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**4**

*Australia*

**Neuroblastoma: A Malignancy Due**

Andrew Tee, Pei Y. Liu, Glenn M. Marshall and Tao Liu

Neuroblastoma originates from precursor neuroblasts of the sympathetic nervous system. Unlike most other cancer types, neuroblastoma is characterized by a unique capacity for spontaneous complete regression, at least partly through neuronal differentiation, in a proportion of patients, and is therefore regarded as a cancer due to cell differentiation block. The first demonstration of *in vitro* differentiation of human neuroblastoma cells was published 30 years ago, when human SK-N-SH and SH-SY5Y neuroblastoma cells were shown to differentiate morphologically (neurite outgrowth) and biochemically in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment (Påhlman et al., 1981). The differentiated cells showed an increased expression of noradrenaline, adrenaline and neuron-specific enolase (NSE), differentiation markers which are employed for the diagnosis of neuroblastoma in patients. In 1982, retinoic acid (RA) was shown to induce concentrationdependent morphologic differentiation and growth inhibition in the LA-N-1 human neuroblastoma cell line (Sidell, 1982). The RA-induced morphologic differentiation and growth inhibition persisted despite removal of the drug. These observations demonstrate that RA promotes the differentiation of LA-N-1 neuroblastoma cells and results in a reduced expression of the malignant phenotype, and suggest that patients with advanced neuroblastoma may be successfully treated by RA to induce their tumour cells to differentiate and to undergo growth inhibition. In the last three decades, a number of factors, such as up-regulation of the expression of retinoic acid receptors, have been identified as crucial for the induction or blockage of neuroblastoma cell differentiation. Importantly, naturally occurring and synthetic retinoids have shown great promise in the clinic when used as differentiation agents in neuroblastoma patients with minimal residual disease, while the mechanism of retinoid anticancer signalling in neuroblastoma is still not

fully understood (Liu et al., 2005; Reynolds et al., 2003).

**2. Neurotrophin and neurotrophin receptor-induced neuroblastoma cell**

Among the best characterized factors involved in pre-cancerous neuroblast and neuroblastoma cell differentiation are the tyrosine kinase receptor (TRK), its ligands nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-5). Neuroblastoma cell lines generally lack functional neurotrophin receptors of the TRK gene family and therefore do not differentiate when stimulated with

**1. Introduction**

**differentiation**

*Children's Cancer Institute Australia for Medical Research*

 **to Cell Differentiation Block**

## **Neuroblastoma: A Malignancy Due to Cell Differentiation Block**

Andrew Tee, Pei Y. Liu, Glenn M. Marshall and Tao Liu *Children's Cancer Institute Australia for Medical Research Australia*

## **1. Introduction**

78 Neuroblastoma – Present and Future

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Nogo-A stabilizes the architecture of hippocampal neurons. *J. Neurosci*. 30(40):

Caspase-3 activation via mitochondria is required for long-term depression and

Neuroblastoma originates from precursor neuroblasts of the sympathetic nervous system. Unlike most other cancer types, neuroblastoma is characterized by a unique capacity for spontaneous complete regression, at least partly through neuronal differentiation, in a proportion of patients, and is therefore regarded as a cancer due to cell differentiation block. The first demonstration of *in vitro* differentiation of human neuroblastoma cells was published 30 years ago, when human SK-N-SH and SH-SY5Y neuroblastoma cells were shown to differentiate morphologically (neurite outgrowth) and biochemically in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment (Påhlman et al., 1981). The differentiated cells showed an increased expression of noradrenaline, adrenaline and neuron-specific enolase (NSE), differentiation markers which are employed for the diagnosis of neuroblastoma in patients. In 1982, retinoic acid (RA) was shown to induce concentrationdependent morphologic differentiation and growth inhibition in the LA-N-1 human neuroblastoma cell line (Sidell, 1982). The RA-induced morphologic differentiation and growth inhibition persisted despite removal of the drug. These observations demonstrate that RA promotes the differentiation of LA-N-1 neuroblastoma cells and results in a reduced expression of the malignant phenotype, and suggest that patients with advanced neuroblastoma may be successfully treated by RA to induce their tumour cells to differentiate and to undergo growth inhibition. In the last three decades, a number of factors, such as up-regulation of the expression of retinoic acid receptors, have been identified as crucial for the induction or blockage of neuroblastoma cell differentiation. Importantly, naturally occurring and synthetic retinoids have shown great promise in the clinic when used as differentiation agents in neuroblastoma patients with minimal residual disease, while the mechanism of retinoid anticancer signalling in neuroblastoma is still not fully understood (Liu et al., 2005; Reynolds et al., 2003).

## **2. Neurotrophin and neurotrophin receptor-induced neuroblastoma cell differentiation**

Among the best characterized factors involved in pre-cancerous neuroblast and neuroblastoma cell differentiation are the tyrosine kinase receptor (TRK), its ligands nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-5). Neuroblastoma cell lines generally lack functional neurotrophin receptors of the TRK gene family and therefore do not differentiate when stimulated with

Neuroblastoma: A Malignancy Due to Cell Differentiation Block 81

**5. Histone deacetylase 1 and tissue transglutaminase in neuroblastoma cell**

The transamidation activity of tissue transglutaminase (TG2) has been shown to be essential for the neuroblastoma and leukemia cell differentiation response to retinoid therapy (Balajthy et al., 2006; Tucholski, Lesort, and Johnson, 2001), and TG2 overexpression alone induces neuronal differentiation in neuroblastoma cells (Tucholski, Lesort, and Johnson, 2001). We have recently shown that repression of TG2 gene expression and reduction in transamidation activity are essential for N-Myc-induced neuroblastoma cell differentiation block (Liu et al., 2007). Importantly, dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays show that N-Myc acts as a transrepressor by recruiting HDAC1 protein to an Sp1-binding site in the TG2 gene core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites (Liu et al., 2007). Histone deacetylase inhibitor treatment blocks the N-Myc-mediated HDAC1 recruitment and TG2 repression *in vitro*. In neuroblastoma-bearing N-Myc transgenic mice, histone deacetylase inhibitor treatment induces TG2 expression and demonstrates marked antitumor activity *in vivo*. Taken together, these data indicate the critical roles of HDAC1 and TG2 in N-Mycinduced oncogenesis and have significant implications for the use of histone deacetylase

**6. Opposing effects of two tissue transglutaminase protein isoforms in**

Uniquely, two isoforms of TG2 mRNA and protein, a short form (TG2-S) and a full length form (TG2-L), have been characterized (Begg et al., 2006). We have shown that TG2-L and TG2-S exert opposing effects on cell differentiation (Tee et al., 2010). Repression of TG2-L with small interfering RNA, which does not affect TG2-S expression, induces dramatic neuronal differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-bindingdeficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induces neuroblastoma cell differentiation, which is blocked by an inhibitor of transamidase activity. Whereas N-Myc represses and retinoid activates both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhances neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induces neuroblastoma cell differentiation, and VIP can be up-regulated by TG2-L, but not TG2-S. Taken together, these data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP

Conventional therapy of neuroblastoma patients now includes the differentiation agent retinoid. Unlike chemo-radiotherapy, retinoid differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. The effects of retinoids are mediated by two classes of non-steroid nuclear hormone receptors, the retinoic acid (RAR α, β, γ) and the retinoic X (RXR α, β, γ) receptors (Reynolds et al., 2003). The naturally occurring all-trans-retinoic acid (atRA), 9-cis-retinoic acid and the synthetic 13-cis-retinoic acid (13-cis-RA) are examples of retinoids studied in neuroblastoma. It has long been established that atRA stimulation of neuroblastoma cells results in growth inhibition and neuronal differentiation as indicated by neurite outgrowth, increased NSE

**differentiation**

inhibitor therapy in N-Myc-driven neuroblastoma.

binding and modulation of VIP gene transcription (Tee et al., 2010).

**neuroblastoma cell differentiation**

**7. Retinoid differentiation therapy**

neurotrophins including NGF, BDNF, NTF3 or NTF5, and absence of TRKA and TRKC gene expression in neuroblastoma tissues correlates with poor prognosis in neuroblastoma patients (Kogner et al., 1993; Suzuki et al., 1993; Yamashiro et al., 1996). When transfected with either TRKA or TRKC, neuroblastoma cell lines can differentiate into sympathetic neurons in response to treatment with their cognate ligands NGF and NT-3 (Edsjo et al., 2001; Lavenius et al., 1995). Moreover, treatment with growth inhibition agents such as aphidicolin and all-trans RA (atRA) results in NGF responsiveness through induction of TRKA gene expression, demonstrating that TRK-negative cells retain a capacity to respond to TRK-mediated cell differentiation (Hishiki et al., 1998; Poluha, Poluha, and Ross, 1995). Taken together, these data show that many neuroblastoma cell lines have the capacity to respond to physiological differentiation stimuli, although they have lost this capacity with regard to stimulation with NGF or with other neurotrophins unless the neuroblastoma cells are triggered to express the cognate receptors [reviewed in (Edsjö, Holmquist, and Påhlman, 2007)].

While TRKA- and TRKC-mediated signalling decreases neuroblastoma cell proliferation and aggressiveness, BDNF/TRKB signalling corresponds with poor outcome in neuroblastoma patients [reviewed in (Edsjö, Holmquist, and Påhlman, 2007)]. BDNF treatment of neuroblastoma cells transfected with a TRKB over-expressing construct does not induce differentiation (Ho et al., 2011), and BDNF stimulation of neuroblastoma cell lines expressing TRKB as a result of RA treatment does not affect proliferation, but increases survival and invasiveness (Matsumoto et al., 1995). In addition to these effects of TRKA and TRKB signalling, recent data also suggest a difference with respect to therapy resistance, invasiveness, angiogenesis, and possibly also genomic stability (Schramm et al., 2005).

atRA treatment of neuroblastoma cells can also induce the expression of the proto-oncogene RET, which codes for a tyrosine kinase growth factor receptor specifically binding glial cell linederived neurotrophic factor (GDNF) (Airaksinen and Saarma, 2002). GDNF is required for the proper development of enteric and parasympathetic neuroblasts (Airaksinen and Saarma, 2002), and atRA-treated neuroblastoma cells differentiate further in response to GDNF (Hishiki et al., 1998). Additionally, activation of RET by GDNF leads to differentiation of neuroblasts and neuroblastoma cells into ganglia cells (D'Alessio et al., 1995), and GDNF synergizes with ciliary neurotrophic factor (CNTF) to enhance TRKA receptor expression, thereby strengthening NGFmediated cell differentiation signal (Peterson and Bogenmann, 2004).

## **3. Fyn and Fyn kinase induce neuroblastoma cell differentiation**

Other factors involved in neuroblastoma cell differentiation include the Fyn nonreceptor kinase. Expression of active Fyn induces differentiation and growth arrest in neuroblastoma cells (Berwanger et al., 2002). High expression of Fyn and high Fyn kinase activity are restricted to low-stage human neuroblastoma tissues, and expression of Fyn predicts longterm survival independently of MYCN gene amplification (Berwanger et al., 2002).

