invasion assays. The graphs show the average +/- SD of triplicate experiments. The asterisks indicate a statistically significative difference between

through *Matrigel*.

chambers of a FluoroBlok transwell was covered with *Matrigel* (5μg/cm2), simulating the basal membrane and 5% FBS was added to the lower chamber as chemoattractant (Fig. 3C). Similarly to what occurred with the migration assay, a transient increase on the invasive capacity of neuroblastoma cells was detected on *Matrigel* invasion assays after 24 h of RA treatment. Nevertheless, treatment with RA for 72 h resulted in strong reduction on the invasiveness of SH-SY5Y neuroblastoma cells (Fig. 3D). In the human neuroblastoma cell line LA-N-1 the effects of RA on migration/haptotaxis and in *in vitro* invasion were equivalent to those reported for SH-SY5Y cells (data not shown).

#### **3.4 The 67-KDa Laminin receptor is involved in migration towards and invasion through Matrigel**

The 67LR binds to the sequence CDPGYIGSR from the Laminin 1 chain (Graf et al., 1987b), and a peptide with that sequence (P11 peptide) impaired the activity of 67LR *in vitro* in

laminin-mediated cell attachment and haptotaxis experiments (Graf et al., 1987a) and reduced the formation of experimental metastases *in vivo* (Iwamoto et al., 1987), probably by blocking the Laminin binding sites on 67LR. To assess the involvement of 67LR on the haptotaxis and invasiveness capacities of neuroblastoma cells, we have used P11 peptide to specifically block 67LR. Neuroblastoma cells were incubated with P11 peptide (60 g/ml), or a scrambled peptide of the same chemical composition but different amino acid sequence. Afterwards the cells were used either on migration/haptotaxis experiments or in *in vitro* invasion assays as above. The results obtained showed that incubation of SH-SY5Y cells with P11 peptide significatively reduced migration towards *Matrigel*, whereas addition of the scrambled control peptide had no relevant effect. (Fig. 4A).

Similarly, addition of peptide P11 also reduced the invasion of neuroblastoma cells through *Matrigel*, whereas incubation with the control peptide had no remarkable effect on invasion (Fig. 4B). These results indicate the involvement of 67LR on laminin-induced migration/haptotaxis and in invasion through *Matrigel*-coated filters. Taken together, our results strongly support that RA-induced downregulation of 67LR contributes to the observed reduction of the biological aggressiveness of neuroblastoma cells after RA treatment.

## **3.5 Expression of the 67-KDa Laminin receptor in human neuroblastoma tumors**

The expression of 67LR in human neuroblastoma primary tumors was tested by immunocytochemistry with the same 67LR antibody used for Western Blots and Expression of 67LR in neuroblastoma tumor samples was analyzed by immunohistochemistry with anti-67LR antibodies. The graph shows the percentage of the analyzed neuroblastoma samples (n=60) showing weak or no signal (empty sector), moderate intensity signal (hatched sector) or strong signal (black sector).

immunofluorescence. The antibody provided good results with paraffin sections, showing specific staining on epithelial tissues and invasive carcinomas used as positive controls (data not shown). Paraffin sections of a tissue microarray including 49 high-risk primary neuroblastoma tumor samples, including40 cases with *MYCN* amplification (100 to 500 copies per haploid genome) and 9 cases with *MYCN* gain (2 to 4 copies per haploid genome), and complete sections from 11 additional poorly differentiated neuroblastoma tumors were analyzed. From these 60 different human primary neuroblastoma samples 17 showed no or weak signal (28.33%), 22 showed positive staining with moderate intensity signal (36.67%), and 21 (35%) showed strong signal (Fig. 5).

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor

**4. Discussion**

effective therapeutic effects in that context.

1999; Satoh et al., 1999; Tanaka et al., 2000)

et al., 2002; Lopez-Carballo et al., 2002).

Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells 227

conceivable that artefactual location of shed 67LR molecules could occur during the fixation and inclusion procedures. In summary, over 71% of the assayed neuroblastoma samples showed expression of 67LR by immunocytochemistry, with a full range of expression levels.