## **4. N-Myc blocks neuroblastoma cell differentiation**

The most powerful endogenous blocker of neuroblastoma cell differentiation is the *MYCN* oncogene. N-Myc is well-known to repress neuroblastoma cell differentiation partly through repressing TRKA gene expression. A recent study demonstrates that N-Myc blocks TRKA gene expression by recruiting histone deacetylase 1 (HDAC1) to TRKA gene promoter and repressing its transcription (Iraci et al., 2011). Additionally, N-Myc represses the expression of growth arrest specific1 (GAS1), leading to increased RET tyrosine 1062 phosphorylation and decreased GDNF signaling (Lopez-Ramirez et al., 2008).

neurotrophins including NGF, BDNF, NTF3 or NTF5, and absence of TRKA and TRKC gene expression in neuroblastoma tissues correlates with poor prognosis in neuroblastoma patients (Kogner et al., 1993; Suzuki et al., 1993; Yamashiro et al., 1996). When transfected with either TRKA or TRKC, neuroblastoma cell lines can differentiate into sympathetic neurons in response to treatment with their cognate ligands NGF and NT-3 (Edsjo et al., 2001; Lavenius et al., 1995). Moreover, treatment with growth inhibition agents such as aphidicolin and all-trans RA (atRA) results in NGF responsiveness through induction of TRKA gene expression, demonstrating that TRK-negative cells retain a capacity to respond to TRK-mediated cell differentiation (Hishiki et al., 1998; Poluha, Poluha, and Ross, 1995). Taken together, these data show that many neuroblastoma cell lines have the capacity to respond to physiological differentiation stimuli, although they have lost this capacity with regard to stimulation with NGF or with other neurotrophins unless the neuroblastoma cells are triggered to express the

While TRKA- and TRKC-mediated signalling decreases neuroblastoma cell proliferation and aggressiveness, BDNF/TRKB signalling corresponds with poor outcome in neuroblastoma patients [reviewed in (Edsjö, Holmquist, and Påhlman, 2007)]. BDNF treatment of neuroblastoma cells transfected with a TRKB over-expressing construct does not induce differentiation (Ho et al., 2011), and BDNF stimulation of neuroblastoma cell lines expressing TRKB as a result of RA treatment does not affect proliferation, but increases survival and invasiveness (Matsumoto et al., 1995). In addition to these effects of TRKA and TRKB signalling, recent data also suggest a difference with respect to therapy resistance, invasiveness, angiogenesis, and possibly also genomic stability (Schramm et al., 2005). atRA treatment of neuroblastoma cells can also induce the expression of the proto-oncogene RET, which codes for a tyrosine kinase growth factor receptor specifically binding glial cell linederived neurotrophic factor (GDNF) (Airaksinen and Saarma, 2002). GDNF is required for the proper development of enteric and parasympathetic neuroblasts (Airaksinen and Saarma, 2002), and atRA-treated neuroblastoma cells differentiate further in response to GDNF (Hishiki et al., 1998). Additionally, activation of RET by GDNF leads to differentiation of neuroblasts and neuroblastoma cells into ganglia cells (D'Alessio et al., 1995), and GDNF synergizes with ciliary neurotrophic factor (CNTF) to enhance TRKA receptor expression, thereby strengthening NGF-

cognate receptors [reviewed in (Edsjö, Holmquist, and Påhlman, 2007)].

mediated cell differentiation signal (Peterson and Bogenmann, 2004).

**4. N-Myc blocks neuroblastoma cell differentiation**

and decreased GDNF signaling (Lopez-Ramirez et al., 2008).

**3. Fyn and Fyn kinase induce neuroblastoma cell differentiation**

term survival independently of MYCN gene amplification (Berwanger et al., 2002).

Other factors involved in neuroblastoma cell differentiation include the Fyn nonreceptor kinase. Expression of active Fyn induces differentiation and growth arrest in neuroblastoma cells (Berwanger et al., 2002). High expression of Fyn and high Fyn kinase activity are restricted to low-stage human neuroblastoma tissues, and expression of Fyn predicts long-

The most powerful endogenous blocker of neuroblastoma cell differentiation is the *MYCN* oncogene. N-Myc is well-known to repress neuroblastoma cell differentiation partly through repressing TRKA gene expression. A recent study demonstrates that N-Myc blocks TRKA gene expression by recruiting histone deacetylase 1 (HDAC1) to TRKA gene promoter and repressing its transcription (Iraci et al., 2011). Additionally, N-Myc represses the expression of growth arrest specific1 (GAS1), leading to increased RET tyrosine 1062 phosphorylation

## **5. Histone deacetylase 1 and tissue transglutaminase in neuroblastoma cell differentiation**

The transamidation activity of tissue transglutaminase (TG2) has been shown to be essential for the neuroblastoma and leukemia cell differentiation response to retinoid therapy (Balajthy et al., 2006; Tucholski, Lesort, and Johnson, 2001), and TG2 overexpression alone induces neuronal differentiation in neuroblastoma cells (Tucholski, Lesort, and Johnson, 2001). We have recently shown that repression of TG2 gene expression and reduction in transamidation activity are essential for N-Myc-induced neuroblastoma cell differentiation block (Liu et al., 2007). Importantly, dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays show that N-Myc acts as a transrepressor by recruiting HDAC1 protein to an Sp1-binding site in the TG2 gene core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites (Liu et al., 2007). Histone deacetylase inhibitor treatment blocks the N-Myc-mediated HDAC1 recruitment and TG2 repression *in vitro*. In neuroblastoma-bearing N-Myc transgenic mice, histone deacetylase inhibitor treatment induces TG2 expression and demonstrates marked antitumor activity *in vivo*. Taken together, these data indicate the critical roles of HDAC1 and TG2 in N-Mycinduced oncogenesis and have significant implications for the use of histone deacetylase inhibitor therapy in N-Myc-driven neuroblastoma.

## **6. Opposing effects of two tissue transglutaminase protein isoforms in neuroblastoma cell differentiation**

Uniquely, two isoforms of TG2 mRNA and protein, a short form (TG2-S) and a full length form (TG2-L), have been characterized (Begg et al., 2006). We have shown that TG2-L and TG2-S exert opposing effects on cell differentiation (Tee et al., 2010). Repression of TG2-L with small interfering RNA, which does not affect TG2-S expression, induces dramatic neuronal differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-bindingdeficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induces neuroblastoma cell differentiation, which is blocked by an inhibitor of transamidase activity. Whereas N-Myc represses and retinoid activates both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhances neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induces neuroblastoma cell differentiation, and VIP can be up-regulated by TG2-L, but not TG2-S. Taken together, these data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription (Tee et al., 2010).

## **7. Retinoid differentiation therapy**

Conventional therapy of neuroblastoma patients now includes the differentiation agent retinoid. Unlike chemo-radiotherapy, retinoid differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. The effects of retinoids are mediated by two classes of non-steroid nuclear hormone receptors, the retinoic acid (RAR α, β, γ) and the retinoic X (RXR α, β, γ) receptors (Reynolds et al., 2003). The naturally occurring all-trans-retinoic acid (atRA), 9-cis-retinoic acid and the synthetic 13-cis-retinoic acid (13-cis-RA) are examples of retinoids studied in neuroblastoma. It has long been established that atRA stimulation of neuroblastoma cells results in growth inhibition and neuronal differentiation as indicated by neurite outgrowth, increased NSE

Neuroblastoma: A Malignancy Due to Cell Differentiation Block 83

Cerchia, L., D'Alessio, A., Amabile, G., Duconge, F., Pestourie, C., Tavitian, B., Libri, D., and

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activity and accumulation of norepinephrine (Pahlman et al., 1984; Sidell, 1982) . Even though atRA induces neuronal differentiation, the outcome differs depending on the neuroblastoma cell line studied. While some cell lines develop a sympathetic noradrenergic phenotype, a cholinergic switch has been suggested in other cell lines (Handler et al., 2000; Hill and Robertson, 1997). Mechanism studies have revealed that the down-regulation of the proto-oncogenes N-Myc, MYB and HRAS precedes the morphological differentiation, with changes in the expression of other proto-oncogenes following thereafter (Thiele, Deutsch, and Israel, 1988; Thiele, Reynolds, and Israel, 1985). As discussed in the previous sections, atRA treatment can also result in neuroblastoma cell differentiation by up-regulating the expression of the neurotrophin receptors TRKA, TRKB and RET, leading to responsiveness to their ligands NGF, BDNF and GDNF (Cerchia et al., 2006; Hishiki et al., 1998).

Phase I clinical trials show that higher and more sustained drug levels can be obtained with 13-cis-RA relative to atRA, and a phase III randomized clinical trial shows that high-dose, pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improved event-free survival in high-risk neuroblastoma patients (Reynolds et al., 2003) (Matthay et al., 1999). Moreove, *In vitro* as well as clinical data indicate that synthetic retinoids such as Fenretinide might be effective against neuroblastoma cells resistant to 13-cis-RA therapy. This effect seems to be independent of retinoid receptors, does not involve differentiation but is involved in the induction of programmed cell death (Reynolds et al., 2003).

### **8. Combination differentiation therapy with retinoids and histone deacetylase inhibitors**

Although 13-cis-RA significantly improves event-free survival in high-risk neuroblastoma patients (Matthay et al., 1999), resistance to retinoid therapy eventually develops in more than 50% of neuroblastoma patients. Combination therapies with retinoids and other anticancer agents have therefore been studied extensively. Interestingly, during screening a small-molecule library for compounds enhancing histone deacetylase inhibitor-induced neuroblastoma cell differentiation, retinoids were found to be the top hit compounds (Hahn et al., 2008). Secondary assays confirmed greater neuroblastoma differentiation with the combination therapy of histone deacetylase inhibitors and retinoids. In a xenograft model of neuroblastoma, animals treated with the combination therapy with the histone deacetylase inhibitor SAHA and atRA had the longest survival. This work suggests that combination therapy with histone deacetylase inhibitors and retinoids could be a promising new strategy for differentiation therapy of children with neuroblastoma (Hahn et al., 2008).

#### **9. References**

activity and accumulation of norepinephrine (Pahlman et al., 1984; Sidell, 1982) . Even though atRA induces neuronal differentiation, the outcome differs depending on the neuroblastoma cell line studied. While some cell lines develop a sympathetic noradrenergic phenotype, a cholinergic switch has been suggested in other cell lines (Handler et al., 2000; Hill and Robertson, 1997). Mechanism studies have revealed that the down-regulation of the proto-oncogenes N-Myc, MYB and HRAS precedes the morphological differentiation, with changes in the expression of other proto-oncogenes following thereafter (Thiele, Deutsch, and Israel, 1988; Thiele, Reynolds, and Israel, 1985). As discussed in the previous sections, atRA treatment can also result in neuroblastoma cell differentiation by up-regulating the expression of the neurotrophin receptors TRKA, TRKB and RET, leading to responsiveness

Phase I clinical trials show that higher and more sustained drug levels can be obtained with 13-cis-RA relative to atRA, and a phase III randomized clinical trial shows that high-dose, pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improved event-free survival in high-risk neuroblastoma patients (Reynolds et al., 2003) (Matthay et al., 1999). Moreove, *In vitro* as well as clinical data indicate that synthetic retinoids such as Fenretinide might be effective against neuroblastoma cells resistant to 13-cis-RA therapy. This effect seems to be independent of retinoid receptors, does not involve differentiation but is

**8. Combination differentiation therapy with retinoids and histone deacetylase**

Although 13-cis-RA significantly improves event-free survival in high-risk neuroblastoma patients (Matthay et al., 1999), resistance to retinoid therapy eventually develops in more than 50% of neuroblastoma patients. Combination therapies with retinoids and other anticancer agents have therefore been studied extensively. Interestingly, during screening a small-molecule library for compounds enhancing histone deacetylase inhibitor-induced neuroblastoma cell differentiation, retinoids were found to be the top hit compounds (Hahn et al., 2008). Secondary assays confirmed greater neuroblastoma differentiation with the combination therapy of histone deacetylase inhibitors and retinoids. In a xenograft model of neuroblastoma, animals treated with the combination therapy with the histone deacetylase inhibitor SAHA and atRA had the longest survival. This work suggests that combination therapy with histone deacetylase inhibitors and retinoids could be a promising new strategy

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**5**

**Interplay Between Protein Kinase C**

 **Neurofibromin, and the Ras/MAPK**

Shalini Johnson1, Chengjun Li1, Andreas Florakis1,

*1Neurosciences, Biomedical Research Foundation of the*

 **Pathway in Neuroblastoma Differentiation**

Konstantinos Dimas2, Nikos Sakellaridis2 and Dimitra Mangoura1,\*

Neuroblastoma (NB) 1 is the most common extracranial tumor in childhood and accounts for nearly half of neoplasms diagnosed during infancy (Maris, 2010; Brodeur, 2003). A striking feature of these tumors has been their heterogeneous course, which ranges from spontaneous regression to inevitable progression and death (Brodeur, 2003). Current pharmacological approaches in the treatment of NBs include standard combination chemotherapy using dose-intensive cycles of carboplatin, etoposide, cyclophosphamide, and doxorubicin, with the addition of topoisomerase I inhibitors. For intermediate-risk NB, a high rate of survival among patients may still be achieved with significant reduction of doses and duration of chemotherapy (Baker et al., 2010). The retinoic acid analogue isotretinoin (13-cis-retinoic acid) is additionally used in high-risk NB patients with

Animal cancer models have offered valuable preclinical testing systems for studying the impact of specific genes in the appearance and the progress of the disease as well as the efficacy of novel therapeutic regimes. Animal models of NB have been developed by subcutaneous inoculation (xenografting) of established human NB cell lines in immunocompromised mice; for instance, the cell line SK-N-BE2c was successfully used to develop an animal model and test the effects of imatinib (Meco et al., 2005). Yet, the major

1 Abbreviations used: ERK, extracellular signal-regulated kinase; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; DAG, diacylglycerol; TH, tyrosine hydroxylase; GAP43, growthassociated protein 43; NPY, neuropeptide Y; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; dBcAMP,

progressive or recurrent disease (Maris, 2010; Reynolds et al., 2003).

\* Corresponding Authors (George Leondaritis, Dimitra Mangoura)

dibutyryl cAMP; BrdU, 5-bromo-2-deoxyuridine.

**1. Introduction**

 **Isoforms Alpha and Epsilon,**

George Leondaritis1,\* , Xenia Koliou1,

*2Department of Pharmacology, Medical School,*

 *Academy of Athens (BRFAA) Athens,*

*University of Thessaly, Larissa,*

*Greece*

## **Interplay Between Protein Kinase C Isoforms Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation**

George Leondaritis1,\* , Xenia Koliou1, Shalini Johnson1, Chengjun Li1, Andreas Florakis1, Konstantinos Dimas2, Nikos Sakellaridis2 and Dimitra Mangoura1,\* *1Neurosciences, Biomedical Research Foundation of the Academy of Athens (BRFAA) Athens, 2Department of Pharmacology, Medical School, University of Thessaly, Larissa, Greece*

## **1. Introduction**

84 Neuroblastoma – Present and Future

Liu, T., Tee, A. E., Porro, A., Smith, S. A., Dwarte, T., Liu, P. Y., Iraci, N., Sekyere, E., Haber, M.,

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Thiele, C. J., Deutsch, L. A., and Israel, M. A. (1988). The expression of multiple proto-

Thiele, C. J., Reynolds, C. P., and Israel, M. A. (1985). Decreased expression of N-myc

Tucholski, J., Lesort, M., and Johnson, G. V. (2001). Tissue transglutaminase is essential for

Yamashiro, D. J., Nakagawara, A., Ikegaki, N., Liu, X. G., and Brodeur, G. M. (1996). Expression of TrkC in favorable human neuroblastomas. *Oncogene* 12(1), 37-41.

human neuroblastoma cells in vitro. *J Natl Cancer Inst* 68(4), 589-96. Suzuki, T., Bogenmann, E., Shimada, H., Stram, D., and Seeger, R. C. (1993). Lack of high-

neuroblastoma cell differentiation. *J Biol Chem* 285(6), 3561-7.

human neuroblastoma cell lines. *Oncogene* 3(3), 281-8.

neuroblastoma. *Nature* 313(6001), 404-6.

Biological effects of TrkA and TrkB receptor signaling in neuroblastoma. *Cancer Lett*

affinity nerve growth factor receptors in aggressive neuroblastomas. *J Natl Cancer*

T. (2010). Opposing effects of two tissue transglutaminase protein isoforms in

oncogenes is differentially regulated during retinoic acid induced maturation of

precedes retinoic acid-induced morphological differentiation of human

neurite outgrowth in human neuroblastoma SH-SY5Y cells. *Neuroscience* 102(2), 481-91.

and invasiveness of human neuroblastoma cells. *Cancer Res* 55, 1798-806. Matthay, K. K., Villablanca, J. G., Seeger, R. C., Stram, D. O., Harris, R. E., Ramsay, N. K.,

Children's Cancer Group. *N Engl J Med* 341(16), 1165-73.

regulate neuroblastoma differentiation. *Oncogene* 23(1), 213-25.

differentiation of neuroblastoma cells. *Oncogene* 10(1), 185-9.

of high-risk neuroblastoma. *Cancer Lett* 197(1-2), 185-92.

signaling. *Int J Dev Neurosci* 26(5), 497-503.

acetate. *Int J Cancer* 28, 583-9.

228, 143-53.

*Inst* 85(5), 377-84.

Norris, M. D., Diolaiti, D., Della Valle, G., Perini, G., and Marshall, G. M. (2007). Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis. *Proc Natl Acad Sci U S A* 104(47), 18682-7.

reduces Ret tyrosine 1062 phosphorylation and alters GDNF-mediated intracellular

brain-derived neurotrophic factor and p145TrkB affects survival, differentiation,

Swift, P., Shimada, H., Black, C. T., Brodeur, G. M., Gerbing, R. B., and Reynolds, C. P. (1999). Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid.

of human neuroblastoma cells in culture induced by 12-Otetradecanoyl-phorbol-13-

Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation. *Cell Differ* 14(2), 135-44. Peterson, S., and Bogenmann, E. (2004). The RET and TRKA pathways collaborate to

> Neuroblastoma (NB) 1 is the most common extracranial tumor in childhood and accounts for nearly half of neoplasms diagnosed during infancy (Maris, 2010; Brodeur, 2003). A striking feature of these tumors has been their heterogeneous course, which ranges from spontaneous regression to inevitable progression and death (Brodeur, 2003). Current pharmacological approaches in the treatment of NBs include standard combination chemotherapy using dose-intensive cycles of carboplatin, etoposide, cyclophosphamide, and doxorubicin, with the addition of topoisomerase I inhibitors. For intermediate-risk NB, a high rate of survival among patients may still be achieved with significant reduction of doses and duration of chemotherapy (Baker et al., 2010). The retinoic acid analogue isotretinoin (13-cis-retinoic acid) is additionally used in high-risk NB patients with progressive or recurrent disease (Maris, 2010; Reynolds et al., 2003).

> Animal cancer models have offered valuable preclinical testing systems for studying the impact of specific genes in the appearance and the progress of the disease as well as the efficacy of novel therapeutic regimes. Animal models of NB have been developed by subcutaneous inoculation (xenografting) of established human NB cell lines in immunocompromised mice; for instance, the cell line SK-N-BE2c was successfully used to develop an animal model and test the effects of imatinib (Meco et al., 2005). Yet, the major

 \* Corresponding Authors (George Leondaritis, Dimitra Mangoura)

<sup>1</sup> Abbreviations used: ERK, extracellular signal-regulated kinase; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; DAG, diacylglycerol; TH, tyrosine hydroxylase; GAP43, growthassociated protein 43; NPY, neuropeptide Y; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; dBcAMP, dibutyryl cAMP; BrdU, 5-bromo-2-deoxyuridine.

Interplay Between Protein Kinase C Isoforms

**2.1 The PKC/NF1/Ras/ERK pathway**

**neuroblastoma cells**

family (Durgan et al., 2008).

Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation 87

The PKC family of serine/threonine protein kinases consists of at least ten isoforms that are classified into three subgroups based on their structure and specific cofactor requirements. Conventional PKCs (cPKCs: α, βI, βII and γ) are activated by the second messengers Ca2+ and DAG, while novel PKCs (nPKCs: δ, ε, η and θ) are activated only by DAG. In contrast to conventional and novel, atypical PKCs (aPKCs: ζ and λ/ι) are insensitive to both Ca2+ and DAG, but responsive to the Par6–Cdc42 complex (Parker & Murray-Rust, 2004; Rosse et al., 2010). PKCs are considered hubs for the transduction of signals from G protein-coupled and tyrosine kinase receptors (Parker & Murray-Rust, 2004; Griner and Kazanietz 2007), having long been recognized as a link between receptor-dependent generation of DAG by phospholipases C and D, and the key event for enganging the ERK pathway, the activation of Ras and Raf (Mangoura and Dawson, 1993; Marais et al., 1998). Attesting to the widespread effects of PKC activation, numerous studies have investigated the involvement of all PKC isoforms in oncogenesis and cellular differentiation, proliferation, polarity, migration, apoptosis, and survival (Bosco et al., 2011; Rosse et al., 2010; Larsson, 2006). PKCs are founding members of the AGC kinase group and share a common structure consisting of a conserved C-terminal kinase catalytic domain and a more divergent Nterminal regulatory region. The regulatory region contains C1 and C2-domains (cPKC and nPKCs) that recognize the second messengers DAG/phospholipids or Ca2+/phospholipids, respectively, and a pseudosubstrate sequence that serves in autoinhibition by interacting with the substrate-binding pocket of the catalytic domain. Binding of second messengers or allosteric effectors on the C1/C2-domains of the regulatory region results in stabilized interaction with the plasma membrane and activation (Parker & Murray-Rust, 2004; Griner and Kazanietz 2007). PKCs are additionally regulated by specific phosphorylation events that "prime" the kinase for activation. As for all AGC kinases, PDK1 phosphorylates threonines (Thr) in the activation loop (Thr566 in PKCε). Residues in the turn motif (Thr710 in PKCε) and the C-terminal hydrophobic motif of c/nPKCs (Ser729 in PKCε) are often phosphorylated by the mTORC2 complex (Freeley et al., 2011). These phosphorylations critically depend on the occupation of the catalytic site by ATP, stabilize the active conformation of PKCs, and result in a fully primed kinase (Cameron et al., 2009). Additional auto- or in-trans phosphorylation events by other PKC isoforms may have more subtle effects, such as modulation of catalytic activity, protein stability, or binding to other proteins (Freeley et al., 2011). Specifically for PKCε, phosphorylation on Ser368 in the regulatory region by cPKCs suggests a high level of functional crosstalk within members of the PKC

By the acute and reversible post-translational modification of phosphorylation, PKCs regulate the activity and subcellular localization of several of their protein substrates. PKCs phosphorylate an array of substrates that include transcription factors, receptors, ion channels, and cytoskeletal proteins (Mangoura and Dawson 1993; Mangoura, 1997; Parker & Murray-Rust, 2004; Larsson, 2006; Mangoura et al., 2006a). In many cases, however, proof of direct PKC phosphorylation of substrates in vivo has remained limited, with several molecules representing remote "PKC targets". PKCs also act as scaffols, as they interact with other proteins independently of their enzymatic properties as kinases (e.g. Saurin et al.,

**2. PKC isoforms, NF1, and the Ras/ERK pathway in differentiation of**

**2.1.1 The PKC family: Structure, regulation, and substrates**

drawback was the lack of metastases. Pseudometastatic models were next developed by tail-vein injections of human NB cell lines in athymic mice (Pastorino et al., 2010). A clinically relevant model should, however, reflect the characteristics of advanced NB in children, that is large adrenal gland tumors and multiple small metastatic lesions. Such models have been generated only in nude mice with murine NB cells (NXS2) or by allografting human NB cell lines (Pastorino et al., 2010). Spontaneous adrenal neuroblastomas may be developed, yet rarely, in experimental animals (Rice, 2004), while, neither carcinogen-induced nor genetically engineered animal NB models have been successfully generated thus far.