The 67LR has been involved in the biological aggressiveness of tumor cells, by helping to laminin remodeling and favoring its degradation by proteolytic enzymes (Ardini et al., 2002; Berno et al., 2005; Vande Broek et al., 2001). Remodeling of the tumor microenvironment and degradation of basement membrane components are two crucial steps in the development of metastases (Wang et al., 2005b). Here we show that RA treatment of neuroblastoma cells results in a reduction of the expression of 67LR. Concomitant to the downregulation of 67LR, RA treatments result in a reduction in cell migration towards *Matrigel* and an impairment in neuroblastoma cells invasiveness in *in vitro* assays. Finally, the involvement of 67LR in migration and invasion of neuroblastoma cells is supported by experiments in which impairment of the 67LR activity by laminin-derived P11 peptide results in reduced migration and invasion capacities for neuroblastoma cells. Taken together all these results support the idea that RA-induced downregulation of 67LR would account for the observed reductions of the migration capacity and invasiveness *in vitro*, and therefore would contribute of a marked reduction of the biological aggressiveness of neuroblastoma cells. RA treatments have been shown to be effective for the therapy of human neuroblastoma, especially in avoiding relapse when applied in a context of minimal residual disease after intensive therapies (Matthay & Reynolds, 2000; Reynolds et al., 2003) The reduction of the ability of migrating towards the basement membrane and degrading it would fit into the range of actions that would lead to

Several lines of evidence suggest that the 67LR could be a useful therapeutic target for cancer treatment. The anti-cancer cytokines TNF and IFN downregulate the expression of 67LR in transformed keratinocytes (Clausse et al., 1998). Blocking of 67LR with specific antibodies or laminin peptides, or downregulating its expression by anti-sense RNA leads to decreased migration and invasiveness *in vitro* to reduced metastatic and angiogenic potentials in different experimental metastasis models (Iwamoto et al., 1987; Narumi et al.,

Remarkably, short term RA treatments induce a transient increase in neuroblastoma cell migration and invasion *in vitro*. The increase in migration appears to be nonspecific, since also occurs in the absence of laminin chemoattractant. Similar RA-induced rapid transient increases in migration and invasion in neuroblastoma cells have been reported (Joshi et al., 2006; Meseguer et al., 2011), and correlated to the expression of tissue transglutaminase (Joshi et al., 2006). Blocking of RA signaling *in vivo* also impaired neuroblast migration from the subventricular zone of the brain to the olfactory bulb (Wang et al., 2005a), suggesting that short term RA-induced increases in migration probably reflects a physiological feature of RA action on the regulation of neurogenesis. Coupling of differentiation with migration has been described for mouse cortical neurons and the involvement of proneural bHLHs transcription factors in migration has been proposed (Ge et al., 2006). Remarkably, RA treatment of neuroblastoma cells results in changes in the expression patterns of bHLH factors including induction of proneural bHLH factors like *NEUROD6* and *NEUROD1* (Jogi

Another important aspect is the mechanism through which RA induces the downregulation of *LAMR1* gene, which appears to be complex. It has reported that *LAMR1* is downregulated by p53, through an AP-2 binding element located in its first intron (Modugno et al., 2002). It

Fig. 5. Expression of 67LR in neuroblastoma tumor samples.

Photomicrographs of representative neuroblastoma tumor sections stained for 67LR are shown in Figure 6, including sections showing no signal (Fig. 6A), weak signal (Fig 6B), moderate intensity signal (Fig. 6 C and D), and strong signal (Fig. 6 E and F). Nuclear staining was detected in these strongly labeled tumor sections, which is an unexpected location for 67LR. Nevertheless, it has to be noted that shedding of 67LR by tumor cells has been reported (Berno et al., 2005; Karpatova et al., 1996; Moss et al., 2006), and it appears

Fig. 6. (a-f) Immunohistochemical detection of 67LR in neuroblastoma tumor samples.

Photomicrographs of representative neuroblastoma tumor paraffin sections stained for 67LR antibody and counterstained with hematoxylin, including sections showing no signal (A), weak signal (B), moderate intensity signal (C and D), and strong signal (E and F).\

conceivable that artefactual location of shed 67LR molecules could occur during the fixation and inclusion procedures. In summary, over 71% of the assayed neuroblastoma samples showed expression of 67LR by immunocytochemistry, with a full range of expression levels.

## **4. Discussion**

226 Neuroblastoma – Present and Future

Photomicrographs of representative neuroblastoma tumor sections stained for 67LR are shown in Figure 6, including sections showing no signal (Fig. 6A), weak signal (Fig 6B), moderate intensity signal (Fig. 6 C and D), and strong signal (Fig. 6 E and F). Nuclear staining was detected in these strongly labeled tumor sections, which is an unexpected location for 67LR. Nevertheless, it has to be noted that shedding of 67LR by tumor cells has been reported (Berno et al., 2005; Karpatova et al., 1996; Moss et al., 2006), and it appears

Fig. 6. (a-f) Immunohistochemical detection of 67LR in neuroblastoma tumor samples.

weak signal (B), moderate intensity signal (C and D), and strong signal (E and F).\

Photomicrographs of representative neuroblastoma tumor paraffin sections stained for 67LR antibody and counterstained with hematoxylin, including sections showing no signal (A),

Fig. 5. Expression of 67LR in neuroblastoma tumor samples.