Therefore, the development of novel therapeutic strategies, including both new targets and models, is urgently needed. Progress is expected through elucidation of the key molecular pathways that drive NB proliferation, differentiation, or apoptosis. Clinical trial data have also suggested that induced differentiation may be an alternative therapeutic approach, and retinoic acid analogues have been introduced in the clinical practice (Reynolds & Lie, 2000; Brodeur, 2003). The clinical observation of spontaneous differentiation into benign ganglioneuromas has provided the basis for studying neuronal differentiation of NB cells in culture (Reynolds & Lie, 2000; Edsjo et al., 2003; Hahn et al., 2008), and such studies have provided invaluable mechanistic insight into the fundamental mechanisms of neuronal differentiation and neurotransmitter phenotype acquisition (Mangoura et al., 2006b; Edsjo et al., 2007). The agents, mostly used to study NB cell differentiation in culture are phorbol esters, membrane permeable non-hydrolyzable cAMP analogues, and the clinically relevant vitamin A metabolite retinoic acid, alone or in combination with specific neurotrophic and growth factors (Table 1). These agents target distinct proximal signalling pathways: phorbol esters activate novel and conventional protein kinase C (PKC) isoforms, cAMP analogues activate protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs), while retinoic acid acts as a ligand for nuclear hormone receptors/transcription factors (RARs) (Table 1). Yet, a significant level of crosstalk amongst these agents has been demonstrated in the induction of NB differentiation. Activation of PKCs and the ensuing Ras/ERK signalling cascade have been highlighted as central modulators of NB differentiation, with novel (PKCε) and conventional (PKCα) PKC isoforms critically controlling the signalling output and dynamics of MAPKs (Griner & Kazanietz, 2007). The importance of the PKC/Ras/ERK pathway is further emphasized by recent studies showing that neurofibromin, a prominent tumor suppressor and a neuronal RasGAP protein (a) is a PKCα and PKCε substrate (Mangoura et al., 2006a) actively phosphorylated during phorbol ester-induced differentiation (Leondaritis et al., 2009) and (b) provides responsiveness to retinoic acid (Holzel et al., 2010). In these studies, the role of PKCα and PKCε may be viewed as differential and even opposing, with PKCε emerging as a crucial, neuronal differentiationspecific PKC isoform.

In this chapter, we introduce the basic aspects of the PKC/NF1/Ras/ERK pathway and its implications in neuronal differentiation, we discuss critical findings from studies with NB cells that highlight the importance of this pathway in NB differentiation, and present original experiments that further expand current knowledge. Finally, we propose that agents promoting NB differentiation via distinct primary targets may actually converge on establishing a balance between PKCα and PKCε activities that coordinates neurofibromin (NF1)-dependent Ras/ERK activation and NB differentiation.

## **2. PKC isoforms, NF1, and the Ras/ERK pathway in differentiation of neuroblastoma cells**

## **2.1 The PKC/NF1/Ras/ERK pathway**

86 Neuroblastoma – Present and Future

drawback was the lack of metastases. Pseudometastatic models were next developed by tail-vein injections of human NB cell lines in athymic mice (Pastorino et al., 2010). A clinically relevant model should, however, reflect the characteristics of advanced NB in children, that is large adrenal gland tumors and multiple small metastatic lesions. Such models have been generated only in nude mice with murine NB cells (NXS2) or by allografting human NB cell lines (Pastorino et al., 2010). Spontaneous adrenal neuroblastomas may be developed, yet rarely, in experimental animals (Rice, 2004), while, neither carcinogen-induced nor genetically engineered animal NB models have been

Therefore, the development of novel therapeutic strategies, including both new targets and models, is urgently needed. Progress is expected through elucidation of the key molecular pathways that drive NB proliferation, differentiation, or apoptosis. Clinical trial data have also suggested that induced differentiation may be an alternative therapeutic approach, and retinoic acid analogues have been introduced in the clinical practice (Reynolds & Lie, 2000; Brodeur, 2003). The clinical observation of spontaneous differentiation into benign ganglioneuromas has provided the basis for studying neuronal differentiation of NB cells in culture (Reynolds & Lie, 2000; Edsjo et al., 2003; Hahn et al., 2008), and such studies have provided invaluable mechanistic insight into the fundamental mechanisms of neuronal differentiation and neurotransmitter phenotype acquisition (Mangoura et al., 2006b; Edsjo et al., 2007). The agents, mostly used to study NB cell differentiation in culture are phorbol esters, membrane permeable non-hydrolyzable cAMP analogues, and the clinically relevant vitamin A metabolite retinoic acid, alone or in combination with specific neurotrophic and growth factors (Table 1). These agents target distinct proximal signalling pathways: phorbol esters activate novel and conventional protein kinase C (PKC) isoforms, cAMP analogues activate protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs), while retinoic acid acts as a ligand for nuclear hormone receptors/transcription factors (RARs) (Table 1). Yet, a significant level of crosstalk amongst these agents has been demonstrated in the induction of NB differentiation. Activation of PKCs and the ensuing Ras/ERK signalling cascade have been highlighted as central modulators of NB differentiation, with novel (PKCε) and conventional (PKCα) PKC isoforms critically controlling the signalling output and dynamics of MAPKs (Griner & Kazanietz, 2007). The importance of the PKC/Ras/ERK pathway is further emphasized by recent studies showing that neurofibromin, a prominent tumor suppressor and a neuronal RasGAP protein (a) is a PKCα and PKCε substrate (Mangoura et al., 2006a) actively phosphorylated during phorbol ester-induced differentiation (Leondaritis et al., 2009) and (b) provides responsiveness to retinoic acid (Holzel et al., 2010). In these studies, the role of PKCα and PKCε may be viewed as differential and even opposing, with PKCε emerging as a crucial, neuronal differentiation-

In this chapter, we introduce the basic aspects of the PKC/NF1/Ras/ERK pathway and its implications in neuronal differentiation, we discuss critical findings from studies with NB cells that highlight the importance of this pathway in NB differentiation, and present original experiments that further expand current knowledge. Finally, we propose that agents promoting NB differentiation via distinct primary targets may actually converge on establishing a balance between PKCα and PKCε activities that coordinates neurofibromin

(NF1)-dependent Ras/ERK activation and NB differentiation.

successfully generated thus far.

specific PKC isoform.

## **2.1.1 The PKC family: Structure, regulation, and substrates**

The PKC family of serine/threonine protein kinases consists of at least ten isoforms that are classified into three subgroups based on their structure and specific cofactor requirements. Conventional PKCs (cPKCs: α, βI, βII and γ) are activated by the second messengers Ca2+ and DAG, while novel PKCs (nPKCs: δ, ε, η and θ) are activated only by DAG. In contrast to conventional and novel, atypical PKCs (aPKCs: ζ and λ/ι) are insensitive to both Ca2+ and DAG, but responsive to the Par6–Cdc42 complex (Parker & Murray-Rust, 2004; Rosse et al., 2010). PKCs are considered hubs for the transduction of signals from G protein-coupled and tyrosine kinase receptors (Parker & Murray-Rust, 2004; Griner and Kazanietz 2007), having long been recognized as a link between receptor-dependent generation of DAG by phospholipases C and D, and the key event for enganging the ERK pathway, the activation of Ras and Raf (Mangoura and Dawson, 1993; Marais et al., 1998). Attesting to the widespread effects of PKC activation, numerous studies have investigated the involvement of all PKC isoforms in oncogenesis and cellular differentiation, proliferation, polarity, migration, apoptosis, and survival (Bosco et al., 2011; Rosse et al., 2010; Larsson, 2006).

PKCs are founding members of the AGC kinase group and share a common structure consisting of a conserved C-terminal kinase catalytic domain and a more divergent Nterminal regulatory region. The regulatory region contains C1 and C2-domains (cPKC and nPKCs) that recognize the second messengers DAG/phospholipids or Ca2+/phospholipids, respectively, and a pseudosubstrate sequence that serves in autoinhibition by interacting with the substrate-binding pocket of the catalytic domain. Binding of second messengers or allosteric effectors on the C1/C2-domains of the regulatory region results in stabilized interaction with the plasma membrane and activation (Parker & Murray-Rust, 2004; Griner and Kazanietz 2007). PKCs are additionally regulated by specific phosphorylation events that "prime" the kinase for activation. As for all AGC kinases, PDK1 phosphorylates threonines (Thr) in the activation loop (Thr566 in PKCε). Residues in the turn motif (Thr710 in PKCε) and the C-terminal hydrophobic motif of c/nPKCs (Ser729 in PKCε) are often phosphorylated by the mTORC2 complex (Freeley et al., 2011). These phosphorylations critically depend on the occupation of the catalytic site by ATP, stabilize the active conformation of PKCs, and result in a fully primed kinase (Cameron et al., 2009). Additional auto- or in-trans phosphorylation events by other PKC isoforms may have more subtle effects, such as modulation of catalytic activity, protein stability, or binding to other proteins (Freeley et al., 2011). Specifically for PKCε, phosphorylation on Ser368 in the regulatory region by cPKCs suggests a high level of functional crosstalk within members of the PKC family (Durgan et al., 2008).

By the acute and reversible post-translational modification of phosphorylation, PKCs regulate the activity and subcellular localization of several of their protein substrates. PKCs phosphorylate an array of substrates that include transcription factors, receptors, ion channels, and cytoskeletal proteins (Mangoura and Dawson 1993; Mangoura, 1997; Parker & Murray-Rust, 2004; Larsson, 2006; Mangoura et al., 2006a). In many cases, however, proof of direct PKC phosphorylation of substrates in vivo has remained limited, with several molecules representing remote "PKC targets". PKCs also act as scaffols, as they interact with other proteins independently of their enzymatic properties as kinases (e.g. Saurin et al.,

Interplay Between Protein Kinase C Isoforms

**2.1.3 NF1 and PKCε in neuronal differentiation**

differentiation.

Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation 89

documented later in cultured neurons, and neuroblastoma and glioma cell lines (Mangoura et al., 2006a). Moreover, phosphorylation of neurofibromin results in both its increased association with actin and enhancement of its GAP activity (Mangoura et al., 2006a). PKCphosphorylation sites on neurofibromin are present in the N-terminal CSRD domain (Mangoura et al., 2006a) and in the C-terminal domain (Leondaritis et al., 2009). The residue Ser2808 in the C-tail of neurofibromin is indeed a PKC-specific site and its phosphorylation correlates well with enhanced signalling through the Ras/ERK pathway in TPA-treated SH-SY5Y cells (Leondaritis et al., 2009). Interestingly, studies with glioblastoma cell lines or tumors have indicated that PKCα may promote proteasome-dependent neurofibromin proteolysis, hence insufficient neurofibromin RasGAP activity to control Ras and accelerated cellular proliferation (McGillicuddy et al., 2009). Whether neurofibromin phosphorylation on Ser2808 (or other sites) is directly involved is, however, unknown. In sharp contrast, phosphorylation of neurofibromin in-non tumor cellular contexts has been correlated with increased stability of the protein, at least in melanocytes (Kaufmann et al., 1999) and neurons (Mangoura et al., 2006a). In conclusion, PKCs have the potential to directly impact on the activation state of Ras by modulating the activity of both GEFs and GAPs in a variety of ways. It should be also noted that in certain cell lines, PKC-dependent ERK activation may occur independently of Ras, since the dominant-negative form of Ras (RasS17N) that resists GEF-dependent activation does not inhibit TPA-induced ERK activation (Ueda et al., 1996; Rubio et al., 2008 and refs. therein). Overall, c/nPKCs like PKCα, PKCη, PKCδ and PKCε and aPKCs have been suggested to provide an activatory input on ERK pathway mostly at the level of Raf (Ueda et al., 1996; Schönwasser et al., 1998; Paruchuri et al., 2002).

All major transduction molecules of the Ras/ERK pathway, namely Ras and Raf, are also potent inducers of differentiation and neuritic outgrowth in several neuronal cell lines (Wood et al., 1993; Olsson & Nanberg, 2001; Hynds et al., 2003). Moreover, it is the intensity and duration of Ras/ERK activation that determines the biological outcome. Seminal observations were first made in PC12 cells treated with EGF or NGF. Both growth factors activate the same Ras/ERK pathway, yet, transient ERK activation by EGF induces cell proliferation, whereas a sustained ERK activation by NGF induces neurite outgrowth and differentiation (Marshall, 1995 and refs. therein). The reasons for these fundamental differences are still debated (Santos et al., 2007; von Kriegsheim et al., 2009). At any rate, both nPKCs (for example PKCδ, Santos et al., 2007) and neurofibromin (von Kriegsheim et al., 2009) may actively mediate the long-lasting ERK activation that drives PC12 cell

Neurofibromin is highly expressed in neurons with lower levels of expression detected in oligodendrocytes, Schwann cells, astrocytes, and other cell types (Li et al., 2001 and refs therein). Support for a specific role of neurofibromin in neuronal differentiation derives from several studies which show that its expression is developmentally regulated in the CNS and dorsal root ganglia in mouse and chick embryos, and that its peaks in expression coincide with the onset of neuronal differentiation (Vogel et al., 1995; Li et al., 2001). Studies in mice where neurofibromin has null expression after genetic ablation of central exons that render the protein unstable (functional knockout) have revealed an essential role for neurofibromin in the dependence of PNS neurons to neurotrophins (Vogel et al., 1995). Specifically, larger numbers of sensory and sympathetic neurons survive and continue to

2008; Valcova et al., 2007). Mechanistically and pharmacologically important PKC-binding proteins are the RACKs (receptors for activated C-kinases), which stabilize the interaction of activated PKCs with plasma membrane (Schechtman & Mochly-Rosen, 2001). Studies of the PKC-RACK interactions by Mochly-Rosen and co-workers, have shown that isoform-specific activators or inhibitors may be rationally designed, from unique 7-8 amino acid sequences in each PKC or in their respective RACKs (Budas et al., 2007; Csukai & Mochly-Rosen, 1999). As such, εV1-2 and ψεRACK peptides, specific inhibitor and activator peptides for PKCε, respectively, and analogous peptides for other PKCs have been used in numerous studies in culture and in vivo with great success (Asimaki and Mangoura, 2011; Asimaki et al., 2011).

#### **2.1.2 The Ras/ERK pathway and modulation by PKCs**

Ras GTPases, are key molecules for growth and neurotrophic factor signalling through the three kinase MAPK module, consisting of Rafs (MAPKKK), MEK1/2 (MAPKK), and ERK1/2 (MAPK) (Raman et al., 2007 and refs. therein). All Ras proteins, namely H-, N- and K-Ras, cycle between active GTP-bound and inactive GDP-bound states; activation of guanine nucleotide exchange factors (GEFs) after membrane receptor stimulation promotes the exchange of GDP for GTP and activation of Ras (Buday & Downward, 2008). The opposite event is controlled by GTPase-activating proteins (GAPs) that activate the intrinsic GTPase activity of Ras and lead to GTP hydrolysis and Ras inactivation (Scheffzek et al., 1998). Ras-GTP recruits Raf to the membrane and together with other kinases activates Raf kinase activity (Stokoe and McCormick, 1997). Subsequently, relay phosphorylations through MEKs result in phosphorylation and activation of ERK, which is considered as the terminal effector of the pathway (Raman et al., 2007). The Ras/ERK pathway controls various cellular processes such as proliferation, migration, and differentiation. A recent review by Katz et al., provides an overview of the Ras/ERK pathway impact on cellular differentiation and oncogenesis (Katz et al., 2007). Our focus is the PKC input on the regulation and dynamics of activation of Ras and ERK.

Mechanistically, PKC activation is necessary for the formation of Ras and activated Raf-1 complexes (Marais et al. 1998; Hamilton et al., 2001). PKCs may directly phosphorylate Raf (Ueda et al., 1996), RasGEFs (Ebinu et al. 1998; Roose et al., 2005; Zheng et al., 2005), and RasGAPs (Izawa et al., 1996; Mangoura et al., 2006a; Leondaritis et al., 2009), hence regulating the output of Ras/ERK signalling at multiple levels. PKCs on GEFs: Members of the GRP family of RasGEFs possess C1-domains that "recognize" DAG produced by phospholipases in the membrane upon membrane receptor stimulation, and through these interactions translocate to the membranes too (Ebinu et al. 1998). The GEF activity of RasGRP1,3 is greatly enhanced by concurrent phosphorylation by DAG-activated nPKCs, at least in T- and B-cells (Roose et al., 2005; Zheng et al., 2005). PKCs may also activate SOS1, the other major RasGEF, directly by phosphorylation (Rubio et al., 2006) or indirectly, by recruiting Grb2/SOS1 complexes via the Syk tyrosine kinase (Kawakami et al., 2003). PKCs on RasGAPs: Most of the cellular RasGAP activity is attributed to p120GAP and neurofibromin, and earlier studies have suggested a PKC-dependent inhibition of RasGAP activity in certain cell types (Downward et al., 1990). PKCs indirectly modulate the GAP activity of p120GAP via its interaction with RACK1 (Koehler & Moran, 2001), and neurofibromin, the main neuronal RasGAP, is directly regulated by PKC. Neurofibromin is phosphorylated by PKCs in vitro, particularly at the C-terminal domain (Izawa et al., 1996). Evidence for direct PKCα and PKCε-dependent neurofibromin phosphorylation was documented later in cultured neurons, and neuroblastoma and glioma cell lines (Mangoura et al., 2006a). Moreover, phosphorylation of neurofibromin results in both its increased association with actin and enhancement of its GAP activity (Mangoura et al., 2006a). PKCphosphorylation sites on neurofibromin are present in the N-terminal CSRD domain (Mangoura et al., 2006a) and in the C-terminal domain (Leondaritis et al., 2009). The residue Ser2808 in the C-tail of neurofibromin is indeed a PKC-specific site and its phosphorylation correlates well with enhanced signalling through the Ras/ERK pathway in TPA-treated SH-SY5Y cells (Leondaritis et al., 2009). Interestingly, studies with glioblastoma cell lines or tumors have indicated that PKCα may promote proteasome-dependent neurofibromin proteolysis, hence insufficient neurofibromin RasGAP activity to control Ras and accelerated cellular proliferation (McGillicuddy et al., 2009). Whether neurofibromin phosphorylation on Ser2808 (or other sites) is directly involved is, however, unknown. In sharp contrast, phosphorylation of neurofibromin in-non tumor cellular contexts has been correlated with increased stability of the protein, at least in melanocytes (Kaufmann et al., 1999) and neurons (Mangoura et al., 2006a). In conclusion, PKCs have the potential to directly impact on the activation state of Ras by modulating the activity of both GEFs and GAPs in a variety of ways. It should be also noted that in certain cell lines, PKC-dependent ERK activation may occur independently of Ras, since the dominant-negative form of Ras (RasS17N) that resists GEF-dependent activation does not inhibit TPA-induced ERK activation (Ueda et al., 1996; Rubio et al., 2008 and refs. therein). Overall, c/nPKCs like PKCα, PKCη, PKCδ and PKCε and aPKCs have been suggested to provide an activatory input on ERK pathway mostly at the level of Raf (Ueda et al., 1996; Schönwasser et al., 1998; Paruchuri et al., 2002).

### **2.1.3 NF1 and PKCε in neuronal differentiation**

88 Neuroblastoma – Present and Future

2008; Valcova et al., 2007). Mechanistically and pharmacologically important PKC-binding proteins are the RACKs (receptors for activated C-kinases), which stabilize the interaction of activated PKCs with plasma membrane (Schechtman & Mochly-Rosen, 2001). Studies of the PKC-RACK interactions by Mochly-Rosen and co-workers, have shown that isoform-specific activators or inhibitors may be rationally designed, from unique 7-8 amino acid sequences in each PKC or in their respective RACKs (Budas et al., 2007; Csukai & Mochly-Rosen, 1999). As such, εV1-2 and ψεRACK peptides, specific inhibitor and activator peptides for PKCε, respectively, and analogous peptides for other PKCs have been used in numerous studies in culture and in vivo with great success (Asimaki and Mangoura, 2011; Asimaki et al., 2011).

Ras GTPases, are key molecules for growth and neurotrophic factor signalling through the three kinase MAPK module, consisting of Rafs (MAPKKK), MEK1/2 (MAPKK), and ERK1/2 (MAPK) (Raman et al., 2007 and refs. therein). All Ras proteins, namely H-, N- and K-Ras, cycle between active GTP-bound and inactive GDP-bound states; activation of guanine nucleotide exchange factors (GEFs) after membrane receptor stimulation promotes the exchange of GDP for GTP and activation of Ras (Buday & Downward, 2008). The opposite event is controlled by GTPase-activating proteins (GAPs) that activate the intrinsic GTPase activity of Ras and lead to GTP hydrolysis and Ras inactivation (Scheffzek et al., 1998). Ras-GTP recruits Raf to the membrane and together with other kinases activates Raf kinase activity (Stokoe and McCormick, 1997). Subsequently, relay phosphorylations through MEKs result in phosphorylation and activation of ERK, which is considered as the terminal effector of the pathway (Raman et al., 2007). The Ras/ERK pathway controls various cellular processes such as proliferation, migration, and differentiation. A recent review by Katz et al., provides an overview of the Ras/ERK pathway impact on cellular differentiation and oncogenesis (Katz et al., 2007). Our focus is the PKC input on the

Mechanistically, PKC activation is necessary for the formation of Ras and activated Raf-1 complexes (Marais et al. 1998; Hamilton et al., 2001). PKCs may directly phosphorylate Raf (Ueda et al., 1996), RasGEFs (Ebinu et al. 1998; Roose et al., 2005; Zheng et al., 2005), and RasGAPs (Izawa et al., 1996; Mangoura et al., 2006a; Leondaritis et al., 2009), hence regulating the output of Ras/ERK signalling at multiple levels. PKCs on GEFs: Members of the GRP family of RasGEFs possess C1-domains that "recognize" DAG produced by phospholipases in the membrane upon membrane receptor stimulation, and through these interactions translocate to the membranes too (Ebinu et al. 1998). The GEF activity of RasGRP1,3 is greatly enhanced by concurrent phosphorylation by DAG-activated nPKCs, at least in T- and B-cells (Roose et al., 2005; Zheng et al., 2005). PKCs may also activate SOS1, the other major RasGEF, directly by phosphorylation (Rubio et al., 2006) or indirectly, by recruiting Grb2/SOS1 complexes via the Syk tyrosine kinase (Kawakami et al., 2003). PKCs on RasGAPs: Most of the cellular RasGAP activity is attributed to p120GAP and neurofibromin, and earlier studies have suggested a PKC-dependent inhibition of RasGAP activity in certain cell types (Downward et al., 1990). PKCs indirectly modulate the GAP activity of p120GAP via its interaction with RACK1 (Koehler & Moran, 2001), and neurofibromin, the main neuronal RasGAP, is directly regulated by PKC. Neurofibromin is phosphorylated by PKCs in vitro, particularly at the C-terminal domain (Izawa et al., 1996). Evidence for direct PKCα and PKCε-dependent neurofibromin phosphorylation was

**2.1.2 The Ras/ERK pathway and modulation by PKCs**

regulation and dynamics of activation of Ras and ERK.

All major transduction molecules of the Ras/ERK pathway, namely Ras and Raf, are also potent inducers of differentiation and neuritic outgrowth in several neuronal cell lines (Wood et al., 1993; Olsson & Nanberg, 2001; Hynds et al., 2003). Moreover, it is the intensity and duration of Ras/ERK activation that determines the biological outcome. Seminal observations were first made in PC12 cells treated with EGF or NGF. Both growth factors activate the same Ras/ERK pathway, yet, transient ERK activation by EGF induces cell proliferation, whereas a sustained ERK activation by NGF induces neurite outgrowth and differentiation (Marshall, 1995 and refs. therein). The reasons for these fundamental differences are still debated (Santos et al., 2007; von Kriegsheim et al., 2009). At any rate, both nPKCs (for example PKCδ, Santos et al., 2007) and neurofibromin (von Kriegsheim et al., 2009) may actively mediate the long-lasting ERK activation that drives PC12 cell differentiation.

Neurofibromin is highly expressed in neurons with lower levels of expression detected in oligodendrocytes, Schwann cells, astrocytes, and other cell types (Li et al., 2001 and refs therein). Support for a specific role of neurofibromin in neuronal differentiation derives from several studies which show that its expression is developmentally regulated in the CNS and dorsal root ganglia in mouse and chick embryos, and that its peaks in expression coincide with the onset of neuronal differentiation (Vogel et al., 1995; Li et al., 2001). Studies in mice where neurofibromin has null expression after genetic ablation of central exons that render the protein unstable (functional knockout) have revealed an essential role for neurofibromin in the dependence of PNS neurons to neurotrophins (Vogel et al., 1995). Specifically, larger numbers of sensory and sympathetic neurons survive and continue to

Interplay Between Protein Kinase C Isoforms

IGF-1/bFGF ↑ neuronal markers

NGF ↑ neuronal markers

Adenosine ↑ neuronal markers

PACAP ↑ neuronal markers

(Monaghan et al., 2008); 9, (Silei et al., 2000)].

growth arrest, ↑ neuronal markers, morphology

growth arrest apoptosis/necrosis variable differentiation

growth retardation ↑ neuronal markers morphology

growth arrest ↑ neuronal markers morphology

growth inhibition cell cycle arrest ↑ neuronal markers

morphology

growth retardation ↑ neuronal markers morphology

**Retinoids**

RA

all-trans RA/13-cis

4-HPR (fenretinide)

**Phorbol esters**  TPA (12-Otetradecanoylphorbol-13-acetate)

**cAMP analogues**

**Growth factors**

**GPCR agonists**

dBcAMP

GDNF

**Other**

Uridine

Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation 91

**Agent Phenotype/Markers Signalling pathway Cell lines/Comments/**

RAR, PKCs, ATM, ERK (inconclusive)

RAR-independent, ceramide, PKCs, ROS

PKA/Epac, CREB,

PKC, ERK, PKA

Table 1. NB differentiating agents and signalling mechanisms studied; growth retardation refers to decreased proliferation rate; morphology refers to increased neuritic outgrowth measured as a percentage of neurite-bearing cells or average neurite length; neuronal markers refer to increases in neuron-specific enolase, NPY, GAP43, synaptophysin, and TH (mRNA or protein level); signalling pathway implication has been derived mostly by pharmacological and genetic manipulation studies. Only selected pathways are presented to best serve the focus of this chapter. Details of some of the experiments are discussed in the text and further information may be found in the respective references [1-3, (Edsjo et al., 2007; Janardhanan et al., 2009; Reynolds & Lie, 2000); 4-5, (Perez-Juste & Aranda, 1999; Fagerstrom et al., 1996); 6, (Peterson & Bogenmann, 2004); 7, (Canals et al., 2005); 8,

Studies with NB cell lines are subjected to some degree of heterogeneity in the response which may stem from: the passage number, culture conditions, concentration of agents, length of treatment, and certainly the read-out assay system. Thus conflicting reports are not rare, for example the NB cell line IMR32 has been described as resistant (Joshi et al., 2007),

morphology cAMP, ERK/p38 SH-SY5Y (8)

morphology PKCs, ERK, RAR<sup>β</sup> SH-SY5Y (4-5)

morphology TrkA, PKCs TrkA/SH-SY5Y, IMR32 (5)

**References**

Most NB cell lines, resistance, heterogeneity (see text)

RA-sensitive and resistant NB cell lines (1-3)

N-SH, SK-N-SN) (see text)

PKCs ( ERK, RARs) best studied in SH-SY5Y (also SK-

PI3K/ERK Most NB cell lines (see text)

RET/TrkA SH-SY5Y, LA-N-5 (6)

(receptor-specific) SH-SY5Y (7)

PKCε LAN-5 (9)

differentiate in the absence of NGF in culture (Vogel et al., 1995; Vogel et al., 2000; Zhu et al., 2001), in a Ras-depended manner (Vogel et al., 2000). Interestingly, functional ablation of neurofibromin in CNS neurons via a synapsin-Cre approach does not result in altered morphology or survival rates (Zhu et al., 2001). Analysis in the PC12 cell model, utilizing siRNA-downregulation or overexpression of neurofibromin dominant-negative construct approaches, has yielded somewhat contradictory results. In one study, neurofibromin silencing did not affect NGF-induced differentiation, yet, it enabled EGF to partially promote differentiation (von Kriegsheim et al., 2009). In other studies, overexpression of a neurofibromin-based dominant negative construct, known to increase Ras-GTP and p-ERK levels, or siRNA-silencing of neurofibromin resulted in inhibition of NGF-induced differentiation (Ynoue et al., 2003; Patrakitkomjorn et al. 2008). These disparing results may stem from differences in the time period of siRNA silencing, extent of neurofibromin downregulation, and concentrations and times of NGF treatment. Nonetheless, these data attest to a developmental time-dependent function of neurofibromin during neuronal differentiation.

Many studies in neuronal cell lines and primary neurons have implicated most of PKC isoforms in survival and differentiation mechanisms (Mangoura et al., 1993; Hundle et al., 1997; Lallemend et al., 2005; Shirai et al., 2008). In their majority, PKCs have been shown to have a positive role in neuritic outgrowth; yet, in a recent large-scale analysis in hippocampal neurons PKCι and PKCη scored as potent negative neurite growth regulators (Buchser et al., 2010). PKCε on the other hand is regarded as the isoform involved in differentiation of both CNS and PNS neurons (Mangoura et al., 1993; Larsson, 2006; Mangoura, 1997; Hundle et al., 1997; Shirai et al., 2008). In fact, in the developing chick brain, it is the major isoform found in early post-mitotic, just starting to differentiate neurons (Mangoura et al., 1993). A significant number of studies on the pro-differentiation role of PKCε have been actually performed on NB cell lines, as will be presented in more detail in the following sections.

#### **2.2 Neuroblastoma differentiating agents and underlying signalling mechanisms 2.2.1 Retinoids**

Agents, neurotransmitters, growth factors, and neurotrophic factors used in culture models of NB differentiation are summarized in Table 1. Typical differentiating agents are the retinoids all trans-RA, 13-cis RA, and N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretidine) (Reynolds & Lie, 2000), which cause growth arrest concomitantly with downregulation of MYCN expression, upregulation of an array of neuronal markers (neuron-specific enolase, neuropeptide Y, GAP43, MAP2, neurofilament-M, and synaptophysin), induction of neurite outgrowth, and increases in neurotransmitter biosynthetic enzyme activity and expression. The neurotransmitter phenotype induced by RA depends on the cell line, with cells developing sympathetic noradrenergic or cholinergic phenotypes (Edsjo et al., 2007 and refs. therein). Efforts to establish a consensus on what constitutes the RA-induced differentiation has recently led to recognition of a set of 10 genes as a potential signature and a more general yet reliable predictor of differentiation in NBs (Hahn et al., 2008). Moreover, a gene-expression high-throughput screening of small molecule libraries revealed a synergistic action of RA (all trans RA and 13-cis RA) and histone deacetylase inhibitors towards induction of NB differentiation, both in culture and in vivo (Hahn et al., 2008).

differentiate in the absence of NGF in culture (Vogel et al., 1995; Vogel et al., 2000; Zhu et al., 2001), in a Ras-depended manner (Vogel et al., 2000). Interestingly, functional ablation of neurofibromin in CNS neurons via a synapsin-Cre approach does not result in altered morphology or survival rates (Zhu et al., 2001). Analysis in the PC12 cell model, utilizing siRNA-downregulation or overexpression of neurofibromin dominant-negative construct approaches, has yielded somewhat contradictory results. In one study, neurofibromin silencing did not affect NGF-induced differentiation, yet, it enabled EGF to partially promote differentiation (von Kriegsheim et al., 2009). In other studies, overexpression of a neurofibromin-based dominant negative construct, known to increase Ras-GTP and p-ERK levels, or siRNA-silencing of neurofibromin resulted in inhibition of NGF-induced differentiation (Ynoue et al., 2003; Patrakitkomjorn et al. 2008). These disparing results may stem from differences in the time period of siRNA silencing, extent of neurofibromin downregulation, and concentrations and times of NGF treatment. Nonetheless, these data attest to a developmental time-dependent function of neurofibromin during neuronal

Many studies in neuronal cell lines and primary neurons have implicated most of PKC isoforms in survival and differentiation mechanisms (Mangoura et al., 1993; Hundle et al., 1997; Lallemend et al., 2005; Shirai et al., 2008). In their majority, PKCs have been shown to have a positive role in neuritic outgrowth; yet, in a recent large-scale analysis in hippocampal neurons PKCι and PKCη scored as potent negative neurite growth regulators (Buchser et al., 2010). PKCε on the other hand is regarded as the isoform involved in differentiation of both CNS and PNS neurons (Mangoura et al., 1993; Larsson, 2006; Mangoura, 1997; Hundle et al., 1997; Shirai et al., 2008). In fact, in the developing chick brain, it is the major isoform found in early post-mitotic, just starting to differentiate neurons (Mangoura et al., 1993). A significant number of studies on the pro-differentiation role of PKCε have been actually performed on NB cell lines, as will be presented in more

**2.2 Neuroblastoma differentiating agents and underlying signalling mechanisms**

Agents, neurotransmitters, growth factors, and neurotrophic factors used in culture models of NB differentiation are summarized in Table 1. Typical differentiating agents are the retinoids all trans-RA, 13-cis RA, and N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretidine) (Reynolds & Lie, 2000), which cause growth arrest concomitantly with downregulation of MYCN expression, upregulation of an array of neuronal markers (neuron-specific enolase, neuropeptide Y, GAP43, MAP2, neurofilament-M, and synaptophysin), induction of neurite outgrowth, and increases in neurotransmitter biosynthetic enzyme activity and expression. The neurotransmitter phenotype induced by RA depends on the cell line, with cells developing sympathetic noradrenergic or cholinergic phenotypes (Edsjo et al., 2007 and refs. therein). Efforts to establish a consensus on what constitutes the RA-induced differentiation has recently led to recognition of a set of 10 genes as a potential signature and a more general yet reliable predictor of differentiation in NBs (Hahn et al., 2008). Moreover, a gene-expression high-throughput screening of small molecule libraries revealed a synergistic action of RA (all trans RA and 13-cis RA) and histone deacetylase inhibitors towards induction of NB differentiation, both in culture and

differentiation.

detail in the following sections.

in vivo (Hahn et al., 2008).

**2.2.1 Retinoids**

Table 1. NB differentiating agents and signalling mechanisms studied; growth retardation refers to decreased proliferation rate; morphology refers to increased neuritic outgrowth measured as a percentage of neurite-bearing cells or average neurite length; neuronal markers refer to increases in neuron-specific enolase, NPY, GAP43, synaptophysin, and TH (mRNA or protein level); signalling pathway implication has been derived mostly by pharmacological and genetic manipulation studies. Only selected pathways are presented to best serve the focus of this chapter. Details of some of the experiments are discussed in the text and further information may be found in the respective references [1-3, (Edsjo et al., 2007; Janardhanan et al., 2009; Reynolds & Lie, 2000); 4-5, (Perez-Juste & Aranda, 1999; Fagerstrom et al., 1996); 6, (Peterson & Bogenmann, 2004); 7, (Canals et al., 2005); 8, (Monaghan et al., 2008); 9, (Silei et al., 2000)].

Studies with NB cell lines are subjected to some degree of heterogeneity in the response which may stem from: the passage number, culture conditions, concentration of agents, length of treatment, and certainly the read-out assay system. Thus conflicting reports are not rare, for example the NB cell line IMR32 has been described as resistant (Joshi et al., 2007),

Interplay Between Protein Kinase C Isoforms

NADPH oxidase system (Nitti et al, 2010).

**2.2.2 Phorbol esters**

considered.

induction of neuritic outgrowth (see below in section 2.2.2).

Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation 93

SH-SY5Y and LA-N-5 cells, ERK mediates an early (within 1h) RA-induced formation of promyelocytic leukemia nuclear bodies, an event associated with NB differentiation (Delaune et al., 2008). Apparently, ERK activity may impinge differentially on early and late gene regulation and inhibitor studies have not always addressed these issues with sufficient detail (Miloso et al., 2004; Lee & Kim, 2004; Delaune et al., 2008; Holzel et al., 2010). In addition, evidence from the various differentiation protocols and agents suggests that the transcription programs for neuritic outgrowth or expression of neuron-specific genes may be independently regulated in NB cells. In all, ERK activity appears to be crucial for the expression of neuron-specific genes, in the absence of a positive signalling stimulus for the

Besides its role as a differentiating agent, 13-cis-RA antagonizes significantly the cytotoxic effects of agents like etoposide and cisplatin (Hadjidaniel & Reynolds, 2010), which suggests another mechanism of impact of the RA responsiveness, this time involving the Ras/ERK pathway. For example, an inverse correlation between resistance to doxorubicin and ERK pathway activation has been suggested in SK-N-SH cells (Mattingly et al., 2001), while NFkB activation by doxorubicin and etoposide in SH-SY5Y cells, thought to be required for druginduced toxicity, depends also on Ras and MEK activities (Armstrong et al., 2006). With the newly established role of neurofibromin in RA responsiveness and its direct implication in signalling through the Ras/ERK pathway for neuronal differentiation, neurofibromin may indeed contribute to some extent in (the prevention of) cytotoxic responses, an aspect not yet

Experimental evidence from chemical inhibition or overexpression of dominant-negative mutants studies has implicated several PKC isoforms, mostly nPKCs, in RA-induced differentiation. RA-induced ERK activation in SH-SY5Y cells was significantly reduced in the presence of the c/nPKC inhibitor GF1092303X, as well as the RA-induced cell survival and neuritic outgrowth (Miloso et al., 2004). A peptide derived from the actin-binding site of PKCε has been shown to attenuate RA-induced neuritic outgrowth in SK-N-BE(2), while RA induced an increase of the cytoskeleton-associated PKCε pool (Zeidman et al., 2002). Besides PKCε, PKCθ and PKCδ have also been suggested to play a role in RA-induced differentiation (Nitti et al, 2010). In LAN-5 cells, RA-induced differentiation is inhibited by PKCθ-antisense oligonucleotides (Sparatore et al., 2000), while in SH-SY5Y cells, modulation of PKCδ activity by inhibition with rottlerin or overexpression of dominant-negative PKCδ suggested a positive role of PKCδ activity in SH-SY5Y differentiation via the function of

Long-term treatment of SH-SY5Y cells with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has been extensively studied as a NB and human sympathetic neuron differentiation model, providing instrumental insights on the role of PKC in NB differentiation. TPA, acting as a DAG analog, directly binds and activates c/nPKCs, the main transducers of the TPA differentiation signal. Treatment of SH-SY5Y cells with low nanomolar concentrations of TPA results in the acute activation of PKCs and progressively drives cells towards a well-described sympathetic phenotype. This phenotype is characterized by increased neuritic outgrowth and upregulation of neuron-specific genes, such as GAP-43, neuropeptide Y, and tyrosine hydroxylase (Pahlman et al. 1981; Troller et al. 2001; Olsson & Nanberg 2001), increases in noradrenaline content (Pahlman et al., 1984;

sensitive (Holzel et al., 2010), or weakly responsive to RA (Guzhova et al., 2001). Similarly, SK-N-BE2 cells have been characterized as RA-resistant in a long-term 25-30 day colony formation assay (Holzel et al., 2010), and as sensitive when increased neuritic outgrowth was recorded after 2-4 days of exposure (Zeidman et al., 1999a, 2002). For RA, in particular, this is a significant issue, since prediction of resistance to retinoid therapy is very important in the clinical setting. Until recently, no predictive markers of RA responsiveness were established for clinical use. A connection with amplification of MYCN has been suggested (Reynolds et al., 2000), yet, MYCN-overexpressing stable SK-N-SH cells retain their capacity to differentiate in response to a variety of agents, including RA (Edsjo et al., 2004). Several other genes investigated in NBs have not come through as predictors, and even RARs have been questioned, because RA-resistant cells are sensitive to fenretinide (N-(4 hydroxyphenyl) retinamide), the synthetic retinoid that acts independently of RARs (Reynolds et al., 2000; Reynolds and Lie, 2000). More recently, neurofibromin deficiency in NB cells was proposed as a strong predictor of RA responsiveness, as shRNA silencing of neurofibromin was shown to confer resistance to retinoic acid independently of MYCN expression. Neurofibromin-deficient cells continue to proliferate in the presence of low concentrations of RA, bypassing check points for growth arrest and induction of differentiation, and exhibiting reduced expression of RA-target genes, Ret included (Holzel et al., 2010). Expression of Ret, the GDNF receptor, is very important for differentiation of NB cells (Peterson & Bogenmann, 2004; Esposito et al., 2008), especially through its action on upregulation of TrkA, a powerful and favorable prognostic marker in NB tumors (Edsjo et al., 2007; Brodeur, 2003).

The need for prognostic markers and therapeutic targets has strongly driven research on the signalling mechanisms that regulate RA-induced NB differentiation. Apparently RARs constitute a major requirement for the action of RA, as RA confers a significant upregulation of RARβ that drives gains in growth arrest and morphological NB differentiation (Maden, 2007). Besides the well-appreciated role of RARβ, other key signalling pathways appear to contribute to or modulate the RA-induced NB differentiation. These pathways are rapidly engaged upon addition of RA and often persist over a long time period. Thus, RA added to SH-SY5Y or SK-N-BE rapidly (within 20 min) activates the ATM kinase (Fernandes et al., 2007), which is a component of the DNA damage signal transduction pathway. ATM's autophosphorylation persists for over 4 days and correlates with enhanced ATM-dependent CREB phosphorylation and cell differentiation (Fernandes et al., 2007). Importantly, ATM inhibition or depletion does not prevent RA-induced upregulation of target genes, rather impairs cell survival (Fernandes et al., 2007).

A large body of evidence suggests a positive role of Ras, Raf, and prolonged ERK activation in neuronal differentiation in PC12 cells and several other cellular models (see section 2.1.2), but the situation is certainly more complicated when RA-induced NB differentiation is concerned. ERK is invariably activated by RA in the short- (Delaune et al., 2008) or longterm (Lee & Kim, 2004; Miloso et al., 2004), yet inhibition of the ERK activating kinase MEK by U0126 or PD98059 has yielded contradictory results on whether RA-induced ERK activation is necessary for neuritic outgrowth in SH-SY5Y and SK-N-BE(2)C cells (Miloso et al., 2004; Lee & Kim, 2004). Stable overexpression of RKIP (Raf kinase inhibitor protein), a scaffold protein crucial for Raf and ERK pathway activation, accelerated the rate of neuritic outgrowth and increased the expression of neuronal markers in response to RA in SH-SY5Y cells, all concurrent with sustained ERK activation (Hellmann et al., 2010). Furthermore, in SH-SY5Y and LA-N-5 cells, ERK mediates an early (within 1h) RA-induced formation of promyelocytic leukemia nuclear bodies, an event associated with NB differentiation (Delaune et al., 2008). Apparently, ERK activity may impinge differentially on early and late gene regulation and inhibitor studies have not always addressed these issues with sufficient detail (Miloso et al., 2004; Lee & Kim, 2004; Delaune et al., 2008; Holzel et al., 2010). In addition, evidence from the various differentiation protocols and agents suggests that the transcription programs for neuritic outgrowth or expression of neuron-specific genes may be independently regulated in NB cells. In all, ERK activity appears to be crucial for the expression of neuron-specific genes, in the absence of a positive signalling stimulus for the induction of neuritic outgrowth (see below in section 2.2.2).

Besides its role as a differentiating agent, 13-cis-RA antagonizes significantly the cytotoxic effects of agents like etoposide and cisplatin (Hadjidaniel & Reynolds, 2010), which suggests another mechanism of impact of the RA responsiveness, this time involving the Ras/ERK pathway. For example, an inverse correlation between resistance to doxorubicin and ERK pathway activation has been suggested in SK-N-SH cells (Mattingly et al., 2001), while NFkB activation by doxorubicin and etoposide in SH-SY5Y cells, thought to be required for druginduced toxicity, depends also on Ras and MEK activities (Armstrong et al., 2006). With the newly established role of neurofibromin in RA responsiveness and its direct implication in signalling through the Ras/ERK pathway for neuronal differentiation, neurofibromin may indeed contribute to some extent in (the prevention of) cytotoxic responses, an aspect not yet considered.

Experimental evidence from chemical inhibition or overexpression of dominant-negative mutants studies has implicated several PKC isoforms, mostly nPKCs, in RA-induced differentiation. RA-induced ERK activation in SH-SY5Y cells was significantly reduced in the presence of the c/nPKC inhibitor GF1092303X, as well as the RA-induced cell survival and neuritic outgrowth (Miloso et al., 2004). A peptide derived from the actin-binding site of PKCε has been shown to attenuate RA-induced neuritic outgrowth in SK-N-BE(2), while RA induced an increase of the cytoskeleton-associated PKCε pool (Zeidman et al., 2002). Besides PKCε, PKCθ and PKCδ have also been suggested to play a role in RA-induced differentiation (Nitti et al, 2010). In LAN-5 cells, RA-induced differentiation is inhibited by PKCθ-antisense oligonucleotides (Sparatore et al., 2000), while in SH-SY5Y cells, modulation of PKCδ activity by inhibition with rottlerin or overexpression of dominant-negative PKCδ suggested a positive role of PKCδ activity in SH-SY5Y differentiation via the function of NADPH oxidase system (Nitti et al, 2010).

## **2.2.2 Phorbol esters**

92 Neuroblastoma – Present and Future

sensitive (Holzel et al., 2010), or weakly responsive to RA (Guzhova et al., 2001). Similarly, SK-N-BE2 cells have been characterized as RA-resistant in a long-term 25-30 day colony formation assay (Holzel et al., 2010), and as sensitive when increased neuritic outgrowth was recorded after 2-4 days of exposure (Zeidman et al., 1999a, 2002). For RA, in particular, this is a significant issue, since prediction of resistance to retinoid therapy is very important in the clinical setting. Until recently, no predictive markers of RA responsiveness were established for clinical use. A connection with amplification of MYCN has been suggested (Reynolds et al., 2000), yet, MYCN-overexpressing stable SK-N-SH cells retain their capacity to differentiate in response to a variety of agents, including RA (Edsjo et al., 2004). Several other genes investigated in NBs have not come through as predictors, and even RARs have been questioned, because RA-resistant cells are sensitive to fenretinide (N-(4 hydroxyphenyl) retinamide), the synthetic retinoid that acts independently of RARs (Reynolds et al., 2000; Reynolds and Lie, 2000). More recently, neurofibromin deficiency in NB cells was proposed as a strong predictor of RA responsiveness, as shRNA silencing of neurofibromin was shown to confer resistance to retinoic acid independently of MYCN expression. Neurofibromin-deficient cells continue to proliferate in the presence of low concentrations of RA, bypassing check points for growth arrest and induction of differentiation, and exhibiting reduced expression of RA-target genes, Ret included (Holzel et al., 2010). Expression of Ret, the GDNF receptor, is very important for differentiation of NB cells (Peterson & Bogenmann, 2004; Esposito et al., 2008), especially through its action on upregulation of TrkA, a powerful and favorable prognostic marker in NB tumors (Edsjo et

The need for prognostic markers and therapeutic targets has strongly driven research on the signalling mechanisms that regulate RA-induced NB differentiation. Apparently RARs constitute a major requirement for the action of RA, as RA confers a significant upregulation of RARβ that drives gains in growth arrest and morphological NB differentiation (Maden, 2007). Besides the well-appreciated role of RARβ, other key signalling pathways appear to contribute to or modulate the RA-induced NB differentiation. These pathways are rapidly engaged upon addition of RA and often persist over a long time period. Thus, RA added to SH-SY5Y or SK-N-BE rapidly (within 20 min) activates the ATM kinase (Fernandes et al., 2007), which is a component of the DNA damage signal transduction pathway. ATM's autophosphorylation persists for over 4 days and correlates with enhanced ATM-dependent CREB phosphorylation and cell differentiation (Fernandes et al., 2007). Importantly, ATM inhibition or depletion does not prevent RA-induced upregulation of target genes, rather

A large body of evidence suggests a positive role of Ras, Raf, and prolonged ERK activation in neuronal differentiation in PC12 cells and several other cellular models (see section 2.1.2), but the situation is certainly more complicated when RA-induced NB differentiation is concerned. ERK is invariably activated by RA in the short- (Delaune et al., 2008) or longterm (Lee & Kim, 2004; Miloso et al., 2004), yet inhibition of the ERK activating kinase MEK by U0126 or PD98059 has yielded contradictory results on whether RA-induced ERK activation is necessary for neuritic outgrowth in SH-SY5Y and SK-N-BE(2)C cells (Miloso et al., 2004; Lee & Kim, 2004). Stable overexpression of RKIP (Raf kinase inhibitor protein), a scaffold protein crucial for Raf and ERK pathway activation, accelerated the rate of neuritic outgrowth and increased the expression of neuronal markers in response to RA in SH-SY5Y cells, all concurrent with sustained ERK activation (Hellmann et al., 2010). Furthermore, in

al., 2007; Brodeur, 2003).

impairs cell survival (Fernandes et al., 2007).

Long-term treatment of SH-SY5Y cells with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has been extensively studied as a NB and human sympathetic neuron differentiation model, providing instrumental insights on the role of PKC in NB differentiation. TPA, acting as a DAG analog, directly binds and activates c/nPKCs, the main transducers of the TPA differentiation signal. Treatment of SH-SY5Y cells with low nanomolar concentrations of TPA results in the acute activation of PKCs and progressively drives cells towards a well-described sympathetic phenotype. This phenotype is characterized by increased neuritic outgrowth and upregulation of neuron-specific genes, such as GAP-43, neuropeptide Y, and tyrosine hydroxylase (Pahlman et al. 1981; Troller et al. 2001; Olsson & Nanberg 2001), increases in noradrenaline content (Pahlman et al., 1984;

Interplay Between Protein Kinase C Isoforms

as in the maintenance of elongated neurites (Sanchez et al., 2004).

Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation 95

cell lines, like PC12 and NB cells (SH-SY5Y and IMR32), respond to these powerful intracellular signal transducers with increased neuritic outgrowth and upregulation of neuronal markers, such as GAP43 and TH (e.g Christensen et al., 2003; Birkeland et al., 2009). The effect of cAMP analogues may also be reproduced with forskolin, a direct activator of adenylyl cyclase (AC). Forskolin models the established ability of many GPCR agonists coupled to Gs/AC activation to drive differentiation via increased production of cAMP in various NB and other cellular contexts (Monaghan et al., 2008; Canals et al., 2005). Two major pathways serve as proximal transducers of the cAMP signal: the Epac1/2 (exchange protein activated by cAMP)/Rap GTPase and the PKA pathways (Bos, 2006), both of which may contribute to differentiation (Christensen et al., 2003; Birkeland et al., 2009). In addition to PKA and Epac, PI3K has also been implicated in cAMP-induced differentiation of NB cells as well

Fig. 1. Upregulation of PKCε and downregulation of PKCα during cAMP-induced differentiation of IMR32 cells. Cells were incubated in MEM plus 10% heat-inactivated FBS in the

immunostaining, and microscopy (a), as well as Western blotting (b) and RT-PCR (c) were performed as described (e. g., Mangoura et al., 1993; Li et al., 2001; Mangoua et al., 2006b;

A typical example of cAMP-differentiated IMR32 cells is illustrated in Fig. 1a. After 4-6 days of 1 mM dBcAMP and 2.5 μM BrdU cells have acquired pyramidal-shaped cell bodies and

absence (control, C) or presence (D) of dBcAMP+ BrdU. Fixation, antibodies,

Leondaritis et al., 2009)

Heikkila et al. 1993), and development of membrane excitability (Jalonen & Akerman, 1988). Besides SH-SY5Y and the parental cell lines SK-N-SH and SK-N-SN, IMR32 cells also respond to TPA treatment with induction of neuronal genes that specify neurotransmitter phenotypes (Mangoura et al., 2006b). PKC activation may mediate significant crosstalk with the RARs, as TPA induces the upregulation of RARβ expression in an nPKC/Ras/Rafdependent manner (Perez-Juste & Aranda, 1999). Also in IMR32 cells, a synergistic transcriptional action of PKC and Ras with NF1 has been demonstrated in the induction of TH expression by TPA (Mangoura et al., 2006b).

The prominent role of PKCs in TPA-induced SH-SY5Y differentiation has been established in many studies and the most important aspects have been reviewed by Larsson, Pahlman, and co-workers (Larsson, 2006; Edsjo et al., 2007), where they note that PKCε is primarily responsible for TPA-induced differentiation, in particular for the induction of neuritic outgrowth. Characteristically, transfections with PKC constructs have suggested a dominant role of the PKCε regulatory domain as well as of its, unique amongst other PKCs, actinbinding properties in inducing neuritic outgrowth (Zeidman et al., 1999a, Zeidman et al., 2002) 2. Thus, the PKCε pro-differentiating effects may involve mechanisms that do not directly implicate its kinase activity (Zeidman et al., 1999a). The kinase activity is also very important, as c/nPKC inhibitors, such as GF1092303X and Ro-318220, have been repeatedly shown to effectively inhibit TPA-induced ERK activation (Olsson et al., 2000; Troller et al., 2001; Leondaritis et al., 2009), neurofibromin phosphorylation (Leondaritis et al., 2009), and, TPA-induced neuritic outgrowth, expression of neuronal markers, and increase of neurotransmitter content (Heikkila et al., 1993; Fagerstrom et al., 1996; Troller et al., 2001; Olsson et al., 2000). Yet, further elucidation of the roles of individual PKCs would greatly benefit from more specific ATP-targeted inhibitors of cPKCs and nPKCs (Way et al., 2000). In this context, studies that used long-term inhibition of cPKCs with Go6976 to assess proliferation and survival of NB cells need to be re-evaluated, in lieu of recent studies suggesting that (a) Go6976 is a highly potent inhibitor of Aurora A and B kinases (Stolz et al., 2009), (b) Aurora A is overexpressed in most NB cell lines and stage 3-4 NB tumors and (c) Aurora A inhibitors have broad anti-NB activity (Maris, 2010 and refs therein).

An important feature of TPA-induced differentiation of SH-SY5Y cells is the apparent bifurcation of proximal signalling requirements for neuritic outgrowth or neuronal marker expression. Indeed, inhibition of ERK activation by PD98059 abolishes TPA-induced upregulation of neuropeptide Y and GAP-43, but has no effect on neuritic outgrowth (Olsson & Nanberg, 2001). In agreement, inhibition of PKCβI by LY379196 reduces TPAinduced ERK activation, and expression of GAP43 and neuropeptide Y, yet has no effect on neuritic outgrowth (Troller et al., 2001). Collectively, most studies converge on two important aspects: (i) the occurrence of an active cPKCα and cPKCβΙ-ERK axis in regulation of the early (within 24h) induction of neuronal markers and (ii) the mandatory role of PKCε, via both catalytic and scaffolding mechanisms, in the later induction of neuritic outgrowth and maintenance of differentiation.

#### **2.2.3 Cyclic AMP analogues**

cAMP analogues, such as bi-butyryl-cAMP (dbcAMP), have been long used, often in combination with bromodeoxyuridine (BrdU), to induce NB differentiation. Many neuronal

<sup>2</sup>Florakis et al., 2006, Federation of European Neuroscience Societies (FENS) Forum 3:A051.5

Heikkila et al. 1993), and development of membrane excitability (Jalonen & Akerman, 1988). Besides SH-SY5Y and the parental cell lines SK-N-SH and SK-N-SN, IMR32 cells also respond to TPA treatment with induction of neuronal genes that specify neurotransmitter phenotypes (Mangoura et al., 2006b). PKC activation may mediate significant crosstalk with the RARs, as TPA induces the upregulation of RARβ expression in an nPKC/Ras/Rafdependent manner (Perez-Juste & Aranda, 1999). Also in IMR32 cells, a synergistic transcriptional action of PKC and Ras with NF1 has been demonstrated in the induction of

The prominent role of PKCs in TPA-induced SH-SY5Y differentiation has been established in many studies and the most important aspects have been reviewed by Larsson, Pahlman, and co-workers (Larsson, 2006; Edsjo et al., 2007), where they note that PKCε is primarily responsible for TPA-induced differentiation, in particular for the induction of neuritic outgrowth. Characteristically, transfections with PKC constructs have suggested a dominant role of the PKCε regulatory domain as well as of its, unique amongst other PKCs, actinbinding properties in inducing neuritic outgrowth (Zeidman et al., 1999a, Zeidman et al., 2002) 2. Thus, the PKCε pro-differentiating effects may involve mechanisms that do not directly implicate its kinase activity (Zeidman et al., 1999a). The kinase activity is also very important, as c/nPKC inhibitors, such as GF1092303X and Ro-318220, have been repeatedly shown to effectively inhibit TPA-induced ERK activation (Olsson et al., 2000; Troller et al., 2001; Leondaritis et al., 2009), neurofibromin phosphorylation (Leondaritis et al., 2009), and, TPA-induced neuritic outgrowth, expression of neuronal markers, and increase of neurotransmitter content (Heikkila et al., 1993; Fagerstrom et al., 1996; Troller et al., 2001; Olsson et al., 2000). Yet, further elucidation of the roles of individual PKCs would greatly benefit from more specific ATP-targeted inhibitors of cPKCs and nPKCs (Way et al., 2000). In this context, studies that used long-term inhibition of cPKCs with Go6976 to assess proliferation and survival of NB cells need to be re-evaluated, in lieu of recent studies suggesting that (a) Go6976 is a highly potent inhibitor of Aurora A and B kinases (Stolz et al., 2009), (b) Aurora A is overexpressed in most NB cell lines and stage 3-4 NB tumors and

(c) Aurora A inhibitors have broad anti-NB activity (Maris, 2010 and refs therein).

An important feature of TPA-induced differentiation of SH-SY5Y cells is the apparent bifurcation of proximal signalling requirements for neuritic outgrowth or neuronal marker expression. Indeed, inhibition of ERK activation by PD98059 abolishes TPA-induced upregulation of neuropeptide Y and GAP-43, but has no effect on neuritic outgrowth (Olsson & Nanberg, 2001). In agreement, inhibition of PKCβI by LY379196 reduces TPAinduced ERK activation, and expression of GAP43 and neuropeptide Y, yet has no effect on neuritic outgrowth (Troller et al., 2001). Collectively, most studies converge on two important aspects: (i) the occurrence of an active cPKCα and cPKCβΙ-ERK axis in regulation of the early (within 24h) induction of neuronal markers and (ii) the mandatory role of PKCε, via both catalytic and scaffolding mechanisms, in the later induction of neuritic outgrowth

cAMP analogues, such as bi-butyryl-cAMP (dbcAMP), have been long used, often in combination with bromodeoxyuridine (BrdU), to induce NB differentiation. Many neuronal

2Florakis et al., 2006, Federation of European Neuroscience Societies (FENS) Forum 3:A051.5

TH expression by TPA (Mangoura et al., 2006b).

and maintenance of differentiation.

**2.2.3 Cyclic AMP analogues**

cell lines, like PC12 and NB cells (SH-SY5Y and IMR32), respond to these powerful intracellular signal transducers with increased neuritic outgrowth and upregulation of neuronal markers, such as GAP43 and TH (e.g Christensen et al., 2003; Birkeland et al., 2009). The effect of cAMP analogues may also be reproduced with forskolin, a direct activator of adenylyl cyclase (AC). Forskolin models the established ability of many GPCR agonists coupled to Gs/AC activation to drive differentiation via increased production of cAMP in various NB and other cellular contexts (Monaghan et al., 2008; Canals et al., 2005). Two major pathways serve as proximal transducers of the cAMP signal: the Epac1/2 (exchange protein activated by cAMP)/Rap GTPase and the PKA pathways (Bos, 2006), both of which may contribute to differentiation (Christensen et al., 2003; Birkeland et al., 2009). In addition to PKA and Epac, PI3K has also been implicated in cAMP-induced differentiation of NB cells as well as in the maintenance of elongated neurites (Sanchez et al., 2004).